Original Paper
Int Arch Allergy Immunol 2009;149(suppl 1):77–82
DOI: 10.1159/000211377
15-Deoxy-⌬12,14-Prostaglandin J2
Induces IL-8 and GM-CSF in a Human
Airway Epithelial Cell Line (NCI-H292)
Takahito Chiba a Shigeharu Ueki b Wataru Ito b Hikari Kato b
Masahide Takeda b Hiroyuki Kayaba b Masutaka Furue a Junichi Chihara b
a
Department of Dermatology, Kyushu University School of Medicine, Fukuoka, and
b
Department of Clinical and Laboratory Medicine, Akita University School of Medicine, Akita, Japan
Key Words
PGD2 ⴢ 15d-PGJ2 ⴢ Cytokine ⴢ Airway epithelial cells ⴢ CRTH2
Abstract
Background: 15-Deoxy-⌬12,14 -prostaglandin J2 (15d-PGJ2), a
major prostanoid metabolized from prostaglandin D2 (PGD2),
plays an important role in various biological processes in-
cluding inflammatory responses. 15d-PGJ2 exerts its effects
through two major receptors, chemoattractant receptor-
homologous molecule expressed on Th2 cells (CRTH2) and
peroxisome proliferator-activated receptor- ␥ (PPAR␥). The
15d-PGJ2/PPAR␥ system, in particular, regulates numerous
biological processes including adipogenesis, apoptosis, and
inflammation. Although our studies have shown that PGD2
(metabolic precursor of 15d-PGJ2) induces IL-8 and GM-CSF
production, the role of 15d-PGJ2 (metabolite of PGD2) is un-
known in human bronchial epithelial cells. In this study, we
investigated the function of 15d-PGJ2 on a human airway
epithelial cell line: NCI-H292. Method: NCI-H292 cells were cul-
tured in the presence of various stimulants. The resulting
supernatants were used for ELISA analysis. Results: We
demonstrated that 15d-PGJ2 induced production of IL-8 and
GM-CSF from NCI-H292. 13,14-Dihydro-15-keto-PGD2 (DK-
PGD2) (CRTH2 agonist) and troglitazone (PPAR␥ agonist)
failed to increase the production of these cytokines. Pre-
© 2009 S. Karger AG, Basel
1018–2438/09/1495–0077$26.00/0
Fax +41 61 306 12 34
E-Mail karger@karger.ch Accessible online at:
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Published online: June 3, 2009
treatment with ramatroban (CRTH2 antagonist) and GW9662
(PPAR␥ antagonist) did not reduce the production of these
cytokines. 15d-PGJ2 activated the ERK1/2 signaling path-
way in a time-dependent manner. Conclusion: These data
showed that 15d-PGJ2 is a potent inducer of IL-8 and GM-CSF
production through ERK1/2 kinase activation, but this is in-
dependent of CRTH2 or PPAR␥ activation.
Copyright © 2009 S. Karger AG, Basel
Introduction
Mast cells, platelets, and some lymphocytes are large
sources of prostaglandin D2 (PGD2) in the pathogenesis
of inflammation [1, 2], and its metabolite 15-deoxy-
⌬12,14-prostaglandin J2 (15d-PGJ2) has been considered as
an immunoregulatory lipid mediator [3]. At the present
time, 15d-PGJ2 binds mainly two types of receptors to
exert its biological effects: chemoattractant receptor-ho-
mologous molecule expressed on Th2 cells (CRTH2) and
peroxisome proliferator-activated receptor-␥ (PPAR␥).
CRTH2 couples to a Gi-type G protein leading to inhibi-
tion of cAMP [4]. Currently, the signal of CRTH2 on Th2
cells, eosinophils, basophils, and mast cells triggers che-
motaxis and activation [5–7]. These studies indicate that
CRTH2 activation might be relevant to proinflammatory
Correspondence to: Dr. Junichi Chihara
Department of Clinical and Laboratory Medicine
Akita University School of Medicine
1-1-1, Hondo, Akita 010-8543 (Japan)
Tel. +81 18 884 6180, Fax +81 18 836 2624, E-Mail chihara@hos.akita-u.ac.jp
effects. On the other hand, PPAR␥ is a member of the
nuclear hormone receptor superfamily of ligand-activat-
ed transcription factors that regulates lipid metabolism
and glucose homeostasis. PPAR␥ is highly expressed not
only in adipose tissue but also in cells involved in the im-
mune system, and it is implicated in the control of in-
flammation. We and others have previously shown that
human dendritic cells, basophils, and eosinophils ex-
press PPAR␥, and that stimulation of these cells with a
synthetic PPAR␥ agonist inhibits the factor-induced ac-
tivation, such as alteration of cytokine balance, degranu-
lation, and chemotaxis [8, 9]. This evidence suggests that
PPAR␥ agonists may produce anti-inflammatory effects
by negatively modulating immune cell functions [10].
Taken together, 15d-PGJ2 possesses both anti-inflamma-
tory and proinflammatory functions, and activation of
CRTH2 and PPAR␥ plays a key regulatory role in inflam-
matory response by modulating immune cell functions.
A recent finding suggested that PGD2, a precursor
substance of 15d-PGJ2, enhanced the expression of mac-
rophage-derived factor chemokines in airway epithelial
