The Role of Kinin Receptors in Preventing
Neuroinflammation and Its Clinical Severity during
Experimental Autoimmune Encephalomyelitis in Mice
Rafael C. Dutra1, Daniela F. P. Leite1, Allisson F. Bento1, Marianne N. Manjavachi1, Eliziane S. Patrı́cio1,
Cláudia P. Figueiredo1, João B. Pesquero2, João B. Calixto1*
1 Department of Pharmacology, Centre of Biological Sciences, Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil, 2 Department of Biophysics,
Universidade Federal de São Paulo, São Paulo, Brazil
Abstract
Background: Multiple sclerosis (MS) is a demyelinating and neuroinflammatory disease of the human central nervous
system (CNS). The expression of kinins is increased in MS patients, but the underlying mechanisms by which the kinin
receptor regulates MS development have not been elucidated.
Methodology/Principal Findings: Experimental autoimmune encephalomyelitis (EAE) was induced in female C57BL/6 mice
by immunization with MOG35–55 peptide emulsified in complete Freund’s adjuvant and injected with pertussis toxin on day
0 and day 2. Here, we report that blockade of the B1R in the induction phase of EAE markedly suppressed its progression by
interfering with the onset of the immune response. Furthermore, B1R antagonist suppressed the production/expression of
antigen-specific TH1 and TH17 cytokines and transcription factors, both in the periphery and in the CNS. In the chronic phase
of EAE, the blockade of B1R consistently impaired the clinical progression of EAE. Conversely, administration of the B1R
agonist in the acute phase of EAE suppressed disease progression and inhibited the increase in permeability of the bloodbrain barrier (BBB) and any further CNS inflammation. Of note, blockade of the B2R only showed a moderate impact on all of
the studied parameters of EAE progression.
Conclusions/Significance: Our results strongly suggest that kinin receptors, mainly the B1R subtype, play a dual role in EAE
progression depending on the phase of treatment through the lymphocytes and glial cell-dependent pathways.
Citation: Dutra RC, Leite DFP, Bento AF, Manjavachi MN, Patrı́cio ES, et al. (2011) The Role of Kinin Receptors in Preventing Neuroinflammation and Its Clinical
Severity during Experimental Autoimmune Encephalomyelitis in Mice. PLoS ONE 6(11): e27875. doi:10.1371/journal.pone.0027875
Editor: Robyn Klein, Washington University, United States of America
Received September 26, 2011; Accepted October 27, 2011; Published November 22, 2011
Copyright: ß 2011 Dutra et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Conselho Nacional de Desenvolvimento Cientı́fico eTecnológico (CNPq), Coordenação de
Aperfeiçoamento de Pessoal de Nı́vel Superior (CAPES), Programa de Apoio aos Núcleos de Excelência (PRONEX) and the Fundaçãode Apoio à Pesquisa Cientı́fica
Tecnológica do Estado de Santa Catarina (FAPESC), all of Brazil. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript. R.C.D., A.F.B., and M.M.N. are Ph.D. students in pharmacology receiving grants from CNPq and CAPES, respectively.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: calixto@farmaco.ufsc.br
receptors. The B2R is constitutively expressed throughout central
and peripheral tissues, while the B1R is normally up-regulated
following inflammatory, infectious or traumatic stimuli, exerting a
critical role in several chronic diseases [11,12].
Recent reports demonstrated the involvement of the kinins and
their receptors in MS and the experimental autoimmune encephalomyelitis (EAE) model [13,14,15]. For instance, high levels
of the kallikrein-kinin components, namely des-Arg9-bradykinin
(DABK), bradykinin, kallikrein-1 and kallikrein-6, as well as lowmolecular-weight kininogens (KNGL), have been found in the
CNS tissue and cerebrospinal fluid from both animals with EAE
and MS patients [16,17].
Experiments carried out with B2R-knockout mice showed that
the clinical parameters of MOG35–55-induced EAE are reduced
via the modulation of leukocyte recruitment into the CNS [14];
however, the participation of B2R seems to be less important than
B1R in the development of EAE [15,17]. It was recently shown
that B1 mRNA expression positively correlated with the expanded
disability status scale (EDSS) index and the occurrence of clinical
Introduction
Multiple sclerosis (MS) is the most common inflammatory
demyelinating disease of the central nervous system (CNS) that
cause neurological disability in young adults, affecting about two
million people worldwide [1,2]. The hallmarks of MS include
neuronal loss, axonal injury and atrophy of the CNS, due to a
progressive inflammatory reaction involving both the adaptive and
the innate immune system [3,4,5]. During the course of MS,
autoreactive T cells activated in the periphery by viral or infectious
antigens, which show molecular similarity to the CNS antigen [6],
differentiate into TH1 or TH17 cells, migrate across the bloodbrain barrier (BBB) and successively induce inflammatory lesions
distributed throughout the CNS [2].
The CNS of mammals contains all of the components of the
kallikrein-kinin system [7] and accumulating evidence suggests
that these components are altered in neurodegenerative processes
[8,9,10]. The biological activities of kinin are mediated via two Gprotein-coupled receptors, named the B1 (B1R) and B2 (B2R)
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relapse in patients with MS [13]. In addition, B1R was found to be
up-regulated in both the brain endothelial cells [18] and
peripheral T lymphocyte cells in these patients [13].
It is widely accepted that, following their activation, both B1R
and B2R induce inflammation via the release of pro-inflammatory
cytokines and increased vascular permeability [11,19]. In marked
contrast to this, a recent paper suggested exactly the opposite, i.e.
that the activation of B1R protects against encephalitogenic T
lymphocyte recruitment to the CNS [17]. For this reason, we
hypothesized that kinin receptor, mainly B1R subtypes, could
display a dual role in EAE by acting at different phases of disease
progression. We further examined this hypothesis by using B1 and
B2-knockout mice in conjugation with a kinin selective agonist or
antagonist at different time points after the induction of EAE.
19 following immunization (Fig. 1B–G). Interestingly, in the
induction phase of EAE, the blockade of B1R by DALBK
(50 nmol/kg, i.p., twice a day, days 0–5) significantly reduced disease severity when compared to the EAE control group (Fig. 1B–
D). Importantly, the difference in the degree of disease severity
persisted until the end of our study at day 25. In line with our
findings for the DALBK treatment, the genetic deletion of B1R
also drastically reduced the clinical score of EAE and delayed the
disease onset (Fig. 1E–G). In contrast, preventive treatment with
the B2R antagonist HOE-140 (150 nmol/kg, i.p., twice a day,
days 0–5) (Fig. 1B–D), or the use of mice with a deletion of B2R,
only resulted in a moderate inhibition (Fig. 1E–G). These data
suggest a dominant role of kinin B1R in reducing EAE severity
during the induction phase.
Results
Kinin B1R inhibition or its genetic deletion in the disease
induction phase decrease the neuroinflammatory
response and myelin loss in the spinal cord
Dominant role of kinin B1R in the induction phase of EAE
Initially, in order to investigate the role of kinin receptors on the
EAE induction phase, we induced EAE by subcutaneous injections
of MOG35–55 in complete Freund’s adjuvant (CFA), and pertussis
toxin injections. By using this protocol, MOG-reactive T cells
begin to accumulate in regional lymph nodes on day 7, and mice
begin to develop clinical signs between days 10 and 12, with a peak
at around day 17 [20]. Therefore, we defined days 0 to 7 as the
induction phase, days 7–15 as the acute phase and days 15–25 as
the chronic phase of the disease (see scheme in Fig. 1A). Our
results showed that the EAE control group developed a first
relapse with subsequent chronic disease phase, characterized by
manifestation of an ascending paralysis starting from day 13 to day
The hallmarks of MS include multifocal perivascular Tlymphocytes, macrophages and activated microglia infiltrates in
the CNS, which induce oligodendrocyte loss and demyelination
[2,21]. In this set of experiments, we assessed the intensity of
inflammatory cell infiltrates in the naive group, the control group
(EAE), in mice pre-treated with the B1R antagonist DALBK
(50 nmol/kg), in mice pre-treated with the B2R antagonist HOE140 (150 nmol/kg), as well as in B1R2/2 mice and in B2R2/2
mice after 25 days post-immunization. It was found that the
number of inflammatory foci was significantly decreased in
B1R2/2 mice and in mice pre-treated with DALBK (P,0.01)
Figure 1. Kinin B1R inhibition or deletion in the disease induction phase attenuated the development of EAE and reduced the
inflammatory response in the CNS 25 days post-immunization (p.i.). Animals were immunized with MOG35–55 peptide/CFA and pertussis
toxin. (A) Schematic representation of EAE progression. Day 0 to 7: induction phase; day 7 to 15: acute phase and day 15 to 25: chronic phase of the
disease. EAE: experimental autoimmune encephalomyelitis; TH: CD4+ T helper lymphocytes. The clinical score (B, E), area under the curve (AUC) (C, F)
and locomotor activity (D, G) were analyzed in the naive group, the control group (EAE), in mice pre-treated with the selective kinin B1R antagonist
DALBK (50 nmol/kg), in mice pre-treated with the selective kinin B2R antagonist HOE-140 (50 nmol/kg), in B1R2/2 knockout mice and in B2R2/2
knockout mice 25 days p.i. The antagonists were administered intraperitoneally (i.p.), twice a day (12/12 h), for 5 days (day 0–5). The results of clinical
score are expressed as mean or as the AUC. Data are presented as mean 6 SEM of six to nine mice/group and are representative of three
independent experiments. #P,0.05 and ##P,0.001 versus the naive group, *P,0.05 and **P,0.001 versus the EAE group, DP,0.05 and DDP,0.001
versus the DALBK treatment or B1R2/2 mice (one-way ANOVA with the Newmann-Keuls post-hoc test).
