© 2005 Nature Publishing Group http://www.nature.com/natureimmunology

ARTICLESSuppression of allergic inflammation by theprostaglandin E receptor subtype EP3
Tomonori Kunikata1,4, Hana Yamane2,4, Eri Segi1,4, Toshiyuki Matsuoka1,4, Yukihiko Sugimoto2,
Satoshi Tanaka2, Hiroyuki Tanaka3, Hiroichi Nagai3, Atsushi Ichikawa2 & Shuh Narumiya1
Prostaglandins, including PGD2 and PGE2, are produced during allergic reactions. Although PGD2 is an important mediatorof allergic responses, aspirin-like drugs that inhibit prostaglandin synthesis are generally ineffective in allergic disorders,suggesting that another prostaglandin-mediated pathway prevents the development of allergic reactions. Here we show thatsuch a pathway may be mediated by PGE2 acting at the prostaglandin E receptor EP3. Mice lacking EP3 developed allergic
inflammation that was much more pronounced than that in wild-type mice or mice deficient in other prostaglandin E receptor
subtypes. Conversely, an EP3-selective agonist suppressed the inflammation. This suppression was effective when the agonist
was administered 3 h after antigen challenge and was associated with inhibition of allergy-related gene expression. Thus, thePGE2-EP3 pathway is an important negative modulator of allergic reactions.

Allergy, especially type I allergy such as bronchial asthma and allergicrhinoconjunctivitis, is a principal health problem, and the prevalenceof allergy has increased during the past two decades1. Affected people
have a high titer of immunoglobulin E (IgE) antibodies specific forallergens such as those associated with house dust mites and plantpollen2. Exposure of such people to these allergens induces theactivation of mast cells through crosslinking of IgE receptors on thecell surface mediated by antigen-antibody binding. Activated mast
cells release allergic mediators such as histamine, cysteinyl leukotrienes
and various cytokines. These substances not only mediate acuteallergic responses but also induce the late phase of inflammation bystimulating local synthesis of various chemokines such as CCL11(eotaxin) and CCL17 (TARC), which recruit T helper type 2 lymphocytes and eosinophils3–5. Repeated exposure to allergens enhances
inflammation characterized by infiltration of these cells and cancause tissue remodeling. However, how this course of responses isregulated and why it becomes exaggerated in some populations
remains unknown.
Prostaglandins, including PGD2 and PGE2, are produced in substantial amounts during allergen exposure and disease development4–7.
Prostaglandins act at specific cell surface receptors to elicit theirown actions. Whereas PGD2 acts at the prostaglandin D receptor,
PGE2 acts at four subtypes of prostaglandin E receptor, EP1–EP4(ref. 8), which are encoded by Ptger1–Ptger4. Studies examining
the function of PGD2 in allergic asthma using mice deficient in theprostaglandin D receptor found that PGD2 mediates this disease9.
This view has been supported by a genetic study in humans identifying the gene encoding PGD2 as an ‘asthma susceptibility gene’10.

If PGD2 were the only prostaglandin important in allergy, aspirin-likenonsteroidal anti-inflammatory drugs (NSAIDs) that inhibit prostaglandin synthesis would be expected to suppress allergic reactions.
However, these drugs are generally ineffective in the treatmentof allergic disorders. In contrast, they induce life-threatening attacks
in about 10% of adults with asthma11 and aggravate nasal polyposisof allergic rhinitis12. Patients who develop aspirin-induced asthma
also manifest other symptoms of immediate hypersensitivity suchas rhinorrhea, conjunctival irritation and flushing of the head andneck. These observations suggest the existence of a prostaglandindependent process that suppresses type I allergic reactions in general.
Here, we have examined mice deficient in each prostaglandin
E receptor subtype individually and have used an EP3-selective
agonist to identify this protective prostaglandin pathway. We showthat the PGE2-EP3 pathway is an important negative modulator ofallergic reactions.
RESULTS
Enhanced allergic responses in Ptger3/ mice
Because studies have described attenuation by PGE2 in various
asthmatic responses13,14, we subjected mice deficient in each individual prostaglandin E receptor subtype15–17 to the ovalbumin (OVA)–
induced asthma model and examined their responses. We backcrossed
Ptger1/, Ptger2/ and Ptger3/ mice for at least eight generations
onto the C57BL/6 background and used wild-type C57BL/6 mice ascontrols. Ptger4/ mice do not survive on the C57BL/6 backgroundbecause of the development of patent ductus arteriosus17,18; wetherefore intercrossed survivors of F2 progeny and studied the