cells [11]. We, in addition, found that the production of
IL-8 and GM-CSF was observed in airway epithelial cells
stimulated with PGD2 [12]. However, whether PGD2 di-
rectly induces the cytokine production on airway epithe-
lial cells is not yet clear.
To elucidate the role of PGD2 in inflammatory re-
sponses such as allergic inflammation, we explored the
possibility that 15d-PGJ2 derived from PGD2 might be
involved in the production of IL-8 and GM-CSF in air-
way epithelial cells based on the fact that IL-8 and GM-
CSF production is induced by PGD2 in normal human
bronchial epithelial (NHBE) cells [12]. In the present
study, we demonstrated that IL-8 and GM-CSF produc-
tion from bronchial epithelial cell lines was induced by
the PGD2 metabolite 15d-PGJ2 but not by a CRTH2 ago-
nist or PPAR␥ agonist. We also discuss whether an un-
known cell surface receptor is involved in the effect acti-
vated by 15d-PGJ2 via the ERK1/2 pathway.
Materials and Methods
Reagents
The human pulmonary mucoepidermoid carcinoma cell line
(NCI-H292) and NHBE cells were obtained from American Type
Culture Collection (Rockville, Md., USA). 15d-PGJ2 was pro-
cured from Sigma (Poole, Dorset, UK). 13,14-Dihydro-15-keto-
PGD2 (DK-PGD2) (a specific CRTH2 agonist) and GW9662 (a
specific PPAR␥ antagonist) were purchased from Cayman Chem-
icals (Ann Arbor, Mich., USA). Ramatroban (a CRTH2 antago-
78 Int Arch Allergy Immunol
2009;149(suppl 1):77–82
nist) was a gift from Bayer Yakuhin, Ltd. (Kyoto, Japan). ELISA
kits for IL-8 and GM-CSF were purchased from R&D Systems
(Minneapolis, Minn., USA).
Cell Cultures
NCI-H292 cells were cultured in small airway cell basal medi-
um (SABM) supplemented with 7.5 mg/ml of bovine pituitary ex-
tract, 0.5 mg/ml of hydrocortisone, 0.5 ␮g/ml of human recom-
binant epidermal growth factor, 0.5 mg/ml of epinephrine, 10 mg/
ml of transferrin, 5 mg/ml of insulin, 0.1 ␮g/ml of retinoic acid,
6.5 ␮g/ml of triiodothyronine, and 0.5 mg/ml of gentamicin sul-
fate with amphotericin-B (Clonetics, Walkersville, Md., USA) at
37 ° C in a humidified 5% CO2 atmosphere. The cells were then
transferred to a 24-well tissue culture plate (Becton Dickinson
Labware, Franklin Lakes, N.J., USA) and grown until subconflu-
ence. The culture medium was replaced with SABM without any
supplements 24 h before each experiment.
Measurement of 15d-PGJ2 Levels
PGD2 in 100 ␮l of SABM was incubated with or without NCI-
H292 cells for 24 h. 15d-PGJ2 in the supernatants was measured
using the Correlate-EIA 15d-PGJ2 Enzyme Immunoassay kit (As-
say Designs, Ann Arbor, Mich., USA) at the end of each incuba-
tion time.
Measurement of IL-8 and GM-CSF
The NCI-H292 cells were suspended in SABM. The cells were
supplemented with 15d-PGJ2, DK-PGD2, or troglitazone. In some
experiments, after treatment with or without the antagonists for
the indicated times at 37 ° C, the cells were stimulated with 10 ␮ M
15d-PGJ2. The supernatants were separated by centrifugation,
and the concentration of IL-8 and GM-CSF was measured by en-
zyme-linked immunosorbent assay (ELISA).
RT-PCR Analysis
Total RNA was extracted using an Ultraspec RNA kit (Biotex
Laboratories, Inc., Houston, Tex., USA) and reverse-transcribed
by an Omniscript RT kit (Qiagen, Hilden, Germany). One micro-
liter of the cDNA synthesis reaction was used as a template for
PCR amplification with AmpliTaq DNA polymerase (PerkinEl-
mer, Foster City, Calif., USA). The PPAR␥ primers were sense:
5ⴕ-TCTCTCCGTAATGGAAGACC-3ⴕ and antisense: 5ⴕ-GCAT-
TATGAGACATCCCCAC-3ⴕ [13]. The PCR cycle consisted of
45 s of denaturation at 94 ° C, 45 s of annealing at 55 ° C, and 1 min
of extension at 72 ° C for 35 cycles. The PCR products were elec-
trophoresed with a 12.5% PhastGel and PhastSystem, and the gel
was stained using a PhastGel DNA Silver Staining Kit (Amersham
Pharmacia Biotech AB, Uppsala, Sweden).
Preparation of Cytosolic Cell Extracts
The NCI-H292 was stimulated with 15d-PGJ2 for the indicated
times at 37 ° C. The reaction was terminated by the addition of
9 vol of ice-cold HBSS containing 1 mM Na3VO4. The cells were
lysed in a lysis buffer (50 m M Tris-HCl, pH 7.4, 150 mM NaCl,
1 mM Na3VO4, 1 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF,
1% Triton X-100, 10% glycerol, 1 ␮g/ml of aprotinin, leupeptin,
and pepstain). After 20 min on ice, detergent-insoluble materials
were removed by 12,000 g centrifugation at 4 ° C. The whole-cell
lysates were boiled in 2! Laemmli reducing buffer for 4 min.
Chiba /Ueki /Ito /Kato /Takeda /Kayaba /
Furue /Chihara
 Gel Electrophoresis and Western Blotting
SDS-PAGE was performed using Ready Gels J (BioRad, Her-

