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INFLAMMATION AND IMMUNOPHARMACOLOGY

Methylenedioxymethamphetamine Suppresses Production of the Proinflammatory Cytokine Tumor Necrosis Factor- Independent of a -Adrenoceptor-Mediated Increase in Interleukin-10

Department of Physiology, Trinity College Institute of Neuroscience, Trinity College, Dublin, Ireland (T.J.C.).; and Department of Pharmacology, National University of Ireland, Galway, Ireland (T.J.C., A.H., J.P.K.)

Received for publication June 21, 2004
Accepted August 24, 2004.

	Abstract

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Recent data suggest that 3,4-methylenedioxymethamphetamine (MDMA; "Ecstasy") has marked immunosuppressive properties. In this study, we investigate the effect of MDMA on production of the anti-inflammatory cytokine interleukin (IL)-10 in response to an in vivo challenge with bacterial lipopolysaccharide (LPS). Our data demonstrate that both acute and repeated administration of MDMA increases production of LPS-induced IL-10 in vivo, and this increase correlates inversely with the ability of MDMA to suppress the proinflammatory cytokine tumor necrosis factor (TNF)-. Despite this correlation, immunoneutralization of IL-10 does not reverse the suppressive effect of MDMA on LPS-induced TNF- production, indicating that suppression of this proinflammatory cytokine is not mediated by IL-10. In vitro exposure to MDMA does not mimic the immunosuppressive cytokine phenotype induced in vivo, suggesting that these immunosuppressive effects are not mediated by a direct action on monocytes per se. As MDMA activates that hypothalamic pituitary adrenal axis and sympathetic nervous system, we examined the role of glucocorticoids and catecholamines in its immunosuppressive actions. However, the immunosuppressive cytokine phenotype induced by MDMA was not altered by adrenalectomy, sympathetic denervation, or ganglionic blockade, indicating that neither glucocorticoids nor adrenal/sympathetic-derived catecholamines mediate these immunosuppressive effects of MDMA. Interestingly, -adrenoceptor blockade completely inhibited the increase in IL-10 induced by MDMA without altering the suppression of TNF-. Taken together, these data demonstrate a role for -adrenoceptor activation in the ability of MDMA to increase LPS-induced IL-10 and highlight a mechanistic dissociation between the ability of MDMA to increase IL-10 and suppress production of the proinflammatory cytokine TNF-.

We previously demonstrated that MDMA suppresses production of proinflammatory cytokines, particularly tumor necrosis factor (TNF)- following an in vivo challenge with a subseptic dose of bacterial lipopolysaccharide (LPS) (Connor et al., 2000; 2001). This LPS challenge model essentially mimics the initial immune response to bacterial infection and enables us to examine the impact of pharmacological treatments on the immune response. Without stimulation with LPS, basal concentrations of cytokines are low and often undetectable; thus, an immunological challenge is required to stimulate cytokine production. For instance, LPS stimulates production of proinflammatory cytokines, including TNF-, from innate immune cells such as monocytes, macrophages, and dendritic cells (see Suzuki et al., 2003). TNF- is an important signaling molecule in initiating and coordinating a range of immune responses against invading pathogens (Hamblin, 1994); consequently, an inability to produce TNF- can result in defective host resistance to infection (Pasparakis et al., 1996).

In addition to producing proinflammatory cytokines when stimulated with LPS, innate immune cells also produce interleukin (IL)-10, an anti-inflammatory or immunosuppressive cytokine that inhibits several macrophage functions including proinflammatory cytokine production (Bogdan et al., 1992; Gerard et al., 1993). Thus, we suggest that an increase in IL-10 production in response to LPS has the propensity to mediate the suppression of TNF- induced by MDMA. Although the ability of MDMA to alter IL-10 production from cells of the innate immune system has not been examined to date, there is evidence that MDMA increases T-cell-derived IL-10 production (Pacifici et al., 2001b). In addition, another drug of abuse, namely tetrahydrocannabinol, increases innate IL-10 production in a sepsis model (Smith et al., 2000). Consequently, in this study, we examined the impact of MDMA on LPS-induced IL-10 production, and once we established that MDMA increased IL-10, we examined the possibility that the suppression of TNF- induced by MDMA was mediated by IL-10.