1Department of Pharmacology and Faculty of Medicine, and 2Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University,

Kyoto 606-8501, Japan. 3Department of Pharmacology, Gifu Pharmaceutical University, Gifu 502-0003, Japan. 4These authors contributed equally to this work.
Correspondence should be addressed to S.N. (snaru@mfour.med.kyoto-u.ac.jp).
Published online 3 April 2005; doi:10.1038/ni1188

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resultant Ptger4+/+ and Ptger4/ survivors on the mixed genetic
background of 129/Ola  C57BL/6.
We used OVA-induced allergic asthma as a model of type I allergy9.
We sensitized the mice by intraperitoneal injection of OVA on days 0
and 12 and then exposed them to aerosolized OVA on days 22, 26 and
30. We then killed the mice 24 h after the last antigen challenge andcollected bronchoalveolar lavage fluid (BALF). Antigen inhalationinduced an increase in the number of inflammatory cells in BALF
in all strains of mice examined. However, the BALF of Ptger3/ micecontained significantly more cells than that of wild-type, Ptger1/
or Ptger2/ mice (Fig. 1a). The number of cells in the BALF of
Ptger4/ mice was greater than that in wild-type C57BL/6 mice but
did not differ significantly from that of Ptger4+/+ mice (Fig. 1a).
We next examined whether the exaggerated allergic inflammation in
Ptger3/ mice could be attributed to a higher serum concentration of
IgE. On day 12, after the second OVA injection, the serum concentrations of total IgE were 1,469 7 676 pg/ml and 1,802 7 325 pg/ml andthose of OVA-specific IgE were 149 7 85 U/ml and 112 7 40 U/ml in
wild-type and Ptger3/ mice, respectively (n ¼ 5 of each). On day 31,
the concentration of total IgE in Ptger3/ mice was significantly
higher than that of wild-type mice (1,706 7 542 pg/ml versus 592 7
286 pg/ml; n ¼ 5; P o 0.05). However, the concentration ofOVA-specific IgE did not differ significantly between the two groups(41 7 25 U/ml versus 72 7 28 U/ml for wild-type and Ptger3/
mice, respectively; n ¼ 5; P ¼ 0.28). Ptger3/ mice thus demonstrated exaggerated allergic inflammation, as assessed by cellular

infiltration, despite serum concentrations of antigen-specific IgE
similar to those of wild-type mice, indicating that EP3 deficiency
enhanced the allergic response at a step or steps subsequent to IgEsynthesis. We confirmed this conclusion using a passive cutaneous
anaphylaxis (PCA) test, which monitors the allergic response to fixedamounts of exogenous dinitrophenyl and dinitrophenyl-specific IgE.
The exudation reaction of Ptger3/ mice from the PCA test wassignificantly greater than that of wild-type mice in this assay (Fig. 1b).
Allergic inflammation is characterized by infiltration of eosinophilic
leukocytes and T helper type 2 lymphocytes3–5. Eosinophils constituted the main fraction of the inflammatory cells in BALF of both
Ptger3/ and wild-type mice subjected to OVA sensitization andchallenge. However, their number was significantly higher in
Ptger3/ mice than in wild-type mice (Fig. 2a). Ptger3/ micealso had more lymphocytes and neutrophils in BALF, whereas thenumbers of macrophages were similar for the two groups. Concentrations of the T helper type 2 cytokines interleukins 4, 5 and 13 in BALF
were increased by antigen challenge in both groups (Fig. 2b). However, Ptger3/ mice had much higher concentrations than did wildtype mice, whereas the concentration of the T helper type 1 cytokine
interferon-g in BALF did not differ significantly between the two
genotypes (P ¼ 0.103, wild-type versus Ptger3/). Histologicalexamination showed that lymphocytes, eosinophils and other leukocytes accumulated in the perivascular and peribronchial regions of thelungs of both groups of mice, but quantitative analysis showed thatthe cell infiltration was more extensive in Ptger3/ mice (Fig. 2c).

