Changes in interleukin-1 signal modulators induced by 3,4-methylenedioxymethamphetamine (MDMA): regulation by CB2 receptors and implications for neurotoxicity.

Abstract

3,4-Methylenedioxymethamphetamine (MDMA) produces a neuroinflammatory reaction in rat brain characterized by an increase in interleukin-1 beta (IL-1β) and microglial activation. The CB2 receptor agonist JWH-015 reduces both these changes and partially protects against MDMA-induced neurotoxicity. We have examined MDMA-induced changes in IL-1 receptor antagonist (IL-1ra) levels and IL-1 receptor type I (IL-1RI) expression and the effects of JWH-015. The cellular location of IL-1β and IL-1RI was also examined. MDMA-treated animals were given the soluble form of IL-1RI (sIL-1RI) and neurotoxic effects examined.
Dark Agouti rats received MDMA (12.5 mg/kg, i.p.) and levels of IL-1ra and expression of IL-1RI measured 1 h, 3 h or 6 h later. JWH-015 (2.4 mg/kg, i.p.) was injected 48 h, 24 h and 0.5 h before MDMA and IL-1ra and IL-1RI measured. For localization studies, animals were sacrificed 1 h or 3 h following MDMA and stained for IL-1β or IL-1RI in combination with neuronal and microglial markers. sIL-1RI (3 μg/animal; i.c.v.) was administered 5 min before MDMA and 3 h later. 5-HT transporter density was determined 7 days after MDMA injection.
MDMA produced an increase in IL-ra levels and a decrease in IL-1RI expression in hypothalamus which was prevented by CB2 receptor activation. IL-1RI expression was localized on neuronal cell bodies while IL-1β expression was observed in microglial cells following MDMA. sIL-1RI potentiated MDMA-induced neurotoxicity. MDMA also increased IgG immunostaining indicating that blood brain-barrier permeability was compromised.
In summary, MDMA produces changes in IL-1 signal modulators which are modified by CB2 receptor activation. These results indicate that IL-1β may play a partial role in MDMA-induced neurotoxicity.

Full text

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RESEARCHOpen Access
Changes in interleukin-1 signal modulators induced
by 3,4-methylenedioxymethamphetamine (MDMA):
regulation by CB2 receptors and implications for
neurotoxicity
Elisa Torres, Maria D Gutierrez-Lopez, Andrea Mayado, Ana Rubio, Esther O’Shea and Maria I Colado*
Abstract
Background: 3,4-Methylenedioxymethamphetamine (MDMA) produces a neuroinflammatory reaction in rat brain
characterized by an increase in interleukin-1 beta (IL-1b) and microglial activation. The CB2 receptor agonist JWH-
015 reduces both these changes and partially protects against MDMA-induced neurotoxicity. We have examined
MDMA-induced changes in IL-1 receptor antagonist (IL-1ra) levels and IL-1 receptor type I (IL-1RI) expression and
the effects of JWH-015. The cellular location of IL-1b and IL-1RI was also examined. MDMA-treated animals were
given the soluble form of IL-1RI (sIL-1RI) and neurotoxic effects examined.
Methods: Dark Agouti rats received MDMA (12.5 mg/kg, i.p.) and levels of IL-1ra and expression of IL-1RI measured
1 h, 3 h or 6 h later. JWH-015 (2.4 mg/kg, i.p.) was injected 48 h, 24 h and 0.5 h before MDMA and IL-1ra and IL-
1RI measured. For localization studies, animals were sacrificed 1 h or 3 h following MDMA and stained for IL-1b or
IL-1RI in combination with neuronal and microglial markers. sIL-1RI (3 μg/animal; i.c.v.) was administered 5 min
before MDMA and 3 h later. 5-HT transporter density was determined 7 days after MDMA injection.
Results: MDMA produced an increase in IL-ra levels and a decrease in IL-1RI expression in hypothalamus which
was prevented by CB2 receptor activation. IL-1RI expression was localized on neuronal cell bodies while IL-1b
expression was observed in microglial cells following MDMA. sIL-1RI potentiated MDMA-induced neurotoxicity.
MDMA also increased IgG immunostaining indicating that blood brain-barrier permeability was compromised.
Conclusions: In summary, MDMA produces changes in IL-1 signal modulators which are modified by CB2 receptor
activation. These results indicate that IL-1b may play a partial role in MDMA-induced neurotoxicity.
Background
Administration of the recreationally used drug 3,4-
methylenedioxymethamphetamine (MDMA) induces a
long-term toxicity of 5-HT nerve terminals in several
areas of rat brain. The damage is reflected by a substan-
tial decrease in the concentration of 5-HT and its meta-
bolite, (5-HIAA), a reduction in the density of 5-HT
uptake sites labelled with [3H]-paroxetine [1-4], a
decrease in tryptophan hydroxylase activity [5] and a
decrease in the immunoreactivity of fine 5-HT axons in
cortex, striatum and hippocampus [6].