We previously demonstrated that the suppressive effect of MDMA on LPS-induced proinflammatory cytokine production observed in vivo could not be mimicked by in vitro exposure to the drug (Connor et al., 2000). Similarly, in this study, we observed that in vitro exposure to MDMA did not alter LPS-induced IL-10 production. In all, these data indicate that the ability of MDMA to promote an anti-proinflammatory cytokine phenotype is not due to a direct action on immune cells per se and is likely to be due to the release of endogenous immunomodulatory substances. In this regard, MDMA activates the sympathetic nervous system and hypothalamic pituitary adrenal axis (Nash et al., 1988; Grob et al., 1996), and we suggest that the end products of these pathways (catecholamines and glucocorticoids) could mediate MDMA-induced immunosuppression. A number of studies demonstrate that catecholamines increase IL-10 production in response to LPS and suppress LPS-induced TNF- production (Elenkov et al., 1995; Siegmund et al., 1998; Zinyama et al., 2001) and that these effects occur due to -adrenoceptor activation and increased intracellular cAMP production (Siegmund et al., 1998). For instance, in murine macrophages, elevated cAMP suppresses LPS-induced TNF- production by increasing the production of IL-10 (Arai et al., 1995). Therefore, catecholamines present themselves as likely mediators of MDMA-induced suppression of TNF- either directly or via increasing production of IL-10. A vast literature also indicates that glucocorticoids have immunosuppressive properties, including the ability to suppress production of proinflammatory cytokines (Zuckerman et al., 1989). Thus, we suggest that glucocorticoids could potentially mediate the immunosuppressive actions of MDMA. It is also noteworthy that activation of the sympathetic nervous system has been shown to mediate immunosuppressive effects of other drugs of abuse such as morphine and D-amphetamine (Pezzone et al., 1992; Bencsics et al., 1997), and it has been recently demonstrated that glucocorticoids mediate the immunosuppressive actions of cocaine withdrawal (Avila et al., 2003). Consequently, in this study, we examined the role of glucocorticoids and catecholamines in the ability of MDMA to promote an immunosuppressive cytokine phenotype in our model. Although previous studies have examined the central drivers of MDMA-induced immunosuppression (Connor et al., 2001; Pacifici et al., 2004), to our knowledge, this is the first study to investigate peripheral mediators of MDMA-induced immunosuppression.

	Materials and Methods

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Animals
Male Sprague-Dawley rats weighing approximately 250 to 350 g were obtained either from a breeding colony in the Department of Pharmacology (National University of Ireland, Galway, UK) or from Harlan (Bicester, UK) and were housed four per cage. Rats were maintained on a 12-/12-h light/dark cycle (lights on at 8 AM) in a temperature-controlled room (22 ± 2°C), and food and water were available ad libitum at all times. The experimental protocols were in compliance with the European Communities Council directive (86/609/EEC).

Drugs
MDMA was obtained from NIDA (Research Triangle Park, NC). Chlorisondamine was obtained from Novartis (Basel, Switzerland). Lipopolysaccharide (Escherichia coli serotype 0111:B4), 6-hydroxydopamine, and Nadolol were all obtained from Sigma-Aldrich (St. Louis, MO). All drugs were dissolved in 0.89% NaCl and administered via the i.p. route in an injection volume of 1 ml/kg, and 0.89% NaCl was administered alone as a vehicle to control animals.

Normal sheep serum and anti-IL-10 antiserum were obtained from Dr. Steve Poole (NIBSC, Potters Bar, Hertsfordshire, UK).

Experimental Design
Effect of MDMA on Production of the Anti-Inflammatory Cytokine IL-10: Time Course. Rats received either vehicle or MDMA (5 mg/kg i.p.) coadministered with LPS (100 µg/kg i.p.), and were killed 30, 60, 120, or 240 min later by decapitation. Following decapitation, trunk blood was collected from each animal, centrifuged (800g at 4°C for 15 min), and the resultant serum was frozen immediately and stored at –80°C until the IL-10 assay was performed.

We previously found that this dose and route of administration of LPS produces quantifiable increases in circulating IL-10 and TNF- concentrations and that 1 h post-LPS is the optimal time point for simultaneous sampling of these two cytokines (Connor and Kelly, 2002). The dose of LPS used in this study and in our previous studies (Connor et al., 2000, 2001; Connor and Kelly, 2002) is a subseptic dose that is approximately 25-fold lower than the LD₅₀ (Hawes et al., 1992) and provokes modest stimulation of the immune response in rats characterized by a transient increase in cytokine production.

Dose Response. Vehicle or MDMA (1.25–10 mg/kg i.p.) was coadministered with LPS and animals killed by decapitation 60 min later. Serum was prepared and stored for IL-10 analysis.

In Vitro Effect of MDMA. A diluted whole-blood method was used for the assessment of cytokine production ex vivo as previously described (Connor and Kelly, 2002). In diluted whole blood, the natural cell-cell interactions are preserved, whereas the methods used to isolate peripheral blood mononuclear cells modify the lymphocyte/monocyte ratio and eliminate endogenous immunomodulatory agents. Thus, in vivo conditions are better represented using whole-blood culture methods. Aliquots (900 µl) of diluted whole blood obtained from naive rats were pipetted into wells of a sterile flat-bottomed 48-well plate (NUNC A/S, Roskilde, Denmark). To each well was added either 100 µl of RPMI 1640 culture medium alone (control) or 100 µl of MDMA at a final concentration of 0.1, 0.5, 1, 2.5, 5, or 10 µg/ml dissolved in RPMI 1640. Following a 1-h preincubation period with MDMA, 100 µl of LPS (Sigma Chemical, Poole, Dorset, UK) at a final concentration of 10 µg/ml was added to each well, and cultures were incubated for a further 24 h at 37°C in a 5% CO₂ atmosphere. At the end of the culture period, the supernatants were harvested and stored at –80°C until cytokine assays were performed.

