ifn-γ elevates airway hyper-responsiveness via up-regulation of neurokinin a/neurokinin-2 receptor...

IFN-c elevates airway hyper-responsiveness viaup-regulation of neurokinin A/neurokinin-2receptor signaling in a severe asthma model

Minoru Kobayashi�1, Shigeru Ashino�1, Yasuo Shiohama1,

Daiko Wakita1, Hidemitsu Kitamura1 and Takashi Nishimura1,2

1 Division of Immunoregulation, Section of Disease Control, Institute for Genetic Medicine,

Hokkaido University, Sapporo, Japan2 Division of ROYCE’ Health Bioscience, Institute for Genetic Medicine, Hokkaido University,

Sapporo, Japan

The adoptive transfer of OVA-specific Th1 cells into WT mice followed by OVA inhalation

induces a significant elevation of airway hyper-responsiveness (AHR) with neutrophilia

but not mucus hypersecretion. Here, we demonstrate that the airway inflammation model,

pathogenically characterized as severe asthma, was partly mimicked by i.n. administra-

tion of IFN-c. The administration of IFN-c instead of Th1 cells caused AHR elevation but

not neutrophilia, and remarkably induced neurokinin-2 receptor (NK2R) expression along

with neurokinin A (NKA) production in the lung. To evaluate whether NKA/NK2R was

involved in airway inflammation, we first investigated the role of NKA/NK2R-signaling in

airway smooth muscle cells (ASMCs) in vitro. NK2R mRNA expression was significantly

augmented in tracheal tube-derived ASMCs of WT mice but not STAT-1�/� mice after

stimulation with IFN-c. In addition, methacholine-mediated Ca21 influx into the ASMCs

was significantly reduced in the presence of NK2R antagonist. Moreover, the NK2R

antagonist strongly inhibited IFN-c-dependent AHR elevation in vivo. Thus, these results

demonstrated that IFN-c directly acts on ASMCs to elevate AHR via the NKA/NK2R-

signaling cascade. Our present findings suggested that NK2R-mediated neuro-immuno

crosstalk would be a promising target for developing novel drugs in Th1-cell-mediated

airway inflammation, including severe asthma.

Key words: Animal models . Asthma . INF-c . Neurokinin

Supporting Information available online

Introduction

Bronchial asthma, typically recognized as Th2-cell-mediated

airway inflammation, is pathogenically characterized by airway

hyper-responsiveness (AHR), eosinophilic airway inflammation,

mucus hyperproduction in airway epithelium, and elevated

serum levels of IgE [1, 2]. Numerous chemicals and medicines

against immunological cells have been researched and developed

for clinical use in asthma. However, patients suffering from

asthma occasionally develop severe neutrophilia in the lung and

show steroid resistance, which then results in ‘‘severe asthma’’

[3–9]. It has been reported that neutrophil infiltration in the lung

�These authors have contributed equally to this study.Correspondence: Prof. Takashi Nishimurae-mail: [email protected]

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

Eur. J. Immunol. 2012. 42: 393–402 DOI 10.1002/eji.201141845 Immunomodulation 393

was not observed in Th2-cell-associated airway inflammation

models such as immunization of OVA plus aluminium hydroxide

(Al(OH)3) models [10, 11]. Thus, the precise mechanisms of

severe asthma are less understood compared with those of Th2-

cell-mediated airway inflammation.

We previously established Th1- and Th2-mediated airway

inflammation models by the adoptive cell transfer of OVA-specific

Th1 or Th2 cells followed by OVA inhalation. In contrast to Th2

cells, Th1 cells induce strong AHR concomitantly with neutro-

philia in the lung but without mucus hypersecretion [12, 13].

Therefore, our proposed Th1-cell-mediated airway inflammation

model appeared to be suitable for characterizing the pathology of

severe asthma, since little has been done to elucidate how Th1

cells induce elevation of AHR.

Tachykinins such as substance P and neurokinin A (NKA) are

located in the excitatory non-adrenergic and non-cholinergic

(NANC) nerves of the mammalian respiratory tract [14]. Excita-

tion of these nerves results in the release of tachykinins, which

may be involved in the pathogenesis of airway allergy in humans

[14–16]. Because those factors are produced in the airway tissues

during inflammatory responses, tachykinin receptor antagonists

might become good targets for developing therapeutic drugs for

the treatment of allergic inflammation [14, 17]. However, the

precise role of NKA/neurokinin-2 receptor (NK2R) signaling has

not yet been elucidated though tachykinin NK1R was demon-

strated to be crucial for the induction of neutrophilia in the lung

and AHR elevation [18].

In the present work, we established a novel AHR induction

model by i.n. administration of IFN-g and investigated the critical

role of IFN-g in Th1-cell-mediated airway inflammation model.

We found here that (i) Th1-cell-induced AHR elevation was

mimicked by i.n. administration of IFN-g, (ii) IFN-g directly

induced NK2R expression and NKA production in the lung, and

(iii) IFN-g-induced elevation of AHR was significantly inhibited

by specific antagonism of NK2R in our model. Thus, we reveal a

role for IFN-g-induced NKA/NK2R signaling in AHR elevation

during Th1-cell-induced airway inflammation. From the present

results, we propose that NKA/NK2R-mediated neuro-immuno

crosstalk would be a promising target for developing new drugs

in Th1-cell-associated airway inflammation including severe

asthma.