MDMA
reflected as microglial activation and an increase in the
release of interleukin-1b (IL-1b). Within 1-3 h following
MDMA administration to rats there is an acute increase
in IL-1b concentration in the hypothalamus and frontal
cortex. The up-regulation of IL-1b levels appears at an
early time-point after MDMA and is of short duration,
levels returning to basal values 12 h after drug injection
[7]. The IL-1b response is partially a consequence of
MDMA hyperthermia and seems to be involved in the
long-term 5-HT neurotoxicity since the i.c.v. injection
of IL-1b in the rat brain enhances the long-lasting
reduction in 5-HT transporters and 5-HT concentration
induced by MDMA [8]. Nevertheless, the contribution
producessignsofneuroinflammation
* Correspondence: colado@med.ucm.es
Departamento de Farmacologia, Facultad de Medicina, Universidad
Complutense, 28040 Madrid, Spain
Torres et al. Journal of Neuroinflammation 2011, 8:53
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JOURNAL OF
NEUROINFLAMMATION
© 2011 Torres et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
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of IL-1b to MDMA-induced neurotoxicity is not well
established yet since IL-1b potentiates MDMA-induced
damage at doses that exacerbates the immediate
hyperthermic response induced by the drug. Therefore,
actions of IL-1b on body temperature may contribute to
the injury. In addition to increasing IL-1b, MDMA also
increased the density of peripheral benzodiazepine
receptor binding sites, labelled with [3H]-PK11195 ([(1-
(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)3-isoqui-
nolinecarboxamide)], in hypothalamus and frontal cortex
of the rat. This parameter could be reflecting an activa-
tion of microglia as revealed by subsequent immunohis-
tochemical studies which show an increased in OX-42
immunoreactivity in both brain areas which is evident 3
h after drug injection and remains 24 h later [7].
Recent evidence indicates that MDMA increases the
expression of cannabinoid CB2 receptors in frontal cor-
tex and hypothalamus shortly after injection and that
CB2 immunoreactivity co-localizes with the staining for
the microglial cell marker OX-42, indicating the presence
of CB2 receptors in microglial cells. Repeated administra-
tion of the CB2 agonist JWH-015 prevents the MDMA-
induced microglial activation, reduces IL-1b release and
provides partial protection against the serotonergic neu-
rotoxicity induced by the drug [9]. Overexpression of the
cannabinoid CB2 receptor in microglia during non-
immune and immune pathological conditions is thought
to be aimed at controlling the production of neurotoxic
factors such as pro-inflammatory cytokines.
We have now examined the effect of JWH-015 on the
changes induced by MDMA on endogenous IL-1 signal
modulators in the frontal cortex and hypothalamus and
explored the relevance of these actions on the protection
exerted by this compound against MDMA neurotoxicity.
The specific objectives of this study were as follows: 1) to
determine the effects of MDMA on the levels of the IL-1
receptor antagonist (IL-1ra) and on the expression of the
IL-1 receptor type I (IL-1RI) and evaluate the effect of
JWH-015 on these changes; 2) to define the cellular loca-
tion of IL-1b and IL-1RI by immunohistochemical stain-
ing; 3) to study the effect of i.c.v. administration of the
soluble IL-1 receptor type I (sIL-1RI) on MDMA-induced
hyperthermia and neurotoxicity; and 4) to evaluate the
effect of MDMA on blood-brain barrier (BBB) permeabil-
ity. There is evidence showing that BBB integrity is
altered by a number of factors including increased levels
of inflammatory cytokines [10] and free radicals [11-13],
factors/mediators which are increased following MDMA
injection [3,7].
Methods
Animals and drug administration
Male Dark Agouti rats (175-200 g, Harlan Laboratories
Models, Barcelona) were used. MDMA induces a
reproducible acute hyperthermic response in this strain
[14,15] and also a reproducible long-term neurotoxic
loss of 5-HT after a single dose [14]. Rats were housed
in groups of 5 in conditions of constant temperature
(21°C ± 2°C) and a 12 h light/dark cycle (lights on: 08 h
00 min) and given free access to food and water.
MDMA was given at the dose of 12.5 mg/kg (i.p.), ani-
mals being sacrificed 1 h, 3 h, 6 h or 7 days after treat-
ment. Room temperature at the time of MDMA
administration was 21-22°C. The cannabinoid CB2 ago-
nist, JWH-015 (2.4 mg/kg, i.p.), was given 48 h, 24 h
and 0.5 h before MDMA. Doses and protocols of the
cannabinoid agent used have been shown previously to
be effective at modulating microglial functions [9,16,17].
MDMA (LIPOMED, Arlesheim, Suiza) was dissolved
in saline (0.9% NaCl) and given in a volume of 1 mL/kg.
Dose is reported in terms of the base.
JWH-015 was initially dissolved in DMSO (10 mg/
mL), then diluted with 5% BSA in PBS and administered
in a volume of 2 mL/kg.
Studies were performed in two brain areas, frontal
cortex and hypothalamus since previous studies [7,8]
showed that MDMA administration to Dark Agouti rats
induces an increase in IL-1b levels in the hypothalamus
and frontal cortex and that MDMA alters, in a region-
specific manner, the mechanisms which regulate IL-1b
production in the brain of Dark Agouti rats.
All experimental procedures were performed in accor-
dance with the guidelines of the Animal Welfare Com-
mittee of the Universidad Complutense de Madrid
(following European Council Directives 86/609/CEE and
2003/65/CE).
Measurement of rectal temperature
Immediately before and up to 6 h after MDMA injec-
tion, temperature was measured by use of a digital read-
out thermocouple (BAT12 thermometer, Physitemp, NJ,
USA) with a resolution of 0.1°C and accuracy of ± 0.1°C
attached to a RET-2 Rodent Sensor which was inserted
2.5 cm into the rectum of the rat, the animal being
lightly restrained by holding it in the hand. A steady
readout was obtained within 10 s of probe insertion.
