neuroprotective role of bradykinin because of the...

Neuroprotective role of bradykinin because of the attenuation ofpro-inflammatory cytokine release from activated microglia

Mami Noda,* Yukihiro Kariura,* Ulrike Pannasch,� Kaori Nishikawa,� Liping Wang,�Toshihiro Seike,* Masataka Ifuku,* Yuki Kosai,* Bing Wang,* Christiane Nolte,�Shunsuke Aoki,� Helmut Kettenmann� and Keiji Wada�

*Laboratory of Pathophysiology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan

�Department of Cellular Neurosciences, Max-Delbruck Center for Molecular Medicine, Berlin, Germany

�Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and

Psychiatry, Tokyo, Japan

Abstract

Bradykinin (BK) has been reported to be a mediator of brain

damage in acute insults. Receptors for BK have been identi-

fied on microglia, the pathologic sensors of the brain. Here, we

report that BK attenuated lipopolysaccharide (LPS)-induced

release of tumor necrosis factor-alpha (TNF-a) and interleu-

kin-1b from microglial cells, thus acting as an anti-inflamma-

tory mediator in the brain. This effect was mimicked by raising

intracellular cAMP or stimulating the prostanoid receptors EP2

and EP4, while it was abolished by a cAMP antagonist, a

prostanoid receptor antagonist, or by an inhibitor of the indu-

cible cyclooxygenase (cyclooxygenase-2). BK also enhanced

formation of prostaglandin E2 and expression of microsomal

prostaglandin E synthase. Expression of BK receptors and

EP2/EP4 receptors were also enhanced. Using physiological

techniques, we identified functional BK receptors not only in

culture, but also in microglia from acute brain slices. BK re-

duced LPS-induced neuronal death in neuron–microglia co-

cultures. This was probably mediated via microglia as it did

not affect TNF-a-induced neuronal death in pure neuronal

cultures. Our data imply that BK has anti-inflammatory and

neuroprotective effects in the central nervous system by

modulating microglial function.

Keywords: bradykinin, lipopolysaccharide, microglia, pros-

taglandin, tumor necrosis factor-alpha.

J. Neurochem. (2007) 101, 397–410.

Bradykinin (BK) is an endogenous peptide which is producedvia the kallikrein–kinin system and is widespread in the brain(Scicli et al. 1984; Chao et al. 1987; Walker et al. 1995;Raidoo and Bhoola 1998). The effects of BK are mediated bytwo types of receptors, B1 and B2 (Hall 1992, 1997). BothBK receptors belong to the family of seven transmembranereceptors and are coupled to G-proteins (Gq/11 and Gi/o). BKreceptors are not only present on neurons (Delmas et al.2002), but also on all types of glial cells, astrocytes, andoligodendrocytes (Gimpl et al. 1992; Hosli et al. 1992; Linand Chuang 1992; Stephens et al. 1993a,b) and, as recentlydemonstrated, also on microglial cells (Noda et al. 2003,2004). In astrocytes, BK stimulates the release of arachidonicacid (Burch and Kniss 1988; Kaeser et al. 1988) and inducesthe calcium-dependent release of glutamate and IL-6 expres-sion (Parpura et al. 1994; Schwaninger et al. 1999).

After brain trauma or stroke, BK production is up-regulated and the increased BK levels lead to an increase inblood–brain barrier permeability and an accumulation of

leukocytes (Abbott 2000; Lehmberg et al. 2003). BK is thusthought to be involved in secondary brain damage. Byblocking BK receptors with specific antagonists, post-ischemic brain swelling after focal cerebral ischemia isreduced in concert with improved functional neuronal

Received July 6, 2006; revised manuscript received September 20, 2006;accepted October 11, 2006.Address correspondence and reprint requests to Mami Noda, Labor-

atory of Pathophysiology, Graduate School of Pharmaceutical Sciences,Kyushu University, Fukuoka 812-8582, Japan.E-mail: [email protected] used: AH6809, 6-isoproxy-9-osoxanthene-2-carboxylic

acid; BK, braydkinin; CNS, central nervous system; COX-2, cyclo-oxygenase-2; dbcAMP, dibutyryl cyclic AMP; DMEM, Dulbecco’smodified Eagle’s medium; FCS, fetal calf serum; HEPES, N-2-hy-droxyethylpiperazine-N¢-2-ethansulphonic acid; IL-1b, interleukin-1b;LPS, lipopolysaccharide; NS398, N-(2-Cyclohexyloxy-4-nitrophenyl)-Methanesulfonamide; mPGES, microsomal prostaglandin E synthase;RpcAMP, adenosine 3¢,5¢-cyclic monophosphorothioate; TNF-a, tumornecrosis factor-alpha.

Journal of Neurochemistry, 2007, 101, 397–410 doi:10.1111/j.1471-4159.2006.04339.x

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 397–410 397

recovery (Zausinger et al. 2002). In contrast, in an animalmodel of global cerebral ischemia, BK receptor antagonistsincreased mortality (Lehmberg et al. 2003).

In the brain, microglial cells are considered as thepathologic response element (Perry et al. 1993; Kreutzberg1996; Kim and de Vellis 2005). Microglial cells aredispersed throughout the entire central nervous system(CNS) and exhibit a ramified morphology under normalconditions. As recently shown by Davalos et al. (2005) andNimmerjahn et al. (2005), microglial processes are highlydynamic in the intact brain, suggesting that microglial cellsscan the brain parenchyma with their processes andpotentially shield it from injury. Under pathologic condi-tions, such as a lesion, stroke, neurodegenerative disordersor tumor invasion, activated microglia migrate rapidly tothe affected sites of the CNS. At the same time, microglialactivation is accompanied by the release of immunocom-petent molecules such as cytokines or chemokines, andother substances such as growth factors (Hanisch 2002). Inculture, their activation can be stimulated by LPS, a cellwall component of Gram-negative bacteria. Treatment withLPS or cytokines such as interleukin-1b (IL-1b) has beenshown to increase the expression of the B1 receptor in rat orrabbit tissue (Regoli et al. 1981; Galizzi et al. 1994;Nicolau et al. 1996), although in the brain this has notyet been studied.