Results

IFN-c is required for Th1-cell-dependent AHR elevation

To evaluate the precise mechanisms of severe airway inflamma-

tion, we established Th1-cell-mediated asthma model. Adoptive

transfer of OVA-specific Th1 cells into BALB/c mice followed by

OVA inhalation induced significant elevation of AHR (Fig. 1A).

The numbers of cells in BALF and histopathological analysis

revealed that the AHR elevation was induced with severe

neutrophilia and migration of inflammatory cells into the lung

but not eosinophilia as in Th2-type inflammation (Fig. 1B and C).

To elucidate the role of IFN-g, one of the typical cytokines produced

by Th1 cells, we examined pathogenesis of the Th1-cell-dependent

airway inflammation after treatment with neutralizing mAb against

IFN-g. As a result, we found that the elevation of AHR was

significantly suppressed by the administration of anti-IFN-g mAb

(Fig. 1A). However, the migration of inflammatory cells, including

neutrophils and lymphocytes, was not inhibited by anti-IFN-g mAb

(Fig. 1B and C). These results strongly suggest that IFN-g produced

by Th1 cells was involved in the induction of severe AHR but not the

migration of inflammatory cells into the lung.

C

OVA control Th1+ OVA+ αIFN-γ mAb

Th1 + OVA+ control IgG

25A*

20OVA control

Th1 + OVA + control IgG

15Th1 + OVA + αIFN-γ mAb

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BMethacholine (mg/ml)

(x105)18

Total cellMacrophage

12 Lymphocyte

NeutrophilEosinophil

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0

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mb

er o

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lls in

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LF

Th1 + OVA+control IgG

OVAcontrol

Th1 + OVA+αIFN-γ mAb

Figure 1. IFN-g is required for Th1-cell-mediated AHR elevation. OVA-specific Th1 cells (2� 107), generated from naı̈ve CD41 T cells derivedfrom DO11.10 mice, were i.v. injected into WT BALB/c mice. One dayafter the transfer, the mice were exposed to aerosolized OVA proteinsolution for 30 min for 3 consecutive days. (A) 24 h after the final OVAinhalation, AHR of basal values (�) or the response to nebulized PBS (0)and to two concentrations of b-methachloline chloride (Mch) (6 and12 mg/mL) were measured with whole-body plethysmography.(B) Inflammatory cells collected from the BALF were stained withdiaminobenzidine (DAB) solution and hematoxylin. Macrophages,lymphocytes, neutrophils, and eosinophils were differentiated accord-ing to the morphology and stained characteristics. (A, B) Data areshown as mean1SD of n 5 9 mice from three separate experiments.�po0.05 from control mice, ��po0.05 from control IgG-treated Th1-transferred mice, Student’s t-test. (C) Lung tissues were perfused with10% buffered formalin and stained with hematoxylin and eosin forobservation of inflammatory cell migration. Magnification 200� , scalebar represents 100 mm. Data shown are representative of n 5 9 micefrom three separate experiments. Control experiment was performedfor only inhalation of OVA protein without transfer of Th1 cells.

Eur. J. Immunol. 2012. 42: 393–402Minoru Kobayashi et al.394


I.n. administration of IFN-c mimics Th1-cell-mediatedAHR elevation

To determine the direct role of IFN-g in the elevation of AHR, we

administered i.n. IFN-g to WT BALB/c mice. The administration

of IFN-g elevated the AHR (Fig. 2A); moreover, we evaluated

AHR elevation with different doses of IFN-g administration (0.5,

1, 3, and 6 mg). As a result, the IFN-g stimulation elevated the

AHR in a dose-dependent manner and the elevation reached a

plateau at 3mg of IFN-g. AHR induction was significantly higher

after the administration of 3mg of IFN-g compared with the

administration of 1mg IFN-g (Supporting Information Fig. 1).

Therefore, the dose of 3mg/mouse was used in subsequent

experiments. Although the numbers of neutrophils in the lung

increased slightly after IFN-g administration, the total cell

numbers, including neutrophils, in the BALF were not enhanced

compared with those in the control group (Fig. 2B). In addition,

histopathological analysis indicate that severe neutrophilia in the

lung was not observed after the IFN-g treatment (Fig. 2C). These

data indicate that IFN-g remarkably enhanced the AHR elevation,

whereas it did not severely increase the numbers of migrating

inflammatory cells such as neutrophils in vivo.

IFN-c elevates AHR in a T-cell-independent andneutrophil-independent manner

We further examined whether T cells or neutrophils were directly

affected by IFN-g to induce AHR elevation in our airway

inflammation model. CD41 T cells, CD81 T cells, or neutrophils

were depleted by i.p. injection of mAbs (250mg) against CD4,

CD8, and Gr-1 antigen respectively. We confirmed that these

populations were almost completely removed in the airway from

the subjected mice (Supporting Information Fig. 2). As a result,

depletion of CD41 T cells, CD81 T cells, or neutrophils did not

reduce the IFN-g-induced AHR development (Fig. 3A–C). Though

IFN-g induced slight migration of inflammatory cells including

lymphocytes and neutrophils into the lung in our model

(Fig. 2B), the present findings suggested that the migration of

such inflammatory cells into the lung was not the critical process

for IFN-g-dependent AHR elevation.

