T helper 1 cells stimulated with ovalbumin and IL-18
induce airway hyperresponsiveness and lung
fibrosis by IFN-? and IL-13 production
Nobuki Hayashi*†, Tomohiro Yoshimoto*†, Kenji Izuhara‡, Kiyoshi Matsui§, Toshio Tanaka¶, and Kenji Nakanishi*†?
*Departments of Immunology and Medical Zoology and§Internal Medicine, Hyogo College of Medicine, Nishinomiya, Hyogo 663-8501, Japan;‡Division
of Medical Biochemistry, Department of Biomedical Science, Saga Medical School, Saga 849-8501, Japan;¶Department of Molecular Medicine, Osaka
University Graduate School of Medicine, Suita, Osaka 565-0871, Japan; and†Collaborative Development of Innovation Seeds, Japan Science
and Technology Corporation, Tokyo 102-8666, Japan
Communicated by Mark M. Davis, Stanford University School of Medicine, Stanford, CA, July 27, 2007 (received for review October 6, 2006)
We previously reported that ovalbumin (OVA) and IL-18 nasally
administered act on memory type T helper (Th)1 cells to induce
airway hyperresponsiveness (AHR) and inflammation, which is
characterized by peribronchial infiltration with neutrophils and
eosinophils. Here, we report this administration also induces lung
fibrosis in an IL-13-dependent manner. Th1 cells secrete several
cytokines, including IFN-? and bronchogenic cytokine IL-13, when
stimulated with antigen (Ag) and IL-18. However, IL-13 blockade
failed to attenuate AHR, although this treatment inhibited eosin-
ophilic infiltration. To understand the mechanism by which Th1
cells induce AHR after Ag plus IL-18 challenge, we established
‘‘passive’’ and ‘‘active’’ Th1 mice by transferring OVA-specific Th1
cells into naı ¨ve BALB/c mice or by immunizing naı ¨ve BALB/c mice
with OVA/complete Freund’s adjuvant, respectively. Administra-
tion of Ag and IL-18 induced both types of Th1 mice to develop
AHR, airway inflammation, and lung fibrosis. Furthermore, this
treatment induced deposition of periostin, a novel component of
lung fibrosis. Neutralization of IL-13 or IFN-? during Ag plus IL-18
challenges inhibited the combination of eosinophilic infiltration,
lung fibrosis, and periostin deposition or the combination of
neutrophilic infiltration and AHR, respectively. We also found that
coadministration of OVA and LPS into Th1 mice induced AHR and
airway inflammation via endogenous IL-18. Thus, IL-18 becomes a
key target molecule for the development of a therapeutic regimen
for the treatment of Th1-cell-induced bronchial asthma.
bronchial asthma ? LPS ? periostin ? hydroxy proline ? airway inflammation
associated with airway inflammation and remodeling and occa-
sional high serum level of IgE (1–7). Histologically, there are
infiltrates of eosinophils, degranulated mast cells, subbasement
membrane thickening, hyperplasia and hypertrophy of bronchial
smooth muscle, and hyperplasia of airway goblet cells (1, 2). Th2
cells have been recognized as inducing bronchial asthma by pro-
duction of Th2 cytokines (1–12). Particularly, IL-13 is suggested to
play a critical role in induction of AHR, eosinophilic infiltration,
goblet cell metaplasia, and lung fibrosis (9–13). In contrast, Th1
cells previously had been regarded to inhibit bronchial asthma by
virtue of IFN-? (14–16). However, several studies have disclosed
Moreover, a combination of Th1 and Th2 cells or their products
18, 21). Thus, bronchial asthma is a complicated disease induced by
the functions of Th1 and Th2 cells.