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Figure 2 Enhanced inflammatory cell infiltrationin the lungs of Ptger3/ mice. (a) Cell
population analysis. Eosinophils, lymphocytes,
neutrophils, and macrophages were counted in
BALF collected from OVA-sensitized wild-type or
Ptger3/ mice 24 h after the last inhalation of
OVA or saline. Data are means 7 s.e.m. (n ¼ 6).
(b) Cytokine measurement. ELISA of interleukin
4 (IL-4), IL-5, IL-13 and interferon-g (IFN-g) in
BALF collected 8 h after the last inhalation
as described in a. Data are means 7 s.e.m.
(n ¼ 6). (c) Lung histology. The lungs of OVAsensitized wild-type or Ptger3/ mice weredissected 24 h after the last inhalation of
OVA and were stained with hematoxylin andeosin (left). Scale bars, 50 mm. The area ofinflammatory cell infiltration around pulmonary
arterioles was quantified in four mice of eachgroup (right). *, P o 0.05 and **, P o 0.005,
versus OVA-challenged wild-type mice. ND, notdetected. Data are representative of at least three
independent experiments.

Neutrophils (× 10 )© 2005 Nature Publishing Group http://www.nature.com/natureimmunology

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Figure 1 Enhanced allergic responses in Ptger3/ mice. (a) Enhanced
airway inflammation in Ptger3/ mice. Wild-type (WT), Ptger1/,Ptger2/, Ptger3/, Ptger4+/+ and Ptger4/ mice were immunized andwere challenged with OVA. The total number of cells in BALF collected
24 h after the last OVA inhalation was determined. Data are means 7
s.e.m. of values from six mice of each genotype. *, P o 0.05, versus
wild-type, Ptger1/ or Ptger2/ mice. (b) Enhanced exudation reactionin the PCA test in Ptger3/ mice. Data are means 7 s.e.m. (n ¼ 5).
*, P o 0.05, versus sensitized wild-type mice. Data are representativeof at least three independent experiments.

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© 2005 Nature Publishing Group http://www.nature.com/natureimmunology

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Figure 3 Suppression of airway inflammation by an EP3 agonist. (a,b) Effects of an EP3 agonist (AE-248; 10 mg/kg), prednisolone (Pred; 5 mg/kg) anddm-PGE2 (10 mg/kg) on inflammatory cell infiltration in the lungs. Data represent total number of cells (a) and numbers of cell subsets (b) in BALF and aremeans 7 s.e.m. (n ¼ 4–8). (c) Dose-dependent effect of the EP3 agonist. AE-248 was administered at various doses to wild-type mice (n ¼ 7–21) and theeosinophils in BALF were counted. (d) Effect of time of administration of the EP3 agonist. AE-248 was administered to wild-type mice (n ¼ 6–8) on days
22, 26 and 30 either twice at 30 min before and 3 h after OVA challenge (30 min & 3 h), once at 30 min before (30 min) or once at 3 h or 12 h after
the challenge. (e,f) Effects of the EP3 agonist on mucus secretion (e) and airway reactivity (f). Wild-type mice were sensitized with OVA and were treatedwith AE-248 or vehicle 3 h after each challenge with OVA or saline. Data represent the proportion of PAS+ cells in the airway epithelium (e) or assessmentof methacholine (MCh)–induced bronchoconstriction (f) 24 h after the last antigen challenge. (f) Dose-response curves (left) and responses to the maximum
dose of Mch (100 mg/ml; right). Each symbol represents a different mouse. Data are means 7 s.e.m. (n ¼ 6–16). *, P o 0.05, versus vehicle-treated,
OVA-challenged. Data are representative of or means 7 s.e.m of three to five independent experiments.