I.n. administration of IFN-c up-regulates NK2R andNKA levels in the lung

Since it is accepted that neural system is closely related to airway

contraction, we examined the expression of neuro-signaling-

associated ligands and their corresponding receptors in the lung

tissues or airway tracheas during IFN-g-induced AHR elevation.

Although mRNA expression levels of acetylcholine receptor 3

(AchR3) and b2-adrenergic receptor (b2AR) in the lung were not

altered 1 day after the IFN-g administration, NK2R mRNA

expression was greatly elevated in the lung tissues of IFN-g-

administered mice compared with those of control mice (Fig. 4A).

In addition, the levels of NKA, a ligand of NK2R, were significantly

increased by the administration of IFN-g in the BALF (Fig. 4B).

These results suggested that NKA/NK2R-signaling cascade

appeared to be involved in the IFN-g-induced AHR elevation.

IFN-c enhances NK2R mRNA levels of airway smoothmuscle cells (ASMCs) in a STAT-1-dependent manner

To confirm the direct effects of IFN-g on the airway component

cells such as ASMCs, we prepared mouse ASMCs from airway

tracheal and examined their mRNA levels of AchR3, b2AR, and

NK2R, which are related to airway contraction or relaxation.

ASMCs were cultured with or without IFN-g for 1 day or 3 days.

NK2R mRNA expression was substantially increased in ASMCs

compared with non-treated ASMCs after IFN-g treatment.

C

PBS control IFN-γ 1μg IFN-γ 3μg

15

20APBS control

IFN

*

*IFN-γ 1μg

10 *

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Pen

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Methacholine (mg/ml)

15(x104)B

Total cellMacrophage

10

Lymphocyte

NeutrophilEosinophil

5

0

Nu

mb

er o

f ce

lls in

BA

LF

PBS control IFN-γ 3μgIFN-γ 1μg

IFN-γ 3μg

Figure 2. I.n. administration of IFN-g mimics Th1-cell-mediated AHRelevation. IFN-g (1 and 3mg) or PBS was i.n. administered into WT BALB/cmice once daily for 3 consecutive days. (A) 24 h after final IFN-gadministration, AHR of basal values (�) or the response to nebulizedPBS (0) and to three concentrations of Mch (3, 6, and 12 mg/mL) weremeasured with whole-body plethysmography. (B) Inflammatory cellscollected from BALF were stained with DAB solution and hematoxylin.Macrophages, lymphocytes, neutrophils, and eosinophils were differ-entiated according to the morphology and stained characteristics.(A, B) Data are shown as mean1SD of n 5 12 mice from four separateexperiments. �po0.05 from PBS-treated mice, Student’s t-test. (C) Lungtissues were perfused with 10%-buffered formalin and stained withhematoxylin and eosin for observation of inflammatory cell. Magnifi-cation 200� , scale bar represents 100mm. Data shown are representa-tive of n 5 12 mice from three separate experiments.

Eur. J. Immunol. 2012. 42: 393–402 Immunomodulation 395


However, mRNA levels of AchR3 and b2AR in ASMCs did not

increase in the same condition (Fig. 5A). Moreover, we confirmed

that the IFN-g-induced NK2R expression was remarkably abol-

ished in STAT1-deficient ASMCs (Fig. 5B). In addition

to IFN-g, we also investigated the effects of other cytokines,

associated with neutrophilic airway inflammations or Th1-cell

responses, on NK2R expression levels in ASMCs. IL-17,

TNF-a, or IL-2 was added into the culture of ASMCs for 3 days,

but no significant increase in NK2R mRNA expression was

induced by such cytokines (Fig. 5C). Taken together, it was

suggested that IFN-g, but neither IL-17, TNF-a nor IL-2 was

involved in NK2R expression in ASMCs through the IFN-g/STAT1

signaling pathway.

Blockade of NK2R-mediated signaling significantlysuppresses IFN-c-induced AHR elevation

Finally, we investigated whether NK2R-mediated signaling

cascade was involved in the subsequent airway responsiveness

in vitro and in vivo. Generally, ASMCs alter intracellular Ca21

levels in response to various stimulations and substantially cause

the contraction or relaxation of themselves. Therefore, we

monitored (Ca21)i in ASMCs after b-methacholine chloride

(Mch) stimulation. Pretreatment with NK2R selective antagonist

significantly reduced the peak of (Ca21)i in the IFN-g-treated

ASMCs (Fig. 6). We also found that selective NK2R antagonist

significantly attenuated the IFN-g-induced AHR elevation

(Fig. 7A). In the present study, the dose of NK2R antagonist

was based on the previous study [19]. Because of local

administration, which was different from the previous one

(0.12 mg i.v./kg, namely 2.4 mg i.v./20 g/mouse), we used lower

dose (0.6 mg i.n./20 g/mouse) in our model and confirmed that

this dose of selective NK2R antagonist did not cause adverse

effects including induction of AHR in normal mice (Fig. 7B). In

addition, the numbers of inflammatory cells in BALF collected

from IFN-g alone- or IFN-g plus NK2R antagonist-treated mice

were very little and there was no significant difference from those

in DMSO-treated control mice (data not shown). Thus, these

findings suggested that IFN-g-mediated NK2R expression in the

lung was surely involved in the subsequent AHR elevation.