IL-1ra immunoassays
Brain levels of IL-1ra were determined using a commer-
cially available sandwich enzyme-linked immunosorbent
assay (ELISA) system (human IL-1ra/IL-1F3, Quanti-
kine®Immunoassay, R&D Systems, Minneapolis, MN,
USA). Samples were prepared by homogenization of
frontal cortex and hypothalamus in 6 volumes of ice-
cold buffer containing 50 mM Tris, 320 mM sucrose, 1
mM dithiothreitol, 10 μg/mL leupeptin, 10 μg/mL soy-
bean trypsin, 2 μg/mL aprotinin and 0.2% phenantroline,
pH 7.0. Samples were centrifuged at 14 000 × g for 10
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min at 4°C. Protein was determined in the supernatant
[18]. Samples were assayed in triplicate following the
manufacturer’s guidelines. The quantification of IL-1ra
was performed using a standard curve of increasing con-
centrations of IL-1ra (31.2-2000 pg/mL). The optical
density of each well was determined using a microplate
reader (ELX808 IU, Ultra Microplate Reader, Bio-Tek
Instruments, Inc) set to 450 nm (correction wavelength
set at 540 nm). Intra-assay and inter-assay variations
were less than 5% and 15%, respectively.
Immunohistochemistry
Rats were anesthetized with sodium pentobarbital and
perfused transcardially through the left ventricle with
200 mL of phosphate buffered saline (0.1 M PBS, pH
7.4) followed by 200 mL of 4% paraformaldehyde-PBS.
Brains were removed, postfixed in the same solution for
4 h at room temperature and cryoprotected by immer-
sion in 30% sucrose-PBS at 4°C. Brains were then frozen
in powdered dry ice and stored at -80°C. The brains
were sliced at 40 μm in the coronal plane through the
whole frontal cortex and hypothalamus, and stored in
cryoprotectant solution. Immunohistochemical studies
were performed in hypothalamus and the sections were
localized using a rat brain stereotaxic atlas [19].
For double labelling studies, cerebral free-floating sec-
tions were blocked by incubation with 0.5% BSA, 10%
normal goat serum and 0.1% Triton X-100 for 30 min
and incubated at 4°C with the appropriate primary anti-
bodies (OX-42, Serotec, 1:400; anti-NeuN, Millipore,
1:100; anti rat-IL-1b, R&D Systems, 1:200; anti IL-1RI,
Santa Cruz Biotechnology, 1:200) followed by the Alexa
Fluor™ 488 donkey anti-mouse IgG (1:200), Alexa
Fluor™ 594 donkey anti-rabbit IgG or Alexa Fluor™
555 donkey anti-goat IgG (Invitrogen, Spain) secondary
antibodies (1:500) and mounted in ProLong®Gold (Invi-
trogen). Images were acquired sequentially on a Leica
TCS-SP2AOBS confocal microscope (Leica Microsys-
tems, Heidelberg, Germany) for each fluorophore to
avoid any cross-signal between them. Control experi-
ments were performed in which sections were stained
with each of the secondary antibodies or with the com-
bination of them to rule out the possibility of reaction
between them and images were taken using the same
settings for each antibody staining.
IgG leakage from serum into the brain was assessed as
a marker of vasculature damage. Rats were anesthetized
with sodium pentobarbital and sacrificed. Brains were
then frozen in powdered dry ice and stored at -80°C.
The brains were sliced at 20 μm in the coronal plane
through the hypothalamus. After three washes with 0.1
M PBS, sections were blocked by incubation with 0.5%
BSA, 10% horse serum and 0.1% Triton X-100 for 60
min, incubated at 4°C overnight with the antibody Alexa
FluorTM 488 donkey anti-rat IgG (1:500) and covered
with ProLong®Gold (Invitrogen). Images were acquired
as described above and ImageJ (NIH) software was used
for image analysis. All images were converted to gray
scale and blood vessels were outlined to provide an inte-
grated gray scale value for statistical analysis.
Western blot for IL-1RI immunoreactivity
Rats were killed by cervical dislocation and decapitation
1 h, 3 h or 6 h after MDMA administration, the brains
rapidly removed and frontal cortex and hypothalamus
dissected out on ice. Tissue was homogenized by sonica-
tion in ice-cold buffer containing 50 mM Tris, 320 mM
sucrose and a number of protease inhibitors (Complete
Mini, Roche, Madrid, Spain). Samples were centrifuged
at 14 000 × g for 15 min at 4°C and protein determined
in the supernatant [18].
The samples were boiled in laemmli buffer and pro-
teins were resolved by 10% SDS-polyacrylamide gel elec-
trophoresis (SDS-PAGE) and transferred to PVDF
membranes. Nonspecific binding was blocked by incuba-
tion for 2 h in TBS buffer containing 5% skimmed milk.
Membranes were incubated overnight at 4°C with the
polyclonal anti IL-1RI as primary antibody (1:200) fol-
lowed by incubation with goat anti-rabbit IgG-horserad-
ish peroxidase (Amersham GE; 1:2000) for 2 h. Equal
protein sample loading was confirmed by quantification
of the b-actin signal.
Quantification of IL-1RI immunoreactivity
Immunoreactivity was detected with an enhanced chemi-
luminescence (ECL) western blot detection system
(Amersham Bioscience), followed by exposure to Amer-
sham Hyperfilm ECL for 1-5 min. Different film exposure
times were used to ensure that bands were not saturated.
Quantification of specific bands on the film was per-
formed using the Quantity One (USA) program. Each
IL1-RI band density was normalised for protein content
by referring it to its b-actin band density and then the
IL1-RI expression in the different experimental condi-
tions was expressed as a percentage of the control group.