on adoptively transferred memory type Th1 cells to induce airway
express ovalbumin (OVA)-specific T cell antigen receptor and
IL-18 receptor (22, 23). They produce IFN-? in response to OVA
and increase further IFN-? production in response to additional
IL-18 stimulation (22, 23). Most surprisingly, they simultaneously
produce Th2 cytokines (e.g., IL-9 and IL-13), granulocyte–
macrophage colony-stimulating factor and chemokines (e.g.,
RANTES and macrophage inflammatory protein 1?) when stim-
ulated with OVA and IL-18 (22). Human Th1 cells also produce
(24). Recently, we demonstrated Th1 cells induce intrinsic atopic
(25). Thus, IL-18 has added its new function to its growing
functional list (26–29). Based on this unique function of Ag- plus
IL-18-stimulated Th1 cells, we proposed to designate them as
‘‘super Th1 cells’’ (25). It is important to determine the mechanism
by which super Th1 cells induce bronchial asthma by production of
both Th1 and Th2 cytokines. However, as we reported previously
(22), IL-13 blockade fails to attenuate Ag- plus IL-18-induced
AHR, although this treatment markedly diminishes eosinophilic
infiltration. These results prompted us to examine the role of Th1
cytokine in induction of AHR and airway inflammation.
In our previous report, we established ‘‘passive Th1 mice’’ by
transferring OVA-specific memory Th1 cells (1 ? 107cells per
mouse) into naı ¨ve BALB/c mice (22). Here, we prepared ‘‘active
Th1 mice’’ by immunizing BALB/c mice with OVA in complete
Freund’s adjuvant (CFA) 2 weeks before experimentation. Both
types of Th1 mice develop AHR, airway inflammation, and lung
fibrosis after challenge with OVA and IL-18. Furthermore, they
express periostin, a novel component of lung fibrosis under the
Ab almost completely inhibited AHR and neutrophilic infiltration,
whereas IL-13 neutralization inhibited lung fibrosis and eosino-
philic infiltration without affecting AHR. Thus, Th1 cells become
very harmful super Th1 cells when stimulated with OVA and IL-18
by production of IFN-? and IL-13 in the lungs. Of interest,
OVA/CFA-immunized mice develop AHR and airway inflamma-
tion upon challenge with OVA and LPS via endogenous IL-18.
Importantly, administration of anti-IL-18 almost completely inhib-
ited this OVA/LPS-induced AHR, thereby rationalizing the devel-
opment of reagents that down-regulate IL-18 for the treatment of
Th1-cell-induced bronchial asthma.
Author contributions: N.H. and K.N. designed research; N.H. and T.Y. performed research;
K.I. contributed new reagents/analytic tools; T.Y., K.I., K.M., and T.T. analyzed data;
and K.N. wrote the paper.
The authors declare no conflict of interest.
Abbreviations: Ag, antigen; AHR, airway hyperresponsiveness; BALF, bronchoalveolar
lavage fluid; Cdyn, dynamic compliance; CFA, complete Freund’s adjuvant; OVA, ovalbu-
min; Th, T helper.
?To whom correspondence should be addressed at: Department of Immunology and
Medical Zoology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo
663-8501, Japan. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
September 11, 2007 ?
vol. 104 ?
no. 37 ?
OVA and IL-18 Induce AHR in Th1 Cell-Bearing Mice in an IFN-?-
Dependent Manner. We previously reported that mice receiving
memory Th1 cells develop AHR and airway inflammation after
IFN-?, IL-9, IL-13, granulocyte–macrophage colony-stimulating
factor, and chemokines in response to Ag, IL-2, and IL-18 in vitro
(22, 25). Among the cytokines produced, IL-13 is most broncho-
genic and participates in AHR (7–13). However, neutralization of
IL-13 in the lungs fails to inhibit Th1-cell-induced AHR (22). Thus,
We first examined the relevant role of IFN-? in induction of
bronchial asthma. For this purpose, we constructed a convenient
mice with OVA in CFA (OVA/CFA) to actively induce OVA-
specific Th1 cells in vivo. Indeed, this treatment strongly induced
OVA-specific Th1 cells in vivo [supporting information (SI) Fig.
8A]. We designated these OVA/CFA-primed mice as active Th1
mice and tested their development of bronchial asthma after their
those of naı ¨ve mice receiving adoptively transferred OVA-specific
of Th1 mice developed AHR. Consistent with our previous report
(22), each stimulus alone did not induce AHR (SI Fig. 8B).