doi:10.1371/journal.pone.0027875.g001
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Figure 2. Kinin B1R inhibition or genetic deletion decreased the level of inflammatory cell infiltration and the demyelination area in
experimental EAE. The lumbar spinal cords were histologically analyzed on day 25 p.i. in the different experimental groups for inflammation by
H&E staining (A, B, E) and for demyelination by luxol fast blue staining (C, D, F). The degree of inflammatory infiltrates and demyelination was
quantified from an average of four ocular field 5-mm sections of lumbar spinal cord white matter transverse sections per mouse for a total of six to
nine mice/group in the naive group, the control group (EAE), in mice pre-treated with the B1R antagonist DALBK (50 nmol/kg), in mice pre-treated
with the B2R antagonist HOE-140 (150 nmol/kg), in B1R2/2 knockout mice and in B2R2/2 knockout mice. Scale bar corresponds to 25 mm and applies
throughout. Data are presented as mean 6 SEM of six to nine mice/group and are representative of three independent experiments. #P,0.05 and
##
P,0.001 versus the naive group, *P,0.05 and **P,0.001 versus the EAE group (one-way ANOVA with the Newmann-Keuls post-hoc test).
doi:10.1371/journal.pone.0027875.g002
that CREB phosphorylation by itself is not a limiting event in the
recovery of animals with EAE.
(Fig. 2A, B, E). In order to further evaluate whether or not reduced
inflammation in the CNS in the absence of B1R could preserve
tissue integrity, we investigated the loss of myelin, the number of T
lymphocytes and reactive astrogliosis. The demyelination index
(Fig. 2C, D, F), number of CD3+ T cells (Fig. 3A–F) and GFAP
immunoreactivity (Fig. 3G–L) were all strikingly reduced when
assessed 25 days post-immunization in DALBK-treated animals
during induction phase of EAE and in B1R2/2 mice. In contrast,
in mice pre-treated with HOE-140 (150 nmol/kg, i.p., days 0–5)
and in B2R2/2 mice, these parameters showed no significant
changes (P.0.05) (Fig. 2A–F and Fig. 3A–L). Once again, these
data indicate that kinin B1R shows a dominant effect on EAE
neuroinflammation.
Recent studies also suggested that, at the peak of EAE, the
transcription factor cyclic AMP response element-binding protein
(CREB) is highly phosphorylated in the spinal cord [22], contributing
to the activation of macrophages and microglia, up-regulating the
MHC and co-stimulatory molecules [22,23]. Notably, pre-treatment
with the B1R antagonist (induction phase of EAE) or its genetic
depletion resulted in a marked reduction in CREB activation at the
lumbar white/grey matter of the spinal cord (Fig. 3M–R), according
to the assessment 25 days post-immunization. Pre-treatment with
HOE-140 or the genetic deletion of B2R also significantly prevented
the induction of CREB phosphorylation (P,0.01) (Fig. 3M–R).
However, the blockade of B2R did not significantly reduce the
demyelination area nor the inflammatory cell infiltrates, suggesting
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Blockade of kinin B1R in the EAE induction phase limits
CD4+ activation/expansion and effector cytokine
production in the peripheral lymphoid tissue
Several pieces of evidence now suggest that the extent of CNS
damage in EAE is associated with peripheral T cell activation [5].
We next assessed whether B1R or B2R exert a role on peripheral
lymphocyte activation. Lymph node (LN) obtained from B1R2/2
mice, B2R2/2 mice or mice previously treated with B1R or B2R
antagonists were re-stimulated with MOG and analyzed for cytokine production and CD69 expression, a major T cell activation
marker. We found a pronounced reduction in TNF-a (Fig. 4 A,B),
IFN-c (Fig. 4 C,D) and IL-17 (Fig. 4 E,F) levels produced by
MOG-reactive LN cells isolated from mice pre-treated with
DALBK and from B1R2/2 mice. In addition, pre-treatment with
HOE-140 resulted in a discrete inhibition of TNF-a in LN cells
(Fig. 4 A,B). Of great relevance, the blockade of B1R in the
induction phase restored IL-4 levels in LN after in vitro restimulation with MOG (Fig. 4 G,H), suggesting that the inhibition
of B1R may cause a shift from TH1/TH17 towards the TH2
phenotype. Notably, T CD4+ and T CD8+ MOG-reactive cells
LN from B1R2/2, B2R2/2 mice and from mice pre-treated with
DALBK showed a significant reduction in CD69 expression, while
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Figure 3. The blockade of kinin B1R in the disease induction phase by pharmacological treatment or genetic deletion ameliorated
EAE pathology. The spinal lumbar cords obtained on the 25th day after immunization from the different experimental groups were processed for
immunohistochemistry assays: T cell infiltration by CD3 immunoreactivity (A–F); astrocytes activation by GFAP immunoreactivity (G–L); and CREB
phosphorylation (M–R). Specifically, four 5-mm sections of lumbar spinal cord white matter (six to nine mice/group)<150 mm apart were obtained
between L4 and L6 from the naive group (A, G and M), the EAE group (B, H and N), from mice pre-treated with the B1R antagonist DALBK (50 nmol/
kg) (C, I and O), from mice pre-treated with the B2R antagonist HOE-140 (150 nmol/kg) (D, J and P) and from mice deficient in B1R (E, K and Q) and B2R
(F, L and R). The antagonists were administered i.p. twice a day (12/12 h), during day 0–5 p.i. Representative sections from three independent
experiments are shown. Scale bar corresponds to 25 mm and applies throughout.
doi:10.1371/journal.pone.0027875.g003
(Fig. 5F), IFN-c (Fig. 5G) and T-bet (Fig. 5H) in the lumbar spinal
cord, 25 days post-immunization. In addition, treatment with
DALBK during induction phase of EAE prevented the upregulation of B1R (Fig. 5I).
pre-treatment with HOE-140 only decreased CD69 expression in
T CD8+ cells (Fig. 4 I,L).
A recent study demonstrated that CD4+ T cells are activated in
the periphery by the MHC class II+ APC, which in turn increases
the proliferative response and migration to the subarachnoid space,
resulting in the formation of large T cell aggregates in the CNS [24].
In order to investigate whether or not B1R or B2R influences T cell
proliferative responses, we evaluated the incorporation of thymidine
in lymph node and spleen cells from WT, B1R2/2 and B2R2/2
immunized mice. In WT animals with EAE, a significant increase
was observed in the proliferative response towards MOG35–55 restimulation (Fig. 5 A,C), whereas in splenocytes (Fig. 5 A,B) and
lymph node cells (Fig. 5C) from B1R2/2 mice this proliferation was
markedly decreased. Taken together, these results suggest that the
blockade of B1R in the induction phase of EAE modulates the
activation and/or differentiation of TH1 and TH17-MOG reactive
cells.
Blockade of B1R after disease onset reduces clinical
symptoms and neuroinflammation induced by the EAE
model
Since MS patients urgently need new and more efficacious
therapies to be used after disease outcome, we next investigated
whether therapeutic treatment with the B1R antagonist DALBK
(50 nmol/kg, i.p.) or the B2R antagonist HOE-140 (150 nmol/kg,
i.p.) might be effective in controlling EAE. The mice were treated
for 5 days, twice a day, as soon as clinical signs of the disease
appeared (chronic phase: day 15 to day 20 post-immunization).