Examination of the Role of Increased IL-10 Production in the Suppressive Effect of MDMA on TNF-
Correlation between Increased IL-10 and Reduced TNF-. Rats were treated with either an acute (one dose) or repeated (nine doses) regimen of MDMA (2 mg/kg i.p). Rats in the control group received nine injections of 0.89% saline, and rats in the acute MDMA group received eight injections of 0.89% saline and a final injection of MDMA (2 mg/kg i.p.), whereas rats in the repeated MDMA group received nine injections of MDMA (2 mg/kg i.p.). Each animal received three injections per day with an interval of 3 h between each injection, and this treatment regimen continued for 3 consecutive days. The last dose of vehicle or MDMA was coadministered with LPS (100 µg/kg i.p.), and animals were killed by decapitation 60 min later. Serum was prepared and stored as described in study I prior to IL-10 and TNF- analysis.

Immunoneutralization. In this study, rats were pretreated with either anti-IL-10 antisera or normal sheep serum (1 ml/rat s.c.) immediately prior to administration of MDMA (5 mg/kg i.p.) and LPS (100 µg/kg i.p.), and blood samples were obtained by cardiac puncture 1 h later under halothane anesthesia. Serum was prepared and stored for analysis of IL-10 and TNF-.

The anti-IL-10 antiserum used in the present study was previously shown to neutralize IL-10 in the rat (Cartmell et al., 2003; Souza et al., 2003).

Examination of the Role of Glucocorticoids and Catecholamines in the Ability of MDMA to Increase IL-10 and Suppress TNF-
Adrenalectomy. Sham-operated and adrenalectomized rats were purchased from Charles River (Margate, Kent, UK). Following arrival in the laboratory, adrenalectomized rats were provided with saline solution (0.5%) supplemented with corticosterone (25 µg/ml in 0.2% ethanol) to restore basal corticosterone concentrations (see Houghtling and Bayer, 2002). Sham-operated rats were provided with drinking water containing 0.2% ethanol. Rats were given 10 days to acclimate to the laboratory prior to experimentation. In this experiment, vehicle or MDMA (2 mg/kg i.p.) was coadministered with LPS, and animals were killed 60 min later by decapitation. Serum was prepared and stored for analysis of IL-10 and TNF- analysis.

Adrenalectomy was verified at the end of the experiment by measuring serum corticosterone concentrations using a commercially available OCTEIA corticosterone enzyme immunoassay kit (IDS, Boldon, UK) and was performed as per manufacturer's instructions.

Chemical Sympathectomy. 6-hydroxydopamine was dissolved in phosphate-buffered saline supplemented with 0.01% ascorbic acid. On the 1st day, animals received 6-hydroxydopamine at a dose of 40 mg/kg. The following 2 days, animals received an 80 mg/kg dose of 6-hydroxydopamine; thus, over a 3-day period, animals received a total of 200 mg/kg 6-hydroxydopamine. The regime of 6-hydroxydopamine treatment used in the present study was previously shown to induce a 90% depletion in splenic noradrenaline concentrations (see Exton et al., 2002). Three days after the last 6-hydroxydopamine treatment, vehicle or MDMA (2 mg/kg i.p.) was coadministered with LPS and animals killed 60 min later by decapitation. Serum was prepared and stored for IL-10 and TNF- analysis.

The degree of sympathectomy was determined by measuring splenic noradrenaline concentrations. Noradrenaline was measured using a high-performance liquid chromatography system coupled with electrochemical detection as previously described. A piece of spleen tissue was homogenized in 1 ml of high-performance liquid chromatography mobile phase spiked with 20 ng/20 µl of N-methyl serotonin as internal standard. Mobile phase contained 0.1 M citric acid, 0.1 M sodium dihydrogen phosphate, 0.1 M EDTA, 1.4 mM octane-1-sulfonic acid, and 10% (v/v) methanol and was adjusted to pH 2.8 using 4 N sodium hydroxide. Homogenates were centrifuged at 15,000g for 15 min, and a 15-µl sample of the resultant supernatant was injected onto a reverse phase column (LI Chrosorb RP-18, 25-cm x 4-mm internal diameter, particle size 5 µm) for separation of the neurotransmitters (flow rate 1 ml/min). Noradrenaline concentrations were quantified by electrochemical detection (Shimadzu, Kyoto, Japan), and chromatograms were generated using a Merck-Hitachi D-2000 integrator. Protein concentrations were determined using the Bradford dye-binding assay, and noradrenaline concentrations were expressed as nanograms per milligram of protein.

Ganglionic Blockade. In this study, rats were pretreated with either vehicle or chlorisondamine (0.5 or 1 mg/kg i.p.) 30 min prior to administration of MDMA (2 mg/kg i.p.) and LPS (100 µg/kg i.p.) and were killed 60 min later by decapitation. Serum was prepared and stored for IL-10 and TNF- analysis.