Furthermore, both noninvasive and invasive measurements of
AHR provided basically identical results, excluding a contribution
from the upper airway component to the induction of AHR (Fig.
2). Invasive measurement also indicated that OVA- plus IL-18-
challenged Th1 mice reduced lung compliance. Bronchoalveolar
lavage fluid (BALF) examination revealed that administration of
OVA and IL-18 induced increases in the numbers of eosinophils,
active and passive Th1 mice showed similar responses to OVA plus
IL-18 in terms of AHR and airway inflammation.
To evaluate the effects of IFN-? and IL-13 on AHR and airway
inflammation, we treated Th1 mice with an Ab against IFN-? or
sIL-13R?2-Fc chimera (sIL-13R?2). As shown in Fig. 1 A and B,
neutralization of IFN-? almost completely inhibited Ag- plus
IL-18-induced AHR, whereas neutralization of IL-13 failed to do
so, suggesting a contribution from IFN-? but not from IL-13 to the
eosinophilic infiltration, excluding a contribution from eosinophilic
infiltration to the induction of AHR in Th1-cell-induced bronchial
asthma. To understand the mechanism whereby only IFN-? neu-
tralization inhibited AHR in both types of Th1 mice, we compared
the cellular components in BALFs from Th1 mice either receiving
these treatments or not. As shown in Fig. 1 C and D, neutralization
of IFN-? or IL-13 selectively diminished neutrophilic or eosino-
philic infiltration, respectively, suggesting their differential regula-
tion by IFN-? and IL-13.
Histological analysis revealed that both types of Th1 mice ex-
pressed peribronchial and perivenular infiltrations composed
mainly of eosinophils and neutrophils after OVA plus IL-18 treat-
ment (Fig. 3). The degree of eosinophilic infiltrations in the lungs
of the Th1-cell-bearing mice is comparable to that of Th2-cell-
of Fig. 1 C and D, neutralization of IFN-? and IL-13 appeared to
the lungs, respectively (Fig. 3). These results strongly indicated that
Th1 cells induce AHR and eosinophilic infiltration in response to
Ag and IL-18 by production of IFN-? and IL-13, respectively.
IL-4R??/?Mice Normally Develop Bronchial Asthma After OVA Plus
IL-18 Challenge. It is important to exclude entirely the contribution
from IL-13 to AHR because residual IL-13 might collaborate with
IFN-? for the induction of AHR. For this purpose, we primed
BALB/c background IL-4R??/?mice with OVA/CFA and subse-
quently challenged them with OVA and IL-18. As expected,
IL-4R??/?mice normally developed AHR (Fig. 4A), which for-
mally excluded a contribution from IL-13 to the induction of AHR
in Th1-cell-bearing mice.
We simultaneously examined the cell components in the BALFs
(Fig. 4B). We also performed histological evaluation of the lungs
(Fig. 4C). As expected from the BALFs, results of sIL-13R?2-
treated mice (Fig. 1 C and D), OVA/CFA-primed, and OVA/IL-
18-challenged IL-4R??/?mice markedly reduced the numbers of
D) Th1 mice were exposed to daily intranasally administered PBS (50 ?l) or OVA (100 ?g per 50 ?l of PBS) plus IL-18 (0.5 ?g per 50 ?l of PBS) for 3 days. A total
of 20 ?g of sIL-13R?2-Fc chimera (sIL13R?2) was used for IL-13 blockade in vivo. For the blockade of IFN-? in vivo, 50 ?g of anti-IFN-? Ab was intranasally
?-methacholine and inflammatory cell composition of BALF was examined. (C and D) Cell differential percentages were determined by light microscopic
evaluation of cytospin preparation. Representative results of five to seven animals per group are shown.
Anti-IFN-? Ab treatment protected against Ag- plus IL-18-induced AHR and accumulation of neutrophils in Th1 mice. Passive (A and C) and active (B and
or OVA (100 ?g per 50 ?l of PBS) plus IL-18 (0.5 ?g per 50 ?l of PBS) for 3 days.
concentrations of inhaled ?-methacholine was determined by noninvasive
[specific airway resistance (sRaw)] (A) and invasive [pulmonary resistance
(cmH2O/ml?sec?1) and Cdyn (ml/cmH2O) were 4.58 for PBS, 4.65 for OVA plus
IL-18, 0.0119 for PBS, and 0.0133 for OVA plus IL-18. Representative results of
five to seven animals per group are shown.