Here, we provide evidence that BK receptors in microgliamediate anti-inflammatory or neuroprotective effects, namelythe inhibition of LPS-induced release of tumor necrosisfactor-alpha (TNF-a) and IL-1b.

Materials and methods

Cell culture

The study was approved by the Animal Research Committee of

Kyushu University and Max-Delbruck Center for Molecular

Medicine, and carried out in accordance with the National Institutes

of Health Guide for the Care and Use of Laboratory Animals.

Rat microglial cells were isolated from mixed cultures of

cerebrocortical cells in postnatal day 3 Wistar rats (Kyudo,

Kumamoto, Japan) as described previously (Sastradipura et al.1998; Noda et al. 2000). In brief, for rat microglia, the cerebral

cortex was minced, and treated twice with papain (90 U) and DNase

(2000 U) at 37�C for 15 min. Dissociated cells were seeded into

300 cm2 plastic flasks at a density of 107 per 300 cm2 in Eagle’s

medium (MEM) with 0.17% NaHCO3 and 10% fetal calf serum

(FCS), and maintained at 37�C in 10% CO2/90% air. Medium was

changed twice per week. After 10–14 days, floating cells and

weakly attached cells on the mixed primary cultured cell layer were

obtained by gently shaking for 5–8 min. The resulting cell

suspension was seeded on plastic dishes, with or without glass

coverslips, or 96-well plates and allowed to adhere for 30 min at

37�C. Microglial cells were then isolated as strongly adhering cells

after unattached cells were removed.

Mouse microglial cells were isolated from NMRI mice (Tierzucht

Schonwalde, Schonwalde, Germany) or C57BL/6 mice (Kyudo,

Kumamoto, Japan), as described previously (Prinz et al. 1999). Inbrief, cortical tissue was trypsinized for 2 min, dissociated with a

fire-polished pipette and washed twice. Mixed glial cells were

cultured for 9–12 days in Dulbecco’s modified Eagle’s medium

(DMEM) supplemented with 10% FCS, 2 mmol/L L-glutamine,

100 U/mL penicillin, and 100 lg/mL streptomycin, with medium

changes every third day. Microglial cells were then separated from

the underlying astrocytic layer by gentle shaking of the flask for 1 h

at 37�C in a shaker–incubator (100 rpm). The cells were seeded on

glass coverslips in 35 mm dish. Cells were used for experiments 1–

5 days after plating. The purity of both rat and mouse microglia, as

measured by binding of isolectin, was >99%.

Neuronal cell cultures were prepared from cerebral cortices of

embryonic day 14–16 (E14–16) C57BL/6 mice brains. The cortices

were trypsinized together with DNaseI and dissociated with a fire-

polished pipette and washed twice. Cells were plated on 4-well

culture slides (BD Falcon, Bedford, MA, USA) that had been

previously coated overnight with poly-D-lysine (50 lg/mL, Sigma,

St Louis, MO, USA) and incubated in culture medium (high-glucose

DMEM; Invitrogen, Carsbad, CA, USA) supplemented with

110 lg/mL pyruvic acid, 3.7 mg/mL NaHCO3, 5 lg/mL insulin,

2 mmol/L L-glutamine, 100 U/mL penicillin, 100 lg/mL strepto-

mycin, 2% B27 supplement (Invitrogen). Cells were used after

7 days. The density of neuronal cells was about 5 · 104 cell/well.

Mixed neuron–microglia cultures were obtained by seeding

mouse microglia onto a neuronal cell culture at a density of 5 · 104

cell/well with 10% microglial medium.

Assay of TNF-a, IL-1b, IL-6, and IL-10

Microglial cells isolated from Wistar rats were seeded in a 96-well

plate at a density of 4 · 104 cells/well and were treated with or

without BK and/or LPS for 4, 6, and 18 h for measuring TNF-a, IL-1b, IL-6, and IL-10, respectively, according to the time-dependent

release of each cytokine. The amount of each cytokine released into

the culture medium was measured using an ELISA Kit following the

manufacturer’s protocol (Biosource, Camarillo, CA, USA). The

absorbence at 450 nm was measured with a Microplate Reader

(ImmunoMini NJ-2300, Nalge Nunc International, KK, Tokyo,

Japan).

Microglial cell counting

The number of live microglial cells with or without drug application

was checked by using Cell Counting Kit-8 (Dojindo, Kumamoto,

Japan), according to the manufacturer’s instructions.

Assay of PGE2

Microglial cells isolated from postnatal day 3 Wistar rats were

seeded in a 96-well plate at a density of 5 · 104 cell/well and were

treated with or without BK and/or LPS for 24 h. The serum was

reduced to 0.1%. Amount of PGE2 in the supernatant fluid was

measured with a PGE2 EIA kit from Amersham (Piscataway, NJ,

USA), according to the manufacturer’s instructions.

Immunocytochemistry

Rat primary cultured microglia were treated with or without 100–

300 nmol/L BK or 100 ng/mL LPS, and fixed with 4% parafor-

398 M. Noda et al.

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 397–410� 2007 The Authors

maldehyde. The fixed cells were incubated with rabbit anti-

microsomal prostaglandin E synthase (mPGES) polyclonal antibody

(1/500, Cayman Chemical, Ann Arbor, MI, USA), goat polyclonal

anti-B1 receptor (1/200, Santa Cruz Biotechnology Inc., Santa Cruz,

CA, USA), or mouse monoclonal anti-B2 receptor (1/500, Research

Diagnostics Inc., Flanders, NJ, USA) together with goat serum (1/10

volume, Cedarlane, Ontario, Canada) overnight at 4�C. After fourwashes, the cells were incubated with the fluorescence-conjugated

secondary antibody together with goat serum (10% in volume) at

20–25�C for 1 h. Control cells were incubated with nonimmune rat

IgG. For the identification of microglia in the staining of mPGES,

the same cells were treated with FITC-labeled isolectin-B4 (1/200,

Santa Cruz). For identification of neurons, monoclonal anti-MAP2

(1/1000, Sigma) was used. Each treatment was followed by the

washing with PBS for a couple of times. Then the cells were

mounted in the anti-fading medium Vectashield (Vector Laborator-

ies, Burlingame, CA, USA). For mPGES, the cells were examined

with a digital, high-resolution camera system (AxioCam, Carl Zeiss,

Obenkochen, Germany) mounted to a fluorescence microscope

(Axioscope2 plus, Carl Zeiss). For B1 and B2 receptors, series of

images were sequentially acquired to avoid signal cross-over, and

were examined with a confocal laser scanning microscope

(LSM510META, Carl Zeiss).