20

15 *IFN-γ + αCD4 mAb

IFN-γ + control lgG

PBS control

*

10

Pen

h

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PBS controlIFN-γ + control IgG

B

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15 IFN-γ + αCD8 mAb * *

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20PBS control

C

15IFN-γ + control IgG

**

IFN-γ + αGr-1 mAb

*10

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h

*

5 *

0(-) 0 3 6 12


Figure 3. I.n. administration of IFN-g elevates AHR in a CD41 T-cell,CD81 T-cell, and neutrophil-independent manner. IFN-g (3 mg) or PBSwas i.n. administered into WT BALB/c mice once daily for consecutive3 days. 24 h after the final IFN-g administration, the mice treated with(A) anti-CD4 mAb, (B) anti-CD8a mAb and (C) anti-Gr-1 mAb weresubjected to analysis of AHR. Each mAb was i.p. injected (250 mg/mouse) at half a day before first IFN-g administration. AHR of basalvalues (�) or the response to nebulized PBS (0) and to threeconcentrations of Mch (3, 6, and 12 mg/mL) were measured withwhole-body plethysmography. Data are shown as mean7SD of n 5 9mice from three separate experiments. �po0.05 from PBS-treated mice,Student’s t-test.

5

AchR3NK2R *

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B

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80

100

60

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roki

nin

A le

vel (

pg

/ml)

0Control Day1 Day2 Day3

** *

Figure 4. NK2R mRNA and NKA levels into the lung are up-regulatedduring IFN-g-induced AHR. IFN-g (3 mg) or PBS was i.n. administeredinto WT BALB/c mice once daily for consecutive three days. 24 h afterthe final IFN-g administration, lung tissues or BALF were collected fromPBS-treated (control) and IFN-g-treated mice. (A) mRNA expression ofAchR3, b2AR, and NK2R into the lung tissues was evaluated by real-time PCR. (B) NKA levels in BALF were measured by ELISA. Data areshown as mean1SD of n 5 9 mice from three separate experiments.�po0.05 from PBS-treated mice, Student’s t-test.



Discussion

Previously, allergic model by adoptive cell transfer of antigen-

specific Th2 cells revealed that Th2 cells played a pivotal role in

airway hypersensitivity and eosinophilia at effector phase

[20, 21]. In addition, many investigators have reported that

most asthmatic patients have allergic symptoms such as

eosinophil infiltration into the lung, mucus hypersecretion from

airway epithelia, and elevated serum IgE levels, which were

closely related with Type 2 cytokines including IL-4, IL-5, IL-9,

and IL-13 produced by Th2 cells [22–26]. Based on these

findings, numerous anti-inflammatory agents as inhaled and

systemic corticosteroids have been developed for the clinical

usage for the therapy of asthma. Especially, such anti-inflamma-

tory agents are useful for the therapy of eosinophilic asthma by

attenuation of Type 2 immune responses [27–29].

However, it has been reported that some asthma patients

refractory to steroid treatment were classified as patients with

‘‘severe asthma’’. Several investigators indicated that neutrophils

are increased in the lungs of patients with severe asthma [3–9],

but detailed cellular and molecular mechanisms for this

phenomenon remain poorly understood.

Th1 cells, producing IFN-g, IL-2, and TNF-a, mediate protec-

tive immunity through the activation of macrophages, dendritic

cells, natural killer cells, and cytotoxic T lymphocytes, with the

subsequent production of various effector molecules. However, it

has been well known that the excess activation of Type 1

immunity also promotes pathogenic inflammatory responses in

liver injury and certain autoimmune diseases [30]. Previously, we

demonstrated that the adoptive cell transfer of antigen-specific

Th1 cells severely induced airway inflammation with neutrophilia

in the lung (Fig. 1). Furthermore, we have already confirmed that

this severe airway inflammation model is resistant to steroids

(data not shown). In the present study, we indicated that

blockade of IFN-g significantly reduced Th1-cell-induced

AHR without inhibiting infiltration of inflammatory

cells in the lung (Fig. 1). In addition, the elevation of AHR was

not altered by depletion of neutrophils in this model (data not

shown). These findings strongly suggested that factors from Th1

cells played critical roles in the AHR elevation. Previous works

indicated that the IFN-g production was closely related with the

pathogenesis of Th1- or Th17-mediated asthma [13, 31, 32]. In

consistent with those findings, we found here that i.n. adminis-

tration of IFN-g alone significantly induced AHR elevation (Fig.

2). In contrast to Th1-cell-induced AHR model, there was less

neutrophil infiltration into the lung during IFN-g-induced AHR

elevation. These results suggest that IFN-g but not neutrophilic

infiltration would be a key factor for Th1-cell-mediated AHR

elevation.

Our data support previous findings that IFN-g secreted by Th1

cells was the critical mediator for the induction of AHR [33].

However, the previous study using LPS stimulation required

transferred Ag-specific Th1 cells and IFN-g to induce the AHR.

