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RESEARCH ARTICLE | APRIL 01 2012
Stimulation of Dopamine Receptor D5 Expressed on Dendritic Cells
Potentiates Th17-Mediated Immunity
Carolina Prado; ... et. al
J Immunol (2012) 188 (7): 3062–3070.
https://doi.org/10.4049/jimmunol.1103096
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The Journal of Immunology
Stimulation of Dopamine Receptor D5 Expressed on
Dendritic Cells Potentiates Th17-Mediated Immunity
Carolina Prado,*
,†
Francisco Contreras,*
,†
Hugo Gonza
´lez,*
,†
Pablo Dı
´az,*
Daniela Elgueta,* Magaly Barrientos,* Andre
´s A. Herrada,* A
´lvaro Lladser,*
Sebastia
´n Bernales,* and Rodrigo Pacheco*
,‡
Dendritic cells (DCs) are responsible for priming T cells and for promoting their differentiation from naive T cells into appropriate
effector cells. Emerging evidence suggests that neurotransmitters can modulate T cell-mediated immunity. However, the involve-
ment of specific neurotransmitters or receptors remains poorly understood. In this study, we analyzed the role of dopamine in the
regulation of DC function. We found that DCs express dopamine receptors as well as the machinery necessary to synthesize, store,
and degrade dopamine. Notably, the expression of D5R decreased upon LPS-induced DC maturation. Deficiency of D5R on the
surface of DCs impaired LPS-induced IL-23 and IL-12 production and consequently attenuated the activation and proliferation
of Ag-specific CD4
+
T cells. To determine the relevance of D5R expressed on DCs in vivo, we studied the role of this receptor in the
modulation of a CD4
+
T cell-driven autoimmunity model. Importantly, D5R-deficient DCs prophylactically transferred into wild-
type recipients were able to reduce the severity of experimental autoimmune encephalomyelitis. Furthermore, mice transferred
with D5R-deficient DCs displayed a significant reduction in the percentage of Th17 cells infiltrating the CNS without differences in
the percentage of Th1 cells compared with animals transferred with wild-type DCs. Our findings demonstrate that by contributing
to CD4
+
T cell activation and differentiation to Th17 phenotype, D5R expressed on DCs is able to modulate the development of an
autoimmune response in vivo. The Journal of Immunology, 2012, 188: 3062–3070.
Dendritic cells (DCs) are the most efficient type of APCs
and are specialized in the initiation of immune responses
by directing the activation and differentiation of naive
T cells (1). In the absence of foreign Ags, immature DCs can
present self-antigens on MHC molecules and, by stimulating
CD4
+
regulatory T cells (Tregs), induce peripheral tolerance (2).
In contrast, maturation induced by recognition of pathogen-
associated molecular patterns allows DCs to migrate into lymph
nodes and promote the differentiation of naive CD8
+
T cells and
CD4
+
T cells into CTLs and the appropriate effector Th subset,
respectively (1). Depending on the cytokine milieu, DCs can
stimulate the polarization of naive CD4
+
T cells into Th1, Th2,
and Th17 cells (3, 4). In particular, the release of IL-12 by DCs
can induce CD4
+
T cells to adopt the IFN-g–secreting Th1 phe-
notype. However, the production of IL-6 or IL-23 by DCs can
promote CD4
+
T cells to acquire the IL-17–producing Th17 fate.
Because of the key role of DCs in modulating the interface be-
tween immunity and tolerance, their function must be tightly
regulated (5). Traditionally, it has been thought that the functions
of immune cells such as T cells and DCs are regulated mainly by
cytokines. However, a number of more recent studies have shown
that immune system cells can also be regulated by neurotrans-
mitters (6–11). Consistent with this, both primary and secondary
lymphoid organs are highly innervated by sympathetic ends that
store dopamine (DA) (12, 13).
DA is an important neurotransmitter in the CNS and is involved
in the control of locomotion, emotion, cognition, and neuroen-
docrine secretion (14). The first and rate-limiting step in DA
biosynthesis is the conversion of L-tyrosine to (S)-2-amino-3-(3,4-
dihydroxyphenyl)propanoic acid (L-DOPA), which is catalyzed by
the enzyme tyrosine hydroxylase (TH). This compound is subse-
quently metabolized by aromatic amino acid decarboxylase to
produce cytosolic DA (15). Cytosolic DA can also be taken up
from the extracellular environment through plasma-membrane
dopamine transporters (DATs) (16). Once inside the cell, DA
can either be inactivated by monoamine oxidases (MAO-A and
-B) (17) or can be stored in intracellular vesicles. Storage is me-
diated by type 1 and type 2 vesicular monoamine transporters
(VMATs 1 and 2) that mobilize cytosolic DA toward vesicular
stores (18). In some catecholaminergic cells, DA can adopt a third
fate and be further processed by dopamine b-hydroxylase (DbH)
to yield norepinephrine (19).
DA exerts its effects by stimulating dopamine receptors (DARs)
expressed on the cell surface. Five DARs have been identified to
date: D1R, D2R, D3R, D4R, and D5R (20, 21). All of these re-
ceptors are hepta-spanning membrane proteins that belong to the
superfamily of G protein-coupled receptors. Based on their se-
*Fundacio
´n Ciencia y Vida, Santiago, Chile;
†
Universidad Andre
´s Bello, Santiago,
Chile; and
‡
Universidad San Sebastia
´n, Santiago, Chile
Received for publication October 27, 2011. Accepted for publication January 31,
2012.
This work was supported by Grants 1095114 and 11110525 from Fondo Nacional
de Desarrollo Cientı
´fico y Tecnolo
´gico, PFB-16 and “Insertion to the Academy”
791100038 from Comisio
´n Nacional de Investigacio
´n Cientı
´fica y Tecnolo
´gica,
and 2011-0001-R from Universidad San Sebastia
´n. C.P., F.C., and H.G. hold gradu-
ated fellowships from Comisio
´n Nacional de Investigacio
´n Cientı
´fica y Tecnolo
´gica.