SYBR green-based real-time quantitative RT-PCR

Total RNAs were prepared from 2 · 106 rat microglial cells and a

rat brain with RNeasy� RNA purification kit (QIAGEN, Valencia,

CA, USA), according to the manufacturer’s protocol. First-strand

cDNA synthesized from 1 lg total RNA with random hexamer

primers was used as template for each reaction. SYBR Green-based

real-time quantitative RT-PCR was performed as described (Aoki

et al. 2002). 7700 Sequence Detection System (Applied Biosys-

tems, Foster City, CA, USA) was used for the signal detection and

the PCR was performed in 1· SYBR Green Master mix (Applied

Biosystems) and 50 nmol/L of each primer. For standardization and

quantification, rat b-actin was amplified simultaneously. Primer

sequences were designed with Primer Express� Software (Applied

Biosystems). The following primer pairs were employed: 5¢-CTGTGCCAAGTCAATCAAGC-3¢ (upper, 387–407) and 5¢-TCCCTTTCAGATCCCACTTCA-3¢ (lower, 487–467) for amplifi-

cation of rat EP1 receptor (GenBank accession No. NM_013100):

5¢- TTGCTCTTCTGTTCTCTGCCG-3¢ (upper, 538–558) and 5¢-CAGCTGAAGGTATGCGGTCC-3¢ (lower, 642–623) for amplifi-

cation of rat EP2 receptor (GenBank accession No. NM_031088):

5¢-ACTCGGTTGAAGCGCACAG-3¢ (upper, 111–129) and 5¢-CGACACAAGCAACATGGCC-3¢ (lower, 238–220) for amplifica-

tion of rat EP3 receptor (GenBank accession No. NM_012704): 5¢-GTGACCATTCCCGCAGTGA-3¢ (upper, 61–79) and 5¢-ACA-GCCAGCCCACATACCA-3¢ (lower, 188–170) for amplification

of rat EP4 receptor (GenBank accession No. NM_032076): 5¢-ATCGCTGACAGGATGCAGAAG-3¢ (upper, 925-945) 5¢- AGA-

GCCACCAATCCACACAGA-3¢ (lower, 1032–1012) for amplifi-

cation of rat b-actin. PCR conditions were: 95�C for 10 min,

followed by 40 cycles at 95�C for 15 s and 60�C for 1 min. The

threshold cycle of each gene was determined as the PCR cycle at

which an increase in fluorescence was observed above the baseline

signal in an amplification plot (Wada et al. 2000). The ‘normalized

expression level of target’ (dCt) was calculated as the difference in

threshold cycles for target and reference (b-actin). Subtraction of

dCt for microglial cells from dCt for the rat brain provided the ddCt

value. The formula, 2-ddCt, was used to calculate relative expression

levels for microglial cells compared with the rat brain. To reduce

possible error, RT-PCR reaction was performed three times and

averaged 2-ddCt values were obtained.

Electrophysiological measurements

Whole-cell slice patch recordings were made as reported previ-

ously (Brockhaus et al. 1993, 1996). The forebrain hemispheres of

young NMRI mice (aged postnatal day 6–9) were cut into 150 lmthin coronal slices using a vibratome (LEICA VT1000S, Leica,

Nussloch, Germany). Slices were stored in 5% CO2 and 95% O2-

gassed solution at 20–25�C prior to use. For the patch-clamp

experiments, a slice was placed into a recording chamber mounted

on the stage of a Zeiss upright microscope; the slice was held in

place with a grid of nylon threads framed by a U-shaped platinum

wire. The chamber was continuously perfused with external

solution (in mmol/L: NaCl, 150; KCl, 5.4; CaCl2, 2; MgCl2, 1;

N-2-hydroxyethylpiperazine-N¢-2-ethansulphonic acid (HEPES), 5;

and glucose, 10, pH 7.4). Substances were introduced by changing

the perfusate. Cells in the corpus callosum near to or on the

surface of the slice were viewed with 60· water immersion bright-

field optics. While approaching a cell with the patch pipette,

positive pressure was applied to the pipette, thereby blowing aside

any material between the slice surface and the cell. The

composition of patch-pipette was (in mmol/L): KCl, 130; MgCl2,

2; CaCl2, 0.5; ethyleneglycol-bis-N, N, N’, N’-tetraacetic acid, 4;

HEPES, 11, Mg2ATP3, 2, pH 7.3.

Membrane currents were measured with the patch-clamp tech-

nique in whole-cell recording configuration (Hamill et al. 1981).Current signals were amplified with conventional electronics (EPC-

9 amplifier, HEKA electronics, Lambrecht/Pfalz, Germany), and

filtered at 0.1 kHz during recording. Measured data was filtered at

12 kHz by an interface connected to an AT-compatible computer

system, which also served as stimulus generator.

Assessment of neuronal death

To induce inflammatory toxicity, LPS (10 lg/mL) or TNF-a (1 ng/

mL) was applied for 48 h to induce neuronal toxicity in neuron–

microglia cultures or neuron. BK at concentrations of 100 and

300 nmol/L were added to cells for 30 min before treatment of

neuron–microglia cultures with LPS. Cells were fixed and stained

with MAP2 antibodies as described above. The average number of

MAP2-positive cells/mm2 was calculated by counting cells at a 200-

fold magnification in four visual fields in each well, using a

fluorescence microscope (Axioscope2 plus; Carl Zeiss) equipped

with a digital camera system (AxioCam; Carl Zeiss).