Noninvasive or invasive measurement of Ag- plus IL-18-induced AHR.
www.pnas.org?cgi?doi?10.1073?pnas.0706378104 Hayashi et al.
their BALFs (Fig. 4B). Histological examination revealed that
WT mice, IL-4R??/?mice markedly increased the cell infiltrates
composed mainly of neutrophils (Fig. 4C), substantiating further
the differential induction of neutrophils and eosinophil by IFN-?
of AHR in OVA- plus IL-18-administered Th1 mice.
Coadministration of Ag and LPS Induces AHR in Th1-Immunized Mice.
Patients with bronchial asthma often develop AHR and airway
reported that infected animals often display an increase in serum
levels of IL-18 after viral or bacterial infection (27). Thus, we
assumed that pathogen or pathogen-associated molecular pattern
might induce AHR by the induction of IL-18. To examine this
possibility, we treated Th1 mice with intranasal administration of
OVA and LPS instead of OVA and IL-18. We found that mice
receiving such treatment developed AHR and severe airway in-
flammation (Fig. 5 A and B).
We next tried to determine whether OVA and LPS induced
production from Th1 cells. Thus, we examined the capacity of
anti-IL-18 or anti-IFN-? Ab treatment to inhibit this OVA plus
LPS-induced AHR (Fig. 5). Each Ab treatment markedly dimin-
ished AHR (Fig. 5A). Furthermore, IFN-??/?or IL-18?/?mice
were resistant to the sequential treatment with OVA/CFA priming
and OVA/LPS challenge (Fig. 5C), substantiating further the
importance of IL-18-dependent IFN-? production for AHR. How-
ever, this OVA plus LPS challenge only increased the number of
neutrophils in BALFs (Fig. 5B). Thus, OVA plus LPS partially
replaced the action of OVA and IL-18. However, anti-IFN-? Ab
treatment modestly increased the number of eosinophils and lym-
phocytes but markedly reduced the number of neutrophils (Fig.
5B). These results strongly indicated that OVA plus LPS induced
Th1 cells to produce IFN-? via endogenous IL-18, resulting in
induction of neutrophilic infiltration and inhibition of eosinophilic
Ag- and IL-18-Induced Lung Fibrosis Depends on Endogenous IL-13.
IL-13 is indispensable for eosinophilic inflammation (7, 9–13, 34).
Furthermore, IL-13 has the potential to induce lung fibrosis by
activating macrophages, bronchoepithelial cells, and eosinophils to
produce fibrogenic cytokine TGF-? (35, 36). Therefore, we tested
the pathological effect of IL-13 derived from Ag- plus IL-18-
stimulated Th1 cells on lung fibrosis. We compared the degree of
lung fibrosis in the lungs of Th1 and Th2 mice after challenge with
OVA plus IL-18 or OVA alone, respectively. Both Th1 and Th2
mice similarly developed lung fibrosis (Fig. 6). Development of
fibrosis was proven to depend on the function of endogenous IL-13
because blockade of endogenous IL-13 inhibited lung fibrosis
(Fig. 6 Ad and Bh). Thus, OVA- plus IL-18-stimulated Th1 cells
or OVA-stimulated Th2 cells induced lung fibrosis by production
To examine further the extent of fibrosis, we measured the lung
hydroxyproline content (Table 1). Th1 mice challenged with OVA
plus IL-18 significantly increased hydroxyproline content in their
lungs, whereas Th1 mice challenged identically but under IL-13
fibrosis by acting on Th1 cells to produce IL-13. Furthermore,
to OVA and IL-18. Both passive and active Th1 mice were exposed daily to
described in the legend of Fig. 1. At 24 h after final exposure, lungs from each
group of mice were prepared for histological examination by perfusing the
animal via the right ventricle with 10 ml of PBS; the lungs were then fixed in
and Methods. [Original magnification, ?40 (Insets, ?200).]