Address correspondence and reprint requests to Dr. Rodrigo Pacheco, Fundacio
´n
Ciencia y Vida, Avenida Zan
˜artu 1482, Santiago 7780272, Chile. E-mail address:
rpacheco@cienciavida.cl
The online version of this article contains supplemental material.
Abbreviations used in this article: DA, dopamine; DAR, dopamine receptor; DAT,
dopamine transporter; DC, dendritic cell; DbH, dopamine b-hydroxylase; D5RKO,
D5R-knockout; EAE, experimental autoimmune encephalomyelitis; iDC, immature
dendritic cell; MAO, monoamine oxidase; mDC, mature dendritic cell; MFI, mean
fluorescence intensity; MS, multiple sclerosis; NET, norepinephrine transporter; pAb,
polyclonal Ab; pMOG, myelin oligodendrocyte glycoprotein 35–55 peptide; SCH,
SCH23390; SERT, serotonin transporter; SKF, SKF38393; TH, tyrosine hydroxylase;
Treg, regulatory T cell; VMAT, vesicular monoamine transporter; WT, wild-type.
Copyright Ó2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103096
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quence homology, signal transduction machinery, and pharmaco-
logical properties, DARs have been classified into two subgroups.
D1R and D5R are type I DARs, which couple with stimulatory Ga
subunits, and D2R, D3R, and D4R are type II DARs, which couple
to inhibitory Gasubunits (20).
Much evidence regarding the functional relevance of DAR ex-
pression and DA signaling in immune cells has amassed over the
years. Some immune cells such as Tregs have been found to contain
substantial amounts of DA and other catecholamines. These cells
also constitutively express TH, an enzyme necessary to synthesize
DA. Effector T cells, in contrast, contain only trace amounts of DA
(22, 23). In Tregs, endogenous DA signals through D1R/D5R
in a paracrine/autocrine manner resulting in the downregulation
of Treg function (23). In DCs, recent pharmacological evidence
suggests that the antagonism of D1R/D5R expressed on DCs
could impair the polarization of naive CD4
+
T cells toward the
Th17 phenotype (24, 25). However, as the D1R/D5R antagonist
used in these studies is also an agonist for serotonin receptors (26,
27) and serotonin receptors are also expressed on DCs (28), the
individual contributions of specific DARs and serotonin receptors
in this phenomenon are not clear (24).
The involvement of Th1/Th17 T cells and Tregs in autoimmunity
and the possible connection between type I DAR signaling in DCs
and the biasing of T cell fate suggest that type I DARs expressed on
immune cells are involved in the balance between autoimmunity
and tolerance. Notably, alterations in components of the dopa-
minergic system have been correlated with multiple sclerosis (MS),
an inflammatory and demyelinating neurodegenerative disease of
the CNS mediated mainly by Th17 and Th1 autoreactive T cells.
In this regard, patients suffering from MS show decreased ex-
pression of D5R in PBMCs compared with that in healthy indi-
viduals (29). Furthermore, increased local levels of DA in the CNS
were detected in mice induced with experimental autoimmune
encephalomyelitis (EAE), an animal model of MS, compared with
those in controls (30). Taken together, this evidence suggests a
possible modulatory role for DA via type I DARs in this auto-
immune disease. However, further efforts are necessary to identify
specific receptors and cells involved in DA-mediated regulation
of this autoimmune response.
In this study, we investigate the contribution of DA in modu-
lating the pivotal role of DCs during adaptive immune response.
We report that DCs express several DARs and the components
required to synthesize, to store, and to degrade DA. Our data
suggest an autocrine modulatory role of DA in DC function and
illustrate that DA stimulates D5R expressed on DCs thereby fa-
cilitating strong CD4
+
T cell activation and differentiation toward
the Th17 phenotype, thus contributing to the development of
a CD4
+
T cell-mediated autoimmune response.
Materials and Methods
Animals
Six- to eight-week-old mice of the C57BL/6 background were used for all
experiments. Wild-type (WT) C57BL/6 mice were purchased from The
Jackson Laboratory (Bar Harbor, ME). D5R-knockout (D5RKO) mice
were kindly donated by Dr. David Sibley (31). OVA-specific OT-I trans-
genic mice expressing specific TCRs for H-2K
b
/OVA
257–264
were obtained
from Taconic. OVA-specific OT-II transgenic mice expressing specific
TCRs for I-A
b
/OVA
323–339
were kindly donated by Dr. Marı
´a Rosa Bono
(32). All mice were maintained and manipulated according to institutional
guidelines at the pathogen-free facility of the Fundacion Ciencia y Vida.
Generation of DCs
Bone marrow-derived DCs from WT and D5RKO mice were prepared as
previously described (33). Briefly, DCs were grown in RPMI 1640 medium
(Hyclone, Logan, UT) supplemented with 5% heat-inactivated FBS (Bi-
ological Industries, Beit Haemek, Israel) and 10 ng/ml recombinant mouse
GM-CSF (PeproTech, Rocky Hill, NJ). On day 5, differentiation of DCs
was routinely assessed obtaining .80% CD11c
+
cells. In some experi-
ments, day 5 DCs were either left unstimulated (immature dendritic cells;
iDCs) or stimulated (mature dendritic cells; mDCs) with 100 ng/ml LPS
(Sigma Chemical Co., St. Louis, MO) for 24 h and used for further
experiments.
Immunostaining and Western blots
To determine expression levels of key surface molecules, DCs were im-
munostained with the following fluorochrome-conjugated mAbs for 30
min: allophycocyanin-conjugated anti-CD11c (clone HL3), PE-conjugated
anti–I-A
b
(clone AF6-120.1), FITC-conjugated anti-CD80 (clone 16-
10A1), PE-conjugated anti-CD86 (clone GL1), FITC-conjugated anti–H2-
K
b
(clone AF6-88.5), and FITC-conjugated anti-CD40 (clone 3/23), all of
them from BD Pharmingen (San Diego, CA). For DAR detection, DCs
were incubated with unconjugated, rabbit anti-D2R, rabbit anti-D3R, or
goat anti-D4R polyclonal Abs (pAbs) specific for extracellular epitopes for
1 h, followed by FITC-conjugated anti-rabbit IgG or anti-goat IgG Abs
(Santa Cruz Biotechnology, Santa Cruz, CA). In the case of D1R-like
receptor and TH detection, Abs against intracellular epitopes were used.
