activation of kinin receptor b1 limits encephalitogenic … · activation of kinin receptor b1...

Nature Medicine: doi:10.1038/nm.1980

Supplementary methods

Autopsy Material. We obtained brain tissue from multiple sclerosis patients in collaboration with The

Netherlands Brain Bank (coordinator Dr. Huitinga, approval by the Ethical Committee of the VU University

Medical Center, Amsterdam). Classification of the lesions was based on standard histopathologic stainings for

inflammatory cells and on the presence of myelin breakdown products. Immunofluorescence of the kinin B1

receptor was performed as described previously8 with the following modifications: stainings with rat anti-CD3

(MCA 1477, Serotec, 1:200), subsequently anti-rat antibody conjugated to biotin (Vector Labs) and

streptavidin-FITC (BD Bioscience), as well as rabbit anti-B1 receptor (H-90, Santa Cruz, 1:200), subsequently

anti-rabbit secondary antibody conjugated to Alexa Fluor 568 (Molecular Probes). Nuclei were stained with

Hoechst 33258 (Sigma).

RNA isolation and real-time PCR. Total RNA extracted from cell lysates using the RNeasy mini kit was

treated with RNase-free DNase Set (Qiagen). First-strand cDNA was synthesized using 1 μg of total RNA and

TaqMan reverse transcription reagents (Applied Biosystems). Primer sets designed with Primer Express

Software spanned an intron to ensure discrimination between cDNA and genomic DNA, and PCR was

performed in duplicates with an ABI PRISM 7,000 and Platinum SYBR Green qPCR SuperMix-UDG

(Invitrogen). The relative amount of specific mRNA was normalized to GAPDH. Expression of Bdkrb1 and

Bdkrb2 was additionally confirmed by Taqman Gene Expression Assays (Bdkrb1 (Mm00432059_s1); Bdkrb2

(Mm01339907_m1); Applied Biosystems) and Universal PCR Master Mix (Applied Biosystems). Specificity

of signal/PCR reactions was confirmed for each sample by melting-curve analysis or loading PCR products on

an agarose gel. Negative controls (without reverse transcriptase) were used for each sample and primer pairs to

exclude contamination with genomic DNA.

Isolation of CNS derived mononuclear cells. Isolation of CNS mononuclear cells was performed as

previously described9 with minor adaptations: tissue digestion was performed in IMDM (Gibco) containing

200 U ml-1 DNase I (Roche) and 363 U ml-1 clostridiopeptidase A (Sigma) for 30 min at 37°C under

continuous rotation. Homogenate was resuspended in 27% Percoll (Amersham Pharmacia) and underlain with

73% Percoll. The gradient was centrifuged for 30 min. Finally, CNS infiltrate was analyzed by a FACS

Calibur (BD). For analysis of intracellular cytokines, isolated CNS-derived lymphocytes were stimulated for 5

h using 3 μg ml-1 anti-CD3, 3 μg ml-1 anti-CD28 and 5 μg ml-1 Breferdin A.


Flow Cytometry. Single cell suspensions were incubated with anti-CD16/CD32 (1:100) to prevent unspecific

antibody binding. Cell suspensions were stained with anti-CD4/anti-CD8 (1:100) and their activation state was

analyzed using antibodies against CD69, CD25 (1:100) and CD44 (1:50). Intracellular cytokine production of

CNS derived lymphocytes was analyzed by monitoring the expression of IFN-γ, and IL-17 (1:100) (all

antibodies from BD). FoxP3 staining was performed according to the manufacturer’s protocol (eBioscience).

For flow cytometric analysis of polymerized actin (F-actin) cells were stained with 50 ng ml-1 FITC-phalloidin

(Sigma-Aldrich) and gating was applied on activated T cells.

FACS on Bdkrb1 was performed by incubating the cells with the Bdkrb1 antibody8 at a dilution of 1:25 for 30

min in FACS buffer. After washing, cells were incubated with a goat anti-rabbit Alexa 488 at a concentration

of 2 μg ml-1 (from Invitrogen) for 30 min.

Immunoblotting. Cells were solubilized in lysis buffer (50 mM Tris-HCl, pH 7.4, 0.5% Triton-X 100, 150

mM NaCl, 5 mM EDTA, 1mM DTT, 10 µg ml-1 leupeptin, 10 µg ml-1 aprotinin, 10 µg ml-1 sodium

orthovanadate and 0.1 mM phenylmethylsulfonyl fluoride) at 4°C. The lysate was centrifuged at 13,000g for

15 min at 4°C and dissolved in SDS sample buffer. The samples were subjected to 12% SDS-PAGE,

electroblotted onto nitrocellulose membranes, and then stained with affinity-purified anti-Bdkrb1 receptor

antibody raised against C-terminal part of human receptor (Santa Cruz, H-90, 1:400) or anti-β-actin antibody

(Sigma, 1:100,000). Immunoreactive bands were visualized with a chemiluminescence immunodetection ECL-

Plus kit (Amersham Biosciences) using peroxidase-labeled anti-rabbit and anti-mouse antibody (Dako) and

protein bands were analyzed densitometrically (Adobe Photoshop™) from two independent runs.