Remarkably, only the therapeutic treatment with DALBK
consistently blocked disease progression and motor deficits (Fig. 6
A,B). On day 25, the lumbar spinal cord from animals treated in
the chronic phase of EAE with DALBK or HOE-140 showed a
significant decrease in levels of cell infiltration (Fig. 6 C,E),
demyelination (Fig. 6 D,F), astrocytes (Fig. 6G), microglial
activation (Fig. 6H) and CREB phosphorylation (Fig. 6I). In order
to further evaluate the loss of spinal cord axons, we assessed the
level of neurofilament heavy proteins (NF-H) [29]. In the EAE
control group there was a significant loss of axons, while mice
treated in the chronic phase of EAE with DALBK (50 nmol/kg,
i.p.) or HOE-140 (150 nmol/kg, i.p.) showed a significantly
attenuated loss of axons (Fig. 6J). Together, our data suggest that
blockade of B1R in the CNS showed a dominant role in
hampering EAE progression, mainly by affecting the function of
astrocytes/microglia, which could lead to neuronal dysfunction.
Inhibition of B1R in the induction phase of EAE
suppresses TH1 and TH17 autoimmunity in the CNS
Increasing evidence suggest a role for TH17 and TH1 in CNS
damage, especially the observation of these subpopulations and
their signature cytokines in the brains of individuals with MS
[25,26,27]. The TH1 cells are characterized by their expression of
IFN-c and its differentiation is orchestrated by the transcription
factor T-bet, whereas TH17 mainly produces IL-17 and the
transcription factor involved in its differentiation is the retinoid
orphan receptor (ROR-cT) [28]. Therefore, we next investigated
whether or not the blockade of B1R in the induction phase could
reduce the expression of IL-17 (Fig. 5D), ROR-cT (Fig. 5E), TNF-a
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Figure 4. Kinin B1R inhibition or its deletion reduced the production of inflammatory cytokines and the activation of isolated CD4+
T lymphocytes. Lymphocytes (16106/well) obtained 25 days after immunization from the naive group, the control group (EAE), from mice pretreated with the B1R antagonist DALBK (50 nmol/kg), from mice pre-treated with the B2R antagonist HOE-140 (50 nmol/kg), from B1R2/2mice and
from B2R2/2 mice were cultured in the presence or absence of MOG35–55 (10 mg/ml) for 3 days; the supernatants were collected and measured for the
concentrations of TNF-a (A, B), IFN-c (C, D), IL-17 (E, F) and IL-4 (G, H) using ELISA assays. After culture supernatants were collected, the cells were
analyzed by flow cytometry for CD4+CD69+ (I, J) and CD8+CD69+ (K, L) T cells. Each column represents the mean 6 SEM of six to nine mice per group
and is representative of three independent experiments. #P,0.05, ##P,0.001 versus the naive group, *P,0.05, **P,0.001 versus the EAE group
(one-way ANOVA with the Newmann-Keuls post-hoc test).
doi:10.1371/journal.pone.0027875.g004
best therapeutic strategy is the inhibition of B1R (Fig. 7 A,B),
which corroborates previous data [15].
Corroborating and extending previously published data, the
treatment of animals with the selective B1R agonist DABK
(300 nmol/kg, i.p.) from day 7 to 17 (acute phase) strikingly inhibited EAE progression (Fig. 7 C,D), whereas the B1 antagonist
DALBK (50 nmol/kg, i.p.) did not alter the course of the disease
(Fig. 7 C,D). Herein, in order to elucidate this peculiar data, we
considered which process would be more likely to happen in the
acute phase of the disease. A pivotal step in triggering CNS
inflammation is disruption of the blood-brain barrier (BBB) [30].
In order to investigate whether the activation of B1R in the acute
phase influenced BBB permeability and neuroinflammation, we
used the Evan’s blue assay and TH1- and TH17-cytokine expression in the lumbar spinal cord, respectively. On day 21 after
immunization, the EAE group showed a significant increase in
Evan’s blue extravasations, demonstrating the disruption of BBB
permeability (Fig. 7E). Interestingly, the B1R agonist DABK
The dual role of kinin B1R
So far, our data and other recent reports in the literature have
demonstrated a protective role of kinin receptor blockade in EAE
progression [13,14,15]. However, an unexpected report showed
that the activation of B1R ameliorates the disease [17]. In order to
clarify this discrepancy, we investigated whether or not the B1R
agonist (DABK) could exert a protective action over EAE
throughout the three different stages of the disease (see scheme
in Fig. 1A). Treatment with the B1R agonist during the induction
phase (days 0–5) delayed the onset of clinical signs of EAE by 3
days, as observed previously [17]. However, in our hands the same
treatment (DABK, 300 nmol/kg, i.p.) resulted in a severe disease
that was similar to the severity of disease observed in the EAE
control group (Fig. 7A), and this slight delay did not reach
significant difference when calculated based on the area under the
curve (Fig. 7B). These results indicate that activation of B1R
during the EAE induction phase does not improve locomotor
activity induced by EAE. This data suggests that in this phase the
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Figure 5. B1R inhibition affects the peripheral and central MOG-specific immune responses during EAE pathology. Splenocytes (A, B)
and drained lymph node (C) were isolated from immunized mice (EAE group) and B1R2/2 and B2R2/2 mice on days 14 and 25 p.i. The cells (16106/
well) were cultured in the presence or absence of MOG35–55 (10 mg/ml) for 72 h to assess proliferation by [3H] thymidine incorporation. Total RNA was
extracted from the lumbar spinal cord of mice in the naive group, the control group (EAE), DALBK (50 nmol/kg) mice, HOE-140 (50 nmol/kg) mice,
B1R2/2 mice and B2R2/2 mice on day 25 p.i. The mRNA levels of IL-17 (D), ROR-cT (E), TNF-a (F), IFN- c (G), T-bet (H) and B1R (I) were measured.
GAPDH mRNA was used to normalize the relative amounts of mRNA. The data are presented as mean 6 SEM of six to nine mice/group and are
representative of three independent experiments. #P,0.05, ##P,0.001 versus the naive group, *P,0.05, **P,0.001 versus the EAE group (one-way
ANOVA with the Newmann-Keuls post-hoc test).
doi:10.1371/journal.pone.0027875.g005
clinical score (Fig. 7 I,J) and locomotor deficits (data not shown)
induced by EAE. The study of this disease stage has huge
importance since therapies applied after disease outcomes are
mostly clinically relevant. Our work solved many conflicting data
in the literature and indicates that in the chronic phase of EAE the
best therapeutic option would be the blockade of B1R. Taken
together, these results suggest that, in the acute phase of
immunization, the kinin B1 agonist (DABK, 300 nmol/kg, i.p.)
(300 nmol/kg, i.p.) markedly reduced Evan’s blue extravasations
in the spinal cord, whereas the blockade of B1R with DALBK
induced an increase in the BBB permeability (Fig. 7E). In addition,
in the acute phase of EAE, DABK treatment (300 nmol/kg, i.p.)
significantly reduced IL-17 (Fig. 7F), IFN-c (Fig. 7G) and TNF-a
(Fig. 7H) mRNA in the spinal cord on day 21. Of note, therapeutic
treatment with the B1R agonist DABK (300 nmol/kg, i.p.) during
the chronic phase of EAE (day 15 to 20) failed to inhibit the
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Figure 6. Blockade of kinin B1R in the chronic disease phase decreased the clinical symptoms and neuroinflammation in the EAE
model. The clinical score (A) and locomotor activity (B) were analyzed 25 days after the animals were immunized with MOG35–55 peptide/CFA. The
inflammatory infiltrates (H&E staining; C, E), demyelination area (LFB staining; D, F), immunoreactivity of GFAP (G), Iba-1 (H), phospho-CREB (I) and
neurofilament-H (J) were analyzed 25 days p.i. in the lumbar spinal cords of mice in the naive group, the control group (EAE) and in mice treated in
the chronic phase (days 15–20) with B1R (DALBK, 50 nmol/kg, i.p.) or B2R (HOE-140, 150 nmol/kg, i.p.) antagonists. Specifically, four alternate 5-mm
sections (six to nine animals/group) of the white matter (E–I) and grey matter (J) of the lumbar spinal cord were obtained between L4–L6. Scale bar
corresponds to 25 mm and applies throughout. The data are presented as mean 6 SEM of six to nine mice/group and are representative of three
independent experiments. #P,0.05, ##P,0.001 versus the naive group, *P,0.05 and **P,0.001 versus the EAE group (one-way ANOVA with the
Newmann-Keuls post-hoc test).
doi:10.1371/journal.pone.0027875.g006
inhibits disruption of the BBB permeability, prevents the entry of
both TH17 and TH1 cells into the CNS and consequently suppresses neuroinflammation. Unlike in the induction and chronic
phases, in the acute phase B1R activation positively regulates EAE
progression, suggesting a dual role of this receptor at different time
points of EAE progression.
treatment, either a selective B1 agonist or a selective B1 antagonist
is able to block EAE progression through different mechanisms
(see proposed scheme in Fig. 8).