-Adrenoceptor Antagonism. In this study, rats were pretreated with either vehicle or nadolol (0.25 mg/kg i.p.) 30 min prior to administration of MDMA (2 mg/kg i.p.) and LPS (100 µg/kg i.p.) and were killed 60 min later by decapitation. Serum was prepared and stored for IL-10 and TNF- analysis.

Cytokine Determinations. TNF- concentrations were determined using specific rat enzyme-linked immunosorbent assays (ELISAs) performed using antibodies and standards obtained from Dr. S. Poole (NIBSC) as previously described (Connor and Kelly, 2002). Similarly, IL-10 concentrations were determined by ELISA performed using antibodies and standards (rat IL-10 cytoset) obtained from BioSource International (Camarillo, CA) as previously described (Connor and Kelly, 2002).

Statistical Analysis of Data. Data were analyzed using a Student's t test or a one- or two-way analysis of variance (ANOVA) followed by post hoc comparisons using Newman-Keuls test. Data were deemed significant when P < 0.05 and expressed as group means with S.E.M.

	Results

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Effect of MDMA on Production of the Anti-Inflammatory Cytokine IL-10
To determine whether MDMA could alter production of the anti-inflammatory cytokine IL-10, we examined the effect of in vivo and in vitro exposure to MDMA on LPS-induced IL-10 production.

In Vivo Administration of MDMA
Administration of MDMA (5 mg/kg) to rats significantly increased LPS-induced IL-10 production 30 min to 2 h following LPS challenge. When the areas under the curves were analyzed, it was revealed that the overall IL-10 response was increased almost 3-fold by treatment with MDMA (Fig. 1a). In the dose-response study, it was observed that MDMA (1.25–10 mg/kg) produced a significant increase in circulating IL-10 concentrations following the in vivo LPS challenge (Fig. 1b). A maximal response was induced by MDMA (2.5 mg/kg), with no additional increase observed following administration of MDMA (5–10 mg/kg). We also examined the ability of MDMA to alter circulating IL-10 concentrations under basal conditions, i.e., in the absence of an immunological challenge. When MDMA (5 mg/kg) was administered to rats in the absence of LPS, it produced a modest but statistically insignificant (P = 0.082; Student's t test) increase in circulating IL-10 concentrations 1 h later: vehicle, 7.5 ± 4.2 pg/ml; and MDMA, 17.4 ± 3.2 pg/ml (n = 7).

View larger version (20K): [in this window] [in a new window]   Fig. 1. MDMA stimulates a time-related and dose-dependent increase in IL-10 production in response to an in vivo LPS challenge. a, MDMA (5 mg/kg) was coadministered with LPS (100 µg/kg) and animals were killed 30, 60, 120, and 240 min following LPS challenge for the measurement of IL-10. ANOVA demonstrated a significant MDMA x time interaction [F(4,65) = 27.81, P < 0.0001]. b, MDMA (1.25–10 mg/kg) was coadministered with LPS and animals were killed 60 min following LPS challenge for the measurement of IL-10. ANOVA demonstrated a significant effect of MDMA treatment [F(4,34) = 22.10, P < 0.0001]. Data expressed as means ± S.E.M. (n = 6–8). *, P < 0.05; **, P < 0.01 versus vehicle (Newman-Keuls test).

In Vitro Administration of MDMA
In contrast to the robust increase in LPS-induced IL-10 production observed in vivo, in vitro exposure to MDMA (0.1–10 µg/ml) failed to alter LPS-induced IL-10 production in diluted whole-blood culture (Fig. 2).

View larger version (13K): [in this window] [in a new window]   Fig. 2. In vitro exposure of diluted whole blood to MDMA fails to alter LPS-induced IL-10 production. Diluted whole-blood cultures were preincubated with MDMA (1–10 µg/ml) for 1 h prior to stimulation with LPS (10 µg/ml), and cell culture supernatants were harvested 24 h later for the measurement of IL-10. Data expressed as means ± S.E.M. (n = 7–8).

Examination of the Role of Increased IL-10 Production in the Suppressive Effect of MDMA on TNF-
To determine whether the observed increase in IL-10 could account for the ability of MDMA to suppress production of the proinflammatory cytokine TNF- (Connor et al., 2000, 2001), we first determined if there was a correlation between the effect of MDMA on IL-10 and TNF- production following both acute and repeated treatment. The results indicate that acute and repeated treatment with MDMA produced a significant increase in IL-10 that was accompanied by a suppression of TNF- production. Moreover, correlational analysis demonstrated a significant (P < 0.01) inverse correlation (R = –0.62) between LPS-induced IL-10 and TNF- production (Fig. 3).