Thus, the allergic inflammation in Ptger3/ mice was much more
pronounced than that in wild-type mice.
Antiallergic effects of an EP3 agonist in vivo
Our results suggested that PGE2 acts at the EP3 receptor duringelicitation of allergy and downregulates allergic reactions. To verify thisconclusion, we examined the effects of an EP3-selective agonist,
ONO-AE-248 (AE-248)19, and of a nonselective stable PGE2 analog,
16,16-dimethyl-PGE2 (dm-PGE2), on OVA-induced asthma in wildtype mice. We administered these compounds subcutaneously to
OVA-sensitized mice beginning on day 21. As a positive control, we
administered the glucocorticoid prednisolone, the drug used mostprevalently for clinical asthma therapy, once a day from day 21 to day
30. We collected BALF 24 h after the last OVA challenge and assessed
the effects of these compounds on cell infiltration. The EP3 agonist
AE-248 significantly inhibited the antigen-induced infiltration of
inflammatory cells (Fig. 3a). This inhibitory effect was absent fromPtger3/ mice, confirming that it was mediated by EP3. In contrast,
dm-PGE2 did not significantly affect cell infiltration compared withthat resulting from vehicle treatment at this dose (P ¼ 0.219). The
inhibitory effect of AE-248 at a dose of 10 mg/kg was similar to thatobtained with prednisolone at a dose of 5 mg/kg for reducing
asthmatic symptoms. Cell population analysis showed that theEP3 agonist significantly suppressed infiltration of eosinophils,
lymphocytes and neutrophils to an extent similar to that noted withprednisolone; although prednisolone also inhibited macrophage infiltration, AE-248 did not (Fig. 3b). The suppressive effect of AE-248 on

526eosinophil infiltration was apparent at a dose of 0.1 mg/kg and wassignificant at doses of 1 mg/kg and 10 mg/kg (Fig. 3c).
We next attempted to optimize the regimen of AE-248 treatment
with regard to suppression of the allergic reaction. We limited
administration of the compound to the days of antigen challenge
only (days 22, 26 and 30), either twice at 30 min before and 3 h after
OVA challenge or once at either 30 min before or 3 or 12 h after thechallenge. We compared the antiallergic effects at 24 h after the last
antigen inhalation. We noted suppression of inflammatory cell infiltration similar to that obtained in the original treatment protocol inmice treated with the EP3 agonist either twice at 30 min before and3 h after or once 3 h after antigen challenge on days 22, 26 and 30 only
(Fig. 3d). Treatment with the compound once at 30 min before
antigen challenge tended to reduce the number of inflammatory cells(P ¼ 0.165, 30 min before versus vehicle), but treatment 12 hafter challenge had no effect. In addition to the anti-inflammatory
effect, administration of the EP3 agonist 3 h after OVA challenge
tended to reduce the number of periodic acid Schiff (PAS)–positive
cells in the lungs and airway hypersensitivity (Fig. 3e,f). These
results indicate that administration of the EP3 agonist suppresses
allergic inflammation.
EP3 agonist suppresses allergen-induced mediator release
Our results showed that PGE2 acts at EP3 during allergy to suppress
allergic inflammation. To provide mechanistic insight into this EP3mediated action, we examined the effects of the EP3 agonist on
antigen-induced allergic mediator release in vitro. We isolated cells

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Figure 4 Inhibition by the EP3 agonist of antigen-induced mediator release.
(a,b) Pieces of lung tissue obtained on day 19 from OVA-sensitized wild-type(a) or Ptger3/ (b) mice were challenged in vitro with OVA in the absence() or presence of indomethacin or PGE2 or the prostaglandin E receptor–
selective agonists ONO-DI-004 (EP1), ONO-AE1-259 (EP2), ONO-AE-248(EP3) or ONO-AE1-329 (EP4). Histamine release was then measured.
(c,d) Lung tissue from wild-type (c) or Ptger3/ (d) mice was incubatedas described for a and b, after which cysteinyl leukotriene (CysLT) release
was measured. Data are means 7 s.e.m. (n ¼ 8–10). #, P o 0.05, versus
vehicle-treated; *, P o 0.05, versus indomethacin-treated, OVA-challenged
lung. Data are means 7 s.e.m of at least three independent experiments.