Furthermore, both the genetic and pharmacological data
presented in this study showed that blockade of the B2Rmodulated activation of T CD4+ and, particularly, CD8+ MOGreactive cells in the peripheral lymphoid tissue decreased CREB
phosphorylation in the spinal cord, decreased neuropathic pain
and also attenuated the loss of axons in the CNS. These findings
largely extend those of a previous study which indicated that
B2R2/2 mice showed a reduction in the clinical parameters of
MOG-induced EAE through the modulation of leukocyte recruitment into the CNS [14]. Nonetheless, unlike with B1R, B2R
inhibition was not able to significantly reduce the size of the
demyelination area, nor the central inflammatory cell infiltration,
justifying the moderate impact found for all of the parameters of
EAE progression studied. These results confirmed the dominant
role of B1R in the development of EAE [15,17].
Indeed, it is widely accepted that the activation of both B1R and
B2R positively contribute to pain generation and inflammation [11].
Besides this, many studies imply the involvement of the kallikreinkinin system in MS and the EAE model [13,14,15]. Nonetheless two
recent studies using MOG-induced EAE presented contradictory
Discussion
The main results to emerge from the present study are, to the
best of our knowledge, the first pieces of evidence to show that the
blockade of kinin B1R, and, to a lesser extent, the B2R subtype,
either in the EAE induction or chronic phase, prevented disease
progression by modulating the onset of the immune response and
by affecting the functioning of astrocytes/microglia cells, respectively. However, we also found that the systemic administration of
the B1 agonist but not the antagonist, given in the acute phase of
the disease, markedly reduced disease severity and inhibited the
increase in BBB permeability, blocking neuroinflammation. Our
last findings confirm and also extend previous data which
indicated that the activation of B1R can ameliorate EAE severity
by controlling the migration of pro-inflammatory T cells across the
BBB into the CNS [17]. Our data provide what we believe is new
evidence to demonstrate that, depending on the phase of
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B1 and B2 Kinin Receptors and Multiple Sclerosis
Figure 7. Activation of kinin B1R in the acute disease phase improved the pathology of EAE. The clinical score (A, C and I) and area under
the curve (AUC) (B, D and J) were analyzed in the induction (day 0–5), acute (day 7–17) and chronic (day 15–20) phases of EAE, respectively. The
animals were separated into different groups: naive group, EAE group, mice treated with the B1R agonist DABK (300 nmol/kg, i.p.) and mice treated
with the B1R antagonist DALBK (50 nmol/kg, i.p.). The agonists/antagonists were administered intraperitoneally (i.p.), twice a day (12/12 h), during
different time-points of EAE. In the acute phase of EAE, the pharmacological activation of B1R (DABK treatment: 300 nmol/kg, i.p.) significantly
decreased Evan’s blue extravasations in the spinal cord on day 21 compared to the EAE control group (E) and the mRNA levels of IL-17 (F), IFN-c (G)
and TNF-a (H), as measured by RT-PCR. The GAPDH mRNA was used as an endogenous control in the RT-PCR assay. The data are presented as mean
6 SEM of six to nine mice/group and are representative of four independent experiments. #P,0.05, ##P,0.001 versus the naive group, *P,0.05 and
**P,0.001 versus the EAE-treated group (one-way ANOVA with the Newmann-Keuls post-hoc test). N.D. not detected.
doi:10.1371/journal.pone.0027875.g007
offspring breed mutant, which can often characterize an inbred
substrain [31,32,33]. In addition, some gene-by-environment risk
factors may be responsible for the appearance of phenotypic
differences between similar strains [34,35,36]. Another relevant
point is that our animals were immunized twice with MOG on days
0 and 7 in order to increase the incidence of EAE, as previously
described (24). However whether the double MOG immunization
could have induced some degree of tolerance in our B1R2/2 mice
needs to be further investigated. Here, we hypothesize that this
apparent discrepancy could reside in the unusual role played by
B1R throughout the different phases of EAE progression. We
further examined this hypothesis by using a kinin agonist or
antagonist at different time points of EAE progression.
Accumulated evidence now suggests that in the induction phase
of EAE and MS disease T cells in the periphery become activated
by a viral or another infectious antigen [6]. Herein, we show that
blockade of kinin B1R in the induction phase of EAE consistently
results; i.e., Gobel and collaborators showed that mice with the
deletion of B1R presented a significantly reduced disease severity
compared to the EAE control group, whereas Schulze-Topphoff
and colleagues demonstrated a greater clinical disease severity in
these animals. The discrepancies between our findings and those
reported by Schulze-Topphoff et al. could be explained by certain
genetic differences and the method of maintenance of our B1R2/2
mutant mice, as well as by the gene-environment risk factor.
Schulze-Topphoff et al. used B1R2/2 mice after they were
backcrossed to C57BL/6 to produce F10 offspring with a SV129
background. Our B1R2/2 knockout animals were obtained by the
same way, although after the F10 offspring we maintained the
mutant line by crossing them with each other. A necessary caveat
with this design is that time, isolation and constant generations
among homozygous mutants increase the risk of de novo mutations,
which could lead to biasing genetic difference between the
congenics, the parental recipient strain, and, particularly, the F10
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B1 and B2 Kinin Receptors and Multiple Sclerosis
Figure 8. Schematic representation of the mechanism via which kinin regulates the physiopathology of the EAE model. The blockade
of kinin B1R in either the induction or the chronic phase of EAE suppressed disease progression with the concomitant suppression of TH1 and TH17myelin-specific cell development in at least two different stages: (1) during onset of the peripheral immune response, through the modulation of
differentiation and/or expansion of auto-aggressive TH cells in the MOG35–55-specific immune responses; and (2) during neuroinflammation by
affecting the auto-aggressive function of T cells and astrocytes. However, the blockade of B1R in the acute phase of EAE only had a slight effect,
whereas that of the B1 agonist also given at this time markedly reduced disease severity through the inhibition of increased BBB permeability and cell
migration, and consequently blocked CNS inflammation. Altogether, we found that kinins, especially B1R subtypes, have a dual role during the
progression of EAE by distinct mechanisms of action at each stage of disease progression. APC: antigen-presenting cell; DALBK: des-Arg9-[Leu8]-BK;
HOE-140: D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK; DABK: des-Arg9-BK; MOG: myelin oligodrendrocyte glycoprotein; B1R: kinin B1 receptor; B2R: kinin B2
receptor; THP: precursor T cell; BBB: blood-brain barrier; CXCL1/KC: keratinocyte-derived chemokine, TNF-a; tumour necrosis factor-alpha; IFN-c:
interferon-gamma; TGF-b: transforming growth factor beta; PMN: polymorphonuclear leukocytes; GFAP: glial fibrillary acidic protein; Iba1: ionized
calcium binding adaptor molecule 1; CREB: cAMP response element-binding; NF-H: neurofilament-H. (
), inhibition;
), stimulation.
doi:10.1371/journal.pone.0027875.g008
proteolipid protein (PLP)139–151-reactive lymphocytes significantly
reduced clinical disease severity when compared with the EAEcontrol group [17]. Interestingly, it is worth highlighting the fact
that the period of treatment of animals in this previous work using
the passive induction protocol (day 0 to day 10 after transfer) [17]
can be compared with our acute phase of EAE (day 7–17) after the
active induction protocol, since the immune response had been
already triggered. During the above mentioned period it is possible
to evaluate initiation of the myelin-reactive immune attack, as well
as TH1 and TH17 cell migration to the CNS [42]. Based on these
results, it is tempting to suggest that activation of B1R in the acute
phase of EAE seems to affect CNS inflammation and control the
migration of pro-inflammatory T cells across the BBB into the CNS,
as previously described [17].