Statistical analysis

Data are presented as mean ± SEM of 3–8 experiments. Statistical

significance was determined by Student’s t-test or ANOVA using

ORIGIN program (Microcal, Northampton, MA, USA).

Drugs and reagents

Papain and DNase were purchased from Worthington Biochemical

(Freehold, NJ, USA). DMEM was obtained from Nissui (Tokyo,

Japan). FCS was from Hyclone Laboratories Inc. (Logan, UT,

Attenuation of cytokine release from microglia by bradykinin 399

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 397–410

USA). Isolectin-B4, LPS, dibutyryl cyclic AMP (dbcAMP), cholera

toxin, and 6-isoproxy-9-osoxanthene-2-carboxylic acid (AH6809)

were purchased from Sigma. Bradykinin, and Des-Arg9-Bradykinin

(bradykinin B1 receptor agonist) were from PEPTIDE Institute

Inc. (Osaka, Japan). The cyclooxygenase-2 (COX-2) inhibitor,

N-(2-Cyclohexyloxy-4-nitrophenyl)-Methanesulfonamide (NS398),

was from Biomol Research Labs (Plymouth Meeting, PA, USA).

The membrane-permeable cAMP antagonist adenosine 3¢,5¢-cyclicmonophosphorothioate (RpcAMP) was from Calbiochem (San

Diego, CA, USA). Specific EP2 and EP4 agonists (ONO-AE1-

259-01 and ONO-AE1-329) were generous gifts from Ono

Pharmaceutical Inc. (Osaka, Japan).

Results

Bradykinin inhibits lipopolysaccharide-induced TNF-arelease in cultured microglia

Activation of microglia in pathologic conditions stimulatesthe release of TNF-a (Brosnan et al. 1988; Fillit et al. 1991).When microglial cells were treated with LPS (100 ng/mL)for 24 h, we could measure TNF-a levels in the supernatantranging between 1003 and 3241 pg/mL, while in untreatedcontrols levels were very low (9 pg/mL). When BK(100 nmol/L to 1 lmol/L) was co-applied with LPS, TNF-a release was reduced in a dose-dependent manner (Fig. 1a).The relative amount of LPS-induced TNF-a releasedecreased to 60.4 ± 9.7% (n = 3) and 48.0 ± 11.1%(n = 3) by addition of 300 nmol/L and 1 lmol/L BK,respectively. The inhibition of LPS-induced TNF-a releasewas also observed after addition of a bradykinin B1 receptoragonist (Des-Arg9-Bradykinin: 1 lmol/L) leading to areduction of TNF-a release to 53.5% ± 7.9% (n = 3).Application of BK alone did not significantly increaseTNF-a levels in the supernatant even at concentrations up to1 lmol/L (15–48 pg/mL).

Inhibition of LPS-induced TNF-a release by BK was notbecause of a decrease in the cell number, because the cellviability did not change by treatment of cells with both LPSand BK or B1 agonist (Fig. 1b).

Inhibition of LPS-induced TNF-a release by bradykinin

was mimicked by an increase in intracellular cAMP and

stimulation of prostanoid receptors

We investigated whether an experimentally induced increasein cAMP would mimic the effect of BK on the LPS-inducedTNF-a release. We co-applied the membrane-permeablecAMP analogue, dbcAMP in concert with LPS. Whenmicroglial cells were treated with 100 ng/mL LPS, therelease of TNF-a was increased from 30.5 ± 16.4 to373.9 ± 18.9 pg/mL. Co-treatment of the cells with LPSand dbcAMP (1 mmol/L) decreased the LPS-induced TNF-arelease to 55.7 ± 6.3 pg/mL (Fig. 2a), thus leading to areduction of 85%. Co-application of cholera toxin, which hasbeen reported to stimulate cAMP accumulation and PGE2

synthesis in murine macrophages (Burch et al. 1988), alsodose-dependently inhibited the LPS-induced TNF-a release.TNF-a release induced by 100 ng/mL LPS was reduced from980.5 ± 15.2 (n = 3) to 280 ± 19.2 (n = 3) and 194 ±39.5 pg/mL (n = 3) in the presence of 100 ng/mL and1 lg/mL cholera toxin, respectively (Fig. 2b). Conversely,the inhibition of LPS-induced TNF-a release by BK wasprevented by 50 lmol/L RpcAMP, a membrane-permeablecAMP antagonist (Fig. 2c). TNF-a release induced by100 ng/mL LPS was also inhibited by specific agonists forGs-coupling prostanoid receptors, EP-2 and EP-4 (Fig. 2d),suggesting the activation of microglia by prostaglandins. Theamount of TNF-a release was reduced from 2910 ± 12.6 to263 ± 34 and to 2019 ± 73 pg/mL (n = 3) by EP-2 agon-ist (ONO-AE1-259-01, 1 lmol/L) and EP-4 agonist

(a)

(b)

Fig. 1 Inhibition of lipopolysaccharide (LPS)-induced TNF-a release

by bradykinin (BK). (a) The relative inhibition rates of LPS-induced

TNF-a release by addition of 100, 300, and 1000 nmol/L BK or bra-

dykinin B1 receptor agonist (B1 ago). *p < 0.05, **p < 0.01 (ANOVA). (b)

The number of live cells was not changed by the treatment of LPS with

or without BK or B1 agonist. Drugs were added for 4 h.

400 M. Noda et al.


(ONO-AE1-329, 10 lmol/L), respectively. The involve-ment of prostaglandins and their receptors were confirmedby the facts that both COX-2 inhibitor (NS398, 10 lmol/L) and PGE2 antagonist (AH6809, 10 lmol/L) (Capehartand Biddulph 1991) antagonized the effect of BK onLPS-induced TNF-a release (Fig. 2c). The non-specificCOX inhibitor indomethacin (10 lmol/L) had an effectsimilar to, but no greater than, that of NS398 (data notshown).