from the lungs from OVA-sensitized wild-type mice on day 19 after
sensitization, which we then challenged by adding OVA to the cell
cultures, and then measured the release of histamine and cysteinyl
leukotrienes. The OVA challenge induced a significant increase inhistamine release from the sensitized lung tissue cells (Fig. 4a). Thiseffect of OVA was augmented by pretreatment of the tissue withindomethacin, and this effect of indomethacin was blocked by PGE2
or AE-248 but not by agonists selective for EP1, EP2 or EP4.
To confirm the function of EP3 in this phenomenon, we repeatedthe experiments with lung tissue from OVA-sensitized Ptger3/ mice.
Stimulation of the tissue with OVA alone induced the release of
a substantially more histamine than that found in wild-type
mice. Moreover, this response was not affected by indomethacin,
PGE2 or AE-248 (Fig. 4b). We obtained essentially identical resultsby analysis of OVA-induced cysteinyl leukotriene release from lungtissue of wild-type and Ptger3/ mice (Fig. 4c,d). Given that most of
the histamine and cysteinyl leukotrienes released in response to
antigen challenge is derived from mast cells, these results suggestthat PGE2 acts at EP3 in mast cells to suppress mediator release. The
augmentation of mediator release by indomethacin further indicatesthat such inhibition occurs in a feedback way through endogenous
PGE2 production.
EP3 agonist inhibits allergy-related gene expression
Although our results showed that the EP3 agonist inhibits mediatorrelease, this effect alone cannot explain the action of the compoundin vivo, given that the agonist suppressed allergic inflammation evenwhen administered 3 h after antigen inhalation (Fig. 3d). To further
address the mechanism of action of the EP3 agonist, we did DNA
microarray analysis. We subjected OVA-sensitized wild-type mice(n ¼ 3 per group) to inhalation of either OVA or saline on days 22,