Recent data from the literature have shown that bradykinin
(BK), a preferential B2R agonist, is able to selectively open the
BBB, since BK accelerate the release of TNF-a [43,44]. Of note,
in animal models, B2R agonist like CereportH (RMP-7) has been
used by the intracarotid route in order to increase the BBB permeability and consequently enhance the delivery of chemotherapeutic agents to the brain tumor area [45,46,47]. More recently,
a very interesting study conducted by Liu et al. demonstrated that
BK increases the BBB permeability by down-regulating the expression levels of tight junction (TJ)-associated proteins, such as
zonula occluden-1 (ZO-1), occludin, and claudin-5 and rearranging cytoskeleton protein filamentous actin (F-actin) [48]. However,
additional studies are needed to assess the precise mechanisms by
inhibited onset of the immune response by modulating the
activation of TH1 and TH17-MOG reactive cells during the
presentation of myelin antigens in peripheral lymphoid organs.
Consequently, these cells failed to differentiate, proliferate and
migrate to the CNS effectively, an effect that abrogated the
development of EAE. In fact, our data extends the results of recent
reports which indicated that modulation of the microenvironment
during antigen presentation and cell activation altered the immune
response and then the course of the disease, mainly by diminishing
the response of MOG-specific T cells [37,38,39]. In agreement
with our data, Aliberti et al. showed that kinin can serve as a
danger signal that triggers dendritic cell activation, driving T cell
polarization, which means that kinins can modulate the immune
response at the very beginning of the disease [40].
Another interesting aspect investigated in the present study was
the fact that after peripheral activation, CD4+ T cells effectively
migrated to the CNS during the acute phase of EAE. Here, we
found that in the acute phase of EAE, the blockade of B1R occurred
after the presentation of antigens in peripheral lymphoid organs
and, at the same time, treatment of animals with the B1 antagonist
had no effect. In marked contrast, the activation of B1R, assessed via
treatment with the B1 agonist DABK, inhibited the progression of
EAE and neuroinflammation by modulating the permeability of the
BBB, a pivotal step that triggers CNS inflammation, an event which
is directly related to the development of MS [30,41]. A recent report
showed that in the passive induction of EAE, activation of B1R with
the agonist R838 (day 0–10) after the adoptive transfer of
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B1 and B2 Kinin Receptors and Multiple Sclerosis
30–45 min, and removing the unattached ES cells. Two B2Rtargeted ES clones, KO-5 and KO-24, were injected into recipient
C57BL/6 3-day-old blastocysts; following this, they were reimplanted into day 3 pseudo-pregnant Tac:SW(fBR) mice and
allowed to develop to term. The B12/2 and B22/2 mice used in
the experiments originated from ten generations of backcrossing of
mice with an initially mixed genetic background (129/SvJ and
C57BL/6) with C57BL/6 mice (Taconic, Germantown, NY).
Following the F10 offspring the line is 99% genetically identical to
the recipient strain (C57BL/6) and is considered congenic with it;
we maintained this linage by crossing them with each other. The
C57BL/6 animals were used as controls. The mice were kept
in groups of six to nine animals per cage, maintained under
controlled temperature (2261uC) and humidity (60–80%) conditions with a 12 h light/dark cycle (lights on at 7:00 a.m.) and were
given free access to food and water. All procedures used in the
present study followed the Guide for the Care and Use of
Laboratory Animals (NIH publication No. 85-23) and were
approved by the Animal Ethics Committee of the Universidade
Federal de Santa Catarina (CEUA-UFSC, protocol number
23080038266/2008-43).
which BK modulate the BBB permeability during neuroinflammation conditions, such as MS.
Concerning the chronic phase of EAE, it has been reported that
CD4+ T cells enter the subarachnoid space by crossing the bloodcerebrospinal fluid (CSF) barrier in either the choroid plexus or in
the meningeal venules, and they are re-activated by MHC class IIexpressing macrophages/microglia and DCs. Afterwards, macrophage and glial cells secrete soluble mediators that trigger
demyelination and attract further inflammatory cells into the
CNS [5]. Of note, our data revealed that the blockade of B1R in
the chronic phase of EAE consistently improved the clinical signs
and neuroinflammation induced by EAE. Moreover, in vitro pretreatment with the B1R antagonist (DALBK) blocked the proinflammatory release/expression induced by IFN-c in primary
astrocytes cultures (data not shown). At this same point in time,
due to the peptide nature of these antagonists, it is highly unlikely
that, given systemically, they could cross the BBB in opposite
directions to block central kinin B1R or B2R. However, several
studies suggested that when under neuroinflammatory conditions,
the activation or damage of cellular components of the BBB could
facilitate leukocyte infiltration leading to oligodendrocyte death,
axonal damage, demyelination and lesion development [30]. Likewise, a recent report showed that B1R antagonist (R715) treatment, which started once the animals were already displaying the
first clinical symptoms of EAE (chronic phase), markedly inhibited
the EAE score [15]. Therefore, we can suggest that after disruption of the BBB in the chronic phase of EAE, the kinin B1R
antagonists most likely penetrate the CNS and mainly hamper
EAE progression by affecting the functioning of astrocytes cells.
Altogether, the present study identified B1R as a key mediator in
EAE disease and suggested that kinin B1R displays a dual role in
the progression of EAE via distinct mechanisms at each stage of
the disease, mainly through the modulation of TH1 and TH17myelin-specific lymphocytes and glial cell-dependent pathways
(Fig. 8). Our findings open up important options for the development of clinically relevant therapies for the management of
MS, as well as for other immune diseases in which TH1 and TH17
cells play a key role.
Drug treatment protocol
The following series of experiments were designed to evaluate
how kinin receptors regulate some of the biological processes that
occur in the EAE model. (1) In order to determine the effect of the
kinin receptor on the origin of the autoimmune response induced
by EAE (Induction phase), different groups of animals were treated
with the selective B1R and B2R antagonists and/or agonists twice
a day, for 5 days (starting on day 0 until day 5 post-immunization).
(2) In order to assess the ability of kinin B1R to modulate the
development of neuroinflammation induced by EAE (Acute
phase), the animals were treated for 10 days (starting on day 7
until day 17 post-immunization). (3) In order to evaluate the effect
of B1R and B2R after the clinical signs of EAE had already been
observed (Chronic phase), the animals were treated for 5 days as
soon as the first clinical signals appeared (from day 15 to 20 postimmunization). The B1R antagonist Des-Arg9-[Leu8]-BK
(DALBK, 50 nmol/kg), the B1R agonist Des-Arg9-BK (DABK,
300 nmol/kg) or the B2R antagonist HOE-140 (HOE, 150 nmol/
kg), or their vehicles, were administered intraperitoneally (i.p.) The
choice of the dose for each drug was based on pilot experiments
(data not shown) or on data previously described in the literature
[8,52].
Materials and Methods
Experimental animals
The experiments were conducted using female C57BL/6, kinin
B1R-knockout (B1R2/2) and kinin B2R-knockout (B2R2/2) mice
(6–10 weeks old). The B1R gene was cloned from a genomic
library of 129/SvJ mice in lFIXII [49]. The targeting vector was
generated by flanking the neomycin resistance gene with a 1.0-kb
genomic 59 fragment of the B1-coding region and a 7.0-kb 39
fragment of the B1-coding region. The construct was linearized
with NotI and transfected into E14–1 embryonic stem cells by
electroporation as previously described [50]. Gancyclovir- and
G418-resistant clones were selected and identified by polymerase
chain reaction (PCR). Two positive clones were microinjected into
C57BL/6 blastocysts, which gave rise to two germ-line chimeras
with offspring that were heterozygous for the targeted mutation.
The generation of B2R mice and gene targeting was performed in
the embryonic stem (ES) cell line AB 2.1, derived from 129/SvJ
mice [51]. The mouse genomic DNA utilized to construct the
targeting vector was obtained from a cosmid clone isolated from a
library constructed by Dr John Mudgett (Merck Research Laboratory, Rahway, NJ) from an ES cell line, J1, derived from 129/
SvJ mice. The ES cell clones containing the targeted disruption of
the B2R gene were separated from SNL feeder cells by treating the
cell culture with trypsin, allowing the feeder cells to reattach for
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EAE induction
The experimental autoimmune encephalomyelitis (EAE) was
induced by subcutaneous (s.c.) immunization into the flanks with
200 ml of an emulsion containing 200 mg of MOG35–55 peptide
and 500 mg of Mycobacterium tuberculosis extract H37Ra (Difco
Laboratories, Detroit, MI, USA) in incomplete Freund’s adjuvant
oil (Sigma Chemical Co., St. Louis, MO, USA). This procedure
was repeated after 7 days in order to increase the incidence of
EAE, as previously described [42]. In addition, the animals
received 300 gg of pertussis toxin (Sigma Chemical Co., St. Louis,
MO, USA) i.p. on day 0 and on day 2 post-immunization. Nonimmunized (naive) and EAE-group animals were used as the
control groups. The animals were monitored daily and neurological impairment was quantified using a clinical scale after day 7
post-immunization [42]. Mice were weighed and observed daily
for clinical signs of EAE for up to 25 days post-immunization.