View larger version (15K): [in this window] [in a new window]   Fig. 3. The increase in IL-10 induced by MDMA is observed following both acute and repeated treatment and is correlated with a suppression of TNF- production. Rats were treated with either an acute (one dose) or repeated (nine doses) regimen of MDMA (2 mg/kg/dose). The last dose of MDMA was coadministered with LPS (100 µg/kg), and animals were killed 60 min following LPS challenge. Concentrations of IL-10 (a) and TNF- (b) were determined in serum, and a correlational analysis was performed (c). ANOVA demonstrated a significant effect of MDMA on LPS-induced IL-10 [F(2,15) = 24.51, P < 0.001] and TNF- [F(2,15) = 7.26, P < 0.01] production. Data expressed as means ± S.E.M. (n = 6). **, P < 0.01 versus Vehicle (Newman-Keuls test).

Because a strong inverse correlation was observed, we then determined if immunoneutralization of IL-10 could inhibit the suppressive effect of acute MDMA administration on LPS-induced TNF- production. The results demonstrate that MDMA induced a significant increase in LPS-induced IL-10 production, and this was abrogated in those animals treated with the anti-IL-10 antiserum (Fig. 4a). However, MDMA induced a significant decrease in TNF- production in both control and IL-10-depleted animals (Fig. 4b). Thus, immunoneutralization of IL-10 fails to block the suppressive effect of MDMA on LPS-induced TNF- production. The data presented in Fig. 4a demonstrate that the ELISA could not detect IL-10 in control animals pretreated with the anti-IL-10 antiserum. In contrast, in the MDMA-challenged rats treated with the anti-IL-10 antiserum, IL-10 was clearly detectable (360 ± 180 pg/ml). These data indicate that the anti-IL-10 antisera did not interfere with the IL-10 assay per se, because the assay could detect an increase in circulating IL-10 following MDMA administration.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Immunoneutralization of IL-10 fails to block the suppressive effect of MDMA on LPS-induced TNF- production. Rats were treated with either normal sheep serum or anti-IL-10 antiserum immediately before MDMA (5 mg/kg) and LPS (100 µg/kg). Blood was collected 60 min following LPS challenge and concentrations of IL-10 (a) and TNF- (b) were determined in the serum. ANOVA demonstrated a significant effect of MDMA [F(1,16) = 14.36, P < 0.01] and a significant effect of the anti-IL-10 antiserum [F(1,16) = 11.12, P < 0.01] on IL-10 production. There was also a significant effect of MDMA treatment on LPS-induced TNF- production [F(1,16) = 49.15, P < 0.001]. Data expressed as means ± S.E.M. (n = 5). *, P < 0.05 versus saline-treated counterparts (Newman-Keuls test).

Examination of the Role of Glucocorticoids and Catecholamines in the Ability of MDMA to Increase IL-10 and Suppress TNF-
Adrenalectomy. To test the hypothesis that MDMA promotes an immunosuppressive cytokine phenotype (characterized by increased IL-10 and reduced TNF- production) via release of glucocorticoids or catecholamines from the adrenal gland, we examined the effect of MDMA on LPS-induced IL-10 and TNF- production in adrenalectomized rats. The results demonstrate that MDMA-induced a significant increase in serum IL-10 and a significant decrease in TNF- levels in both sham and adrenalectomized animals (Fig. 5, a and b). Thus, adrenalectomy failed to block the ability of MDMA to promote an immunosuppressive cytokine phenotype.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Adrenalectomy fails to block the ability of MDMA to increase IL-10 and suppress TNF- production. Either sham or adrenalectomized rats were treated with MDMA (2 mg/kg) coadministered with LPS (100 µg/kg) and were killed 60 min following LPS challenge. Concentrations of IL-10 (a) and TNF- (b) were determined in the serum. Adrenalectomy was verified by measuring plasma corticosterone (c). ANOVA demonstrated a significant effect of MDMA on serum IL-10 [F(1,25) = 97.61, P < 0.0001] and on serum TNF- [F(1,25) = 14.55, P < 0.001] concentrations and a significant effect of adrenalectomy on circulating corticosterone concentrations [F(1,25) = 99.48, P < 0.0001]. Data expressed as means ± S.E.M. (n = 6–8). **, P < 0.01 versus vehicle; ++, P < 0.01 versus sham-operated counterparts (Newman-Keuls test).

Adrenalectomy was verified by measuring serum corticosterone concentrations. The results show that adrenalectomy drastically reduced circulating corticosterone concentrations in both vehicle- and MDMA-treated rats, in comparison with their sham-operated counterparts. There was also a modest but nonsignificant elevation in circulating corticosterone concentrations in the MDMA-treated sham group, in comparison with their vehicle-treated counterparts (Fig. 5c).

Chemical Sympathectomy. The results revealed that MDMA induced a significant increase in serum IL-10 levels in both control (nonsympathectomized) and 6-hydroxydopamine-treated (sympathectomized) animals (Fig. 6a). MDMA provoked a characteristic decrease in serum TNF- concentrations in nonsympathectomized animals. However, pretreatment with 6-hydroxydopamine alone also significantly suppressed LPS-induced TNF- production. Although MDMA also suppressed TNF- in sympathectomized animals, this effect was not significant due to the suppression of TNF- production induced by 6-hydroxydopamine alone (Fig. 6b). Therefore, chemical sympathectomy using 6-hydroxydopamine fails to block the ability of MDMA to promote an immunosuppressive cytokine phenotype.