I.c.v. sIL-1RI administration
Rats were anesthetized with a mixture of isoflurane
(3.5% for induction, 1-2% for maintenance; flow rate 1.5
L/min) and nitrous oxygen/oxygen mixture (30/70%) in
air and placed in a stereotaxic frame secured in a Kopf
stereotaxic frame with the tooth bar 3.3 mm below
interaural zero. A 22 G guide cannula was implanted in
the right lateral ventricle (according to the following
coordinates: 7.9 mm rostral to the interaural line, 0.8
mm lateral to the midline and 3.1 mm from the skull
surface; [19]). The cannula was secured to the skull as
previously described [20]. I.c.v. injections took place 5
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days after surgery through a 28 G injector (Plastics One,
Roanoke, VA, USA) fitted in the guide cannula which
protruded 1 mm beyond the guide. sIL-1RI (Sigma-
Aldrich, Madrid, Spain) (3 μg/animal in 5 μl over 5
min) or PBS containing 0.1% BSA were administered i.c.
v. 5 min before and 3 h after MDMA (12.5 mg/kg, i.p.)
or saline (1 mL/kg, i.p.).
Quantification of 5-HT transporter density by [3H]-
paroxetine binding
[3H]-Paroxetine binding was measured in fresh hypotha-
lamical and cortical tissue [2]. Briefly, tissue was homo-
genized in ice-cold Tris-HCl (50 mM; pH 7.4)
containing NaCl (120 mM) and KCl (5 mM) using an
Ultra-Turrax. The homogenate was centrifuged at 30
000 × g for 10 min at 4°C. The supernatant was dis-
carded and the wash procedure repeated twice more.
The pellet was finally resuspended in the Tris buffer at
a concentration of 10 mg tissue/mL. Aliquots of tissue
(800 μL) were incubated with [3H]-paroxetine (1 nM)
for 90 min at room temperature in the absence and pre-
sence of 5-HT (100 μM) for determination of total and
non-specific binding, respectively. Assays were termi-
nated by rapid filtration through glass fibre filters and
radioactivity determined by scintillation spectrometry.
Protein was determined by the method of Lowry [21].
Measurement of 5-HT concentration
Seven days after MDMA administration, rats were killed
by cervical dislocation and decapitation, the brains
rapidly removed and frontal cortex dissected out on ice.
Tissue was homogenized and 5-HT measured by HPLC
with electrochemical detection [3]. Briefly, the mobile
phase consisted of KH2PO4(0.05 M), octanesulfonic
acid (0.16 mM), EDTA (0.1 mM) and methanol (16%),
and was adjusted to pH 3 with phosphoric acid, filtered
and degassed. The flow rate was 1 mL/min and the
working electrode potential was set at +0.4 V.
The HPLC system consisted of a pump (Waters510)
linked to an automatic sample injector (Loop 200 μL,
Waters 717 plus Autosampler), a stainless steel
reversed-phase column (Spherisorb ODS2, 5 μm, 150 ×
4.6 mm) fitted with a precolumn, and a coulometric
detector (Coulochem II, Esa, USA). The current pro-
duced was monitored by using an integration software
package (Unipoint, Gilson).
Statistics
Data from the ELISA, quantification of immunoreactiv-
ity and serotonergic parameters were analyzed using
one-way ANOVA followed by the Newman-Keuls multi-
ple-comparisons test when a significant F value was
obtained. Statistical analyses of the temperature mea-
surements were performed by two-way ANOVA with
repeated measures using treatment as the between sub-
jects factor and time as the repeated measure followed
by Bonferroni as post test (GraphPad Prism 5).
Results
Effect of MDMA on brain IL-1ra levels
One-way ANOVA revealed a significant effect of treat-
ment in hypothalamus (Figure 1b; F3,20= 15.14, p <
0.001) but not in frontal cortex (Figure 1a; F3,23= 2.28,
p = 0.11, n.s.). Post-hoc analysis indicated that MDMA
produced a substantial increase in IL-1ra levels in
hypothalamus (101%, Figure 1b) 1 h after drug adminis-
tration. This effect was not evident 3 h and 6 h later,
times at which IL-1ra levels were similar to those found
in the saline-treated group.
Effect of MDMA on brain IL-1RI expression
One-way ANOVA revealed a significant effect of treat-
ment in hypothalamus (Figure 2b; F3,16= 7.28, p =
0.0041) but not in frontal cortex (Figure 2a; F3,29= 2.55,
p = 0.078, n.s.). Post-hoc analysis indicated that MDMA
produced a substantial decrease in IL-1RI expression in
hypothalamus 1 h (28.8%), 3 h (41.8%) and 6 h (55.1%)
after administration with the greatest reduction occur-
ring at 6 h (Figure 2b, d).
IL-1RI expression in neuronal cells
Saline-treated animals showed immunoreactivity for IL-
1RI in the hypothalamus (Figure 3, red labelling). Double
immunostaining studies using the NeuN antibody to label
neuronal soma (Figure 3b, upper panel) and OX-42 to
stain activated microglia (Figure 3c, upper panel), revealed
that IL-1RI was localized in neuronal cells but not in
microglia. In animals receiving MDMA, IL-1RI was also
found in neurones 3 h after injection although the staining
intensity was lower as expected by the decrease observed
by western blot determinations (Figure 2d).
IL-1b expression in neuronal and microglial cells
Saline-treated animals showed immunoreactivity for IL-
1b in the hypothalamus (Figure 4, red labelling). Double
immunostaining studies using the NeuN antibody to
label neuronal soma (Figure 4a, upper panel) or the
OX-42 marker for microglial cells (Figure 4b, upper
panel) revealed that IL-1b was localized in neuronal
cells in saline-treated animals. Following MDMA injec-
tion, IL-1b staining was also found in microglia, the
immunoreactivity being greater 3 h after MDMA com-
pared with that observed at the 1 h time-point.