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26 and 30 and injected the OVA-challenged mice with either vehicle or
the EP3 agonist 3 h after each antigen challenge; we injected thesaline-challenged control mice with vehicle instead of the EP3 agonist.
We isolated lung tissue from all mice 24 h after the last inhalation andsubjected it to microarray analysis with the MG-U74Av2 gene chipthat includes probes for 12,426 mouse genes.
We then compared expression of each gene in each of the threecontrol mice with that in each of the three vehicle-treated, OVAchallenged mice using Affymetrix GeneChip Expression Analysis
software. We considered a change in the expression of a gene relevant
if it differed in more than six of the nine pairs. The expression of 279
of the 12,426 genes was increased considerably and that of 86 genes
was decreased considerably in the lungs of the OVA-challenged micecompared with that in the lungs of control mice (Fig. 5a). We
then compared gene expression in the EP3 agonist–treated, OVAchallenged mice with that in the vehicle-treated, OVA-challenged micein a similar way. Of the 279 genes that showed an antigen-induced
increase in expression, 195 genes showed a decrease in expression
in response to the EP3 agonist in more than six of the ninecomparison pairs. The expression of the remaining 84 genes was
also decreased by the agonist but in fewer than six of the nine pairs.
Similarly, of the 86 genes that showed an antigen-induced decrease in
expression, 38 genes showed an increase in expression in response to
the EP3 agonist in more than six of the nine pairs (Supplementary
Table 1 online).
Among the genes upregulated by antigen challenge were those for
immunoglobulins (68 of 279) and for receptors or markers oflymphocytes and other immune cells (18 of 279). The genes forchemokines or chemokine receptors accounted for 19 of these 279
genes. The upregulation of the first two groups of genes may represent
recruitment of lymphocytes and other inflammatory cells, and theinduction of the last group of chemokine and chemokine receptor
genes may be responsible for such recruitment. The last group
included genes encoding CCL17 (TARC), CCL11 (eotaxin) andCCL7 (MCP-3), the expression of each of which was upregulated
significantly after antigen challenge and whose induction was completely inhibited by the EP3 agonist. We confirmed these changes by
RT-PCR analysis (Fig. 5b). Each of these chemokines has been linked
to allergic asthma and allergic rhinoconjunctivitis4,5. These results
suggest that the EP3 agonist suppresses allergic inflammation throughinhibition of the induction of chemokine genes.
We also detected induction by antigen exposure and suppression by
the EP3 agonist of the expression of other genes involved in tissue
metaplasia, remodeling and resolution20–24, including those encoding
Clca3 (also called Gob-5), Muc5ac, MMP-12, ADAM-8 and Alox15(Supplementary Table 1 online). Muc5ac encodes a core protein oflarge mucin, and Gob-5 is involved in mucus secretion in theepithelium20. Although the decrease in the number of PAS-positive
cells by EP3 agonist was limited (Fig. 3e), suppression of induction of
the genes encoding Gob-5 and Muc5ac indicates that the EP3 agonist
treatment substantially attenuates goblet cell formation. In contrast,
although transcripts of the genes encoding enzymes that contribute to
PGE2 biosynthesis, including cyclooxygenases 1 and 2 and microsomal
prostaglandin E synthases 1 and 2, were present in normal lung tissue,
their abundance was not substantially affected by OVA challengewith or without treatment with the EP3 agonist. Among the prostaglandin E receptor subtypes, expression of only the gene encoding
EP4 was increased substantially by OVA challenge in a way that wassensitive to the EP3 agonist. These results indicate that EP3 agonist
administered 3 h after the challenge inhibits allergen-induced gene
expression changes.

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Clca3

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Localization of EP3 in the lungs
We next examined the localization of EP3 in the lungs. EP3 mRNAwas detected in the lungs of wild-type mice by RT-PCR analysis, andits abundance was not affected by OVA challenge (Fig. 6a). Consistently, when we enriched BALF from challenged mice for CD11b+
cells, including macrophages, neutrophils and eosinophils, or CD4+ T
cells, and analyzed the cells by RT-PCR, we detected little expression of
EP3 mRNA in these cells (Fig. 6a). Given that antibodies to mouse
EP3 suitable for immunohistochemistry were not available, we used
Ptger3/ mice in which the b-galactosidase gene was ‘knocked-in’at the EP3 gene locus to examine EP3 localization. Staining forb-galactosidase activity showed positive signals in airway epithelial
cells of the engineered mice but not in those of wild-type mice
(Fig. 6b). In contrast, consistent with the PCR analysis reported
above, b-galactosidase signals were barely detectable in the cells
infiltrating in the lung parenchyma (Fig. 6b). To determine thepossible relationship between EP3 localization and its regulation ofgene expression, we examined by immunohistochemistry the site ofallergen-induced chemokine production. CCL17 immunoreactivity
was induced by OVA challenge in the airway epithelial cells of
sensitized wild-type mice, and this effect was inhibited by treatment
with the EP3 agonist (Fig. 6c). We obtained essentially identical resultsfor CCL11 expression (data not shown). These data suggested that
EP3 is constitutively present on the surface of airway epithelial cells
and that EP3 signaling in these cells inhibits the allergen-induced
expression of chemokine genes.