IFN-γ 1day IFN-γ 3day

*

*β2AR

0

1

2

3

4

5

control

Rel

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AchR 3

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*NK2R

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7

A

B

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Figure 5. IFN-g directly up-regulates NK2R but not AchR3 and b2AR inASMCs in a STAT-1-dependent manner. ASMCs were prepared fromWT and STAT-1�/� mice. IFN-g was added into ASMC cultures from(A) WT BALB/c and (B) WT or STAT-1�/� mice on a C57BL/6 backgroundfor 1 or 3 days. (C) IFN-g, IL-17, TNF-a, or IL-2 was added into ASMCcultures derived from WT BALB/c for 3 days. After cytokine stimula-tion, the indicated mRNA expression levels were evaluated by real-time PCR. Data are shown as mean1SD of n 5 9 samples from threeseparate experiments. �po0.05 from PBS-treated (control) mice,Student’s t-test.

Methacholine [M]

Pea

k fl

uo

resc

ence

incr

ease

ΔF/F

o

NK2R antagonist

DMSO control

10-7 10-6 10-5

0.4

0.3

0.2

0.1

0.0*

*

**

Figure 6. NK2R-mediated signaling cascade augments cellularresponses of ASMCs in vitro. Cellular responses of ASMCs by Mchstimulation were evaluated by monitoring the intracellular Ca21

fluorescent signals for 48 h. IFN-g (10 ng/mL) and selective NK2Rantagonist (10 mM) were used for treatment. The cellular responses ofASMCs against Mch were indicated as DF/Fo after calculation from thedata at the peak fluorescence elevated from basal value (Fo). Data areshown as mean1SD of n 5 4 samples and are representative of threeseparate experiments with similar results. �po0.05 from DMSO-treatedASMCs, Student’s t-test.



There were several differences between the two models. In the

previous study, the injected numbers of Th1 cells were 5�106

cells/mouse, whereas 2�107 Th1 cells were transferred into the

recipient mice in our experiment. OVA peptide was i.n. injected in

the previous paper, whereas OVA protein was administered by

inhalation in our model. In addition, 1.5 mg of IFN-g were intra-

tracheally administered in the previous study, whereas 3 mg of

IFN-g was i.n. injected for 3 days in our model. Therefore, we

speculate that the higher levels of IFN-g in our airway inflam-

mation model might be the major cause for inducing higher AHR

responses by cell transfer of Th1 cells with OVA but without

further administration of LPS though commercially available OVA

contained negligible levels of endotoxin. Previous studies

demonstrated that IFN-g was an important factor to induce

AHR [13, 31–37], however, the precise mechanism of AHR

induction by IFN-g remained unclear. As shown in Fig. 2, the

administration of higher dose of endotoxin-free (less than 1 EU/mg)

recombinant IFN-g alone, induced high levels of AHR responses.

This result again indicated that IFN-g induced the elevation of

AHR independently on the existence of endotoxin (LPS). Thus,

we expected that the present IFN-g-induced AHR model could be

a useful tool to demonstrate an important pathway for IFN-g-

dependent AHR elevation.

Several papers described that NKA, one of tachykinins, was

involved in the pathogenesis of asthma via interaction with

NK2R [14–16]. In the present work, we found that NKA

significantly increased in BALF after i.n. administration of IFN-gand that mRNA expression of NK2R, receptor of NKA, was

remarkably up-regulated in the lung (Fig. 4). Moreover, we

confirmed that NK2R was up-regulated when ASMCs were

stimulated with IFN-g in a STAT-1-dependent manner but not by

other cytokines such as IL-17, TNF-a, and IL-2. NK2R mRNA

expression levels of ASMCs were enhanced by IFN-g stimulation

for 3 days compared with 1 day stimulation (Fig. 5). Moreover,

previous report described mRNA levels of NK2R were increased

4-fold in the lung samples from asthmatic as compared with

nonsmoking control subjects [38]. From this evidence, we spec-

ulate that the increase in NK2R mRNA expression would be

clinically relevant. The crucial role of NKA/NK2R is demonstrated

both in vitro and in vivo experimental systems (Figs. 6 and 7). In

the present study, we confirmed that NK2R agonist comes from

FCS composed of medium in the present culture system. Previous

papers have reported that combined administration with multiple

Gq/Gi-coupled receptor agonists has a synergistic effect on

contraction of airway smooth muscles [39, 40]. From this

information, we speculated that ASMCs were constantly stimu-

lated through NK2R at steady state in this system and that the

selective antagonist significantly reduced the peak (Ca21)i in

ASMCs after Mch stimulation (Fig. 6). Here, we revealed that the

blockade of the IFN-g-induced NKA/NK2R-signaling cascade

significantly suppressed the cellular responses of ASMCs by Mch

stimulation in vitro. In addition, severe AHR elevation caused by

i.n. administration of IFN-g was markedly inhibited by treatment

of NK2R antagonist in vivo (Fig. 7). Thus, the present findings

reveal that up-regulation of the NKA-NK2R signaling pathway is

involved in the IFN-g-mediated AHR elevation.