Accordingly, DCs were fixed with 1% paraformaldehyde in PBS for 15
min at room temperature and then treated with permeabilizing buffer
(0.5% saponin, 3% BSA in PBS). Permeabilized cells were incubated with
unconjugated rabbit anti-D1R, rabbit anti-D5R, or rabbit anti-TH pAbs,
followed by FITC-conjugated anti-rabbit IgG Abs (Santa Cruz Biotech-
nology). Nonspecific Ig was included as control in each case, and staining
was analyzed by flow cytometry in the CD11c
+
I-A
b+
population. For
intracellular cytokine staining, cells were stimulated for 4 h with PMA (50
ng/ml; Sigma) and ionomycin (1 mg/ml; Sigma) in the presence of bre-
feldin A (5 mg/ml; Sigma). After staining of surface markers, cells were
fixed, permeabilized, and incubated for 30 min with allophycocyanin-
conjugated anti–IFN-g(clone XMG1.2) and PE-conjugated anti–IL-17
mAbs (clone TC11-18H10), both from BD Pharmingen. All flow cytom-
etry analyses were performed by using a FACSCanto II flow cytometer,
and collected data were analyzed by using FACSDiva software (both from
BD Biosciences). To determine DAT expression, DCs were purified using
CD11c microbeads (MACS; Miltenyi Biotec, Bergisch Gladbach, Ger-
many), and DAT expression was analyzed in whole-cell extracts by im-
munoblot using a rat anti-DAT mAb (clone 6-5G10) and an HRP-
conjugated secondary anti-rat IgG Ab (both from Santa Cruz Biotech-
nology). Immunodetection of b-actin was included as a loading control,
using mouse anti–b-actin mAb (clone AC-15; Sigma) and an HRP-
conjugated anti-mouse IgG Ab (Rockland, Gilbertsville, PA).
RT-PCR
Cells and tissues were lysed, and total RNA was extracted with the
EZNA total RNA kit (VBio-Tek, Norcross, GA), treated with DNase
using TURBO DNA-free kit (Ambion, Austin, TX), and 1 mgRNA
was retrotranscribed using M-MLV reverse transcriptase (Invitrogen,
Carlsbad, CA) according to manufacturer instructions. RT-PCR was
performed using 1 ml cDNA (equivalent to 100 ng), 10 ml Go-Taq Green
Master Mix 23reagent (Promega, Madison, WI), and primers and
water for a final volume of 20 ml. Forward and reverse primers were
used at 0.5 mM. PCR was carried out for 35 cycles with 95˚C melting
(30 s), 57˚C annealing (45 s), and 72˚Cextension (45 s). Primer sequence
were as follows: b-actin forward, 59-CAGCTTCTTTGCAGCTCCTT-39;
b-actin reverse, 59-CCTGGATGGCTACGTACATGGC-39; serotonin trans-
porter (SERT) forward, 59-CTGAGATGAGGAACGAAGAC-39;SERTre-
verse, 59-CTGAGTGATTCCATAGAACCA-39; norepinephrine transporter
(NET) forward, 59-GCTAGATAGTTCAATGGGAGG-39; NET reverse, 59-
CTCACGAACTTCCAACACAG-39; MAO-A forward, 59-GCATGATAA-
TTGAAGATGAGGAGG-39; MAO-A reverse, 59-CGAATCACCCTTCC-
ATAC A G - 3 9; MAO-B forward, 59-GTATGGAATCCTATCACCTACC-39;
MAO-B reverse, 59-AATTTCCTCTCCTGTCCTCC-39;DbHforward,59-
TTCCAATGTGCAGCTGAGTC-39;DbH reverse, 59-GGTGCACTTGT-
CTGTGCAGT-39; VMAT2 forward, 59-TGCCAGCGAGCATCTCTTAT-39;
VMAT2 reverse, 59-CTTCCTTAGCAGGTGGACTT-39. Amplicon sizes
were verified by electrophoresis on a 1.5% agarose gel after ethidium bro-
mide staining.
Analysis of cytokine mRNA production by real-time RT-PCR
Cells were lysed and RNA was extracted using the TRIzol reagent. cDNA
(1 mg) was synthesized using oligonucleotide and M-MLV reverse tran-
scriptase (Promega) according to the manufacturer’s instructions. A 20-ml
real-time PCR reaction included 1 ml cDNA, 10 ml Brilliant SYBR Green
The Journal of Immunology 3063
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QRT-PCR Master Mix (Stratagene), and primers and water as indicated
by the manufacturer’s instructions. PCR was carried out for 40 cycles
with 95˚C melting (30 s), 60˚C annealing (45 s), and 72˚C extension (40 s).
All reactions were performed on a Stratagene Mx3000P. Primer sequences
were as follows: IL-23 forward, 59-TGCTGGATTGCAGAGCAGTAA-39;
IL-23 reverse, 59-GCATGCAGAGATTCCGAGAGA-39; IL-6 forward,
59-AGGATACCACTCCCAACAGACCT-39; IL-6 reverse, 59-CAAGTG-
CATCATCGTTGTTCATAC-39; IL-1bforward, 59-CAAATCTCGCAG-
CAGCACA-39; IL-1breverse, 59-TCATGTCCTCATCCTGGAAGG-39;
TGF-bforward, 59-TGCGCTTGCAGAGATTAAAA-39; TGF-breverse,
59-CTGCCGTACAACTCCAGTGA-39; GADPH forward, 59-TCCGTGT-
TCCTACCCCCAATG-39; GADPH reverse, 59-GAGTGGGAGTTGCTG-
TTGAAG-39. For relative quantification, mRNA expression in each sample
was normalized by comparison with the GAPDH mRNA expression using
the ddCT method as previously described (34).