Clinical signs of EAE were assessed according to the following
scores: 0, no signs of disease; 1 loss of tone in the tail; 2 hindlimb
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B1 and B2 Kinin Receptors and Multiple Sclerosis
Histological analysis
paresis; 3 hindlimb paralysis; 4 tetraplegia; 5 moribund and/or
death.
Twenty-five days after the induction of EAE, the mice were deeply
anesthetized with 7% chloral hydrate (8 ml/kg; i.p.) and intracardially perfused with 4% of the paraformaldehyde fixative solution in
0.1 M phosphate buffer (pH 7.4). The spinal cords were removed
and post-fixed for 24 h in the same solution, and then embedded in
paraffin after dehydration and diaphanization. The histological
analysis of inflammation and demyelination was performed using
haematoxylin-eosin (H&E) and luxol fast blue (LFB), respectively.
The settings used for image acquisition were identical for both control
and experimental tissues. Four ocular fields per section (six to nine
mice per group) were captured and a threshold optical density that
best discriminated the nuclear staining of inflammatory cells
(haematoxylin-eosin) or myelin (luxol fast blue) was obtained using
the NIH ImageJ 1.36b imaging software (NIH, Bethesda, MD, USA)
and applied to all experimental groups The total pixel intensity was
determined, and the data are expressed as optical density (O.D.).
Behavioural experiments
Rotarod test. As a test of locomotor activity and coordination, the mice were placed on a rotarod apparatus at a fixed
rotational speed of 4 rpm. The maximum time for each trial was
set at 60 s. Rotarod training was performed prior to disease
induction and consisted of three consecutive trials in which the
animals became familiar with the task. After disease induction, the
mice were tested every two days, until day 25 post-immunization.
Measurement of cytokine production in lymph node (LN)
and spleen cells
Inguinal LN cells and splenocytes from the mice were prepared
on day 25 after immunization. Briefly, lymph nodes and the spleen
were individually macerated in RPMI 1640 medium supplemented with 10% foetal bovine serum, HEPES (20 nM), 2-mercapto
ethanol, penicillin (100 U/ml) and streptomycin (100 mg/ml) and
the cell suspension was filtered through a 70 mm filter. The
resulting suspension was centrifuged at 1500 g for 7 min at 4uC.
For the spleen tissue, after the initial centrifugation the supernatant was discarded and the cell pellet resuspended in ammonium chloride potassium carbonate buffer (ACK lysis buffer) using
1 ml per donor mouse to lyse red blood cells and incubated on ice
for 5 min. After incubation, 9 ml of the medium was added to stop
the cell lysis. The cell debris was allowed to settle on the bottom of
the tube for 2 min before being transferred to a new 15 ml conical
tube and centrifuged for 5 min at 500 g and 4uC. Finally, the
supernatant was discarded and the cells were resuspended in 2 ml
of the medium. Lymph node cells and splenocytes (16106/well)
were cultured in the presence of MOG35–55 (10 mg/ml) or in the
medium alone. The LN cells and splenocytes were incubated for
48 h at 37uC in a humidified 5% CO2 atmosphere. The culture
supernatants were collected and stored at 270uC until further
analysis. The levels of tumour necrosis factor-a (TNF-a), interferon-c (IFN-c), interleukin-17 (IL-17) and interleukin-4 (IL-4)
were measured using ELISA kits, according to the manufacturer’s
recommendations. After collection of the culture supernatants, the
cells were used in a flow cytometry assay.
Immunohistochemistry assay
Immunohistochemical analysis was performed on paraffinembedded lumbar spinal cord tissue sections (5 mm) using monoclonal mouse anti-CD3+ T cells (1:100), monoclonal mouse antiGFAP (1:300), polyclonal goat anti-Iba1 (1:200), monoclonal mouse
anti-neurofilament H (1:200) and polyclonal rabbit anti-phosphoCREB (1:300), according to the method previously described [53].
After quenching of endogenous peroxidase with 1.5% hydrogen
peroxide in methanol (v/v) for 20 min, high-temperature antigen
retrieval was performed by immersing the slides in a water bath at
95 to 98uC in 10 mmol/l trisodium citrate buffer, pH 6.0, for
45 min. The slides were then processed using the Vectastain Elite
ABC reagent (Vector Laboratories, Burlingame, CA), according to
the manufacturer’s instructions. Following this the immune
complexes were visualized with 0.05% 3,39-diaminobenzidine
tetrahydrochloride (DAB: Dako Cytomation, Glostrup, Denmark)
plus 0.03% H2O2 in PBS (for exactly 1 min). The reaction was
stopped by thorough washing in water and counterstained with
Harris’s haematoxylin. Besides staining untreated animals as
negative controls, sections were also incubated without the primary
antibody (data not shown), and these controls resulted in little or no
staining. To eliminate temporal variations, control and experimental tissues were placed on the same slide and processed under the
same conditions. Images were acquired using a Sight DS-5M-L1
digital camera connected to an Eclipse 50i light microscope (both
from Nikon, Melville, NY, USA). Specifically, four alternate 5-mm
sections of the lumbar spinal cord with an individual distance of
150 mm were obtained between L4 and L6. Images of stained white
matter of the spinal cord were acquired using a Sight DS-5M-L1
digital camera (Nikon, Melville, NY, USA) connected to an Eclipse
50i light microscope (Nikon). For the NF-H analysis, images of the
grey matter of the lumbar spinal cord (dorsal and ventral horns)
were obtained. The optical density threshold that best discriminated
staining from the background was selected using the ‘‘Threshold
Color’’ plug of NIH ImageJ 1.36b imaging software (NIH,
Bethesda, MD, USA) and applied to all experimental groups. For
phospho-CREB, GFAP, Iba-1 and NF-H analyses, the total pixel
intensity was determined and the data were expressed as optical
density (O.D.). In order to analyse the number of T lymphocyte
cells, CD3-positive cells were visually inspected by counting the
number of labelled cells in the white matter of the lumbar spinal
cord section, using a counting grid at a 6400 magnification.
Proliferation assays
Lymph node and spleen cells (16106/well) prepared from
immunized mice (EAE control group) and kinin B1R and B2Rknockout mice were cultured in 96-well flat-bottomed microculture plates in the presence of MOG35–55 (10 mg/ml) or in medium
alone. After 60 h, 0.5 mCi per well [3H] of thymidine was added to
each well and then incubated for 12 h. The cells were then
harvested onto glass fibre filters, and radioactivity was measured in
a Liquid Scintillation/Beta Counter LS5000TD (Beckman
Coulter, Inc., Brea, CA, USA).
Flow cytometric analysis of lymphocytes
After incubation with MOG35–55, the LN cells were washed
with RPMI 1640 medium supplemented with 10% foetal bovine
serum and further incubated with the following antibodies for
20 min at 4uC: anti-CD4-PerCP (clone RM4-5, Caltag Laboratories, Burlingame, CA, USA), anti-CD8a-APC (clone 5H10,
Caltag Laboratories, Burlingame, CA, USA) and anti-CD69-PE
(clone H1.2F3, BD PharmingenTM, San Jose, CA, USA). The data
were collected using FACSCanto II (BD Biosciences) and analysed
using FlowJo (version 7.5) software.
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Real-time quantitative PCR
Lumbar spinal cord (six to nine animals/group) tissues were
removed 25 days post-immunization and the total RNA was
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B1 and B2 Kinin Receptors and Multiple Sclerosis
extracted using the Trizol protocol. The reverse transcription assay
was carried out as described in the M-MLV Reverse Transcriptase
(Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s
instructions. Real-time quantitative PCR analysis of mRNA was
performed in StepOnePlusTM using the TaqManH Universal PCR
Master Mix Kit (Applied Biosystems, Foster City, CA, USA) for
quantification of the amplicons, and 100 ng of cDNA were used in
each reaction. The cDNA was amplified in triplicate using specific
TaqMan Gene Expression target genes, the 39 quencher MGB and
FAM-labelled probes for TNF-a (Mm00443258_m1), IFN-c
(Mm99999071_m1), interleukin-17 (IL-17) (Mm00439618_m1),
kinin B1R (Mm00432059_s1), ROR-cT (Mm00441144_g1), T-bet
(Mm00450960_m1) and GAPDH (NM_008084.2), which were
obtained from Applied Biosystems (Foster City, CA, USA). The
thermocycler parameters were as follows: 50uC for 2 min, 95uC for
10 min, 50 cycles of 95uC for 15 s, and 60uC for 1 min. Samples
without cDNA were analysed as ‘‘no template’’ controls. The mRNA
levels were quantified using the comparative threshold cycle (Ct)
method [54], where the mean Ct values from duplicate measurements were used to calculate the expression of the target gene, with
normalization to the housekeeping gene GAPDH in the samples.