Bradykinin induced expression of microsomal PGE

synthase and the release of PGE2

Prostaglandins are produced by activated microglia whichexpress the mPGES (Ajmone-Cat et al. 2003; Ikeda-Matsuoet al. 2005), a terminal enzyme for PGE2 biosynthesis. LPSand BK have been reported to trigger the release of PGE2

(Minghetti and Levi 1995; Jenkins et al. 2003). BK wasreported to stimulate phospholipase A2 to generate arachi-donic acid (Welsh et al. 1988; Yanaga et al. 1991) and othercytokines (Rang et al. 1991), inducing long-term effects. Toinvestigate whether BK induces the production of prosta-glandin E2 (PGE2) in cultured microglia, we studied the levelof mPGES expression and the release of PGE2.

Immunostaining of microglia using a specific antibodyagainst mPGES showed strong expression of mPGES afterapplication of 300 nmol/L BK and 100 ng/mL LPS for 24 h(not shown) or 42 h (Fig. 3a). The strong staining of mPGESwas not observed when the cells were incubated togetherwith 10 lmol/L NS398, a COX-2 inhibitor. As prostaglan-dins are produced following the sequential oxidation ofarachidonic acid by COX and terminal PGE synthase, theresult suggested that activation of PGE synthase needs theupstream formation of prostaglandin H2 induced by COX.

The released PGE2 was measured using the supernatant ofmicroglial cultures after application of BK (100 and300 nmol/L) for 24 h. Application of 1 lg/mL LPS for thesame period served as a reference. The relative amount ofPGE2 compared with that of untreated microglia (control)increased to 147.7% ± 16.3% (n = 4) by 100 nmol/L BK, to171.5% ± 28.3% (n = 4) by 300 nmol/L BK, and to175.5% ± 1.2% by 1 lg/mL LPS (n = 3), respectively(Fig. 3b). The release of PGE2 was increased further byco-application of LPS and BK. When the microglial cellswere treated with 100 ng/mL LPS for 24 h, the release ofPGE2 increased from 47.3 ± 13.4 to 124 ± 22.5 pg/mL(n = 3). After co-treatment of microglia with LPS and BK

(a)

(c)

(b)

(d)

Fig. 2 Inhibition of lipopolysaccharide (LPS)-induced TNF-a release

by raising intracellular cAMP and via formation of prostaglandin and

prostanoid receptors. (a) Co-treatment of microglial cells with LPS and

dibutyryl cyclic AMP (dbcAMP), a membrane-permeable cAMP ana-

logue, attenuated the LPS-induced TNF-a release. (b) Co-treatment of

microglial cells with LPS and cholera toxin, which accumulates intra-

cellular cAMP, also attenuated the LPS-induced TNF-a release.

***p < 0.005 (Student’s t-test). (c) BK-induced attenuation of LPS-

induced TNF-a release was cancelled either by membrane-permeable

cAMP-inhibitor (RpcAMP, 50 lmol/L), COX-2 inhibitor (NS398,

10 lmpl/L), or prostanoid receptor antagonist (AH6809, 10 lmol/L).

Each experiment was performed individually, therefore each block of

TNF-a release was compared directly with controls and expressed as

% of control. *p < 0.05 (ANOVA). (d) Specific agonists for Gs-coupling

prostanoid receptors, EP2 (ONO-AE1-259-01; 1 lmol/L) and EP4

(ONO-AE1-329; 10 lmol/L), significantly inhibited the LPS-induced

TNF-a release. ***p < 0.005 (Student’s t-test).



(1 lmol/L), the release of PGE2 was further increased to174 ± 25.3 pg/mL. Des-Arg9-Bradykinin, a B1 agonist,mimicked the dose-dependent effect of BK on LPS-inducedPGE2 release; the amount of PGE2 release were 100.1 ±29.1 pg/mL (n = 3), 174.0 ± 31.7 pg/mL (n = 3), and186.0 ± 18.2 pg/mL (n = 3) with 100, 300, and 1000 nmol/LB1 agonist, respectively, together with LPS (Fig. 3c). Theseresults indicate that BK and LPS stimulate PGE2 synthesis andthat B1 receptors may play a more important role than B2.

Up-regulation of prostanoid EP2/EP4 receptors and

bradykinin B1/B2 receptors in rat microglia

Microglia express the EP2/EP4 subtypes of prostanoidreceptors (Caggiano and Kraig 1999; Patrizio et al. 2000).We examined the possibility that the expression of thereceptors is affected by BK or LPS. Prostanoid receptorlevels were determined by quantitative RT-PCR analysis inthe microglial cells. EP1, EP2, and EP4 receptors could beidentified, although at lower levels than those found in

total brain extract. After treatment of the cells with300 nmol/L BK or 100 ng/mL LPS for 24 h, the expres-sion of EP2 receptor increased significantly; the relativeexpression level compared with that of rat brain increasedfrom 4.1 ± 0.1 to 5.9 ± 0.1 and 41.3 ± 6.7 (n = 3) withBK and LPS, respectively. EP4 receptor expressionincreased after the treatment with LPS (from 0.16 ± 0.01to 1.4 ± 0.2 (n = 3)), but not with BK (Fig. 4). EP1receptor was not significantly increased by BK (from0.28 ± 0.05 to 0.56 ± 0.05, n = 3) or by LPS (from0.28 ± 0.05 to 0.31 ± 0.02, n = 3).

Our previous quantitative RT-PCR analysis showed thatmicroglial B1 receptor mRNAs were up-regulated after thetreatment with 100nmol/L BK for 24 h (Noda et al. 2003). Inthe present study, we also showed that expression of B1 andB2 receptor proteins was increased by BK treatment asrevealed by immunolabeling with specific antibodies againstB1 and B2 receptors. In untreated microglia, expression ofboth B1 and B2 receptors was low. However, after the

Fig. 3 Increased expression of micro-

somal PGE synthase (mPGES) and PGE2

release by bradykinin and lipopolysaccha-

ride (LPS). (a) Microglial cells were double-

stained with isolectin-B4 (FITC-labeled:

green) and anti-mPGES antibody (Alexa

Fluor 594-labeled: red). Expression of

mPGES in microglia was increased by

treatment with 100 ng/mL LPS and

300 nmol/L bradykinin (BK) for 42 h, but not

in the presence of 10 lmol/L NS398. (b)

The amount of PGE2 released into the cul-

ture medium was measured by EIA. The

relative amounts of PGE2 released after

treatment for 24 h with 100, 300 nmol/L BK,

and 1 lg/mL LPS were compared with that

in non-treated microglia (control). (c) PGE2

release was further increased after appli-

cation of LPS and BK. LPS, together with

BK induced more release of PGE2 than that

induced by LPS alone. The B1 agonist

(Des-Arg9-Bradykinin) mimicked the

increasing effect of BK on LPS-induced

PGE2 release. *p < 0.05, **p < 0.01 (sig-

nificance from control, ANOVA). #p < 0.05

(significance from 100 nmol/L B1 agonist

together with LPS, ANOVA).