Phosphorylation of ERKs
DCs were incubated with FBS-free medium for 14 h before experiments.
DCs (2 310
6
cells/ml) were washed twice and resuspended in prewarmed
medium either in the presence or absence of 100 ng/ml LPS and either left
untreated or treated with 1 nM SKF38393 (SKF) or 1 nM SCH23390
(SCH; Tocris, Bristol, U.K.) for 10 min. Cells were lysed with ice-cold
lysis buffer 23[2% Triton X-100, 100 mM Tris-HCl pH 7.6, 80 mM
b-glycerophosphate, 50 mM sodium fluoride, 2 mM sodium orthovana-
date and protease inhibitor mixture (Sigma)]. Cell lysates (50 mg/sample)
were resolved by SDS-PAGE, transferred to polyvinylidene difluoride
membranes (Hybond-P; Amersham Biosciences, Uppsala, Sweden),
and diphosphorylated-ERK1/2 was detected using a mouse phospho-
specific ERK1/2 mAb (1:2000; Sigma) followed by HRP-conjugated
goat anti-mouse IgG Ab (1:2000; Rockland). Immunodetection was car-
ried out with SuperSignal West Pico chemiluminescent substrate (Pierce,
Rockford, IL). Membranes were stripped and reprobed with rabbit anti-
ERK1/2-specific pAb (1:30,000; Sigma) followed by HRP-conjugated
goat anti-rabbit Ab (1:4000; Rockland) and detected as described above.
Cytokine ELISA
Levels of IL-6, IL-10, and IL-12 released into the culture supernatant of
iDCs and mDCs and IL-2 secreted by T cells into the coculture supernatant
were quantified by ELISA as previously described (35). Recombinant
murine cytokines were used as standards for quantification. All of the
reagents, recombinant cytokines, and Abs used were purchased from BD
Pharmingen. When indicated, DCs were pretreated with 1 mM reserpine
(Tocris) for 1 h to promote depletion of intracellular stores of DA. Sub-
sequently, cells were washed, resuspended in fresh prewarmed medium,
and stimulated or not with LPS as described earlier.
T cell activation assays
iDCs or mDCs were washed and resuspended in fresh prewarmed medium.
Subsequently, DCs were cocultured at indicated ratios (see the figures and
figure legends that accompany this article) with either purified OT-I or OT-II
T cells (10
5
T cells/well) in the presence of 0.1 ng/ml OVA
257–264
peptide
or 200 ng/ml OVA
323–339
peptide (GenScript, Piscataway, NJ), respec-
tively. Purification of OT-I and OT-II T cells from total splenocytes was
carried out by negative selection using CD8
+
or CD4
+
T cell isolation kits
(Miltenyi Biotec), respectively. T cell activation was determined as IL-2
secretion in the coculture supernatant after incubation for 24 h by ELISA
(35). For determination of T cell proliferation, purified OT-I or OT-II
T cells were stained with 5 mM CFSE (Invitrogen) in the presence of
5% heat-inactivated FBS for 5 min at room temperature. Subsequently,
cells were washed and cocultured with DCs as indicated earlier. After 72 h
of coculture incubation, cells were stained either with allophycocyanin-
conjugated anti-CD8 (clone 53-6.7) or anti-CD4 (clone RM4-5) (both from
BD Pharmingen), and CFSE-associated fluorescence of the CD8
+
or CD4
+
populations were analyzed by flow cytometry.
EAE induction and evaluation
Six- to eight-week-old female C57BL/6 WT or D5RKO mice were injected
s.c. with 50 mg myelin oligodendrocyte glycoprotein 35–55 peptide
(pMOG; Genetel Laboratories, Madison, WI) emulsified in CFA (Invi-
trogen) supplemented with heat-inactivated Mycobacterium tuberculosis
H37 RA (Difco Laboratories, Detroit, MI). In addition, mice received i.p.
injections of 500 ng pertussis toxin (Calbiochem, La Jolla, CA) on days
0 and 2. Clinical signs were assessed daily according to the following
scoring criteria: 0, no detectable signs; 1, flaccid tail; 2, hind limb weak-
ness or abnormal gait; 3, complete hind limb paralysis; 4, paralysis of fore
and hind limbs; and 5, moribund or death. In some EAE experiments, 10
6
bone marrow-derived DCs from WT and D5RKO mice were pulsed with
5mg/ml pMOG for 4 h and then transferred i.v. into WT C57BL/6 re-
cipient mice 14 and 7 d before EAE induction. For the preparation of CNS
mononuclear cells, mice were perfused through the left cardiac ventricle
with cold PBS. The brain and spinal cord were dissected, and CNS tissue
was cut into small pieces and digested by collagenase D (2.5 mg/ml;
Roche Diagnostics) and DNaseI (1 mg/ml; Sigma) at 37˚C for 45 min.
Digested tissue was passed through a 70-mm cell strainer obtaining single-
cell suspension that was subjected to centrifugation in a Percoll gradient
(70%/37%). Mononuclear cells were removed from the interphase and
resuspended in culture medium for further analysis.
Statistical analysis
Statistical significance of differences between groups was evaluated by two-
tailed Student ttest or Mann–Whitney rank sums two-tailed Utest by using
GraphPad Prism software.
Results
DCs express functional D5R whose expression is modulated
during maturation
To determine whether DA could regulate DC function, we first
evaluated the expression of DARs on the cell surface using specific
Abs and flow cytometry. Our results show that both iDCs and mDCs
express D1R, D2R, D3R, and D5R. Notably, only D5R expression
was significantly downregulated on DCs after LPS treatment (Fig.