Effect of JWH-015 on MDMA-induced changes on IL-1ra
release and IL-1R1 expression in hypothalamus
JWH-015 was injected 48 h, 24 h and 0.5 h before
MDMA. With regard to IL-1ra levels, one-way ANOVA
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revealed a significant effect of treatment (Figure 5a; F3,27
= 13.95, p < 0.0001). Post-hoc analysis indicated that
JWH-015 significantly reduced the increase in IL-1ra
levels induced by MDMA (126.6%, Figure 5a) such that
the increase in IL-1ra levels was 32.7%. JWH-015 did
not alter IL-1ra levels in the brain of saline-treated rats.
IL-1RI expression was studied by western blot (Figure
5c). Statistical analysis by one-way ANOVA revealed a
significant effect of treatment in hypothalamus (Figure
5b; F3,25= 4.56, p = 0.013). Post-hoc analysis indicated
that JWH-015 prevented the reduction in IL-1RI expres-
sion induced by MDMA (Figure 5b) such that the
immunoreactivity of IL-1RI was similar to that observed
in the vehicle-treated group. JWH-015 did not alter IL-
1RI expression in the brain of saline-treated rats.
JWH-015 affected neither the hyperthermia of MDMA
nor the rectal temperature of saline-treated animals (Fig-
ure 5d, e). Two-way ANOVA indicated that there was a
significant effect of treatment (Figure 5e: F3,120= 45,7, p
< 0.0001; Figure 5d: F3,154= 152,9, p < 0.0001), time (Fig-
ure 5e: F4,120= 27,3, p < 0.0001; Figure 5d: F6,154= 28,43,
p < 0.0001) and interaction (Figure 5e: F12,120= 10.0, p <
0.0001; Figure 5d: F18,154= 18,9, p < 0.0001). Bonferroni
post test revealed that MDMA produced a hyperthermic
that was not modified by JWH-015.
Effect of sIL-1RI on the MDMA-induced neurotoxicity
Seven days after MDMA administration, the density of
5-HT transporters and the concentration of 5-HT was
significantly reduced in the ipsi- and contralateral fron-
tal cortex (Figure 6a,c). The density of 5-HT transpor-
ters was also reduced in hypothalamus (Figure 6b).
Administration of sIL-1RI (3 μg, i.c.v.) significantly
potentiated the reduction of 5-HT transporters induced
by MDMA in the hypothalamus (Figure 6b) but it did
not alter the 5-HT parameters in frontal cortex (Figure
6a,c). sIL-1RI did not modify the 5-HT levels (Figure 6a,
c) nor density of 5-HT transporters (Figure 6a,b) in the
brain of saline-treated rats.
Effect of sIL-1RI on the MDMA-induced hyperthermia
Two-way ANOVA indicated that there was a significant
effect of treatment (F3,268= 128.2, p < 0.0001), time
(F9,268= 5.69, p < 0.0001) and interaction (F27,268= 5.14,
p < 0.0001). Bonferroni post test revealed that MDMA
produced a hyperthermic response which peaked 30
min after treatment and was sustained for up to 6 h
after treatment. sIL-1RI (3 μg, i.c.v.) affected neither the
hyperthermia of MDMA nor the rectal temperature of
saline-treated animals (Figure 6d).
Effect of MDMA on BBB permeability
BBB permeability was visualized and quantified by
means of IgG immunostaining (Figure 7a). One-way
ANOVA revealed a significant effect of treatment in
hypothalamus (Figure 7b; F3,17= 5.122, p = 0.0113).
Post-hoc analysis indicated that MDMA produced a
substantial increase in hypothalamic IgG leakage (147%,
Figure 7b) 1 h and (212%, Figure 7b) 3 h post-injection.
This effect was not evident at the 6 h time point.
0.0
0.5
1.0
1.5
2.0
Saline
MDMA 1h
MDMA 3h
MDMA 6h
a
IL-1ra levels (pg/mg protein)
0.0
1.5
3.0
4.5
6.0
Saline
MDMA 1h
MDMA 3h
MDMA 6h
???
???
???
b
IL-1ra levels (pg/mg protein)
Figure 1 Time-course of the changes induced by MDMA (12.5
mg/kg, i.p.) on IL-1ra (endogenous antagonist of IL-1 receptor)
levels in a) frontal cortex and b) hypothalamus. IL-1ra levels
were measured 1 h, 3 h and 6 h after drug injection. Results shown
as mean ± SEM (n = 6-8). Different from saline: ***p < 0.001.
Different from MDMA-treated group at 1 h:fffp < 0.001.
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Discussion
The current study demonstrates that MDMA produces
differential regulation of several modulators of IL-1 sig-
nalling in hypothalamus and frontal cortex. Thus, in the
hypothalamus, the drug induces a pronounced and tran-
sient increase in IL-1ra levels and a sustained marked
reduction of IL-1RI density that lasted at least 6 h.
Neither of these two changes is observed in frontal cor-
tex. The differential effects induced by MDMA on IL-
1b signal modulators could be a consequence of the
intensity of action of MDMA on IL-1b release in these
brain areas. Previous studies from our laboratory have
shown that following MDMA, IL-1b levels are much
higher in the hypothalamus than in frontal cortex [7];
the basal levels of IL-1b also being lower in frontal
cortex.