15d-PGJ2 concentration (×10–6 M)

cules, Calif., USA). The concentration of polyacrylamide was 10–
15% depending on the molecular weight of the protein in which 1.2
we were interested. Gels were blotted onto Hybond membranes
for Western blotting using an enhanced chemiluminescence sys-
tem. Blots were incubated in a blocking buffer containing 10% 0.8
bovine serum albumin in a blotting buffer (20 m M Tris-HCl, 137
mM NaCl, pH 7.6, 0.05% Tween 20) for 1 h followed by incubation
in mouse monoclonal anti-phospho-ERK1/2 antibody or rabbit 0.4
polyclonal anti-ERK1 (0.1 ␮g/ml) purchased from Santa Cruz NCI-H292(+)
Biotechnology (Santa Cruz, Calif., USA) for 1–2 h. After washing NCI-H292(–)
three times in the blotting buffer, blots were incubated for 30 min 0
with a rabbit peroxidase-conjugated secondary antibody (0.04 0 1 6 12 18 24
␮g/ml) directed against the primary antibody. The blots were de- Time
veloped with the enhanced chemiluminescence substrate accord-
ing to the manufacturer’s instructions.
Fig. 1. Conversion of PGD2 to 15d-PGJ2 in the presence and ab-
Measurement of Endotoxin Levels sence of NCI-H292 cells. PGD2 (10 –5 M) in SABM was incubated in
The endotoxin concentration of cell culture fluid with each the presence (j) and absence ( $ ) of NCI-H292 cells at 37 ° C. The
agent was measured by Limulus Amebocyte Lysate assay from 15d-PGJ2 levels in the reaction mixture were determined by a
Seikagaku Corp. (Kyoto, Japan). competitive ELISA assay. The data represent means 8 SD of trip-
licate determinations.
Statistical Analysis
Results were expressed as mean 8 SD. Data were analyzed for
statistical significance using ANOVA.

