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ct
ck
de
S
zburg, Jo
ogy e In
c Eberhard Karls University Tuebingen, Medizinische Kli
d Institute of Physiology - Neuropathophysiology, Unive
a r t i c l e i n f o
Article history:
Received 11 October 2010
successively induce disseminated inflammatory lesions within the
CNS [1]. This cell trafficking is a tightly regulated process which on
the trans- and paracellular movement of molecules and cells [3].
Inflammatory events such as binding of immune cells or the release
of solublemediators, however, can rearrange thenormal assembly of
tight-junction proteins and cause further upregulation of adhesion
molecules. As a consequence, the structural integrity of the BBB gets
lost and transendothelial trafficking increases [3,6e10].
Kinins, e.g. bradykinin and kallidin, constitute the end-products
of the so-called kallikrein/kinin-system (KKS). These highly active
proinflammatory peptide hormones are released by kallikreins
* Corresponding author. University of Muenster, Department of Neurology e
Inflammatory Disorders of the Nervous System and Neurooncology, Domagkstr. 13,
48149 Muenster, Germany. Tel.: þ49 251 83 46817; fax: þ49 251 83 46812.
** Corresponding author. Tel.: þ49 931 201 23755; fax: þ49 931 201 23488.
E-mail addresses: christoph.kleinschnitz@uni-wuerzburg.de (C. Kleinschnitz),
sven.meuth@ukmuenster.de (S.G. Meuth).
1
Contents lists availab
Journal of Au
journal homepage: www.els
Journal of Autoimmunity 36 (2011) 106e114Both authors contributed equally.In contrast, blocking B2R had no significant impact on EAE.
We conclude that B1R inhibition can reduce BBB damage and cell invasion during autoimmune CNS
disease and may offer a novel anti-inflammatory strategy for the treatment of MS.
� 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Multiple Sclerosis (MS) is a chronic inflammatory demyelinating
disorder of the central nervous system (CNS) of putative autoim-
mune origin. It is generally believed that during the course of
MS autoreactive T cells activated in the periphery of the immune
compartment migrate across the blood brain barrier (BBB) and
the one hand depends on the activation state of the Tcells [2], but on
the other hand is governed by the BBB via the secretion of various
chemokines, the expression of cellular adhesion molecules and
tight-junction proteins [3,4]. Highly specialized brain microvascular
endothelial cells are key components of the BBB [3,5,6]. Under
physiological conditions these cells express adhesionmolecules only
at low levels and form intercellular tight junctions thereby limitingAccepted 30 November 2010
Keywords:
Multiple sclerosis
EAE
Kallikrein/kinin-system
Bradykinin receptor 1
Leukocyte trafficking0896-8411/$ e see front matter � 2010 Elsevier Ltd.
doi:10.1016/j.jaut.2010.11.004nik III, Department of Cardiovascular Medicine, Tuebingen, Germany
rsity of Muenster, Robert-Koch-Str. 27a, 48149 Muenster, Germany
a b s t r a c t
Disruption of the blood brain barrier (BBB) and transendothelial trafficking of immune cells into the
central nervous system (CNS) are pathophysiological hallmarks of Multiple Sclerosis (MS) and its animal
model, Experimental Autoimmune Encephalomyelitis (EAE). Kinins are proinflammatory peptides
which are released during tissue injury including EAE. They increase vascular permeability and enhance
inflammation by acting on distinct bradykinin receptors, B1R and B2R.
We studied the expression of B1R and B2R and the effect of their inhibition on the disease course, BBB
integrity and T cell migration following myelin oligodendrocyte glycoprotein (MOG35e55)-induced EAE.
B1R, but not B2R expression was markedly enhanced in inflammatory CNS lesions in mice and humans.