To address the physiological meaning, we further evaluated

NK2R and NKA in the Th1-cell-transfer models with or without a

steroid drug, Fluticasone propionate (FP). As a result, NK2R and

NKA levels enhanced in the lung tissue and BALF of our model

with Th1 cells respectively (Supporting Information Fig. 3).

Since NKA is generally secreted in the excitatory NANC nerves

[14], we think NANC nerve cells are one of the NKA sources in

the IFN-g-challenged mice or the mice treated with Th1-cell

transfer and OVA challenge. We are now investigating whether

NKA was secreted from immune cells after IFN-g stimulation. We

also confirmed that the up-regulation of NK2R and NKA level was

resistant to the steroid administration in the present condition

(Supporting Information Fig. 3). Taken together, the NKA–NK2R-

signaling cascade might be related with the AHR induction in the

Th1-cell-mediated steroid-resistant asthma model.

Previous reports demonstrated that neurokinin receptor

antagonists inhibited NKA-induced bronchocontraction in asthmatic

patients [41–44]. On the other hand, one study reported that a

neurokinin receptor antagonist enhanced the allergen-induced early

and late airway responses [41]. The diverse effects of neurokinin

receptor antagonists might be related with the different designs of

the therapy or background of asthma patients, which have different

0

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DMSO controlIFN-γ +DMSO controlIFN-γ +NK2R antagonist

Pen

h *

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NK2R antagonist

DMSO control

Untreated

*

**

*

A

B

Figure 7. Blockade of NK2R-mediated signaling significantly suppressesIFN-g-induced AHR elevation in vivo. Selective NK2R antagonist (0.6mg)was i.n. injected into (A) mice concurrently treated with IFN-g (3mg) or(B) non-treated WT BALB/c mice, for three consecutive days. 24 h afterthe final NK2R antagonist administration, the mice were subjected tothe analysis of AHR. AHR of basal values (�) or the response to nebulizedPBS (0) and to three concentrations of Mch (3, 6, and 12mg/mL) weremeasured with whole-body plethysmography. Data are shown as mean1

SD of n 5 9 mice from three separate experiments. �po0.05 from IFN-g andDMSO-treated mice, Student’s t-test.



immune statuses. Because asthma is a complex disease, affected by

different endogenous and exogenous factors [45], we believe that it

must be very important for asthma treatment to consider such

different interventions, doses, route of administration, schedules,

and timing of samplings in addition to the patient’s characteristics.

It has been proposed that a crosstalk among neuro-immuno-

endocrine super-systems plays a pivotal role for maintaining

homeostasis of our body including immune balance [46–48]. In

the previous work, we initially demonstrated that Th2 cells were

involved in the production of ‘‘immunosteroid’’, which could

control immune balance [49]. Th2 cells were also demonstrated

to produce NT-3, one of neurotropins, which appeared to regu-

late Th1/Th2 immune balance [50].

Here, we clearly demonstrated that NKA, a neuropeptide,

which is located in the sensory nerves of the mammalian

respiratory tract, and produced after excitation of the nerves as

well as substance P, could be involved in the elevation of AHR

induced by severe asthma. It might be possible to consider that

severe symptoms of patients suffering from severe asthma are due

to antigen-induced over-activation of Th1-immunity via IFN-g/

NKA/NK2R signaling, which causes the increase in Ca21-depen-

dent contraction of airway tracheal smooth muscle cells. Thus,

our established IFN-g-induced airway inflammation model will

become a useful tool to elucidate the pathogenesis of severe

asthma and to develop therapeutic drugs for IFN-g-induced

inflammatory diseases via NKA/NK2R signaling.

Materials and methods

Mice

Wild-type BALB/c mice were purchased from Charles River Breeding

Laboratories (Kanagawa, Japan). OVA323–339-specific I-Ad-restricted

TCR-Tg mice (DO11.10) were kindly donated by Dr. K. Murphy

(Washington University School of Medicine, St. Louis, MO, USA).

IFN-g�/� mice on a BALB/c background were kindly provided by

Dr. Y. Iwakura (University of Tokyo, Tokyo, Japan). All mice were

maintained in specific pathogen-free conditions according to the

guidelines of our institute’s animal department.

Reagents

Recombinant IL-2 was kindly donated by Dr. T. Sawada

(Shionogi Pharmaceutical Institute, Osaka, Japan). Recombinant

IFN-g and IL-12 was purchased from Wako Pure Chemical

Industries (Osaka, Japan). PE-conjugated (PE-) anti-IL-4 mAb

(11B11), FITC-conjugated anti-IFN-g mAb (XMG1.2), PE-Cy5-

conjugated anti-CD4 mAb (GK1.5) were purchased from BD

PharMingen (San Diego, CA, USA). Anti-alpha actin mAb (1A4)

was purchased from R&D systems (Minneapolis, MN, USA). Anti-

OVA323–339-specific TCR mAb was purified from ascites fluid of

mice i.p. inoculated with KJ1.26 hybridoma cells in our

laboratory and conjugated with FITC for some experiments.