402 M. Noda et al.


treatment of the cells with 300 nmol/L BK for 24 h, strongimmunolabeling of both B1 and B2 receptors was observed.LPS (100 ng/mL) had a similar effect on BK in increasingBK receptor expression (Fig. 5).

Bradykinin inhibits LPS-induced IL-1b release but not

IL-6 and IL-10 release

We also analyzed whether BK affected the release of otherinflammatory cytokines, namely IL-1b, IL-6, and IL-10. BK

Fig. 4 Quantitative RT-PCR of prostanoid

receptors in rat microglia and brain. The

expression level of each prostanoid recep-

tor (EP1, EP2, EP3, and EP4) mRNA was

normalized to the level of each receptor in

rat brain. The relative values of each

receptor in untreated microglia (control), or

microglia treated for 24 h with bradykinin

(BK: 300 nmol/L) or lipopolysaccharide

(100 ng/mL) are shown. Difference from

controls: *p < 0.05, **p < 0.01 (ANOVA).

Fig. 5 Immunofluorescence of B1 and B2

receptors in rat microglia. Microglial cells

were double-stained with anti-B1 receptor

(FITC labeled: green) and anti-B2 receptor

antibody (Alexa Fluor 594-labeled: red) in

untreated (control), lipopolysaccharide

(100 ng/mL)- or BK (300 nmol/L)-treated

microglia (24 h drug treatment).



application alone did not induce IL-1b release. In contrast,LPS-induced IL-1b release was significantly reduced in thepresence of BK (Fig. 6a). BK (100 nmol/L) decreased theLPS-induced IL-1ß release by 48%. Higher concentrations ofBK were not more effective, indicating that 100 nmol/L wasalready a saturating concentration for inhibiting IL-1ßrelease. On the contrary, 300 nmol/L or 1 lmol/L BKaffected neither LPS-induced IL-6 (Fig. 6b) nor LPS-inducedIL-10 release (Fig. 6c).

Bradykinin receptors in microglia were also functional

in situ

To test for the presence of functional BK receptors in situ,we studied membrane current responses in microglial cellsin acute slices from mouse brain. We focused on asubpopulation of microglial cells which were found on thesurface of the slice in the mouse corpus callosum atpostnatal days 6–9. The microglial cells do not represent theresting form, but are characterized by an amoeboid shapewith a soma diameter in the range of 10–20 lm and migrateactively at this developmental stage (Brockhaus et al. 1993;

Fig. 7a). Previously, we have reported that BK induced anoutward current in cultured rat microglia presumablybecause of the activation of Ca2+-dependent K+ channels(Noda et al. 2003). We therefore tested for the presence ofBK-induced membrane currents in microglia in brain slice.The mean value of the resting membrane potential was)39 ± 15 mV (lowest: )20 mV, highest: )72 mV). At aholding potential of )20 mV, application of 1 lmol/L BKinduced an outward current with a mean amplitude of14.8 ± 0.01 pA in 15 BK-responsive cells out of 52 patchedcells (Fig. 7b). By clamping the membrane to a series ofpotentials ranging from )120 to 60 mV during the BKresponse (Figs 7b and c), we determined the current–voltage curve of the BK-induced current (Fig. 7d). Thesubtracted currents showed the reversal potential of)60 mV, suggesting the induction of a conductance thatwas substantially permeant to K+ (calculated EK and ECl

were about )83 and 10 mV, respectively). The conductanceincrease (measured between 20 and 40 mV) was0.32 ± 0.17 nS. The current–voltage relationship showed aslight outward rectification.

(a)

(b) (c)

Fig. 6 Bradykinin (BK) inhibited lipopoly-

saccharide (LPS)-induced IL-1b release,

but not LPS-induced IL-6 or IL-10 release.

(a) IL-1b release was measured after 6-h

incubation with or without LPS and BK. (b)

IL-6 release was measured after 6-h incu-

bation with or without LPS and BK. (c) IL-10

release was measured after 18-h incubation

with or without LPS and BK. The incubation

time was determined by the time-dependent

assay for each cytokine with or without BK

and was set at the time with maximum re-

lease. *p < 0.05, **p < 0.01, ***p < 0.005

(difference from controls; Student’s t-test).

404 M. Noda et al.


Protective effect of bradykinin on LPS-induced neuronal

damage

To investigate whether BK has a protective effect on neuronsby attenuating LPS-induced TNF-a release, we compared theeffects of LPS alone and LPS together with BK in neuron–microglia co-cultures. Although cortical neurons were quiteresistant to LPS compared with those in substantia nigra (Kimet al. 2000), the MAP2-positive cells decreased significantlyafter incubation of the microglia–cortical neuron co-culture in10 lg/mL LPS for 48 h (Fig. 8a). However, pre-treatmentwith BK protected MAP2-positive cells. The number of theMAP2-positive cells decreased from 317 ± 10 to 157 ± 12cells/mm2 by LPS, but showed a small decrease to 269 ± 9