1A, 1B), suggesting that D5R could be involved in the regulation
of DC function during maturation. Inflammatory signals triggered
by TLR stimulation on DCs involve activation of several signaling
pathways including NF-kB, MAPKs, PI3K, and STATs (36). To
test whether D5R engagement is capable of affecting LPS-induced
intracellular signaling, we studied the phosphorylation of key
signaling molecules involved in cytokine production, ERK1/2,
JNK, and p38 MAPK (37). Stimulation with the selective D1R/
D5R agonist SKF attenuated LPS-induced ERK1/2 phosphoryla-
tion in WT DCs (Fig. 1C, top panel), an effect not observed in
D5RKO DCs (Fig. 1C, bottom panel). No significant differences
were observed in levels of ERK1/2 phosphorylation when WT or
D5RKO DCs were treated with the D1R/D5R antagonist SCH
in the presence (Fig. 1C) or absence of LPS (data not shown). No
changes in phosphorylation levels of p38 and JNK were detected
upon stimulation of D1/D5 receptors in WT or D5RKO DCs (data
not shown). These results indicate that D5R is expressed on DCs,
and its stimulation is coupled to modulation of LPS-triggered
signaling pathways.
DCs express the machinery to synthesize, store, and degrade
DA
Previous studies have shown that some immune cells not only
express neurotransmitter receptors but also have the ability to
synthesize, internalize, and accumulate neurotransmitters in in-
tracellular reservoirs (38, 39). Such properties enable these cells to
release neurotransmitters in the presence of certain stimuli. These
neurotransmitters may act in an autocrine/paracrine manner to
modulate DC function (37, 40). We investigated the expression of
key enzymes necessary for biosynthesis, degradation, uptake, and
storage of DA in DCs. Accordingly, we first analyzed intracellular
TH expression in DCs, an enzyme necessary to synthesize DA. We
found that DCs express TH and that its expression is regulated
during DC maturation (Fig. 2A). Next, transcripts for VMAT2,
which mobilizes cytosolic DA toward vesicular storage, were de-
tected in iDCs, whereas very low to undetectable levels were
found in mDCs (Fig. 2B). These results correlate with the de-
creased expression of TH detected in mDCs compared with that
in iDCs (Fig. 2A). In contrast, expression of DbH mRNA, an
enzyme that oxidizes DA to produce norepinephrine in catechol-
aminergic cells, could not be detected in either iDCs or mDCs
3064 STIMULATION OF D5R FACILITATES DC IMMUNOGENICITY
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(Fig. 2B). Thus, DCs express some components required to syn-
thesize and store DA but do not seem to contain components to
convert DA to norepinephrine.
Some dopaminergic cells can also take up DA from the extra-
cellular environment. According to this notion, we analyzed the
expression of DAT and NET, which can both take up DA from the
extracellular compartment. Neither DAT protein (Fig. 2C) nor NET
mRNA (Fig. 2D) were detected in DCs. The human and rat SERT
were recently described to mediate DA uptake (41). Notably,
SERT mRNA expression was found in both iDCs and mDCs (Fig.
2D), suggesting that DCs could take up DA in a noncanonical
fashion. Next, we assessed the expression of DA-degrading en-
zymes in DCs. We detected the mRNA expression of two mono-
amine oxidase enzymes, MAO-A and MAO-B, in both iDCs and
mDCs (Fig. 2E), indicating that DCs are capable of catabolizing
intracellular DA. Taken together, these results indicate that DCs
express molecular components required to synthesize, store, and
degrade DA.
FIGURE 1. Expression of functional D5R in DCs. DCs obtained from WT mice were either left unstimulated (iDCs) or stimulated with 100 ng/ml LPS
for 48 h (mDCs), and DAR expression was assessed in the CD11c
+
I-A
b+
population by flow cytometry (A,B). (A) Representative histograms of DAR
expression in iDCs (bold line) and mDCs (dotted line) are shown. Filled histograms correspond to nonspecific Ig control for each receptor subtype. (B) The
ratio of the mean fluorescence intensity (MFI) corresponding to specific label over MFI corresponding to unspecific control is represented. Data from four
independent experiments are shown. Values represent mean 6SD. **p,0.01 (unpaired Student ttest). (C) DCs obtained from WT or D5RKO mice were
either left untreated (UT) or treated with 100 ng/ml LPS, alone or in the presence of either the D1R/D5R agonist SKF or the D1R/D5R antagonist SCH for
10 min. Cells were lysed in the presence of phosphatase inhibitors, and the presence of diphosphorylated ERK1/2 (pERK1/2, top panel) or total ERK1/2
irrespective of their phosphorylations (ERK1/2, bottom panel) was analyzed in protein extracts by Western blot. Representative data from one of three
independent experiments are shown.
FIGURE 2. Expression of components of the dopaminergic system in DCs. Bone marrow-derived DCs were either left unstimulated (iDCs) or stimulated
with LPS (mDCs). After 24 h, cells were analyzed for protein expression by flow cytometry (A) or Western blot (C) and for mRNA expression by RT-PCR
(B,D, and E). (A) Intracellular TH expression was analyzed in the CD11c
+
I-A
b+
population. In the left panel, a representative result is shown. Isotype
control is indicated with a solid gray area. In the right panel, the ratio of MFI corresponding to specific label over MFI corresponding to isotype control is
represented. Data from four independent experiments are shown. Values represent mean 6SD. *p,0.05 (unpaired Student ttest). (B) Expression of
enzymes involved in catecholamine biosynthesis (DbH) and storage (VMAT2) was assessed with specific primers. (C) Detection of the plasma membrane
DAT was assessed in total cell extracts by using specific Abs. (D) Transcripts of norepinephrine and serotonin transporters were also analyzed using NET
and SERT specific primers, respectively. (E) Transcripts encoding for DA degrading enzymes, MAO-A and MAO-B, were detected by using specific
primers. Representative data from two experiments are shown. Positive controls (+) correspond to suprarenal gland, except for SERT, for which cerebellum
was used. b-Actin was used as loading control.
The Journal of Immunology 3065
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D5R selectively modulates cytokine secretion by
LPS-stimulated DCs
Next, we determined whether D5R regulates the phenotype of DCs
upon maturation. To do this, we evaluated the secretion of regu-
latory cytokines and expression of key surface markers by WT and
D5RKO DCs either untreated or matured with LPS. Whereas no
differences were detected in the levels of IL-12, IL-10, and IL-6
secreted by WT or D5RKO iDCs, D5RKO mDCs secreted sig-
nificantly less IL-12 compared with that of WT mDCs (Fig. 3A).