Histological examination of the lung tissues from Th1 mice exposed
BALB/c background IL-4R??/?mice, which were immunized with OVA in CFA
2 weeks previously, were exposed to daily intranasal administration of PBS or
for ?-methacholine-induced AHR (A) and BALF (B) and their histological
neutrophil; black arrowhead, eosinophil. Representative results obtained
from five to seven animals per group are shown. (Magnification, ?200.)
IL-4R??/?mice often developed Th1-cell-induced AHR. BALB/c WT or
Hayashi et al.
September 11, 2007 ?
vol. 104 ?
no. 37 ?
in OVA- plus IL-18-challenged Th1 mice. These results allowed us
to conclude that IL-18 induces lung fibrosis by the induction of
We finally examined the expression of periostin, a novel com-
ponent of lung fibrosis developing at the early stage of bronchial
asthma and colocalizing with the extracellular matrix protein in-
volved in lung fibrosis (30). Induction of periostin is shown to
depend on IL-4 and IL-13 but not TGF-? signaling (30). OVA/
IL-18 challenge (Fig. 7). IL-13 blockade inhibited this expression,
suggesting that Th1 cells induce lung fibrosis and periostin depo-
sition by production of IL-13 (Fig. 7). In conclusion, after being
stimulated with Ag and IL-18, Th1 cells became very pathological
super Th1 cells, which induce AHR and lung fibrosis by production
of IFN-? and IL-13, respectively, in this mouse model of bronchial
We have established passive and active Th1 mice by transferring
OVA-specific Th1 cells into or OVA/CFA immunization of naı ¨ve
and airway inflammation associated with lung fibrosis after intra-
nasal challenge with OVA and IL-18. BALF analysis and histolog-
ical evaluation revealed that they increased the number of eosin-
ophils, neutrophils, and lymphocytes in their lungs (Figs. 1 and 3).
Infiltrations of eosinophils and neutrophils in the lungs are regu-
IL-4R??/?mice excluded the potential contribution from IL-13 to
Th1-cell-induced AHR (Fig. 4). Results from anti-IFN-?-treated
mice revealed that IFN-? is a true causative factor responsible for
inducing AHR (Fig. 1). OVA plus LPS replaced partly the effect of
OVA and IL-18 by the induction of endogenous IL-18 (Fig. 5). We
also showed that, like Th2 mice challenged with OVA, Ag- plus
IL-18-challenged Th1 mice develop lung fibrosis associated with
periostin deposition and increased lung hydroxyproline content,
which are induced by the action IL-13 (Table 1 and Figs. 6 and 7).
Thus, Th1 cells become very pathological super Th1 cells when
which in combination induce AHR, peribronchial inflammation,
and lung fibrosis in this mouse model of bronchial asthma.
a dichotomy. However, our previous studies have clearly demon-
and Th2 cells in mice (22, 24, 25). We showed that OVA-specific
monoclonal Th1 cells, which we developed in vitro, have the
IL-13) as well as chemokines (e.g., RANTES and macrophage
spectrum of cytokines and chemokines that are produced by
activated mast cells. In this study, we first examined whether
OVA-specific Th1 cells, which we newly developed in vivo by
immunization of mice with OVA/CFA, can produce IFN-?, IL-9,
IL-13, granulocyte–macrophage colony-stimulating factor, and
we confirmed that they had such potential. Next, we examined
whether OVA/CFA-immunized active Th1 mice developed AHR
and airway inflammation after administration of OVA and IL-18.
Consistent with our previous report obtained from passive Th1
mice (22), active Th1 mice exposed to OVA alone did not develop
AHR (SI Fig. 8B). However, when nasally exposed to OVA and
Table 1. Lung hydroxyproline level in active Th1 mice challenged
with OVA plus IL-18
Active Th1 mouse
Hydroxyproline, ?g per lung
OVA ? IL-18
OVA ? IL-18 ? sIL-13R?2
OVA ? IL-18 ? anti-IFN?