DISCUSSION
Allergic inflammation is initiated by mast cell activation as acutevascular responses and develops into inflammation characterized by
infiltration of T helper type 2 lymphocytes and eosinophils, leading totissue metaplasia and remodeling. This process of progression issimilarly seen in bronchial asthma and allergic rhinitis and conjunctivitis3–5. Using OVA–induced allergic asthma as a model, we haveshown here that PGE2-EP3 signaling negatively regulates this progression of allergic inflammation. This is consistent with published findings that inhaled PGE2 suppresses allergen-induced inflammation inhuman asthmatic patients13,25. Given the similarity of allergic rhinoconjunctivitis to asthma, it is very likely that EP3 signaling also
modulates the process of allergic rhinitis and allergic conjunctivitis.
The PGD2–prostaglandin D receptor pathway mediates allergic
responses9. That pathway and the PGE2-EP3 pathway thus exert
opposing actions in allergic inflammation. Furthermore, mice deficient
Saline

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Figure 5 Effects of OVA challenge and the EP3 agonist on gene expression
in the lungs. (a) Microarray analysis. Wild-type mice were sensitized with
OVA and were treated with either AE-248 or vehicle 3 h after each challengewith OVA or saline. Lung tissue was isolated 24 h after the last challenge,
and polyadenylated RNA was assessed by microarray analysis. Top bar,
proportion of genes whose expression was increased (red) or decreased(blue) in the vehicle-treated, OVA-challenged mice compared with that in
the vehicle-treated, saline-challenged mice. Bottom bars, effects of theEP3 agonist on the expression of genes affected by OVA challenge. Blue or
turquoise, number of genes that decreased in more or less, respectively,
than six of nine pairs by EP3 agonist treatment; red or orange, numberof genes that increased in more or less, respectively, than six of ninepairs by EP3 agonist treatment. (b) RT-PCR analysis of the expression of
Ccl17 (TARC), Ccl11 (eotaxin), Clca3 (Gob-5) and Gapd (glyceraldehyde
phosphodehydrogenase; loading control) in the lungs of vehicle-treated,saline-challenged (Saline), vehicle-treated, OVA-challenged (OVA) or EP3
agonist–treated, OVA-challenged (OVA+EP3) mice. Each lane represents
one mouse. Data are representative of two or three experiments.

Ca Increased (2.2%)Total genes in MG-U74Av2 gene chip (12,426 genes)Decreased (0.7%)

Ptger3
Gapd
Ptger3 –/–

b

WT
X-gal

X-gal

Airway epithelium

Figure 6 Localization of EP3 and CCL17 in the lungs. (a) RT-PCR analysis
of EP3 (Ptger3) mRNA in lungs isolated from OVA-sensitized wild-type
or Ptger3/ mice 24 h after the last challenge with either saline or OVA(left) and in CD11b+ cells and CD4+ cells from BALF (right). Each lane
represents one mouse. Gapd, loading control. (b) Histochemical staining forEP3 (X-gal). Lung tissue from Ptger3/ mice expressing the b-galactosidase
gene at the Ptger3 locus was stained for b-galactosidase activity withthe substrate X-gal. Arrows (top left) indicate positive signals (blue) on
airway epithelial cells. Arrowheads (bottom row) indicate accumulation of
inflammatory cells. Sections were counterstained with eosin (red). A section
sequential to the X-gal staining (bottom left) was stained with hematoxylin
and eosin (bottom right). Scale bars, 50 mm. (c) OVA-induced expressionof CCL17 in airway epithelial cells and its suppression by the EP3 agonist.
The lungs of OVA-sensitized wild-type mice were isolated 24 h after the lastchallenge with saline or OVA; the mice were also treated with AE-248 orvehicle 3 h after each challenge. The tissue was then immunostained for
CCL17. Arrows indicate positive signals. Scale bars, 50 mm. Data arerepresentative of at least three experiments.