We observed no differences in levels of IL-10 and IL-6 released by
WT or D5RKO mDCs. To gain more insight into the role of D5R
in the production of other important cytokines involved in CD4
+
T cell polarization, we determined mRNA levels for IL-23,
TGF-b, and IL-1bproduced by WT or D5R-deficient DCs. Re-
sults show that expression of D5R contributed significantly to the
production of IL-23 mRNA by mDCs but not by iDCs (Fig. 3B).
Conversely, D5R was not relevant for production of TGF-bor
IL-1bby DCs (Fig. 3B). A comparison of the expression of sur-
face maturation markers such as I-A
b
, CD80, and CD86 in WT
and D5RKO mDCs revealed no differences (Supplemental Fig. 1).
Given that DCs express machinery to synthesize and store DA, we
hypothesized that intracellular stores of DA may stimulate D5R in
an autocrine manner and promote IL-12 stimulation upon matu-
ration. To test this possibility, we performed LPS-induced matu-
ration experiments in which WT DCs were previously depleted of
intracellular stores of DA by treatment with reserpine (42). De-
pletion of DA caused a significant decrease in IL-12 secretion in
mDCs without affecting IL-10 or IL-6 production (Supplemental
Fig. 2). These results suggest that during the maturation process,
DC-derived DA selectively facilitates secretion of some regulatory
cytokines by stimulating the D5R in an autocrine manner.
D5R expressed on DCs facilitates a strong CD4
+
T cell
response
Cytokines play an important role in the priming of T cells by DCs.
Because we found that D5R selectively regulates cytokine pro-
duction in DCs (Fig. 3), we assessed the ability of D5R expressed
on DCs to modulate the priming of naive Ag-specific T cells. To
this end, OVA-specific naive T cells were cocultured with WT or
D5RKO DCs pulsed with OVA-derived peptides (see Materials
and Methods), and T cell activation and proliferation were de-
termined by measuring IL-2 secretion and dilution of CFSE-
associated fluorescence, respectively. Whereas no difference in
IL-2 release was observed from CD4
+
T cells cocultured with WT
or D5RKO iDCs, CD4
+
T cells produced much less IL-2 when
cultured with D5R-deficient mDCs than when cultured with WT
mDCs (Fig. 4A). This suggests that D5RKO mDCs are signifi-
cantly less efficient at activating CD4
+
T cells than are WT mDCs.
In contrast, no difference was observed in IL-2 production from
CD8
+
T cells when cocultured with WT or D5RKO DCs, re-
gardless of the maturation state (Fig. 4A).
Because differences in the capability of DCs to promote efficient
T cell activation (Fig. 4A) and cytokine production (Fig. 3) were
observed between WT and D5R-deficient DCs only at the mature
state, we next compared T cell proliferation induced by WT or
D5RKO mDCs. In support of our previous results, we found that
D5R-deficient mDCs were less efficient at inducing CD4
+
T cell
proliferation compared with WT mDCs (Fig. 4B). We observed no
differences in the proliferation rates of CD8
+
T cells when they
were cocultured either with WT or D5RKO mDCs (Fig. 4B),
confirming our data of CD8
+
T cell activation (Fig. 4A). These
results indicate that D5R expressed in DCs facilitates strong
priming of CD4
+
T cells but does not contribute to CD8
+
T cell
activation or proliferation.
Absence of D5R on DCs decreases EAE severity
To study the relevance of the D5R in the modulation of the CD4
+
T cell-mediated response in vivo, we used EAE, a murine model
of autoimmunity mainly mediated by autoreactive CD4
+
T cells
(43). Accordingly, we first compared the susceptibility of WT and
D5RKO mice to develop the disease after EAE induction. Disease
onset and progression were quantified using a clinical score as
described in Materials and Methods. Whereas there were no dif-
ferences in disease incidence between WT and D5RKO mice,
D5R-deficient mice displayed a delayed onset of the disease
compared with WT mice (day 12.80 62.37 versus day 9.54 6
2.18, p,0.001) (Fig. 5A). Additionally, D5RKO mice exhibited
a significantly reduced clinical score or severity of disease com-
pared with that of WT mice (Fig. 5A).
To determine the contribution of the D5R expressed specifically
on DCs to EAE development and progression, pMOG-pulsed WT
or D5R-deficient DCs were transferred into WT recipient mice
FIGURE 3. Lack of D5R selectively impairs cyto-
kine production on LPS-stimulated DCs. (A) DCs ob-
tained from WT (white) or D5RKO (gray) mice were
either left unstimulated (iDCs) or stimulated with 100
ng/ml LPS (mDCs). After 24 h, IL-12, IL-10, and IL-6
secretion was evaluated by ELISA (A), and IL-23,
TGF-b, and IL-1bmRNA expression was evaluated by
semiquantitative real-time RT-PCR (B). Data corre-
spond to the fold increase in cytokine secretion (A)or
cytokine mRNA expression (B) relative to WT iDCs.
PCR amplification of GADPH was used as an internal
control (B). Values represent mean 6SD from at least
three independent experiments. ***p,0.001 (un-
paired Student ttest).
3066 STIMULATION OF D5R FACILITATES DC IMMUNOGENICITY
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14 and 7 d before EAE induction, and disease progression was
monitored daily. We found that mice that received D5RKO DCs
presented a significantly less severe disease than mice that received
WT DCs (Fig. 5B). There were no significant differences in the
disease incidence or kinetics of disease onset when comparing the
development of EAE on WT DC- or D5RKO DC-recipient mice
(data not shown).