View larger version (14K): [in this window] [in a new window]   Fig. 6. Chemical sympathectomy fails to block the ability of MDMA to increase IL-10 and suppress TNF- production. Either vehicle- or 6-hydroxydopamine-treated rats were treated with MDMA (2 mg/kg) coadministered with LPS (100 µg/kg) and were killed 60 min following LPS challenge. Concentrations of IL-10 (a) and TNF- (b) were determined in the serum, and sympathectomy was verified by measuring splenic noradrenaline concentrations (c). ANOVA, a significant effect of MDMA on serum IL-10 concentrations [F(1,20) = 19.36, P < 0.001], and a significant effect of MDMA [F(1,20) = 10.16, P < 0.01] and 6-hydroxydopamine [F(1,20) = 9.46, P < 0.01] on serum TNF- concentrations. There was also significant effect of 6-hydroxydopamine treatment on splenic noradrenaline concentrations [F(1,20) = 15.73, P < 0.001]. Data expressed as means ± S.E.M. (n = 5–7). **, P < 0.01 versus saline-challenged counterparts; +, P < 0.05 versus vehicle + saline; ++, P < 0.01 versus nonsympathectomized counterparts (Newman-Keuls test).

Sympathectomy was verified by measuring splenic noradrenaline concentrations. The results demonstrate that 6-hydroxydopamine dramatically reduced splenic noradrenaline concentrations in both vehicle- and MDMA-treated rats. There was also a modest but nonsignificant reduction in splenic noradrenaline concentrations in the vehicle/MDMA group when compared with the vehicle/vehicle group (Fig. 6c).

Ganglionic Blockade. The results demonstrate that MDMA induced a significant increase in serum IL-10 levels, and this effect was not altered by pretreatment with either dose of chlorisondamine (Fig. 7a). MDMA also provoked a characteristic decrease in serum TNF- concentrations, which was not altered by pretreatment with chlorisondamine (0.5 mg/kg). Pretreatment with chlorisondamine (1 mg/kg) significantly suppressed LPS-induced TNF- production in a similar manner to MDMA; therefore, no significant difference was seen between these two treatment groups (Fig. 7b). Thus, ganglionic blockade with chlorisondamine fails to block the ability of MDMA to promote an immunosuppressive cytokine phenotype.

View larger version (14K): [in this window] [in a new window]   Fig. 7. Ganglionic blockade fails to block the ability of MDMA to increase IL-10 and suppress TNF- production. Vehicle or chlorisondamine treated rats were treated with MDMA (2 mg/kg) coadministered with LPS (100 µg/kg) and were killed 60 min following LPS challenge. Concentrations of IL-10 (a) and TNF- (b) were determined in serum. ANOVA demonstrated a significant effect of MDMA on serum IL-10 concentration [F(1,24) = 24.34, P < 0.0001] and a significant chlorisondamine x MDMA interaction on serum TNF- concentrations [F(2,24) = 5.84, P < 0.01]. Data expressed as means ± S.E.M. (n = 5). **, P < 0.01 versus vehicle-treated counterparts (Newman-Keuls test).

-Adrenoceptor Blockade. Pretreatment with the peripherally acting -adrenoceptor antagonist nadolol completely blocked the increase in serum IL-10 levels induced by MDMA (Fig. 8a). However, although MDMA provoked a characteristic decrease in serum TNF- concentrations, this was not altered by pretreatment nadolol (Fig. 8b). Thus, peripheral -adrenoceptor blockade abolished the MDMA-induced increase in IL-10 production without altering the suppression of TNF-.

View larger version (16K): [in this window] [in a new window]   Fig. 8. -adrenoceptor antagonism blocks the ability of MDMA to increase IL-10 but fails to block the suppressive effect of MDMA on TNF- production. Rats were pretreated with either saline or Nadolol (0.25 mg/kg) 30 min prior to administration of MDMA (2 mg/kg) and LPS (100 µg/kg) and were killed 60 min following LPS challenge. Concentrations of IL-10 (a) and TNF- (b) were determined in the serum. ANOVA demonstrated a significant nadolol x MDMA interaction on serum IL-10 concentrations [F(1,24) = 36.28, P < 0.0001] and a significant effect of MDMA on serum TNF- concentrations [F(1,24) = 27.37, P < 0.0001]. Data expressed as means ± S.E.M. (n = 6–8). **, P < 0.01 versus vehicle; ++, P < 0.01 versus saline + MDMA (Newman-Keuls test).