Brain endothelial cells could be identified as major source of B1R protein. The severity of EAE was
significantly alleviated in B1R�/�mice comparedwithwild-type (WT) controls (P< 0.05). Treatment ofWT
mice with the B1R antagonist R715 before and after disease onset was equally effective (P < 0.05) while
B1R activation by R838 promoted EAE (P < 0.05). B1R inhibition was accompanied by a remarkable
reduction of BBB disruption and tissue inflammation. In vitro analyses revealed that B1R suppression
reverses the upregulation of ICAM-I and VCAM-I at the inflamed BBB thereby limiting Tcell transmigration.aDepartment of Neurology, University of Wuer
bUniversity of Muenster, Department of Neurolsef-Schneider-Strasse 11, 97080 Wuerzburg, Germany
flammatory Disorders of the Nervous System and Neurooncology, Domagkstr. 13, 48149 Muenster, GermanyChristoph Kleinschnitz a,**,1, Sven G. Meuth a,b,d,*,1Blockade of the kinin receptor B1 prote
CNS disease by reducing leukocyte traffi
Kerstin Göbel a,b, Susann Pankratz b, Tilman Schnei
Michael K. Schuhmann a,b, Harald F. Langer c, GuidoAll rights reserved.s from autoimmune
ing
r-Hohendorf b, Stefan Bittner a,
toll a, Heinz Wiendl b,
le at ScienceDirect
toimmunity
evier .com/locate/ jaut imm
Page 2
utoimfrom their precursors, kininogens, during various kinds of
tissue injury including inflammation [4,11,12], ischemia and trauma
[13e15]. The KKS plays an important role in the regulation of
vascular permeability and all components of the KKS have been
identified in the brain [16e18]. Only recently, its suitability as an
endogenous target to combat CNS inflammation [11,19e22] and
other neurological disease states [13,14,23] has been recognized.
The biological activities of kinins are mediated by two different
G-protein-coupled bradykinin receptors: The tissue expression of
bradykinin receptor 2 (B2R) is ubiquitous and constitutive [24,25]
and this receptor subtype mediates the majority of the physiolog-
ical effects of bradykinin [10,13,26e29]. In contrast, bradykinin
receptor 1 (B1R) is present at low levels under normal conditions,
but can be selectively induced by tissue injury and inflammatory
mediators [24]. Previous studies revealed that B1R is expressed
on circulating lymphocytes and infiltrating T cells during active
episodes of MS as well as on endothelial cells within MS lesions
[4,11,12,26]. Following their activation both B1R andB2Rmediate the
classical inflammatory processes after tissue injury such as proin-
flammatory cytokine release, immune cell influx, and increased
vascular permeability [24,28]. Accordingly, genetic disruption or
pharmacological inhibition of kinin receptors are generally believed
to have anti-inflammatory effects during various pathophysiological
conditions and several B1R and B2R blocker are currently under
clinical investigation [23,24,28].
In the present study, we investigated the consequences of
B1R and B2R deficiency or pharmacological blockade on the disease
course, BBB permeability and inflammatory processes after myelin
oligodendrocyte glycoprotein (MOG35e55) peptide-induced Exper-
imental Autoimmune Encephalomyelitis (EAE) in mice, an estab-
lished animal model of MS.
2. Materials and methods
2.1. Mice
B1R-deficient (B1R�/�) mice were generated by Deltagen
(San Mateo, USA). The targeting vector was generated by flanking
the neomycin resistance gene with a 0.7 kb genomic fragment 50 of
the B1R-coding region and a 2.8 kb fragment 30 of the B1R-coding
region. Correctly targeted embryonic stem cells (E14) were used for
the generation of chimeric mice. Transmission of the mutant allele
was determined by Southern Blot analysis (Supplementary Fig. 1A).
Wild-type (WT), heterozygous and homozygous B1R�/� mice
were identified by ‘multiplex’ PCR with the following primers: GS
(E, T), 50-CCAGCAGACCAGGAAGGAGGCTAC-30; NEO (T), 50-GGGTGG
GATTAGATAAATGCCTGCTCT-30; and GS (E1), 50-CTGAACATCTCTG
CCTGCATCCTGC-30. The WT B1R allele was identified by the pres-
ence of a 240ebase pair DNA fragment, whereas the targeted
B1R allele was identified by the presence of a 424ebase pair DNA
fragment. Mice were backcrossed onto a C57Bl/6 background for at
least 10 generations. B1R�/� mice were healthy and bred normally
when maintained in specific pathogenefree conditions.