Rho GTP-loading assay. Rho Assay was performed according to the manufacturer’s instructions (Millipore).

Briefly, cells pretreated for 24 h with 70 U ml-1 TNF-α and IFN-γ to induce Bdkrb1 expression and cultured

under varying conditions, were washed with cold PBS and then lysed in MLB buffer (10 mM MgCl2, 25 mM

HEPES, 150 mM NaCl, 1 mM EDTA, 1% NP40, 10% glycerol, 0.25% Na-deoxycholate, 1 mM Na3VO4, pH

7.4) supplemented with the protease inhibitors. Lysates were clarified by centrifugation at 14,000 g for 5 min

at 4°C. Clarified lysates corresponding to 1 mg protein, as determined by the BCA method (Pierce), were then

gently rotated for 45 min at 4° C with a GST fusion protein of the Rho-binding domain (RBD) of the Rho

effector protein Rhotekin bound to agarose beads, washed three times in MLB buffer and eluted in SDS-PAGE

sample buffer. Before doing the assay an aliquot corresponding to 20 µg protein was removed for


determination of total GTPase. The samples were analyzed by 12% SDS-PAGE and immunoblotting using

RhoA antibodies (Santa Cruz) to detect GTP-bound RhoA.

EAE histology. Histology was performed with conventional stainings (hematoxylin&eosin for visualization of

inflammatory infiltrates, luxol fast blue for demyelinated areas, β-amyloid precursor protein [β-APP, 1:1,000,

Zymed Laboratories] for axonal pathology, anti-Iba1 [1:500, WAKO] for macrophages). Sections were

labeled and developed using the avidin:biotinylated enzyme complex technique (ABC-Elite; Vector) or FITC-

labeled secondary antibodies. Cells positive for β-APP and Iba1 were counted in a blinded manner in spinal

cord as well as in brain stem sections (4 ocular fields per section and 5 mice per group). Immunofluorescence

of Bdkrb1 and CD3 was performed as described above. For further immunostainings, 20 μm thick sections

were collected (Supplementary Fig. S1). Classification of the lesions was based on standard Hoechst 33258

staining for inflammatory regions. Other primary antibodies used for analysis: were rabbit anti-Bdkrb1 (1:100,

SP4077P, Acris), rabbit anti-Bdkrb2 (1:200, SP4078P, Acris), rabbit anti-des-Arg9-BK (1:200, AP01069PU-

N, Acris) and rabbit anti-BK (1:400, GTX14391, GeneTex Inc.). Cy2-or Cy3-conjugated secondary antibodies

(1:1,000, Amersham Bioscience) were applied for 1 h to visualize the signal.

Confocal analysis. Confocal images were recorded with an upright laser microscope (Leica DM 2500)

equipped with a 20× objective (oil-immersion NA 0.7) and a 40× objective (oilimmersion NA 1.15) using a

zoom factor of 3 (for the 40×) and sequential scanning with the 405-nanometer spectral line of a blue diode

laser, the 488-nanometer line of an argon-ion laser and the 532-nanometer line from a green helium-neon

laser. During the processing stage, individual image channels were pseudocolored with RGB values

corresponding to each of the fluorophore emission spectral profiles. All images were collected, measured for

colocalization and compiled with the aid of Metamorph imaging software (Molecular Devices) and Adobe

Photoshop.

BK and des-Arg9-BK in the cerebrospinal fluid (CSF). CSF samples from naïve and mice with EAE were

collected according to the technique described by Fleming et al. (ref. 10). Samples (5 μl) were diluted 20 times

in 50 mmol/ L-1 Tris-HCl buffer, pH 7.4, containing 100 mmol L-1 NaCl and 0.05% Tween-20 and frozen at –

80ºC. The measurement of BK was performed using the Bradykinin EIA Kit (Phoenix Pharmaceuticals. Inc)

with some modifications. Briefly, biotin-labeled antigen was diluted 20 times to increase the sensitivity. The

competitive immunoextraction step was obtained by an incubation of 4 h at room temperature, under agitation.