	Discussion

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Effect of MDMA on Production of the Anti-Inflammatory Cytokine IL-10. We examined the effect MDMA on production of the endogenous immunosuppressive agent IL-10, an anti-inflammatory cytokine that inhibits several macrophage functions (see Moore et al., 2001). We observed that MDMA at a dose as low as 1.25 mg/kg induced a rapid increase in LPS-induced IL-10 production. A limited dose-response relationship was observed because the MDMA-induced increase in IL-10 was maximal at 2.5 mg/kg. Reports indicate that a wide range of doses of MDMA (from 1–30 mg/kg) are ingested by humans (see Connor et al., 2000), indicating that the dose range of MDMA used in the present series of experiments is of relevance to human consumption. In contrast to the ability of MDMA to stimulate LPS-induced IL-10 production in vivo, we clearly demonstrate that MDMA fails to increase IL-10 following in vitro exposure, indicating that the increase in IL-10 induced by MDMA in vivo is not due to a direct action on monocytes per se. We are satisfied that the concentration range of MDMA examined in vitro is reflective of in vivo conditions because it was based on the results of a previous study where we measured plasma MDMA concentrations following in vivo administration in rats (Connor et al., 2000). Specifically, in the current study, we employed in vitro concentrations that were both below, and well in excess, of peak plasma MDMA concentrations observed following in vivo administration of MDMA (5–20 mg/kg).

The capacity of MDMA to rapidly increase production of an endogenous immunosuppressive factor such as IL-10 could serve to elicit a broad spectrum of immunosuppressive actions. For instance, IL-10 has a multitude of immunoregulatory effects such as the ability to suppress proinflammatory cytokine production and down-regulate antigen presenting and the costimulatory molecules on antigen presenting cells (Ding et al., 1993; Gerard et al., 1993). Therefore, it is possible that the large increase in IL-10 induced by MDMA in vivo will have a negative impact on the antigen presenting and/or costimulatory capacity of macrophages or dendritic cells and a consequential downstream effect on T-cell-mediated immunity (see de Waal Malefyt et al., 1991).

Examination of the Role of IL-10 in the Suppressive Effect of MDMA on Production of the Proinflammatory Cytokine TNF-. In parallel to the MDMA-induced increase in IL-10 production, a concomitant suppression of the proinflammatory cytokine TNF- was observed. This is consistent with previous studies in rats (Connor et al., 2000, 2001) and also with studies in humans (Pacifici et al., 2001b, 2004) and demonstrates that acute treatment with MDMA promotes an overall immunosuppressive cytokine phenotype. A strong inverse correlation was observed between the MDMA-induced increase in IL-10 and suppression of TNF-, suggesting that the ability of MDMA to suppress TNF- production may be mediated by IL-10. Despite this correlation, immunoneutralization of IL-10 did not reverse the immunosuppressive effect of MDMA on TNF- production. Therefore, although IL-10 has a well established inhibitory action on TNF- production (Bogdan et al., 1992; Gerard et al., 1993), it is not the key factor in mediating the suppression of this proinflammatory cytokine in response to MDMA. Similarly, other pharmacological agents have recently been shown to suppress proinflammatory cytokine production independently of a concomitant increase in IL-10 (Shames et al., 2001; Connor and Kelly, 2002).

Because recreational drug users often repeatedly ingest MDMA over the course of a weekend and because repeated administration can result in sensitization to its immunosuppressive effects (Pacifici et al., 2001a), we assessed ability of both acute and repeated MDMA administration to promote an immunosuppressive cytokine phenotype. The results demonstrate that acute and repeated administration of MDMA had equivalent effects on IL-10 and TNF- production, indicating that neither tolerance nor sensitization developed to its immunosuppressive actions. These data are at variance with a recent study in humans reporting that repeated administration of MDMA increased the severity of MDMA-induced decrements in circulating CD4⁺ cell numbers and T-cell proliferation (Pacifici et al., 2001a) and may indicate that a differential response to acute versus repeated pretreatment with MDMA could be dependent on the immune parameter(s) being studied or the species under investigation.