B2R-deficient (B2R�/�) mice were purchased from Jackson
Laboratories (002641; Bar Harbor, USA) and backcrossed onto
a C57Bl/6 background for 10 generations. C57Bl/6 (WT) mice
(Charles River; Sulzfeld, Germany) served as controls.
2.2. Induction and evaluation of EAE
All experiments were approved by and conducted in accordance
with the laws and regulations of the regulatory authorities for
animal care and use in Lower Franconia.
EAE was induced by immunization of 6e8 weeks old female
K. Göbel et al. / Journal of AB1R�/�, B2R�/� and WT mice with MOG35e55 peptide (Charite, Berlin,Germany) as previously described [30,31]. Pharmacological modula-
tionwas performed using the selective B1R antagonist R715 (1mg/kg;
Biomatik, Wilmington, USA) and B1R agonist R838 (1 mg/kg; Tocris,
Ellisville, USA) administered by daily intraperitoneal injection.
All animals were kept under standard conditions and had access
to water and food ad libitum. The clinical course of EAE was
monitored by two blinded investigators using the following score
system: grade 0, no abnormality; grade 1, limp tail tip; grade 2, limp
tail; grade 3, moderate hind limbweakness; grade 4, complete hind
limb weakness; grade 5, mild paraparesis; grade 6, paraparesis;
grade 7, heavy paraparesis or paraplegia; grade 8, tetraparesis;
grade 9, quadriplegia or premoribund state; grade 10, death.
Animals, with a score higher than 7, were euthanized and continued
with the accordant score until the end of the experiment.
2.3. Proliferation assay
Splenocytes from WT, B1R�/� and B2R�/� mice were isolated
from immunized animals at the peak of disease and 50 days after
EAE induction. In brief, 1 � 105 splenocytes were cultured in 1 ml
DMEM containing 10 mM HEPES, 25 mg/ml gentamicin, 50 mM
mercaptoethanol, 5% FCS, 2 mM glutamine, and 1% nonessential
amino acids (Cambrex; Verviers, Belgium) for three days and
stimulated with CD3/CD28 beads (cell to bead ratio 2:1; Dynal
Biotech, Hamburg, Germany) or 10 mg/ml MOG35e55. 3H thymidine
(Amerham; Piscataway, NJ) was added for the final 14 h, and
radioactivity was measured on a b-scintillation counter (TopCount
NXT; PerkinElmer, Rodgau-Jügesheim, Germany). Experiments were
performed in quadruplicates.
2.4. Cytokine production
Splenocytes from control WT, B1R�/� and B2R�/� animals were
isolated from immunized mice at the disease maximum and 50
days after EAE induction and stimulated with MOG35e55 peptide
(10 mg/ml) or CD3/CD28 beads (cell to bead ratio 2:1). Supernatants
were assessed for IFNɣ, IL-17, IL-4 and IL-6 protein levels by ELISA
(R&D Systems; Wiesbaden, Germany) according to the manufac-
turer’s instructions.
2.5. Migration assay and analysis of brain microvascular
endothelial cells
2.5.1. Mouse cells
Isolation of murine brain microvascular endothelial cells
(MBMEC) was conducted as previously described [32,33]. In brief,
MBMEC were prepared from brains of WT and B1R�/� mice and
cultured 5 days prior to use on 3.0 mmCollagenIV/fibronectin-coated
Transwell� Pore PolyesterMembrane inserts (Corning; Lowell, USA).
Purity, confluence and cell morphology were checked regularly
by flow cytometry, resistance measurements and microscopy.
To induce upregulation of B1R, MBMEC were in vitro inflamed with
IFNg and TNFa (each 500 U/ml, Peprotech; Hamburg, Germany).