The IL-1b pro-inflammatory effects are balanced by
IL-1ra, a naturally occurring IL-1 receptor antagonist
which is structurally related to IL-1b and binds to the
type I IL-1 receptor (IL-1RI) competitively displacing
IL-1b from the receptor complex without inducing any
biological response [22]. In fact, there is an up-regula-
tion of IL-1ra in various models of neurodegeneration
[23-25] or after infectious stimuli [26]. In line with this,
IL-1ra administration to rodents attenuates the neuroin-
flammatory response and neuronal damage mediated by
IL-1b following several insults [27-32]. The greater IL-
1b content in the hypothalamus compared with frontal
cortex as early as 1 h after MDMA injection could trig-
ger the release of IL-1ra in an attempt to limit the
effects of the released IL-1b. In fact, much evidence
indicates that the effectiveness of IL-1b signal transduc-
tion at the IL-1RI depends on the ratio between IL-1ra
and IL-1b. In vivo studies reveal that relatively high IL-
1ra to IL-1b molecular ratios are necessary for an effec-
tive blockade of IL-1b effects (at least 25:1, [33]). In our
hands, in the hypothalamus, IL-1b levels increased 400%
after MDMA dosing [7], while the increase in IL-1ra
levels was 100% (this paper). This means that although
an increase in IL-1ra levels may contribute to limiting
the effects of IL-1b mediated through IL-1RI, the extent
of this increase might not be sufficient to circumvent
IL-1b signalling.
IL-1b signal transduction occurs via IL-1RI that is
expressed at the cell surface; it binds IL-1b and leads to
intracellular signalling by association with the IL-1














0
50
100
150
200
250
a
Saline
MDMA 1h
MDMA 3h
MDMA 6h
Relative IL-1RI levels
(arbitrary units)
0
50
100
150
??
b
?
Saline
MDMA 1h
MDMA 3h
MDMA 6h
??
Relative IL-1RI levels
(arbitrary units)
Figure 2 Time-course of the changes induced by MDMA (12.5 mg/kg, i.p.) on IL-1RI expression in the frontal cortex (a) and
hypothalamus (b). IL-1RI expression was measured 1 h, 3 h and 6 h after drug injection. Results shown as mean ± SEM (n = 5-13). Different
from saline: *p < 0.05, **p < 0.01. A representative western blot shows immunoreactivity in frontal cortex (c) and hypothalamus (d).
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Figure 3 Fluorescence images (40×) showing IL-1RI immunoreactivity (red staining) in hypothalamus of saline- and MDMA (12.5 mg/
kg, i.p.)-treated rats (3 hours after injection). Double immunofluorescence with IL-1RI and NeuN (green staining, b: upper panel) or OX-42
(green staining, b: lower panel) revealed that IL-1RI is localized on neuronal cells, not on activated microglia. Scale bar = 50 μm. a: image
showing area studied.
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Figure 4 Fluorescence images (40×) showing IL-1b immunoreactivity in the hypothalamus of saline- and MDMA (12.5 mg/kg, i.p.)-
treated rats (1 h and 3 h after injection). Double immunofluorescence with IL-1b (red staining) and NeuN or OX-42 (both green staining,
upper and lower panels respectively) revealed that IL-1b is constitutively expressed in neurons and that following MDMA the immunoreactivity
for IL-1b overlaps with OX-42 immunostaining. Scale bar = 50 μm. Inset shows a stained microglial cell (63×). Same brain sections were studied
as indicated in Figure 3a.
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0.0
0.5
1.0
1.5
2.0
2.5
a
Vehicle+Saline
Vehicle+MDMA
JWH-015+MDMA
JWH-015+Saline
???
??
IL-1ra levels (pg/mg protein)
0
50
100
150
b
Vehicle+Saline
Vehicle+MDMA
JWH-015+MDMA
JWH-015+Saline
?
?
Relative IL-1RI levels
(arbitrary units)
c
-101
23
36
37
38
39
40
e
Time (h)
Rectal temp. (ºC)
-1
0123
36
37
38
39
40
Vehicle+Saline
JWH-015+MDMA
Vehicle+MDMA
JWH-015+Saline
d
Time (h)
Rectal temp. (ºC)
Figure 5 Effect of JWH-015 on the MDMA-induced changes in IL-1ra levels (a) and IL-1RI expression (b) in hypothalamus. JWH-015
(2.4 mg/kg, i.p.) was injected 48 h, 24 h, and 0.5 h before MDMA (12.5 mg/kg, i.p.). For IL-1ra levels determination rats were killed 1 h
after MDMA administration (a). IL-1RI expression was measured 3 h after MDMA (b). The effect of JWH-015 on MDMA-induced hyperthermia is
also shown (d,e). Results shown as mean ± SEM (n = 4-8). Different from vehicle + saline group: *p < 0.05, ***p < 0.001. Different from vehicle +
MDMA-treated group:fp < 0.05,ffp < 0.01. In panels (d, e) different from vehicle + saline group: *p < 0.001. A representative western blot shows
immunoreactivity in hypothalamus (c).
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receptor accessory protein (IL-1RAcP). MDMA also
produces a region-specific response in IL-1RI expression
which may be a consequence of the different relative
levels of IL-1b and IL-1ra in these brain areas. The
more pronounced release of IL-1b in the hypothalamus
after MDMA injection may lead to a down-regulation of
IL-1RI that could be considered an additional defence
mechanism to block the acute effects of IL-1b. Previous
studies using radioligand binding in tissue homogenates
or by autoradiography have reported a reduction in the
neuronal density and affinity of IL-1 receptors after sys-
temic administration of lipopolysaccharide at doses pro-
ducing IL-1b release [34,35]. These findings indicate
that increased levels of IL-1b regulate IL-1R1 receptor
expression resulting in down-regulation.
In the current study we used immunohistochemistry
and double-labelling immunofluorescence to investigate
the cell types expressing IL-1RI. IL-1RI immunoreactiv-
ity was present in a constitutive way in the hypothala-
mus of saline-treated rats and double-staining revealed
neuronal expression of IL-1RI since it shows receptor
co-localization with NeuN. These findings support pre-
vious studies [36-39] and indicate that neurons are
targeted by the endogenous IL-1b produced in the
hypothalamus immediately after MDMA injection. The
immunohistochemistry results show that staining for IL-
1RI decreases following MDMA and reinforce those
obtained by western blot. We could not find IL-1RI
expression in microglial cells. These results are in agree-
ment with those described previously [36,40] but are
not, however consistent with those of previous reports
[37,41,42].