pg/ml
Results 1,600 IL-8
GM-CSF *
Conversion of PGD2 to 15d-PGJ2 1,200 * *
* * *
15d-PGJ2 is recognized to be physiologically formed
through the nonenzymatic conversion of PGD2. We ini- 800
tially investigated the amount of conversion of PGD2 to
15d-PGJ2 in vitro. To examine the extracellular conver- 400
sion of PGD2 to 15d-PGJ2, 10 ␮M PGD2 was incubated in
NCI-H292 cells or cell-free medium, after which the for- 0
mation of 15d-PGJ2 was examined. As shown in figure 1, (–) 1 2 5 10
the concentration of 15d-PGJ2 increased almost linearly 15d-PGJ2 (μM)

from 0 to 8 h and reached a maximum concentration at

24 h. The maximum levels were expected to be main-
tained for the duration of the following experiment. As a
consequence, 10 ␮M PGD2 converted into about 1.2 ␮M
15d-PGJ2. The culture medium in the NCI-H292 cells was
more precipitative than cell-free medium toward the pro-
duction of 15d-PGJ2 from PGD2.
Fig. 2. Effects of 15d-PGJ2 on IL-8 and GM-CSF protein level in
the culture media after 24 h incubation of NCI-H292 cells. IL-8
and GM-CSF were measured by ELISA. Data are expressed as
means 8 SD (n = 6). * p ! 0.001 vs. control (DMSO alone) (ANO-
VA with Bonferroni correction).

Cytokine Production from 15d-PGJ2-Stimulated

Bronchial Epithelial Cells from a Human Airway
Epithelial Cell Line (NCI-H292)
We measured cytokine production from NCI-H292
cells to investigate the functional role of 15d-PGJ2. The
cells were stimulated with 15d-PGJ2 for 12 h. The cyto-
kine concentration in the supernatants was measured by
ELISA. As shown in figure 2, IL-8 and GM-CSF were pro-
duced by 15d-PGJ2 in a dose-dependent manner. Endo-
toxins have been reported to increase IL-8 production in
airway epithelial cells [14]. However, endotoxins were not
detectable in any cell culture fluid used in this study (!10
pg/ml).

Role of 15d-PGJ2 in Airway Epithelium Int Arch Allergy Immunol 79

2009;149(suppl 1):77–82
 used as a positive control. As shown in figure 4a, PPAR␥
pg/ml * mRNA was detectable by RT-PCR in both NCI-H292 and
1,200 IL-8
GM-CSF
NHBE cells. We also decided to investigate whether a
* PPAR␥ agonist (troglitazone) would mimic the 15d-PGJ2-
800
mediated induction of IL-8 and GM-CSF. However, tro-
glitazone did not affect the production of IL-8 and GM-
400
CSF, and a PPAR␥-selective antagonist (GW9662) did not
reduce 15d-PGJ2-induced IL-8 and GM-CSF production
0
Control 10 μM 1 μM 0.1 μM 15d-PGJ2 (fig. 4).
a DK-PGD2 (10 μM)

Kinetics of ERK1/2 Phosphorylation

To investigate the kinetics of ERK1/2, NCI-H292 cells
pg/ml
* were incubated with medium or stimulated with 15d-
*
1,200 IL-8 * * PGJ2 (10 ␮M) for 1, 3, 5, 10, or 30 min. Western blotting
GM-CSF * with anti-phospho-ERK antibody revealed that the ty-
800
* * * rosine/threonine phosphorylation of ERK1/2 reached a
maximum at 5 min (fig. 5).
400