110.17 ? 6.4
149.9 ? 12.3*
118.48 ? 3.5
158.83 ? 8.3†
Active Th1 mice were exposed daily to intranasal administration of PBS (50
?l), OVA (100 ?g per 50 ?l of PBS), or OVA (100 ?g per 50 ?l of PBS) plus IL-18
(0.5 ?g per 50 ?l of PBS) for 3 days. sIL-13R?2 or anti-IFN-? antibody was used
for IL-13 or IFN-? blocking, respectively, in vivo. At 24 h after the final
exposure, lung total hydroxyproline levels were measured.*, P ? 0.01 (vs. PBS
treatment or OVA ? IL-18 ? sIL-13R?2 treatment).
†Not significant (vs. OVA ? IL-18 treatment).
background IFN-??/?or IL-18?/?mice immunized with OVA/CFA 2 weeks previously were challenged intranasally with 5 ?g of LPS and 100 ?g of OVA. To block
endogenous IL-18 or IFN-?, Ab against IL-18 or IFN-? mixed with OVA and LPS was coadministered daily. At 24 h after final administration, ?-methacholine-
are shown. The results shown in A Left and C Left were obtained from the same experiment.
Neutralization of IFN-? or IL-18 abolished OVA plus LPS-induced AHR and neutrophilic infiltration in active Th1 mice. BALB/c WT mice or BALB/c
or Ag-administered Th2 mice. Active Th1 mice or Th2 were induced by immu-
nization with OVA/CFA (A) or OVA/alum (B), respectively. Immunized mice
were exposed daily to intranasally administered OVA or OVA plus IL-18 with
or without sIL-13R?2 as described in the legend of Fig. 1. At 24 h after final
b, e, and f) or Azan–Mallory (c, d, g, and h), and used for histological
examination. (Original magnification, ?40.)
www.pnas.org?cgi?doi?10.1073?pnas.0706378104Hayashi et al.
IL-18, both passive and active Th1 mice equally developed AHR
and severe airway inflammation (Figs. 1–3). Histological examina-
tion revealed that additional IL-18 administration induced massive
cell infiltrates composed of eosinophils, lymphocytes, and neutro-
phils (Fig. 3).
In our previous report, we tried to understand the mechanism by
which Th1 cells induced AHR and airway inflammation when they
were stimulated with Ag and IL-18 in vivo. We initially envisaged
that IL-13 from Ag- plus IL-18-stimulated Th1 cells was a causative
factor because IL-13 has been shown to induce AHR in Th2-cell-
bearing mice (9–13). However, neutralization of IL-13 did not
affect Th1-cell-induced AHR, although this treatment strongly
in PBS-treated mice even if we performed daily administration of
the effects of this treatment on lung tissue eosinophils or neutro-
phils were less effective than those on BALF levels of these cells
(Figs. 1 and 3). IL-13 neutralization in BALF might be more
efficient than that in lung tissue.
In this study, we formally could exclude the contribution from
IL-13 to AHR by showing the capacity of IL-4R??/?mice to
normally develop AHR after OVA/CFA priming and subsequent
OVA/IL-18 challenge (Fig. 4). Because Ag- plus IL-18-stimulated
Th1 cells produced IFN-?, IL-9, and IL-13 but not IL-5, we could
assume that eosinophilic infiltration was induced by the action of
IL-13. Of interest, compared with WT BALB/c mice, IL-4R??/?
mice augmented neutrophilic infiltration in their lungs, suggesting
that IL-13 down-regulates neutrophilic infiltration but up-regulates
eosinophilic infiltration. We recently demonstrated that IL-13
recruits eosinophils in the lungs by the induction of eotaxin from
lung epithelial cells (34).
We tried to determine the causative factor. Here, we could
demonstrate that IFN-? from Ag- plus IL-18-stimulatd Th1 cells
was responsible for inducing AHR and airway inflammation (Figs.
1 and 3). Neutralization of IFN-? inhibited AHR and neutrophilic
not know how IFN-? induces recruitment of neutrophils in the
lungs. It is quite reasonable to speculate that IFN-? induces
production of some chemokines, which have the capacity to recruit
neutrophils in the lungs.