Main, Germany). Formaldehyde, formamide, NH4Cl and KHCO3
were obtained from Merck (Frankfurt, Darmstadt, Germany). The
MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) was
obtained from EZBiolab, Carmel, IN 46032, USA; and M.
tuberculosis extract H37Ra from Difco Laboratories, Detroit, MI,
USA. The RPMI 1640 and foetal bovine serum were purchased
from GIBCO (Carlsbad, CA, USA). [3H] Thymidine (0.5 mCi/ml)
was provided by Amersham Biosciences (Piscataway, NJ, USA).
The anti-mouse-TNF-a, IFN-c, IL-17 and IL-4 DuoSet kits were
obtained from R&D Systems. The monoclonal mouse anti-CD3+ T
cells were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA, USA) and the polyclonal goat anti-ionized calcium binding
adaptor molecule 1 (Iba-1) was purchased from Abcam (Cambridge, MA, USA). The monoclonal mouse anti-astrocytes marker
glial fibrillary acidic protein (GFAP), monoclonal mouse antineurofilament H (NF-H) and polyclonal rabbit anti-nuclear
phospho-cyclic AMP response element binding protein (CREB)
were purchased from Cell Signaling Technology (Danvers, MA,
USA). Secondary antibody Envision Plus, streptavidin-HRP
reagent and 3,3-diaminobenzidine chromogen were purchased
from Dako Cytomation (Carpinteria, CA, USA). The primers and
probes for mouse TNF-a, IFN-c, IL-17, kinin B1R, ROR-cT, T-bet
and GAPDH were purchased from Applied Biosystems (Warrington, UK). The other reagents used were of analytical grade and
obtained from different commercial sources.
Measurement of BBB permeability
The BBB permeability was assessed by measuring the extravasations of Evan’s blue (EB) dye, as previously described [55]. Briefly,
on day 21, the mice were i.v. injected with 0.1 ml 2% EB, which was
allowed to circulate for 60 min. Following this period of circulation,
the animals were transcardially perfused with 0.9% phosphate
buffered saline (PBS) to remove the intravascular EB dye. The
lumbar spinal cord (L4–L6) was further dissected and weighed.
Tissue was incubated in 600 ml formamide at 60uC for 24 h. At the
end of incubation, the tissues were removed and the formamide
solution was centrifuged at 20,000 g for 20 min. The supernatant
solution was collected and the optical density was measured at
620 nm to determine the relative amount of EB dye in each sample.
Statistical analysis
All data are presented as mean 6 SEM of six to nine mice/
group and are representative of three/four independent experiments. A statistical comparison of the data was performed by twoway ANOVA followed by Bonferroni’s post-hoc test or one-way
ANOVA followed by Bonferroni’s or Newman-Keuls’s test,
depending on the experimental protocol; p-values less than 0.05
(p,0.05 or less) were considered significant. The statistical
analyses were performed using GraphPad Prism 4 software
(GraphPad Software Inc., San Diego, CA, USA).
Materials
Pertussis toxin, PBS, H&E, H2O2, Incomplete Freund’s adjuvant
oil, kinin B1R antagonist des-Arg9-[Leu8]-BK, kinin B1R agonist
des-Arg9-BK, HEPES, penicillin, streptomycin, Na-EDTA, trypsin,
deoxyribonuclease I (DNase) and bovine serum albumin (BSA) were
purchased from Sigma Chemical Co. (St. Louis, MO, USA). The
kinin B2R antagonist HOE-140 was donated by Aventis (Frankfurt
Author Contributions
Conceived and designed the experiments: RCD DFPL AFB JBC.
Performed the experiments: RCD DFPL AFB MNM ESP CPF. Analyzed
the data: RCD DFPL AFB ESP CPF JBC. Contributed reagents/
materials/analysis tools: JBP. Wrote the paper: RCD DFPL CPF JBC.
References
1. Frohman EM, Racke MK, Raine CS (2006) Multiple sclerosis–the plaque and its
pathogenesis. N Engl J Med 354: 942–955.
2. McFarland HF, Martin R (2007) Multiple sclerosis: a complicated picture of
autoimmunity. Nat Immunol 8: 913–919.
3. Hafler DA (2004) Multiple sclerosis. IFN-c, TNF-a J Clin Invest 113: 788–794.
4. Kawakami N, Odoardi F, Ziemssen T, Bradl M, Ritter T, et al. (2005)
Autoimmune CD4+ T cell memory: lifelong persistence of encephalitogenic T
cell clones in healthy immune repertoires. J Immunol 175: 69–81.
5. Goverman J (2009) Autoimmune T cell responses in the central nervous system.
Nat Rev Immunol.
6. Sospedra M, Martin R (2005) Immunology of multiple sclerosis. Annu Rev
Immunol 23: 683–747.
7. Walker K, Perkins M, Dray A (1995) Kinins and kinin receptors in the nervous
system. Neurochem Int 26: 1–16; discussion 17–26.
8. Prediger RD, Medeiros R, Pandolfo P, Duarte FS, Passos GF, et al. (2008)
Genetic deletion or antagonism of kinin B(1) and B(2) receptors improves
cognitive deficits in a mouse model of Alzheimer’s disease. Neuroscience 151:
631–643.
9. Danielisova V, Gottlieb M, Nemethova M, Kravcukova P, Domorakova I,
et al. (2009) Bradykinin postconditioning protects pyramidal CA1 neurons
against delayed neuronal death in rat hippocampus. Cell Mol Neurobiol 29:
871–878.
10. Khan TK, Nelson TJ, Verma VA, Wender PA, Alkon DL (2009) A cellular
model of Alzheimer’s disease therapeutic efficacy: PKC activation reverses
PLoS ONE | www.plosone.org
11.
12.
13.
14.
15.
16.
17.
12
Abeta-induced biomarker abnormality on cultured fibroblasts. Neurobiol Dis 34:
332–339.
Calixto JB, Medeiros R, Fernandes ES, Ferreira J, Cabrini DA, et al. (2004)
Kinin B1 receptors: key G-protein-coupled receptors and their role in
inflammatory and painful processes. Br J Pharmacol 143: 803–818.
Campos MM, Leal PC, Yunes RA, Calixto JB (2006) Non-peptide antagonists
for kinin B1 receptors: new insights into their therapeutic potential for the
management of inflammation and pain. Trends Pharmacol Sci 27: 646–651.
Prat A, Biernacki K, Saroli T, Orav JE, Guttmann CR, et al. (2005) Kinin B1
receptor expression on multiple sclerosis mononuclear cells: correlation with
magnetic resonance imaging T2-weighted lesion volume and clinical disability.
Arch Neurol 62: 795–800.
Dos Santos AC, Roffe E, Arantes RM, Juliano L, Pesquero JL, et al. (2008)
Kinin B2 receptor regulates chemokines CCL2 and CCL5 expression and
modulates leukocyte recruitment and pathology in experimental autoimmune
encephalomyelitis (EAE) in mice. J Neuroinflammation 5: 49.
Gobel K, Pankratz S, Schneider-Hohendorf T, Bittner S, Schuhmann MK,
et al. (2011) Blockade of the kinin receptor B1 protects from autoimmune CNS
disease by reducing leukocyte trafficking. J Autoimmun 36: 106–114.
Germain L, Barabe J, Galeano C (1988) Increased blood concentration of des-Arg9bradykinin in experimental allergic encephalomyelitis. J Neurol Sci 83: 211–217.
Schulze-Topphoff U, Prat A, Prozorovski T, Siffrin V, Paterka M, et al. (2009)
Activation of kinin receptor B1 limits encephalitogenic T lymphocyte
recruitment to the central nervous system. Nat Med 15: 788–793.
November 2011 | Volume 6 | Issue 11 | e27875
B1 and B2 Kinin Receptors and Multiple Sclerosis
18. Prat A, Biernacki K, Pouly S, Nalbantoglu J, Couture R, et al. (2000) Kinin B1
receptor expression and function on human brain endothelial cells.