Our results also show that IL-1b is constitutively
expressed in hypothalamic neurons supporting previous
studies showing that IL-1mRNA co-localize with large
neuronal cell bodies [43]. There was no constitutive
expression of IL-1b in microglial cells but it appeared in
these cells coinciding with an increase in the number of
OX-42 positive cells produced after MDMA administra-
tion. These data are in agreement with those showing
that IL-1b is produced and released by microglia [36,44]
during the early phase of activation and suggest that
microglia is the main cell involved in propagating IL-1b
effects in neuronal cells. In fact, we found that the
intensity of IL-1b labelling in microglia was higher 3 h
after MDMA compared with 1 h samples as expected
IpsiContra
0
50
100
150
200
??
??
???
a
Vehicle+Saline
Vehicle+MDMA
sIL-1RI+MDMA
sIL-1RI+Saline
??
[3H]-Paroxetine binding
(fmol/mg protein)
0
50
100
150
200
??
???
b
?
[3H]-Paroxetine binding
(fmol/mg protein)
IpsiContra
0
50
100
150
200
c
??????
?
???
5-HT conc. (ng/g tissue)
-10123456
36
37
38
39
40
Vehicle+Saline
Vehicle+MDMA
sIL-1RI+MDMA
sIL-1RI+Saline
?
d
Time (h)
Rectal temp. (ºC)
Figure 6 Effect of intracerebroventricular injection of sIL-1RI on the MDMA-induced reduction of 5-HT transporters in frontal cortex
and hypothalamus (a,b), cortical 5-HT concentration (c) and hyperthermia (d). sIL-1RI (3 μg) was administered 5 min before and 3 h after
MDMA (12.5 mg/kg, i.p.), rats being killed 7 days later. Results shown as mean ± SEM (n = 5-9). Different from vehicle + saline group: *p < 0.05,
** p < 0.01, ***p < 0.001. Different from vehicle + MDMA-treated group:fp < 0.05. In panel (d) different from vehicle + saline group: *p < 0.001.
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according to the greater release of IL-1b based in our
previous studies of time-course IL-1b levels after
MDMA administration [7].
With respect to the role of IL-1b in the neurotoxicity
induced by MDMA we have previously shown that i.c.v.
administration of the cytokine potentiates the neurotoxi-
city produced by MDMA; however the exact role is dif-
ficult to assess since the IL-1b injection also increased
the drug’s hyperthermic response [8]. The data obtained
following i.c.v. administration of sIL-1RI support at least
a partial role for IL-1b in the neurotoxicity of MDMA
in the hypothalamus. In addition to IL-1ra, IL-1b
signalling is balanced by the soluble forms of its recep-
tors which serve to capture the active molecules and
thus reduce the levels available for signal transduction.
IL-1b has very low affinity for IL-1sRI which, however,
binds IL-1ra with high affinity [45,46]. Human sIL-1RI
displays higher affinity for IL-1ra than membrane-bound
IL-1RI, human sIL-1RI binds IL-1ra with a Kd of 14
pM, i.e. an affinity which is 200 times that of mem-
brane-bound IL-1RI [47]. Both types of IL-1 inhibitor, i.
e., sIL-1R and IL-1ra, may simultaneously be present at
the inflammatory site and may interfere with each
other’s activity, one of which acts at the receptor level
and the other at the ligand level thus leaving IL-1b
activity unimpaired [46]. In fact, it has been reported
that the inhibitory activity of IL-1ra is reduced by the
simultaneous presence of sIL-1RI. Thus, in our study
the binding of IL-1ra by sIL-1RI may be allowing the
IL-1b-mediated effects.
Activation of the IL-1RI receptor by IL-1b may elicit a
number of different events. Binding of the cytokine to
the type I receptor allows the recruitment of IL-1RAcP,
an action which is necessary for the initiation of the sig-
nalling cascade. Typically the resulting response involves
the activation of NF-?B and mitogen-activated protein
kinase pathways [48]. In fact, we have observed NF-?B
activation following MDMA [49]. Activation of this
transcription factor regulates the transcription of target
genes that mediate the inflammatory response including
IL-1b in a cyclic mechanism involving further IL-1b
release and inflammation [50]. In addition, activation of
NF-?B is associated with increased expression of several
enzymes such as COX-2 and iNOS [51,52] which may
in turn lead to the production of free radicals, elements
which have been implicated both by us and by others in
the 5-HT neurotoxicity produced by the drug [3,53-55].
Thus, activation of the IL-1RI receptor may ultimately
lead to a potentiation of the neuroinflammatory
response and an increase in oxidative stress.
Although these data point to a potential role for IL-1b
in the neurotoxicity produced by MDMA, the studies
with the CB2 agonist JWH-015 indicate that it cannot
be the sole factor mediating damage. JWH-015 adminis-
tration before MDMA prevented the increase in IL-1ra
levels and the reduction of IL-1RI density in the
hypothalamus. Previous studies showed that this com-
pound also prevents the increase in IL-1b levels in
hypothalamus [9]. Interestingly, the effects induced by
JWH-015 on the MDMA-induced changes on IL-1
modulators are produced using a dosing regimen of
JWH-015 that does not protect against MDMA-induced
neurotoxicity [9]. It is worth noting that JWH-015 did
protect against MDMA-induced neurotoxicity following
regimens which in addition to inhibiting IL-1b produced
inhibition of microglial activation. Thus, it appears that
a
salinesaline saline
MDMA 3hMDMA 3hMDMA 3h
MDMA 6h MDMA 6hMDMA 6h
MDMA 1hMDMA 1hMDMA 1h
0
100
200
300
400
500
600
Saline
MDMA 1h
MDMA 3h
MDMA 6h
?