Bronchial asthma is often induced by viral or bacterial infection
(2, 31–33). We tested whether pathogen-associated molecular pat-
tern (LPS) can induce IL-18 production from alveolar macrophage
and/or lung epithelial cells. The lung epithelial cell line
TGMBE02-3 cells (37) express Toll-like receptor 4 and produce
IL-18 upon LPS stimulation in vitro (SI Fig. 9). Furthermore,
OVA/CFA-immunized and OVA/LPS-challenged mice increased
their serum levels of IL-18 (SI Fig. 10). IL-18 is also involved in
human bronchial asthma. Serum levels of IL-18 are elevated in
patients with bronchial asthma (SI Fig. 10), and a significant
correlation between IL-18 serum levels and the disease severity of
bronchial asthma has been reported (38).
In this study, we could demonstrate the unique capacity of
OVA-specific Th1 cells to induce AHR after intranasal adminis-
tration of OVA and LPS. Neutralization of IL-18 or IFN-? atten-
uated AHR and decreased the number of neutrophils in BALF.
Furthermore, OVA/CFA-immunized IL-18?/?or IFN-??/?mice
These results clearly indicated that endogenous IL-18 played a
critical role in induction of this mouse model of bronchial asthma
by activation of Th1 cells to produce IFN-?.
We have shown that IL-13 induces lung fibrosis associated with
periostin deposition (Figs. 6 and 7), which binds to extracellular
matrix proteins to form a reticular structure (30). Indeed, neutral-
ization of IL-13 inhibited lung fibrosis (Fig. 6), lung hydroxyproline
content (Table 1), and periostin deposition (Fig. 7) without inhib-
iting AHR (Fig. 1). Thus, Ag- plus IL-18-stimulated Th1 cells
exhibit two pathological effects on the development of bronchial
lung fibrosis. Here, we also demonstrated that LPS induces Th1-
cell-induced asthma by the induction of IL-18 production. It is well
known that Th2 cell-induced asthma can be controlled by the
treatment with anti-Th2 cytokines or anti-IgE (2, 39). However,
there are no appropriate treatments for infection- or Th1-cell-
induced bronchial asthma, in which super Th1 cells play very
pathological roles. Our present results clearly indicated that we
could regulate AHR and lung fibrosis by down-regulation of IL-18.
Thus, IL-18 becomes rational target for the treatment of Th1-cell-
induced bronchial asthma. Because difficult asthma or refractory
asthma is often induced by bacterial or viral infection, anti-IL-18
therapy might be applicable for its treatment.
Materials and Methods
Animals and Reagents. Specific pathogen-free female BALB/c mice
and IL-4R??/?mice (BALB/c background) were purchased from
The Jackson Laboratory (Bar Harbor, ME). BALB/c background
IFN-??/?mice were kindly provided by Y. Iwakura (University of
Tokyo, Tokyo, Japan). BALB/c background IL-1?/?mice were
established in our laboratory (40) and used for experimentation.
Mice transgenic for ?? T cell antigen receptor recognizing
OVA323–339(DO11.10; BALB/c background) (41) were generously
provided by D. Loh (Washington University, St. Louis, MO). All
experiments were performed on the line heterozygous for the
transgene. All mice were bred under specific pathogen-free con-
ditions at the animal facilities of Hyogo College of Medicine
(Nishinomiya, Japan) and were used at 6 to 10 weeks of age.
Recombinant mouse IL-12 and IL-18 were purchased from R & D
Systems (Minneapolis, MN) and MBL (Nagoya, Japan), respec-
tively. LPS from Salmonella minnesota Re-595 was purchased from
Sigma–Aldrich (St Louis, MO). Anti-CD3 (2C11) and anti-IL-4
(11B11) were prepared by Harlan Bioproducts for Science (Indi-
in our laboratory.
Generation of Th1 Cells in Vivo. We induced Th1 cells by immuni-
in OVA was removed by END-X 15 (Seikagaku America, Cape
Cod, MA). After removal, endotoxin level was ?0.5 pg/ml in 1
of our experiments.
after exposure to OVA and IL-18. Active Th1 mice were exposed daily to
intranasally administered OVA plus IL-18 or OVA plus IL-18 plus sIL-13R?2 as
described in the legend of Fig. 1. At 24 h after final exposure, lungs from each
group of mice were fixed and stained with H&E or Azan–Mallory. Immuno-
(Original magnification, ?40.)