Naïve or in vitro inflamed (100 U/ml IFNg and TNFa) CD4 T cells
from WT, B1R�/� and B2R�/� mice isolated by magnetic-activated
cell sorting (MACS, Miltenyi; Bergisch-Gladbach, Germany) were
loaded in the upper chamber. B1R protein expression under in vitro
inflamed conditions was confirmed using immuncytochemistry
(Supplementary Fig. 1B) [11]. After an incubation period of 18 h
migrated cells from the lower chamber of two compartments
were collected and Calibrite� beads (BD Biosciences; Heidelberg,
Germany) were added. The relative cell number of CD4 T cells was
determined by flow cytometry (BD Biosciences).
RNA isolation and RT-PCR was performed as previously described
munity 36 (2011) 106e114 107using following TaqManGene ExpressionAssays (Applied Biosystems;
Page 3
toimFoster City, USA): Claudin-5 (Mm00727012_s1), ICAM-I (Mm00
516023_m1), Occludin (Mm00500912_m1), PECAM-I (Mm01242
584_m1), VCAM-I (Mm01320970_m1), ZO-1 (Mm00493699_m1),
eukaryotic 18S RNA (Hs99999901_s1). Datawere calculated usingDct,
DDct and relative quantification (2�DDCT).
2.5.2. Human cells
Human Brain Microvascular Endothelial Cells (HBMEC, ScienCell
Research Laboratories; Carlsbad, USA) were cultured on 3.0 mm
Transwell� Pore Polyester Membrane inserts (Corning) until conflu-
ence. For induction of B1R, HBMECs were treated in vitro with 100 U/
ml IFNg and TNFa (Peprotech) for 24 h and incubated with B1R
agonist R838 or B1R antagonist R715 (each 500 nM) for additional 3 h
(Supplementary Fig.1C). MACS-isolated CD4 Tcells (5�105) from the
peripheral blood of healthy donorswere added to the upper chamber.
After 18 h incubation, the number of migrated T cells was analyzed as
described above.
2.6. Static adhesion assay
CD4 T cells fromWTand B1R�/� micewere activated with 100 U/
ml IFNg and TNFa for 12 h. Tcellswere then incubatedwith 40 mg/ml
6-carboxyfluorescein diacetate (6-CFDA, Sigma; Munich, Germany)
in RPMI at 37 �C for 30 min. After washing, cells were suspended at
2�106/ml in standard adhesion buffer containing PBSwith 2mg/ml
BSA, 10 mM HEPES, 1 mM CaCl2, 1 mM MgCl2. Adhesion assays were
performed on 96-well plates coated overnight with mouse ICAM-I
or mouse VCAM-I (each 2 mg/ml; both R&D Systems). 100 ml of cell
suspension were added to the wells and incubated for 10 min.
After washing, fluorescence intensity was immediately measured.
All experiments were performed in triplicates.
2.7. Immunophenotyping and analysis of integrin expression
For analysis of T cell subtype distribution and integrin expres-
sion flow cytometry was performed as previously described [30,31]
using appropriate antibodies or isotype controls (all by BD Biosci-
ences): rat anti-mouse CD4-PerCP (no. 553052), rat anti-mouse
CD25-APC (no. 557192), rat anti-mouse CD44-FITC (no. 553133),
rat anti-mouse CD62L-APC (no. 553152), rat anti-mouse CD69-FITC
(no. 557392), rat anti-mouse CD49d-PE (no. 557420) and rat anti-
mouse CD11a-PE (no. 553121).
2.8. Brain tissue specimen
For immunohistochemical staining, human autopsy and biopsy
material from MS patients and healthy controls was received from
The UK Multiple Sclerosis Tissue Bank (Division of Neuroscience and
Mental Health, London, UK, n¼ 5). For the present studyfiveMS cases
were analyzed in which cryo-fixed brain tissue was available. The
lesions fulfilled the morphological criteria of an inflammatory demy-
elinating process consistentwithMSwhen stainedwithH&E, LFB-PAS
myelin stain and Bielschowsky’s silver impregnation for axons.