Examination of the Role of Glucocorticoids and Catecholamines in the Ability of MDMA to Increase IL-10 and Suppress TNF-. Based on the in vitro data presented in this study (Fig. 2) and in our previous study (Connor et al., 2000), we conclude that the ability of MDMA to increase IL-10 and suppress TNF- following in vivo administration is not due to a direct action on monocytes per se. In addition to having a direct effect on immune cells, MDMA has the propensity to elicit its immunosuppressive actions by releasing endogenous immunoregulatory substances. For instance, MDMA activates both the sympathetic nervous system and hypothalamic pituitary adrenal axis (Nash et al., 1988; Grob et al., 1996; Connor et al., 1998). In contrast to their in vitro counterpart, in vivo immunopharmacological studies take into account the contribution of circulating endogenous immunomodulatory substances such as glucocorticoids and catecholamines, the end products of the hypothalamic pituitary adrenal axis and sympathetic nervous system, respectively. Because it is well established that glucocorticoids and catecholamines have the ability to promote an immunosuppressive cytokine phenotype (Zuckerman et al., 1989; Elenkov et al., 1995; Siegmund et al., 1998), we tested the hypothesis that increased circulating glucocorticoid or catecholamine concentrations mediated the immunosuppressive cytokine phenotype induced by MDMA. To this end we examined the effect of MDMA on LPS-induced IL-10 and TNF- production in adrenalectomized rats. However, adrenalectomy failed to attenuate the MDMA-induced suppression of TNF- and increase in IL-10, indicating that neither glucocorticoids nor adrenal-derived catecholamines mediated these responses. In addition to the ability of circulating catecholamines to alter immune cell function, sympathetic innervation and local release of noradrenaline within immune organs such as the spleen is known to have immunomodulatory properties (see Elenkov et al., 2000). Therefore, we examined the ability of sympathetic denervation induced by peripheral administration of the noradrenergic neurotoxin 6-hydroxydopamine to block the immunosuppressive cytokine phenotype induced by MDMA. In this regard, we hypothesized that following an in vivo LPS challenge that splenic cytokine outflow would be a significant contributor to circulating cytokine concentrations. However, although 6-hydroxydopamine caused an 83% depletion of splenic noradrenaline, it failed to block MDMA-induced increase in IL-10 and suppression of TNF-, indicating that its ability to alter production of these cytokines is independent of sympathetic innervation. This assertion is reinforced by the fact that pretreatment with the ganglionic blocker chlorisondamine failed to block the immunosuppressive cytokine phenotype induced by MDMA. The dose range of chlorisondamine used in the present study blocks sympathetic outflow and has previously been found to block the immunomodulatory actions of morphine (Houghtling and Bayer, 2002). Interestingly, animals treated with 6-hydroxydopamine alone or with chlorisondamine (1 mg/kg) also had a reduced capacity to produce TNF-, indicating that a basal level of noradrenaline is required to mount a TNF- response to LPS.

Although the results of the adrenalectomy, sympathectomy, and ganglionic blockade studies argue against a role for catecholamines in the immunosuppressive cytokine phenotype induced by MDMA, we observed that pretreatment with nadolol, a -adrenoceptor antagonist that does not cross the blood brain barrier, completely blocked the increase in IL-10 induced by MDMA, yet failed to alter its suppressive actions on TNF-. Moreover, the ability of nadolol to block the MDMA-induced increase in IL-10 production is shared by the related -adrenoceptor antagonist propranolol (T. J. Connor, unpublished data). The fact that -adrenoceptor blockade inhibits the MDMA-induced increase in IL-10 production without altering its suppressive effects on TNF- production reinforces the immunoneutralization studies, further highlighting a mechanistic dissociation between the ability of MDMA to increase IL-10 and suppress TNF- production. These data also point toward an alternative source of catecholamines in mediating this response. One possibility is that peripheral dopamine may play a role; for instance, a recent study demonstrated that dopamine increased IL-10 and suppressed IL-12 production via -adrenoceptor activation (Hasko et al., 2002). This is not an entirely surprising finding because it has been previously demonstrated that dopamine has both affinity for, and efficacy at, -adrenoceptors (Ruffolo et al., 1984). In addition, a recent report indicates that plasma dopamine levels are elevated in a cohort of human MDMA users (Stuerenburg et al., 2002). Another possibility is that metabolism of MDMA could yield agents that have activity at -adrenoceptors. For instance, a major metabolite of MDMA is -methyldopamine (see Monks et al., 2004), and evidence suggests that this compound can be transformed into a -adrenergic effector following metabolism by monoamine oxidase (Langeneckert and Palm, 1968). Nevertheless, although the precise identity and source of the effector that mediates the MDMA-induced enhancement of IL-10 production is not known, these data clearly demonstrate that it is a -adrenoceptor-mediated event.

Conclusion. MDMA increases LPS-induced IL-10 production via -adrenoceptor activation. However, neither MDMA-induced IL-10 production nor MDMA-induced glucocorticoid or adrenaline/noradrenaline secretion mediates the suppressive effect of MDMA on production of the proinflammatory cytokine TNF-. Further studies are needed to elucidate the exact mechanisms that underlie MDMA-induced suppression of TNF- and the functional consequences of MDMA-induced increases in IL-10.

	Acknowledgements

 
We thank Ivan Sullivan for technical assistance.

	Footnotes

 
This work was supported by fellowships from the Health Research Board of Ireland (to T.J.C.) and by a research grant from Trinity College Dublin (to T.J.C.).

This work was presented as a poster at the International Cytokine Society meeting, Sep 20–24 2003, Dublin. Abstract citation: Connor TJ, Harkin A, and Kelly JP (2003) Methylenedioxymethamphetamine (MDMA; "Ecstasy") increases LPS-induced IL-10 production via -adrenoceptor activation. Eur Cytokine Netw 14 (Suppl):47.

doi:10.1124/jpet.104.073023.

ABBREVIATIONS: MDMA, methylenedioxymethamphetamine; TNF, tumor necrosis factor; LPS, bacterial lipopolysaccharide; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; ANOVA, analysis of variance.

¹ Current address: Department of Pharmacology and Therapeutics, School of Pharmacy, University College Cork, Ireland.

Address correspondence to: Dr. Thomas Connor, Department of Physiology, Trinity College, Dublin 2, Ireland. E-mail: connort{at}tcd.ie

	References

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