0 Discussion
Control 10 μM 1 μM 0.1 μM 15d-PGJ2
(10 μM)
Ramatroban +
15d-PGJ2
(10 μM)
b lergic asthma and atopic dermatitis [1, 2]. In fact, PGD2
Fig. 3. a Effect of DK-PGD2 (CRTH agonist) on IL-8 and GM-CSF
release. The NCI-H292 cells were stimulated with DK-PGD2 or
15d-PGJ2 for 24 h. b Effect of ramatroban (CRTH2 antagonist) on
15d-PGJ2-induced IL-8 and GM-CSF release. The NCI-H292 cells
were stimulated with 15d-PGJ2 (10 ␮ M) for 24 h. After incubating
epithelial cells with ramatroban for 20 min, the cells were stimu-
lated with 15d-PGJ2 for 24 h. The concentration of IL-8 and GM-
CSF in the supernatants was measured by ELISA. Data are ex-
pressed as means 8 SD (n = 4). * p ! 0.001 vs. control (DMSO
alone) (ANOVA).
Effect of Multiple Signaling Pathway on the
15d-PGJ2-Stimulated Bronchial Epithelial Cells from
a Human Airway Epithelial Cell Line (NCI-H292)
To elucidate the signaling pathway that elicits IL-8 and
GM-CSF production, we examined the effect of DK-
PGD2 (CRTH2 agonist) and ramatroban (CRTH2 antag-
onist) on the function. As shown in figure 3a and b, DK-
PGD2 did not influence the production of IL-8 and GM-
CSF. Ramatroban did not abrogate the effect of 15d-PGJ2
on IL-8 and GM-CSF production.
It has been reported that 15d-PGJ2 has the ability to
bind and activate PPAR␥ [15]. Thus, we first demonstrat-
ed PPAR␥ expression at the mRNA level on NCI-H292
cells or NHBE cells. Eosinophils expressing PPAR␥ were
PGD2 synthesized by activated effector cells is impli-
cated in the pathogenesis of allergic diseases such as al-
was detected in bronchoalveolar lavage fluid of asthmat-
ics locally challenged with allergen [16]. Although airway
epithelial cells are expected to have a function on PGD2,
little is known about it. Therefore, in our previous report
[12], using NHBE cells, we showed that PGD2 induced the
production of IL-8 and GM-CSF. However, PGD2 is bro-
ken down into 15d-PGJ2 in vivo, which could act directly
on nearby cells including airway epithelial cells. Consis-
tent with postulated mechanism, production of IL-8 in
activated T cells or endothelial cells stimulated with 15d-
PGJ2 has been reported [17, 18]. Thus, it is important to
investigate whether PGD2 metabolite 15d-PGJ2 is in-
volved in allergic inflammation with respect to cytokine
production in the airway.
First, we demonstrated that 15d-PGJ2 induced IL-8
and GM-CSF production in a human airway epithelial
cell line. In an inflammatory focus, IL-8 augments a va-
riety of cellular functions, especially cell migration. It has
chemotactic activity not only for neutrophils but also eo-
sinophils [19, 20]. GM-CSF also has priming or direct ef-
fects on eosinophil functions such as survival, adhesion,
and degranulation. GM-CSF expression is upregulated in
asthmatic bronchial epithelial cells and is clearly corre-
lated with bronchial hyperresponsiveness. Moreover, it
was reported that IL-8 enhanced eosinophil migration
induced by GM-CSF [21]. Analysis of the results showed

80 Int Arch Allergy Immunol Chiba /Ueki /Ito /Kato /Takeda /Kayaba /
2009;149(suppl 1):77–82 Furue /Chihara
 500 bp
PPAR␥