2.9. Immunohistochemistry
Immunohistochemistry was performed as previously described
[30,31,34]. Primary antibodies used for this study were anti-CD11b
(1:100, Serotec, Düsseldorf, Germany) and antibodies against phos-
phorylated neurofilament (1:5000, SMI31) and non-phosphorylated
neurofilament (1:5000, SMI32, both Sternberger Monoclonals,
Lutherville, USA). Secondary antibodies were horse anti-mouse IgG-
biotin or horse anti-rat IgG-biotin (Vector Laboratories, Burlingame,
USA). H&E and Luxol fast blue (LFB) staining were done according to
K. Göbel et al. / Journal of Au108standard procedures.Immunofluorescence stainings were performed as described
earlier. Primary antibodies against B1R (1:100, Santa Cruz; Heidel-
berg, Germany), PECAM-I (1:200, Abcam; Cambridge, UK), VCAM-I
(1:200, Abcam) and ICAM-I (1:200, Abcam) were used. Secondary
antibodies were Alexa Fluor 488-coupled goat anti-mouse (1:100,
BD Biosciences), Cy3-coupled goat anti-rabbit or Cy3-coupled
goat anti-rat (both 1:300, Dianova; Hamburg, Germany). Negative
controls were obtained by either omitting the primary or secondary
antibody and revealed no detectable signal (not shown).
For quantification, stainings were examined in a blinded fashion
by microscopy (Axiophot2, Zeiss; Oberkochen, Germany) with
a CCD camera (Visitron Systems; Tuchheim, Germany). Inflamma-
tory foci (H&E), demyelinated areas (LFB) and cells were counted on
five randomly selected complete spinal cord cross sections from
the lumbar level utilizing MetVue Software (Molecular Devices;
Downingtown, USA). Number of axons was analyzed in defined
lesions. Fluorescence intensity was measured utilizing Image
J (NIH, USA).
2.10. Statistical analysis
All results are presented as mean� SEM. Statistical analysis was
performed using a modified Student t test for parametric data or
ManneWhitney U test for non-parametric datasets (SPSS, Munich,
Germany). P values < 0.05 were considered statistically significant.
3. Results
3.1. B1R is induced during autoimmune CNS inflammation
in mice and humans
In a first set of experiments, we analyzed the expression of B1R
on the protein level in spinal cords fromMOG35e55-immunizedmice
at disease maximum using immunofluorescence staining. While
B1R was undetectable in healthy brain and spinal cord (Fig. 1A;
control brain), clear positive labeling of B1R was seen in vascular
endothelial cells after induction of EAE (Fig. 1A; EAE brain). This was
confirmed by double labeling for PECAM-I (an endothelial marker)
and B1R. Importantly, B1R was also present in active lesions from
MS (MS brain) patients but not in the CNS of healthy donors (control
brain) and again co-localized with vessel (Fig. 1B; n ¼ 5). These data
indicate that B1R is induced during autoimmune CNS inflammation
in mice and men.
3.2. Inhibition of B1R but not B2R ameliorates EAE
and reduces CNS inflammation
We first immunized B1R�/� mice with MOG35e55 peptide and
assessed the disease course of EAE until day 50. B1R�/� mice
showed significantly reduced disease severity at the maximum of
EAE compared to WT controls (Fig. 1C; mean EAE score at day 16:
Con: 6 � 0.3, B1R�/�: 2 � 0.5, P ¼ 0.0002). Importantly, the
difference in the degree of disease severity persisted until the end
of our study at day 50. In contrast, disruption of the B2R gene did
not alter the disease course compared with controls indicating that
B2R is less important for the pathophysiology of EAE (Fig. 1D).
Subsequently, we wanted to examine whether these genetic
insights into the biology of B1R in EAE can be translated into
a therapeutic intervention. In a first approach, MOG35e55-immu-
nized C57Bl/6 mice were treated daily with the B1R antagonist
R715 (1 mg/kg) starting from the day of immunization (day 0).