?
b
Arbitrary Units (% of control)
Figure 7 Time-course of the changes induced by MDMA (12.5
mg/kg, i.p.) on IgG expression in hypothalamus. Fluorecence
images (40×) of representative IgG immunostained sections (a) of
hypothalamus and quantification of intensity of immunostaining (b).
Scale bar = 50 μm. Rats were killed 1 h, 3 h and 6 h after MDMA
injection. Results shown as mean ± SEM (n = 4-6). Different from
saline: *p < 0.05.
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inhibition of microglial activation in addition to IL-1b
production may play essential roles in MDMA
neurotoxicity.
What can be stated unequivocally is that the ability of
JWH-015 to prevent the MDMA-induced changes on
IL-1 modulators is not related to an effect on body tem-
perature. The hyperthermic response immediately fol-
lowing MDMA was not modified in rats also given the
CB2 agonist (this paper; [9]).
What seems to be clear is that the changes induced by
MDMA on IL-1b signal modulators are regulated by
CB2 receptors. Following MDMA there is an increase in
CB2 expression in activated microglia which is pre-
vented in rats receiving concomitantly the CB2 agonist
JWH-015 [9]. Activation of CB2 receptors might inhibit
the conversion of microglia to a phenotype able to
express and release IL-1b [9] and thus is probably
responsible for the inhibition of IL-1b release from this
cell type following MDMA. As a consequence of this,
IL-1ra levels do not rise and IL-1RI are not down-regu-
lated in MDMA-treated animals also given JWH-015.
There is evidence that expression of IL-1b in the CNS
induces reversible breakdown of the BBB [56,57] and
this effect has been linked to increased expression of
matrix metalloproteinases (MMPs), a gene family of
extracellular matrix enzymes which degrade junction
proteins and change the permeability of the BBB [58].
The current study shows that MDMA increases IgG
expression in the hypothalamus at 1 h and was maximal
3 h postinjection. BBB breakdown is often associated
with increased extravasation of plasma proteins and
high levels of immunoglobulin G (IgG) in brain, there-
fore, our results indicate that MDMA induces BBB dis-
ruption. The effect of MDMA on BBB integrity has not
been studied in detail. To the best of our knowledge
there is only one study examining the effects of acute
MDMA on BBB dysfunction, brain edema, and cell
injury in rats and mice [59]. This investigation shows
the leakage of Evans blue dye, particularly in the cere-
bellum, hippocampus, cortex, thalamus, and hypothala-
mus shortly after MDMA and therefore, support the
results reported in the current paper. Further studies are
required in order to investigate possible mechanistic
explanation.
Conclusions
These findings indicate for the first time that MDMA
induces changes in the upstream regulation of IL-1 sig-
nalling, producing an increase in IL-1ra and a decrease
in IL-1RI. These changes are observed selectively in
hypothalamus, but not in frontal cortex where the IL-1b
release following drug injection is less pronounced. The
changes are prevented by CB2 receptor activation. IL-1b
is expressed in microglial cells shortly after MDMA
while IL-1RI is expressed in the neuronal cell body of
neurons suggesting that the release of IL-1b from
microglia plays an important role in the neuroinflamma-
tory response and that hypothalamic neurons are the
main target cells for IL-1b. The fact that sIL-1RI admin-
istration potentiates the MDMA-induced loss of 5-HT
transporters suggests a partial role for IL-1b signalling
in MDMA-induced neurotoxicity.
List of Abbreviations
5-HIAA: 5-hydroxyindoleacetic acid; 5-HT: 5-hydroxytryptamine; BBB: blood-
brain barrier; BSA: bovine serum albumin; DMSO: dimethyl sulfoxide; IL-1β:
interleukin-1beta; IL-1ra: IL-1 receptor antagonist; IL-1RI: IL-1 receptor type I;
MDMA: 3,4-methylenedioxymethamphetamine; PBS: phosphate buffered
saline; sIL-1RI: soluble IL-1 receptor type I.
Acknowledgements
This work was supported by MICINN (Grant no. SAF2007-65175 and PI07/
0892), Plan Nacional sobre Drogas (Grant no. PR75/06-15077 and PR61/08-
16410), Red de Trastornos Adictivos (Grant no. RD06/0001/006), UCM-CAM
(Grant no. CCG07-UCM/SAL-2588 and CCG08-UCM/SAL-3935) and Fundacion
Mutua Madrileña. The authors received predoctoral funding from Ministerio
de Ciencia e Innovación (ET, AM).
Authors’ contributions
ET carried out ELISA and western blot assays and participated in
immunohistochemical studies and design of the study. MDGL carried out
immunohistochemistry, confocal microscopy and participated in the
interpretation of the data. AM participated in western blot analysis and
neurotoxicity study. AR carried out the study on IgG extravasation. EOS
participated in the interpretation of data and helped to draft the manuscript.
MIC participated in the design of the study, data analysis and drafting and
revising the manuscript. All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 28 December 2010 Accepted: 19 May 2011
Published: 19 May 2011
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doi:10.1186/1742-2094-8-53
Cite this article as: Torres et al.: Changes in interleukin-1 signal
modulators induced by 3,4-methylenedioxymethamphetamine (MDMA):
regulation by CB2 receptors and implications for neurotoxicity. Journal of
Neuroinflammation 2011 8:53.
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