Because of the pivotal role of CNS-infiltrating Th1, Th17, and
Treg CD4
+
T cells in the outcome of EAE (43), we examined the
functional phenotypes of CD4
+
T cells infiltrating the CNS at the
peak of the disease. At day 15 after disease induction, we isolated
the infiltrating mononuclear fraction from spinal cords and brains
of mice transferred with either WT or D5R-deficient DCs (see
Materials and Methods for more details). No differences were
detected in the levels of infiltrating Tregs (CD25
+
Foxp3
+
)or
effector CD4
+
T cells (CD25
+
Foxp3
2
) (data not shown). How-
ever, we did detect significant differences in the levels of CD4
+
T cell subtypes (for gating strategy, see Supplemental Fig. 3). We
found a significant reduction in the levels of IL-17–producing
CD4
+
T cells in the CNS of mice that received D5RKO DCs
compared with those in the CNS of mice that received WT DCs
(Fig. 5C, 5D). The proportion of IFN-g
+
CD4
+
T cells were var-
iable between the two groups, but no significant differences were
found (Fig. 5C, 5D). Notably, the percentage of double-positive
IFN-g
+
IL-17
+
CD4
+
T cells infiltrating the CNS was significantly
reduced in mice that received D5RKO DCs compared with those
in the CNS of mice that received WT DCs (Fig. 5C, 5D). Because
DA has also been involved in the polarization of CD4
+
T cells
toward Th2 phenotype (25), we evaluated the presence of IL-4
+
cells in the population of CD4
+
T cells infiltrating the CNS. We
did not detect IL-4–producing cells in the CNS of mice receiving
WT or D5RKO DCs (data not shown), thereby discarding the
participation of Th2 cells. Taken together, these data indicate that
D5R expressed on DCs is able to modulate the development of an
autoimmune response in vivo by biasing the differentiation of
CD4
+
T cells toward the Th17 phenotype.
Discussion
In this work, we present data from in vitro experiments as well
as an in vivo autoimmunity model that illustrate the involvement
of D5R signaling in DCs in the determination of CD4
+
T cell fate.
Our data from in vitro experiments suggest a mechanism triggered
by DC maturation, which involves DA secretion and sub-
sequent autocrine stimulation of D5R promoting selective reg-
ulation on cytokine release, thereby contributing to efficient Ag-
specific CD4
+
T cell response. In agreement with our in vitro data,
in this study we show that D5R expressed on DCs contributes to
the development of a CD4
+
T cell-driven autoimmunity. These
findings contribute to the knowledge of DC physiology and sug-
gest relevant molecular targets for immunotherapy.
Accumulating evidence has pointed to a role for neuro-
transmitters in regulating the immune system and influencing the
activation and differentiation of T cells. In agreement with this, we
show that mouse DCs express receptors for the neurotransmitter
DA and express components necessary to synthesize, store, and
degrade DA itself. The timing of expression of these components
FIGURE 4. DCs lacking D5R show impaired CD4
+
, but not CD8
+
, T cell priming ability. (A) DCs obtained from WT or D5RKO mice were either left
unstimulated (iDCs) or stimulated with LPS (mDCs) for 24 h and subsequently cocultured with CD4
+
(top panels) or CD8
+
(bottom panels) T cells purified
from OT-II or OT-I mice, respectively, in the presence of corresponding OVA-derived peptides. After 24 h, IL-2 release was assessed in the culture su-
pernatant by ELISA. Data from four independent experiments are shown. Values represent mean 6SEM. *p,0.05 (unpaired Student ttest). (B) DCs
obtained from WT or D5RKO mice were stimulated with LPS (mDCs) for 24 h and then cocultured with CFSE-stained CD4
+
(top panels) or CD8
+
(bottom
panels) T cells purified from OT-II or OT-I mice (5 310
3
DCs and 10
5
T cells/well), respectively, in the presence of corresponding OVA-derived peptides.
After 72 h, dilution of CFSE-associated fluorescence was assessed in the CD4
+
or CD8
+
population by FACS. Numbers on histograms represent percentage
of proliferating cells. Data are representative of at least four independent experiments. Values represent mean 6SEM. **p,0.01 (compared with WT by
paired Student ttest).
The Journal of Immunology 3067
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(Fig. 2) and the requirement for intracellular DA to induce se-
lective cytokine secretion from mDCs (Supplemental Fig. 2)
suggest an autocrine mechanism of action of this neurotransmitter.
These results are in agreement with previous observations that
an anti-DA Ab produced positive immunoreactivity in human
monocyte-derived DCs (25). Notably, only D5R expression de-
creased upon LPS-induced DC maturation, suggesting a role for
this receptor during DC maturation. Using a genetic approach, we
were able to analyze the particular effects of D5R on DC function.
In cell culture, D5R appears to be important for both IL-23 and
IL-12 production from mature mDCs and the subsequent activa-
tion of CD4
+
T cells.
A previous study hinted at the involvement of type I DARs in
the amelioration of EAE, the murine model of MS. In this study,
the systemic treatment of mice with the type I DAR antagonist
SCH alleviates the development of EAE by decreasing the Th17
response (24). However, this pharmacologic approach does not
permit discrimination between the effects of D5R or D1R because
the drug inhibits both type I DARs. In fact, SCH not only displays
similar affinities for D1R and D5R (K
i
= 0.2 and 0.3 nM, re-
spectively) (44) but also displays comparable affinities for sero-
tonin receptors 5-HT1C and 5-HT2C (K
i
= 6.3 and 9.3 nM,
respectively) (26, 27). Furthermore, this study could not confine
the cell type responsible for the amelioration effect. EAE is me-
diated mainly by T cells, but B cells also contribute to the initi-
ation and development of the disease (43, 45, 46). Type I DARs as
well as serotonin receptors have been found to be expressed on
many types of immune cells including DCs (47), T cells (23, 48,
49), and B cells (50, 51). Therefore, systemic treatment with SCH
could affect several types of immune cells. To restrict the con-
tribution of DC signaling to the amelioration of EAE, the authors
treated DCs with SCH ex vivo and transferred treated cells into
WT recipient mice (24). A slight decrease in EAE severity was
seen in mice transferred with SCH-treated DCs, but such differ-
ences were not statistically significant (24).