In line with our findings in B1R�/� mice, R715 treatment led to
a significantly lower disease maximum compared to untreated
mice (Fig. 1E). Accordingly, specific activation of B1R by R838
munity 36 (2011) 106e114resulted in a more severe EAE disease (Fig. 1E). In another approach
Page 4
utoimK. Göbel et al. / Journal of Awhich more closely mirrors the clinical scenario, immunized mice
were treated with R715 (1 mg/kg) or vehicle starting at the day of
disease onset when animals already displayed first clinical symp-
toms. Importantly, blockade of B1R post EAE onset still led to
a significantly lower disease maximum (Fig. 1F).
Several studies have demonstrated that B1R deficiency modu-
lates the inflammatory response under various pathophysiological
conditions [35,36]. We therefore analyzed the extent of
Fig. 1. Histopathological evidence and relevance of B1R in vivo. (A) EAE lesions were identifi
staining for PECAM-I (green), B1R (red) and cell nuclei (DAPI, blue) shows enhanced expres
non-immunized mice (control brain). (B) Histopathological stainings for PECAM-I (green), B1
patients with MS (MS brain). Representative images show the presence of B1R in MS lesions
EAE disease course compared to WT littermates after immunization (n ¼ 20 per group). (D
group). (E) Pharmacological modulation of B1R with the B1R antagonist R715 or B1R agonist
Therapeutic treatment with R715, given daily after disease onset, attenuates the disease max
significant.munity 36 (2011) 106e114 109inflammation in spinal cords from B1R�/� mice at the maximum
level of EAE by immunohistochemistry. The number of inflammatory
foci was significantly lower in B1R null mice compared with WT
controls (Fig. 2A; Con: 13 � 1, B1R�/� 6 � 1; P < 0.01). We observed
similar findings in WT mice treated with R715 (10 � 1, P < 0.05),
while application of R838 increased the amount of inflammatory
plaques (16 � 2, P > 0.05) although the difference was not statisti-
cally significant (Fig. 2A). We also quantified the numbers of
ed by H&E staining of brain cryosections of immunized WT mice. Immunofluorescence
sion of B1R on endothelial cells in EAE lesions (EAE brain), but not in control tissue of
R (red) and cell nuclei (DAPI, blue) from CNS of healthy individuals (control brain) and
, but not in control tissue (n ¼ 5). (C) Mice lacking B1R show a significantly attenuated
) B2R deficiency has no influence on the disease course in MOG35e55 EAE (n ¼ 8 per
R838, administered daily in MOG35e55 peptide immunized mice (n ¼ 12 per group). (F)
imum (n ¼ 8 per group). Mean disease courses � SEM are displayed. *P < 0.05, ns ¼ not
Page 5
Fig. 2. Mice lacking B1R show significantly altered histopathological changes at EAE maximum. (AeD) Histological analysis of three spinal cord sections per mouse to assess the
number of inflammatory foci by H&E staining (A, arrows indicate infiltrated cells), number of infiltrated macrophages and microglia (B, CD11b), area of demyelination by LFB
staining (C, arrows indicate demyelinated areas) and axonal damage (D, SMI-31/-32) at disease maximum. Representative images of spinal cord cross sections and corresponding
quantifications are shown for WT (Con), B1R�/� mice (B1R�/�) and pharmacological modulation with B1R antagonist R715 and B1R agonist R838. Scale bars indicate 100 or 10 mm,
respectively. (E, F) Blockade or activation of B1R has no impact on myelin-specific inflammatory responses in the periphery. Proliferation, assessed by thymidine uptake and cytokine
production (IFNg, IL-17, IL-6 and IL-4) in response to MOG35e55 in cells from spleens of immunized WT mice (Con), B1R�/� mice (B1R�/�) and mice treated with B1R antagonist R715
or B1R agonist R838 at disease maximum. (G) Levels of ICAM-I, VCAM-I, PECAM-I, Claudin-5, Occludin and ZO-I of pure primary freshly isolated endothelial cells from control WT
mice (Con) and B1R�/� mice (B1R�/�) assessed by RT-PCR at disease maximum. Expression levels were related to naïve pure primary freshly isolated endothelial cells (naïve WT)
that were normalized to one. Values are mean � SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns ¼ not significant (P > 0.05).
K. Göbel et al. / Journal of Autoimmunity 36 (2011) 106e114110
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