Immunohistochemical staining of periostin in the lungs of Th1 mice
Hayashi et al.
September 11, 2007 ?
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no. 37 ?
In Vivo Intranasal Antigen Challenge.ThepreparationofpassiveTh1 Download full-text
mice has been described previously (22, 42). Briefly, 1 ? 107Th1
cells, which we developed in vitro (22, 42), were injected i.v. into
normal BALB/c mice (10–15 mice per group). At 4 weeks, or even
later, after cell transfer, transplanted Th1 cells expressed the
Ag-specific resting Th1 memory phenotype in host mice. Active
Th1 mice were prepared by immunization of mice with OVA/CFA.
transfer, both types of Th1 mice were exposed to daily intranasal
administration of 50 ?l of PBS or 50 ?l of PBS containing OVA
(100 ?g) or OVA (100 ?g) plus IL-18 (0.5 ?g) for 3 days. In some
LPS (5 ?g) and OVA (100 ?g). Mice were analyzed for their AHR
and airway inflammation at 24 h after the final exposure. sIL-
13R?2-Fc chimera (20 ?g; R & D Systems) was administered daily
intranasally for blocking of IL-13 in vivo as described previously
or 60 ?g of anti-IL-18, as well as its corresponding control Ab (50
?g of rat IgG or 60 ?g of rabbit IgG), was administered daily
intranasally at the time of OVA challenge.
Noninvasive or Invasive Measurement of AHR. Noninvasivemeasure-
34). Briefly, we measured AHR to ?-methacholine inhalation in
mice by using Pulmos-I hardware and software (MIPS, Osaka,
Japan). We placed a mouse in a chamber and first exposed it to
aerosols of saline (baseline) and then to increased concentrations
of ?-methacholine (5, 10, 15, and 20 mg/ml). After each 2-min
reflects changes in amplitude of pressure waveform and expiratory
time (43), for 2 min. Invasive measurement of AHR was assessed
as an increase in pulmonary resistance and a decrease in dynamic
compliance (Cdyn) in response to aerosolized ?-methacholine.
Briefly, mechanical ventilation was achieved by using a MiniVent
Model 845 ventilator (HSE, March–Hugstetten, Germany). Saline
and increased concentrations of ?-methacholine were aerosolized
as noninvasive measurement. Pulmonary resistance and Cdyn
were measured by Pulmos-II (MIPS) hardware and software
Bronchoalveolar Lavage. Bronchoalveolar lavage was performed
with three aliquots of 0.5 ml of PBS per mouse. Total cell counts
Dif-Quik (Baxter Healthcare, Miami, FL), and differentials were
performed based on morphology and staining characteristics.
Histology. Lungs were prepared for histological examination by
then were fixed in 10% buffered formalin, cut into 3-mm sections,
and stained with H&E for H&E stain or azocarmine G and aniline
blue orange G for Azan–Mallory stain. Immunohistochemical
staining for periostin has been described previously (30). Briefly,
lung tissues were fixed with 10% formalin and incubated with
primary Ab or normal IgG (control) overnight. The antigens were
detected by the ENVISION?/HRP (DAB) system (Dako Cyto-
mation, Glostrup, Denmark).
Hydroxyproline. Total hydroxyproline content was measured by
HPLC (SRL, Tokyo, Japan).
We thank Dr. H. Tsutsui of Hyogo College of Medicine for critical
reading of the manuscript; Dr. K. Yasuda, Dr. H. Tanaka, and Dr. M.
Kuroda of Hyogo College of Medicine for enthusiastic discussion; and
Ms. S. Yumikura-Futatsugi and Ms. M. Uemura for excellent technical
assistance. This study was supported by a Grant-in-Aid for Scientific
Research on Priority Areas and Hitech Research Center from the
Ministry of Education, Culture, Sports, Science, and Technology of
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