400 bp
Eo Neg NCI-H292 NHBE
a

Fig. 4. a Expression of PPAR␥ in airway *

epithelial cells. Protein extracts were pre-
*
pg/ml * *
pared from NCI-H292 and NHBE cells and
analyzed using RT-PCR techniques. Eo- 1,200 IL-8
sinophils were used as a positive control. GM-CSF
Neg = Negative control. b Effect of trogli-
tazone (PPAR␥ agonist) on IL-8 and GM- 800 *
CSF release and of GW9662 (PPAR␥ an-
* * *
tagonist) on 15d-PGJ2-induced IL-8 and
GM-CSF release. The NCI-H292 cells were 400
stimulated with troglitazone or 15d-PGJ2
for 24 h. After incubating epithelial cells
with GW9662 for 20 min, the cells were
0
stimulated with 15d-PGJ2 for 24 h. The Control 10 μM 1 μM 0.1 μM 10 μM 1 μM 0.1 μM 15d-PGJ2
concentration of IL-8 and GM-CSF in the (10 μM)
Troglitazone GW9662 +
supernatants was measured by ELISA. 15d-PGJ2
Data are expressed as means 8 SD (n = 4). (10 μM)
* p ! 0.001 vs. control (DMSO alone) b
(ANOVA).

a significant correlation between IL-8 and GM-CSF pro-

duction (data not shown, r = 0.81, p ! 0.02). IL-8, which
acts as a chemoattractant for neutrophils, is found in
bronchoalveolar lavage fluid and serum from patients
with asthma [22]. It may contribute to the increase of the
mechanism of IL-8 and GM-CSF production from bron-
choalveolar lavage fluid in asthmatic patients.
15d-PGJ2 has been elucidated to be physiologically
formed through the nonenzymatic conversion of PGD2.
As shown in figure 1, 10 ␮M PGD2 converted into about
1.2 ␮M 15d-PGJ2. However, IL-8 production by 15d-PGJ2
required a concentration of at least 2 ␮M (fig. 2). One pos-
sible reason for this result is that PGD2 spontaneously
converts into cyclopentenone prostaglandins of the J se-
ries such as PGJ2, ⌬12-prostaglandin J2 (⌬12-PGJ2), and
15d-PGJ2 [3]. Hence, the other J series may be involved in
the cytokine production. Peeraully et al. [23] demonstrat-
ed that the prostaglandin J2-series (PGJ2, ⌬12-PGJ2) stim-
ulated IL-6 and MCP-1 expression and secretion by 3T3-
L1 adipocytes. The action of other PGJ2 series in airway
epithelial cells is currently under intense study.
In summary, we provided a basis for the conclusion
that the induction of IL-8 and GM-CSF by 15d-PGJ2 is
15d-PGJ2 (10 μM)
p-ERK1/2
Total ERK1
0 1 3 5 10 30
Time (min)
Fig. 5. Effect of 15d-PGJ2 on ERK1/2 activation. Cells were treated
with 10 ␮ M 15d-PGJ2 for various lengths of time. Expression of
phospho-ERK1/2 (p-ERK) was determined by Western blot using
a specific antibody. Total ERK1 was used as a loading control.
independent of CRTH2 or PPAR␥ in airway epithelial
cell line NCI-H292, and that 15d-PGJ2 is responsible for
the observed effects by activating ERK1/2 signaling path-
ways. In support of our findings, it has also been reported
that 15d-PGJ2 induces the production of IL-8 through a
PPAR␥-independent mechanism in THP-1 macrophages

Role of 15d-PGJ2 in Airway Epithelium Int Arch Allergy Immunol 81

2009;149(suppl 1):77–82
and human T cells [17, 18]. As one possibility, the exis-
tence of another signal pathway was proposed, including
the possibility of direct intracellular signaling. In fact,
Oliva et al. [24] reported that 15d-PGJ2 directly binds to
and activates H-Ras following MAPK and PI3-kinase ac-
tivation. However, whether the PGJ2 series including 15d-
PGJ2 move into cells through a receptor- or nonreceptor-
mediated pathway in vivo is currently under intense
study. Further study of this mechanism may provide im-
portant information on the regulation of allergic reac-
tions.
Acknowledgments
This study was supported in part by a grant from the 21st Cen-
tury Center of Excellence (COE) Program and by Grants-in-Aid
for Scientific Research supported by the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
Disclosure Statement
The authors declare that no financial or other conflicts of in-
terest exist in relation to the content of the article.

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