and 296 ± 12 cells/mm2 after co-incubation with 100 and300 nmol/L BK, respectively (n = 8 each) (Fig. 8b). Theseresults suggest that BK protects neurons against the toxiceffect of LPS application, namely decrease in TNF-a andIL-1b release. As neurons also possess B2 receptors, toexclude a direct effect of BK on the neurons these experi-ments were repeated using primary neurons cultured in theabsence of microglia. In the CNS, microglia is the major LPS-responsive cell and specific activation of innate immunity byLPS is through a Toll-like receptor 4 (TLR4)-dependentpathway (Lehnardt et al. 2003), i.e., LPS-induced neurotox-icity is because of the activation of TLR4 in microglia andsubsequent release of cytotoxic substances. In fact, we alsoobserved the neuronal cell death induced by LPS in neuron–microglia co-culture but not without microglia (not shown).Therefore we simulated the neuronal death induced by LPSby adding high concentration of TNF-a, as LPS-inducedcytokine release was much greater in TNF-a (from�9 pg/mLin control condition to 1000–3000 pg/mL) (Figs 2b and d)than that in IL-1b (from 40 pg/mL in control condition to75 pg/mL) (Fig. 6). While TNF-a reduced the number ofneurons, co-application of BK did not change the neuronaldeath rate (Fig. 8c). Treatment with TNF-a for 24 hdecreased the number of neuronal cells from 272 ± 17 to165 ± 14 cells/mm2. Addition of 100 or 300 nmol/L BK30 min prior to TNF-a treatment did not significantly affectthis response; the number of MAP2-positive cells was186 ± 19 and 184 ± 14 cells/mm2, respectively (n = 4 each)(Fig. 8d). This result suggests that the neuroprotective effectof BK is not because of an effect on the neurons, but instead ismediated by the microglial cells.

Discussion

The present study demonstrates that BK may have aneuroprotective role in the CNS, mediated by glial cells.During inflammatory diseases, kinins (including BK) mightwell be produced in the brain, or access the brain from theperiphery, and thereby could exert inflammatory effects onneurons analogous to its effects in the periphery. However,when we focus on the effects of BK on glial cells – mainlymicroglia in the present study – then it appears that, on thecontrary, BK has an anti-inflammatory role.

Anti-inflammatory effects of bradykinin mediated by

microglia

Unexpectedly, BK inhibited LPS-induced TNF-a releasefrom microglia (Fig. 1), even although it did not affect restingTNF-a release. It also inhibited LPS-induced release of IL-1b(Fig. 6a). As the release of these cytokines is intimatelyassociated with elements of the acute phase immuneresponses, including fever, and are responsible for LPS-induced fever (Saigusa 1990; Luheshi et al. 1997) and withsome forms of neurodegeneration (Boka et al. 1994; Mogi

(a)

(b)

(d)

(c)

Fig. 7 Bradykinin-induced outward currents in mice microglia in situ.

(a) Phase contrast microscope image of microglia in a brain slice

patched with a pipette (·600). (b) Currents recorded in response to

100 ms depolarizing (0, 20, 40, and 60 mV) and hyperpolarizing ()40,

)60, )80, )100, and )120 mV) voltage commands from a holding

potential of )20 mV (inset below). This clamp protocol was repetitively

applied in 6-s intervals. Bradykinin (BK; 1 lmol/L) was applied as

indicated by the horizontal bar. An outward current was induced at the

holding potential and the currents in response to the voltage step

increased in amplitude. (c) Currents induced by a series of voltage

steps before application (solid circles) and during application (solid

diamonds) indicated in (b) are shown on a faster time scale. (d) Cur-

rent–voltage relationship for BK-induced currents. Current amplitudes

measured before application of BK (solid circles) were subtracted from

those during application of BK (solid diamonds) and plotted against

membrane voltage (solid squares).



et al. 1994; Mehlhorn et al. 2000; Nagatsu et al. 2000), thisimplies that the effect of BK on microglial cells mightpromote anti-inflammatory and neuroprotective effects.Indeed, we show (in Fig. 8) that BK does exert a neuropro-tective action, and that this depends on the presence ofmicroglial cells. These inhibitory effects seems contrary to theview that BK is a well-known pro-inflammatory mediator,although a protective role of BK has previously been reportedin cardiac and renal system (Pinto et al. 2000; Bascands et al.2003), and in the retina (Yasuyoshi et al. 2000). Therefore, itis likely that BK may work as an anti-inflammatory mediatorin the CNS by modulating the brain’s immune system.

Mechanism of the neuroprotective effects of BK

Experiments on other cells suggest that a likely mechanismfor the inhibition of LPS-induced secretion by BK might bethrough the production of PGE2 and subsequent increase inintracellular cAMP (Pyne et al. 1997; Caggiano and Kriag1999; Webb et al. 2003). Our results are consistent with thismechanism. Thus, BK inhibition of LPS-induced TNF-asecretion was replicated by cAMP or an EP2 agonist, andwas antagonized by a COX-2 inhibitor or a prostanoidreceptor antagonist (Fig. 2). It has previously been reportedthat microglia in vitro constitutively express COX-1 and LPSinduces COX-2 expression (Hoozemans et al. 2002). As the

effect of the COX-2 inhibitor (NS398) was almost equal tothat of COX-1/COX2 inhibitor indomethacin, it seems likelythat COX-2 plays the major role in the effect of BK.

Quantitative RT-PCR analysis showed that Gs-coupled EP2receptor was the main prostanoid receptor for PGE2 inmicroglia (Fig. 4). The EP2 receptor has been reported toexert a cAMP-dependent neuroprotective role in cerebralischemia (McCullough et al. 2004). BK receptors link to Gq/11

andGi/o, and do not activate adenylyl cyclase (Pal-Ghosh et al.2003) although Gs-mediated signaling was also reported in(Fang et al. 2005). Instead, in microglia, the BK receptorappears to modulate the production of PGE2/EP2 to increaseintracellular cAMP indirectly, rather than coupling to adenylylcyclase directly. This scenario is supported by several previousobservations. Thus, a dose-dependent production of cAMP byPGE2 and EP2/EP4-specific agonists has previously beenobserved in cultured rat microglia and trabecular meshworkcells (Caggiano and Kriag 1999; Webb et al. 2003), andincubation of the cells with PGE2 in combination with BKresulted in a three- to fivefold enhancement of PGE2-stimu-lated cAMP production (Webb et al. 2003). Further, elevationof intracellular cAMP has been reported to reduce both LPS-induced TNF-a and IL-1b production in rat cultured microglia(Caggiano and Kraig 1999) and to prevent LPS-inducedneuronal death (Kim et al. 2002). In our experiments, we