Neutralizing mAbs against IFN-g (R4-6A2) and IL-4 (11B11)

were purified from ascites fluid of mice in our lab, using

hybridoma donated from Dr. G. Trinchieri (Wistar Institute of

Anatomy and Biology, Philadelphia, PA, USA) and purchased

from ATCC (Manassas, VA, USA), respectively. OVA323–339

peptide was kindly supplied by Dr. H. Tashiro (Fujiya, Hadano,

Japan). OVA protein and Mch were purchased from Sigma-

Aldrich, Japan (Tokyo, Japan). ISOGEN RNA extraction reagent

was purchased from Nippon-gene (Tokyo, Japan). Superscript III

RT and oligo (dT)12–18 primer was purchased from Invitrogen

(Carlsbad, CA, USA). NK2R selective antagonist (GR 159897)

was purchased from TOCRIS Bioscience (Bristol, UK). Fluo-4 AM

was purchased from DOJINDO (Kumamoto, Japan).

In vitro generation of OVA-specific Th1 cells

CD45RB1 naı̈ve CD41 T cells were isolated from DO11.10 TCR-Tg

mouse spleen cells using cell sorter system (FACS Aria; BD

Biosciences, San Jose, CA, USA). Isolated naı̈ve Th cells

were cultured with IL-2 (100 U/mL), IL-12 (20 U/mL), IFN-g(1 ng/mL) and anti-IL-4 mAb (10 mg/mL) in the presence of

mitomycin C-treated spleen cells and 5 mg/mL OVA peptide

(OVA323–339). The CD41 T cells were re-stimulated with

OVA323–339 peptide under the same conditions at 48 h. For

evaluation of the Th1 differentiation, these cells were

analyzed by intracellular staining with mAbs against IFN-g,

IL-4, IL-2, and TNF-a at day 8 by the same method as previous

reports [12, 20, 21]. After washing out the factors at 8–10 days,

the generated Th1 cells were transferred i.v. into syngeneic WT

BALB/c mice.

Th1 adoptive cell-transfer model

A model of Th1-cell-mediated airway inflammation was estab-

lished by the adoptive transfer of Th1 cells as described

previously [12]. Briefly, OVA-specific Th1 cells (2� 107 cells in

0.2 mL PBS) were injected into the tail vein of normal recipient

BALB/c mice. One day after the transfer, mice were daily exposed

with aerosolized OVA protein (2% w/v in PBS, endotoxin:

3.4 EU/mg), which was generated by a nebulizer (omron NE-U07

nebulizer; Omron) driven at 1.0 mL/min atomization of OVA

protein solution, for 30 min during consecutive 3 days. At 24 h

after the third OVA exposure, pulmonary function was tested and

the lung histological examinations were carried out for the mice.

In some experiments, anti-IFN-g mAb (100mg/mouse) or control

rat IgG were i.n. injected into the mice.

IFN-c administration model

IFN-g (3 mg/mouse, endotoxin: less than 1 EU/mg) was i.n.

injected into WT BALB/c mice once daily for 1 day or consecutive



3 days. At 24 h after the IFN-g administration, pulmonary function

was evaluated and the lung histological examinations were carried

out for the mice. Treatment of selective NK2R antagonist (0.6mg

i.n./mouse) or control DMSO was concurrently performed for the

IFN-g i.n. administration to the model mice.

Measurement of AHR

AHR was measured by Mch-induced airflow obstruction as

reported previously [21]. Briefly, the subjected mice were placed

into whole-body plethysmographs (Buxco Electronics, Troy, NY,

USA) interfaced with computers using differential pressure trans-

ducers. Measurements were performed for respiratory rate, tidal

volume, and enhanced pause. Airway resistance was expressed as

Penh 5 [(Te/0.3Tr)�1]� (2Pef/3Pif), where Penh 5 enhanced

pause, Te 5 expiratory time (s), Tr 5 relaxation time (s), Pef 5 peak

expiratory flow (mL) and Pif 5 peak inspiratory flow (mL/s).

Increasing doses of Mch were administered by nebulization (for

3 min), and Penh were calculated over the subsequent 3 min.

Bronchoalveolar lavage and histological examination

After measuring AHR, lungs and tracheas of the subjected mice

were gently lavaged three times with 1 mL PBS containing 0.1%

BSA (Sigma). Cytospins of the bronchoalveolar lavage cells were

prepared with Shandon Cytospin 3 (Thermo Electron), and

stained with DAB/Tris-HCl (pH 7.25) solution and hematoxylin

to evaluate the morphology differentials based on the staining

characteristics. For evaluation of mucus hyperproduction, the

lung tissues were perfused with 10%-buffered formalin. The

sections were stained with hematoxylin and eosin, and periodic

acid-Schiff (PAS), and observed using a microscope (BX50,

OLYMPUS OPTICAL, Tokyo, Japan).

Generation of ASMCs

ASMCs were cultured from explants of excised tracheas using a

modification of previously described methods [30]. The entire

trachea between the larynx and main stem bronchi was removed

from the subjected mice. After additional surrounding

tissues were removed, the tracheal segment was split long-

itudinally, washed twice with PBS, and dissected into 2–3 mm

squares. The segments were then cultured with DMEM (sigma)

supplemented with 20% FCS (Nichirei Biosciences, Tokyo,

Japan) and 25 mM HEPES buffer, 0.05 mM 2-ME, penicillin and

streptomycin in 35-mm cell-culture dish. After 3 days, the

concentration of FCS was reduced to 10%. The cultures were

finally scaled up to twice or three times. The cultured cells were

characterized as AMSCs by intracellular staining analysis with

anti-smooth muscle a-actin mAb (1A4). For some experiments,

ASMCs were cultured in the presence of IFN-g (10 ng/mL) for 1

day or 3 days.