In contrast, our genetic approach allowed us to determine the
contribution of DCs and specific DARs to CD4
+
T cell differen-
tiation and EAE development and progression. By comparing
results obtained from WT DCs and D5RKO DCs, we were able to
determine the effects of D5R signaling on DCs and to resolve the
contributions of D5R signaling to CD4
+
T cell polarization. We
found that D5R signaling in DCs selectively affects IL-23 and IL-
12 production by DCs (Fig. 3) and contributes to CD4
+
T cell
activation and proliferation (Fig. 4). In our in vivo model of EAE,
we showed that signaling through D5R on DCs is important for
the development of this disease. D5R-deficient mice exhibited
delayed EAE progression with reduced severity compared with
normal mice (Fig. 5), presumably due to the reduced proportion of
Th17 (IL-17
+
) T cells present in the CNS of these mice (Fig. 5).
Thus, although data are in agreement with a previous study (24),
we were able to show that D5R specifically expressed on DCs
contributes directly to CD4
+
T cell fate and disease progression. In
addition to the decreased disease severity, we also observed that
the onset of EAE was delayed in D5RKO mice compared with that
in WT mice (Fig. 5A). This delay was not seen in WT mice
transferred with D5RKO DCs (Fig. 5B). Moreover, the decrease
in EAE severity was not as robust in WT mice transferred with
D5RKO DCs (Fig. 5B) as that observed in D5RKO mice (Fig.
5A). These differences in the decrease of EAE severity and in
disease onset observed in D5RKO mice and WT mice transferred
FIGURE 5. The absence of D5R on DCs reduces severity of EAE by decreasing infiltration of IL-17–producing CD4
+
T cells into the CNS. (A) EAE was
induced in WT mice (black line) and D5RKO mice (gray line) by immunization with pMOG in CFA followed by pertussis toxin injection (see Materials
and Methods). (B) WT (black line) or D5RKO (gray line) purified bone marrow-derived DCs were pulsed with pMOG and transferred (10
6
DCs/mice; i.v.
injections) into WT recipient mice at days 14 and 7 prior to EAE induction. Disease severity was evaluated as clinical score (see Materials and Methods)
from day 0 to day 20 postinduction (A,B). Data from six to eight mice in each group, corresponding to a representative from three (A) and two (B) in-
dependent experiments, are shown. Values represent mean 6SEM. *p,0.05, **p,0.01 (Mann–Whitney Utest). Mononuclear cells were isolated from
CNS followed by ex vivo stimulation with PMA/ionomycin in the presence of brefeldin A, and intracellular cytokine staining analysis was carried out by
flow cytometry. (C) Representative dot plots for IL-17 versus IFN-gproduction in the infiltrating CD4
+
gated population are shown. Numbers at the corners
indicate the percentage of IFN-g
+
IL-17
2
, IFN-g
+
IL-17
+
, and IFN-g
2
IL-17
+
cells, respectively. (D) Bar graphs indicate percentage of CNS-infiltrating
CD4
+
T cells producing IFN-g(left panel), IL-17 (central panel), or both cytokines (right panel). Data representative of two independent experiments are
shown. Values represent mean 6SEM. *p,0.05, **p,0.01 (unpaired Student ttest).
3068 STIMULATION OF D5R FACILITATES DC IMMUNOGENICITY
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with D5RKO DCs may suggest that D5R signaling in cell types
other than DCs is involved in the progression and development of
this autoimmune disease. In this regard, Kipnis et al. (52) have
described that Tregs express type I DARs, and stimulation of these
receptors attenuates the suppressive activity of Tregs. Thus, ab-
sence of D5R in Tregs could favor the suppressive activity of these
cells and thereby contribute to decreased EAE manifestation ob-
served in D5RKO mice (Fig. 5A), which should not be observed in
WT mice transferred with D5RKO DCs (Fig. 5B).
Another nonexcluding possibility for explaining the differences
on EAE severity and onset between D5RKO mice and WT mice
transferred with D5RKO DCs is that endogenous DCs in WT
recipients could compete with exogenously administered D5RKO
DCs. Some of the exogenously administered D5RKO DCs bearing
pMOG could die soon after injection and be phagocytosed by
endogenous WT APCs. Thus, the effect due to the pMOG pre-
sentation by D5RKO DCs would be, in part, masked by pMOG
presentation by WT APCs in vivo. Therefore, the occurrence of this
possibility would lead to observation of a subestimated effect
promoted by D5RKO DCs in WT mice (Fig. 5B) compared with
that in D5RKO mice (Fig. 5A).
Considering the in vitro data, our data obtained from in vivo
experiments show both expected and unexpected results. In ag-
reement with the contribution of D5R expressed on DCs to the
production of IL-23 observed in vitro (Fig. 3B), we observed that
mice transferred with D5RKO DCs had fewer Th17 CD4
+
T cells infiltrating the CNS upon EAE (Fig. 5D). In contrast,
despite IL-12 production from DCs being favored by D5R
in vitro (Fig. 3), the proportion of CNS-infiltrating Th1 cells was
not affected by D5R expressed on DCs in vivo (Fig. 5D). This
discrepancy could be due to the presence of other mediators
in vivo (but absent in vitro) that could act on DCs contributing to
IL-12 production, making DA–D5R participation unnecessary
and/or redundant for this effect. However, it seems that the DA–
D5R axis operating in DCs is not redundantly contributing to
strong IL-23 production in vivo.
In summary, the data presented in this study suggest the exis-
tence of an autocrine modulatory mechanism mediated by DA
operating in DCs. DA release triggered by the DC maturation
process could act through D5R and promote the release of
proinflammatory cytokines. This could, in turn, induce a potent
Th17 response in vivo, contributing to the development of auto-
immunity.
Acknowledgments
We thank Dr. Emma McCullagh for critical reading of the manuscript, Dr.
David Sibley for donation of D5RKO mice, Dr. Marı
´a Rosa Bono for
donation of OT-II transgenic mice, and Sebastia
´n Valenzuela for valuable
veterinary assistance at the Fundacion Ciencia y Vida animal facility.
Disclosures
The authors have no financial conflicts of interest.
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