J Neuropathol Exp Neurol 59: 896–906.
19. Marceau F, Bachvarov DR (1998) Kinin receptors. Clin Rev Allergy Immunol
16: 385–401.
20. Yao C, Sakata D, Esaki Y, Li Y, Matsuoka T, et al. (2009) Prostaglandin E2-EP4
signaling promotes immune inflammation through Th1 cell differentiation and
Th17 cell expansion. Nat Med 15: 633–640.
21. Bjartmar C, Wujek JR, Trapp BD (2003) Axonal loss in the pathology of MS:
consequences for understanding the progressive phase of the disease. J Neurol
Sci 206: 165–171.
22. Kim H, Moon C, Ahn M, Lee Y, Kim S, et al. (2007) Increased phosphorylation
of cyclic AMP response element-binding protein in the spinal cord of Lewis rats
with experimental autoimmune encephalomyelitis. Brain Res 1162: 113–120.
23. Gobin SJ, Montagne L, Van Zutphen M, Van Der Valk P, Van Den Elsen PJ,
et al. (2001) Upregulation of transcription factors controlling MHC expression in
multiple sclerosis lesions. Glia 36: 68–77.
24. Kivisakk P, Imitola J, Rasmussen S, Elyaman W, Zhu B, et al. (2009) Localizing
central nervous system immune surveillance: meningeal antigen-presenting cells
activate T cells during experimental autoimmune encephalomyelitis. Ann
Neurol 65: 457–469.
25. Tzartos JS, Friese MA, Craner MJ, Palace J, Newcombe J, et al. (2008)
Interleukin-17 production in central nervous system-infiltrating T cells and glial
cells is associated with active disease in multiple sclerosis. Am J Pathol 172:
146–155.
26. Moldovan IR, Rudick RA, Cotleur AC, Born SE, Lee JC, et al. (2003) Interferon
gamma responses to myelin peptides in multiple sclerosis correlate with a new
clinical measure of disease progression. J Neuroimmunol 141: 132–140.
27. Stromnes IM, Cerretti LM, Liggitt D, Harris RA, Goverman JM (2008)
Differential regulation of central nervous system autoimmunity by T(H)1 and
T(H)17 cells. Nat Med 14: 337–342.
28. Bettelli E, Korn T, Oukka M, Kuchroo VK (2008) Induction and effector
functions of T(H)17 cells. Nature 453: 1051–1057.
29. Trapp BD, Peterson J, Ransohoff RM, Rudick R, Mork S, et al. (1998) Axonal
transection in the lesions of multiple sclerosis. N Engl J Med 338: 278–285.
30. Alvarez JI, Cayrol R, Prat A (2011) Disruption of central nervous system barriers
in multiple sclerosis. Biochim Biophys Acta 1812: 252–264.
31. Gerlai R (2001) Gene targeting: technical confounds and potential solutions in
behavioral brain research. Behav Brain Res 125: 13–21.
32. Crusio WE, Goldowitz D, Holmes A, Wolfer D (2009) Standards for the
publication of mouse mutant studies. Genes Brain Behav 8: 1–4.
33. Kiselycznyk C, Holmes A (2011) All (C57BL/6) Mice are not Created Equal.
Front Neurosci 5: 10.
34. Crusio WE (2004) Flanking gene and genetic background problems in
genetically manipulated mice. Biol Psychiatry 56: 381–385.
35. Carola V, Gross C (2010) BDNF moderates early environmental risk factors for
anxiety in mouse. Genes Brain Behav 9: 379–389.
36. Gresack JE, Risbrough VB, Scott CN, Coste S, Stenzel-Poore M, et al. (2010)
Isolation rearing-induced deficits in contextual fear learning do not require
CRF(2) receptors. Behav Brain Res 209: 80–84.
37. Mycko MP, Cwiklinska H, Walczak A, Libert C, Raine CS, et al. (2008) A heat
shock protein gene (Hsp70.1) is critically involved in the generation of the
immune response to myelin antigen. Eur J Immunol 38: 1999–2013.
38. Ellestad KK, Tsutsui S, Noorbakhsh F, Warren KG, Yong VW, et al. (2009)
Early life exposure to lipopolysaccharide suppresses experimental autoimmune
encephalomyelitis by promoting tolerogenic dendritic cells and regulatory T
cells. J Immunol 183: 298–309.
PLoS ONE | www.plosone.org
39. Terenyi N, Nagy N, Papp K, Prechl J, Olah I, et al. (2009) Transient
decomplementation of mice delays onset of experimental autoimmune
encephalomyelitis and impairs MOG-specific T cell response and autoantibody
production. Mol Immunol 47: 57–63.
40. Aliberti J, Viola JP, Vieira-de-Abreu A, Bozza PT, Sher A, et al. (2003) Cutting
edge: bradykinin induces IL-12 production by dendritic cells: a danger signal
that drives Th1 polarization. J Immunol 170: 5349–5353.
41. Weiss N, Miller F, Cazaubon S, Couraud PO (2009) The blood-brain barrier in
brain homeostasis and neurological diseases. Biochim Biophys Acta 1788:
842–857.
42. Stromnes IM, Goverman JM (2006) Active induction of experimental allergic
encephalomyelitis. Nat Protoc 1: 1810–1819.
43. Zhang H, Gu YT, Xue YX (2007) Bradykinin-induced blood-brain tumor
barrier permeability increase is mediated by adenosine 59-triphosphate-sensitive
potassium channel. Brain Res 1144: 33–41.
44. Qin LJ, Gu YT, Zhang H, Xue YX (2009) Bradykinin-induced blood-tumor
barrier opening is mediated by tumor necrosis factor-alpha. Neurosci Lett 450:
172–175.
45. Elliott PJ, Hayward NJ, Huff MR, Nagle TL, Black KL, et al. (1996) Unlocking
the blood-brain barrier: a role for RMP-7 in brain tumor therapy. Exp Neurol
141: 214–224.
46. Emerich DF, Dean RL, Marsh J, Pink M, Lafreniere D, et al. (2000) Intravenous
cereport (RMP-7) enhances delivery of hydrophilic chemotherapeutics and
increases survival in rats with metastatic tumors in the brain. Pharm Res 17:
1212–1219.
47. Liu Y, Hashizume K, Chen Z, Samoto K, Ningaraj N, et al. (2001) Correlation
between bradykinin-induced blood-tumor barrier permeability and B2 receptor
expression in experimental brain tumors. Neurol Res 23: 379–387.
48. Liu LB, Xue YX, Liu YH, Wang YB (2008) Bradykinin increases blood-tumor
barrier permeability by down-regulating the expression levels of ZO-1, occludin,
and claudin-5 and rearranging actin cytoskeleton. J Neurosci Res 86:
1153–1168.
49. Pesquero JB, Araujo RC, Heppenstall PA, Stucky CL, Silva JA, Jr., et al. (2000)
Hypoalgesia and altered inflammatory responses in mice lacking kinin B1
receptors. Proc Natl Acad Sci U S A 97: 8140–8145.
50. Walther T, Balschun D, Voigt JP, Fink H, Zuschratter W, et al. (1998) Sustained
long term potentiation and anxiety in mice lacking the Mas protooncogene. J Biol
Chem 273: 11867–11873.
51. Borkowski JA, Ransom RW, Seabrook GR, Trumbauer M, Chen H, et al.
(1995) Targeted disruption of a B2 bradykinin receptor gene in mice eliminates
bradykinin action in smooth muscle and neurons. J Biol Chem 270:
13706–13710.
52. Costa R, Motta EM, Dutra RC, Manjavachi MN, Bento AF, et al. (2011) Antinociceptive effect of kinin B(1) and B(2) receptor antagonists on peripheral
neuropathy induced by paclitaxel in mice. Br J Pharmacol.
53. Dutra R, Cola M, Leite D, Bento A, Claudino R, et al. (2011) Inhibitor of
PI3Kgamma ameliorates TNBS-induced colitis in mice by affecting the
functional activity of CD4(+) CD25(+) FoxP3(+) regulatory T cells.
Br J Pharmacol 163: 358–374.
54. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using
real-time quantitative PCR and the 2(2Delta Delta C(T)) Method. Methods 25:
402–408.
55. Zhang M, Mao Y, Ramirez SH, Tuma RF, Chabrashvili T (2010) Angiotensin
II induced cerebral microvascular inflammation and increased blood-brain
barrier permeability via oxidative stress. Neuroscience 171: 852–858.
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