Real-time PCR

Total RNA was extracted from the lung tissues, trachea, and

ASMCs by ISOGEN RNA extraction kit (Nippongene) according to

the manufacturer’s instructions. cDNA was prepared from the

total RNA with reverse transcription with Superscript III RT

(invitrogen) and oligo (dT)12–18 primer and dNTP mixture

(Promega). The prepared cDNA was specifically amplified by

thermal cycler (LightCycler, Roche, Indianapolis, IN, USA) using

the corresponding primer pairs and probes for AchR3, b2AR,

NK2R, and b-actin. The primer sequences used were follows:

AchR3; (sense) 50-AGGACTGGAGTGGGACAGC-30, (antisense)

50-GATGCCATTGCTGGTCATATC-30, (probe) 50-CCTGGACT-30;

b2AR, (sense) 50-TGCTATCACATCGCCCTTC-30, (antisense)

50-ACCACTCGGGCCTTATTCTT-30, (probe) 50-GCCTGCTG-30;

NK2R, (sense) 50-AATGACAACGGAGGCAAGAT-30, (antisense)

50-AAGCTGCAGGAATCACCACT-30, (probe) 50-CTTCCTGC-30;

b-actin, (sense) 50-AAGGCCAACCGTGAAAAGAT-30, (antisense)

50-GTGGTACGACCAGAGGCATAC-30, (probe) 50-GGACAGCA-30.

Sample signals were normalized to the housekeeping gene

b-actin according to the DDCt method: DCt 5DCtsample�DCtreference. Percentages against the WT control sample were

then calculated for each sample.

Measurement of (Ca21Þi

Intracellular Ca21 influx, (Ca21)i was measured by fluorescence

intensity reported previously [51]. Briefly, ASMCs (1�104 cells)

were seeded in 96-well black plates (thermo) and cultured at 371C

for 2 days. The culture media was exchanged and IFN-g (10 ng/mL)

and GR-159897 (10mM) were then added into the fresh ones. Two

days later, ASMCs were washed twice with modified HBSS (pH 7.4)

containing 2.5 mM probenecid and incubated in the modified HBSS

(pH 7.4) containing 4mM fluo-4 AM, 0.0625% Pluronic F-127, and

2.5 mM probenecid at 371C for 30 min. After washing with the

modified HBSS (pH 7.4) containing 2.5 mM probenecid, the cells

were kept for an additional 30 min at room temperature to allow

complete deesterification of the intracellular acetoxy methyl esters.

After washing, the cells were further incubated at 371C for 10 min.

The fluorescence was then continuously recorded at every 5 s by

using a microplate reader with excitation filter of 485/20 nm and

emission filter of 528/25 nm (Synergy 4; BioTek Instruments,

Winooski, VT). Baseline was acquired for 1.5 min before the

addition of Mch in the modified HBSS. The fluorescence signals,

based on the Ca21 influx, were monitored for 5 min. In all

experiments, the fluorescence changes after the stimulation were

indicated as the rates against those of baselines.

Statistical analyses

All experiments were repeated at least three times. Mean

values and standard deviations (SDs) were calculated from all

data obtained in the present experiments. Significant differences in



the results were determined by the Student’s t-test. The po0.05 was

considered as significant in the present experiments.

Acknowledgements: The authors thank Dr. T. Sawada (Shionogi

Pharmaceutical Institute, Osaka, Japan) and Dr. H. Tashiro (Fujiya,

Hadano, Japan) for their kind donations of rhIL-2 and OVA323-339

peptide, respectively. This work was partially supported by a Grant-

in-Aid for Ministry of Education, Culture, Sports, Science and

Technology (D. W., H. K. and T. N.), by a National Project

‘‘Knowledge Cluster Initiative’’ (second stage, ‘‘Sapporo Biocluster

Bio-S’’), Ministry of Education, Culture, Sports, Science and

Technology, Japan (MEXT), and by the Joint Research Program of

the Institute for Genetic Medicine, Hokkaido University.

Conflict of interest: The authors declare no financial or

commercial conflict of interest.

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Abbreviations: AchR: acetylcholine receptor � AHR: airway

hyperresponsiveness � ASMC: airway smooth muscle cell � b2AR: b2-

adrenergic receptor � FP: fluticasone propionate � Mch:

b-methacholine chloride � NANC: non-adrenergic and non-

cholinergic � NKA: neurokinin A � NK2R: neurokinin-2 receptor

Full correspondence: Prof. Takashi Nishimura, Division of

Immunoregulation, Section of Disease Control, Institute for Genetic

Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo, 060-

0815, Japan

Fax: 181-11-706-7546

e-mail: [email protected]

Received: 9/6/2011

Revised: 8/10/2011

Accepted: 16/11/2011

Accepted article online: 22/11/2011



ifn-γ elevates airway hyper-responsiveness via up-regulation of neurokinin a/neurokinin-2 receptor...

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