(a) (b)

(d)(c)

Fig. 8 Protective effect of bradykinin on

lipopolysaccharide (LPS)-induced neuronal

death mediated via microglia. (a) Neuron–

microglia co-cultured cells were treated with

10 lg/mL LPS for 48 h. Bradykinin (BK: 100

or 300 nmol/L) were applied 30 min before

LPS treatment. After 48 h, the cells were

immunostained with MAP2, a neuron-spe-

cific antibody. (b) The numbers of MAP2-

positive neurons in (a) were counted. (c, d)

Without microglia, BK did not rescue the

neuronal cell death induced by 1 ng/mL

TNF-a. Numerical values in (b), (d) are

expressed as number of MAP2-positive

cells in 1 mm2. *p < 0.05, **p < 0.01, and

***p < 0.005 (difference from controls;

ANOVA). #p < 0.05 and ##p < 0.01 (differ-

ence from LPS; ANOVA).

406 M. Noda et al.


confirmed that dbcAMP inhibited LPS-induced TFN-arelease and that the membrane-permeable cAMP antagonist,RpcAMP, prevented the inhibitory effect of BK on thisresponse (Fig. 2). The source of PGE2 would be alsoastrocytes in vivo, because the attenuation of LPS-induceTFN-a release by BK was much greater in mixed glial culture(not shown).

Additionally, we observed that both B1 and B2 receptorsare up-regulated by LPS and BK as determined immunocy-tochemically (Fig. 5). These effects are probably mediatedby nuclear factor-jB (NFjB) and PGE2, respectively. Thus,in other cells, up-regulation of B1 receptors by LPS andTNF-a mediated by the activation nuclear factor-jB (NFjB)has previously been reported (Sardi et al. 1999; Passos et al.2004), while up-regulation of B2 receptors was induced byBK-induced PGE2 production (Castano et al. 1998).

Taken together, a signaling schema explaining how BKmight attenuate the LPS-induced TNF-a release is shown inFig. 9. We propose that the principal effect of BK is toenhance both prostaglandin synthesis and prostanoid receptorexpression, thereby enhancing microglial cAMP production,which in turn inhibits LPS-induced TGF-a release; and thatthis may be amplified by upregulation of BK receptors. Wesurmise that the same mechanism might also apply to theinhibition of IL-1b release, although we have not investi-gated this in detail in the present experiments. This providesan amplified negative feed-back mechanism on TNF-a and

IL-1b production in microglia, thereby accounting for theneuroprotective action of BK shown in Fig. 8.

It seems likely that the principal BK receptor involved inthis anti-inflammatory effect is the B1 receptor, because: (i)B1 receptors are rarely expressed in non-traumatized tissuesbut highly expressed in activated immune cells, (ii) inhibitionof LPS-induced TNF-a release was also induced by B1

agonist (Fig. 1a) and was substantially cancelled by B1

antagonist (not shown), (iii) production of PGE2 was largely,not completely, dependent on B1 receptor activation(Fig. 3c), and (iv) BK-induced microglial migration (viaactivation of Ca2+-dependent K+ channels) was also becauseof the activation of B1 receptors but not B2 receptors (Ifukuet al. 2005). Thus, even although B1 receptors might beresponsible for nerve injury-induced neuropathic pain in theperiphery (Ferreira et al. 2005), in the CNS, microglial B1

receptors confer the opposite (anti-inflammatory and neuro-protective) action.

In conclusion, we suggest that – in addition to its pro-inflammatory action – BK can also work as an anti-inflammatory or neuroprotective mediator in the brain,through its effect on glial cells. The key signaling pathwayappears to be the production of prostaglandine E2 and theconsequential increase in intracellular cAMP in the micro-glia. As BK receptors are expressed in almost all kinds ofcells, it can evoke complex cascades in different cells withinthe central nervous system. However, if we can clarify the

Fig. 9 A proposed schema for the inhibitory effects of bradykinin on

lipopolysaccharide (LPS)-induced TNF-a and IL-1b. Bradykinin (BK)

activates B1 receptors (although B2 receptors were not excluded yet),

activates Gq/11, mobilizes intracellular Ca2+ (inducing a Ca2+-

dependent K+ current IK(Ca)), and releases PGE2. This induces a

negative-feedback inhibition of LPS-induced TNF-a and IL-1b release,

mediated by cyclic AMP, and a positive-feedback increase in B2

receptor expression (Castano et al. 1998). LPS-induced TNF-a re-

lease also up-regulates EP2/EP4 receptors and BK receptors (B1 + B2

receptors), probably through the activation of NF-jB. LPS itself may

exert a negative-feedback inhibition of its own TNF-a release via PGE2

and prostanoid receptors and cAMP. However, it was not proved be-

cause the amount of TNF-a release in the presence of cAMP antag-

onist was not greater than that induced by LPS alone.



inflammatory and anti-inflammatory cascades of BK, it mightbe possible to manipulate inflammation and inflammatorydiseases and could thereby assist in identifying new thera-peutic targets. Our data provide essential information forunderstanding the anti-inflammatory action of kinins withinthe CNS, which could contribute to the treatments againstinfectious diseases, inflammation, trauma, or neurodegener-ation accompanying inflammation.

Acknowledgements

We thank Prof. D. A. Brown (University College London, UK),

Prof. H. Higashida (Kanazawa University, Japan), and Prof. H.

Nakanishi (Kyushu University, Japan) for valuable suggestions.

This work was supported by Grants-in Aid for Scientific Research of

Japan Society for Promotion of Science, Research Grant in Priority

Area Research of the Ministry of Education, Culture, Sports,

Science and Technology, Japan, Kyushu University Foundation,

Grants-in-Aid for Scientific Research of the Ministry of Health,

Labour and Welfare, Japan and the Program for Promotion of

Fundamental Studies in Health Sciences of the National Institute of

Biomedical Innovation (NIBIO).

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