Measurement of des-Arg9-BK was performed as described by Raymond et al. (ref. 11) Labeled antigen was

synthesized from 1 mg ml-1 desArg9-BK (B1542, Sigma) and Sulfo-NHS-LC-Biotin according to the

manufacturer’s protocol (Pierce). To avoid the presence of uncoupled biotin molecules in solution, the

labeling was blocked by incubation with 10 mM BSA for 16 h at 4°C. The resulting solution containing

Biotin-LC-desArg9-BK was used as an original tracer at a final concentration of 5 pmol ml-1. A NUNC 96-

well plate was coated overnight at 4°C with rabbit anti-desArg9-BK antibody (AP01069PU-N, Acris) diluted

1:250 in coating buffer (carbonate 100 mM, pH 9.2). After the removal of free antibody, the plate was washed

three times and saturated for 2 h with incubation buffer (50 mmol L-1 Tris-HCl buffer, pH 7.4, 100 mmol L-1

NaCl, 0.05% Tween-20). The competitive immunoextraction step was obtained by an incubation of 4 h at

room temperature, under agitation. The presence of immune complex was further determined by Bradykinin

EIA Kit (Phoenix Pharmaceuticals. Inc) according to the manufacturer’s protocol. Peptides were detectable at

1–10 pg ml-1 linear range with 0.1 pg ml-1 minimum detectable concentration.

Two-photon laser scanning microscopy. Brain slice cultures were obtained as previously described12. TH17

cells were pre-treated either with R838, R715 (both 500 nM) or PBS for additional 4 h. Cells were visualized

in brain slice cultures by a two-photon system SP2 (Leica, Heidelberg) equipped with an upright microscope

fitted with a 20× water-immersion objective (NA 0.5; Leica). Fluorescent dyes were excited simultaneously by

a mode-locked Ti-sapphire laser (Tsunami; Spectra-Physics) at wavelength 840 nm. Fluorescence from FITC-

dextrane and Celltracker Orange CMTMR (Invitrogen) was collected using two external non-descanned

detectors. XYZ stacks were typically collected in the depth (60 – 120 µm) below the surface over a period of

1– 2 hours (z-stack with a thickness of 60 µm, z-plane distance typically 1.8 µm). For velocity experiments

each cell was tracked for at least 5 min. Cell recognition, movement tracking, 3D presentation and average cell

velocity were calculated using Volocity® (Improvision).


References 1. Pesquero, J.B. et al. Molecular cloning and functional characterization of a mouse bradykinin B1

receptor gene. Biochem. Biophys. Res. Commun. 220, 219-225 (1996).

2. Kintsurashvili, E. et al. Age-related changes of bradykinin B1 and B2 receptors in rat heart. Am. J. Physiol. Heart Circ. Physiol. 289, H202-H205 (2005).

3. Barker, T.A. et al. Angiotensin type 2 receptor expression after vascular injury: differing effects of angiotensin-converting enzyme inhibition and angiotensin receptor blockade. Hypertension 48, 942-949 (2006).

4. Groger, M. et al. Release of bradykinin and expression of kinin B2 receptors in the brain: role for cell death and brain edema formation after focal cerebral ischemia in mice. J. Cereb. Blood Flow Metab. 25, 978-989 (2005).

5. Fortin, J.P., Dziadulewicz, E.K., Gera, L. & Marceau, F. A nonpeptide antagonist reveals a highly glycosylated state of the rabbit kinin B1 receptor. Mol. Pharmacol. 69, 1146-1157 (2006).

6. Kang, D.S., Gustafsson, C., Morgelin, M. & Leeb-Lundberg, L.M. B1 bradykinin receptor homo-oligomers in receptor cell surface expression and signaling: effects of receptor fragments. Mol. Pharmacol. 67, 309-318 (2005).

7. Zhang, X., Tan, F., Zhang, Y. & Skidgel, R.A. Carboxypeptidase M and kinin B1 receptors interact to facilitate efficient B1 signaling from B2 agonists. J. Biol. Chem. 283, 7994-8004 (2008).

8. Prat, A. et al. Kinin B1 receptor expression and function on human brain endothelial cells. J. Neuropathol. Exp. Neurol 59, 896-906 (2000).

9. Aktas, O. et al. Neuronal damage in autoimmune neuroinflammation mediated by the death ligand TRAIL. Neuron 46, 421-432 (2005).

10. Fleming, J.O., Ting, J.Y., Stohlman, S.A. & Weiner, L.P. Improvements in obtaining and characterizing mouse cerebrospinal fluid. Application to mouse hepatitis virus-induced encephalomyelitis. J. Neuroimmunol. 4, 129-140 (1983).

11. Raymond, P. et al. Quantification of des-Arg9-bradykinin using a chemiluminescence enzyme immunoassay: application to its kinetic profile during plasma activation. J. Immunol. Methods 180, 247-257 (1995).

12. Nitsch, R. et al. Direct impact of T cells on neurons revealed by two-photon microscopy in living brain tissue. J. Neurosci. 24, 2458-2464 (2004).


activation of kinin receptor b1 limits encephalitogenic … · activation of kinin receptor b1...

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