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ARTICLESin the PGI2 receptor have higher total and antigen-specific IgE
concentrations than those in wild-type mice and manifest augmented
asthmatic responses26. Cyclooxygenase metabolites and their cognate
receptors thus participate in various steps of allergic asthma, with the
PGD2–prostaglandin D receptor pathway and the PGE2-EP3 pathway
functioning locally at the site of allergy to mediate and to suppress
allergic inflammation, respectively, and the PGI2 receptor pathway
contributing to the primary immune response for IgE production.
PGD2 is produced in substantial amounts by activated mast cells, andour preliminary immunohistochemical study showed that microsomal
prostaglandin E synthase 1 is highly expressed in the airway epithelial
cells of antigen-challenged mice. These observations suggest that thegeneral ineffectiveness of NSAIDs in allergic diseases might be due toinhibition of all of these pathways. Expression in the lung of the geneencoding arachidonate 15-lipoxygenase was enhanced considerably
after OVA challenge and was suppressed by the treatment with
the EP3 agonist (Supplementary Table 1 online). Arachidonate 15lipoxygenase is involved in formation of lipoxins, which are suggested
to work in diminution of various forms of inflammation in the airway
and gastrointestinal tract24,27–29. Because leukocytes are chief sources ofthis enzyme, a decrease in the expression of this gene by the EP3
agonist may be due to inhibition of recruitment of leukocytes to thelung. Thus, various eicosanoids work as anti-inflammatory messengers
in different phases of inflammation.
The aggravation of allergic diseases by aspirin-like NSAIDs isknown as aspirin intolerance and occurs in patients with asthma
and/or nasal polyposis11,12. Despite a high incidence of aspirin
intolerance in these patients, the underlying mechanism has remained
controversial. A prevailing hypothesis has been that NSAIDs shunt thesubstrate arachidonic acid from the cyclooxygenase pathway to thealternative lipoxygenase pathway, resulting in enhanced production ofproinflammatory and proasthmatic leukotrienes. Our finding thatallergic reactions are enhanced in Ptger3/ mice without inhibition of
cyclooxygenase challenges this hypothesis and suggests that the PGE2EP3–mediated suppression of allergic inflammation is a long-soughttarget mechanism of aspirin intolerance. Although it remains unclear
why patients with aspirin-induced asthma are more vulnerable toinhibition of prostaglandin synthesis, it is likely that these people
depend more on protection by the PGE2-EP3 pathway than dopatients without aspirin-induced asthma.
We have shown that endogenous PGE2 acting at EP3 has animportant feedback function in allergic inflammation and thatadministration of an EP3-selective agonist substantially suppresses
elicitation of allergic inflammatory reactions. Notably, the EP3 agonist
was most effective in suppressing allergic inflammation whenadministered 3 h after antigen challenge. Allergic inflammationconsists of two phases: the early-phase reaction, which develops andsubsides within 60 min, and the late phase reaction, which starts at
3–6 h, peaks at 6–8 h and subsides 24 h after the antigen challenge.
Local synthesis of chemokines and cytokines is crucial for the latephase reaction, and the main site of their synthesis is the epithelium3–5.
Notably, EP3 is expressed in airway epithelial cells, where the chemokines CCL11 and CCL17 are also expressed, and administration of theEP3 agonist 3 h after the challenge can inhibit the allergen-induced
synthesis of these chemokines. A variety of mediators released from
activated mast cells can induce expression of the genes encoding
chemokines in the epithelium4,5,30–32, and gene expression is detectable
3 h after antigen inhalation33. In our preliminary study, we found that
the EP3 agonist administered 2 or 3 h after the antigen challenge wasmost effective, whereas agonist administered 1 or 4 h after thechallenge was less effective in suppressing allergic inflammation. The

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time when treatment with the EP3 agonist is most effective thus
coincides with the time of chemokine gene expression. These observations collectively suggest that PGE2 acts at EP3 on airway epithelial cells
and regulates the extent of the late-phase response by attenuating theexpression of these chemokine genes. Notably, the prostaglandin
D receptor is also expressed in airway epithelial cells, where it acts toenhance allergic inflammation9.
Ptger3/ mice demonstrate not only exaggerated asthmatic reactions but also enhancement of the PCA reaction, which is caused
directly by allergic mediators released from mast cells. Consistent withthat observation, we found that the EP3 agonist significantly suppressed the release of histamine and cysteinyl leukotrienes fromantigen-sensitized lung tissue. The effects of PGE2 on the release of
mediators from mast cells have remained unclear. Mast cells derived
from the bone marrow of mice deficient in EP3 have demonstrated theimportance of PGE2-EP3 signaling in potentiating antigen-stimulateddegranulation34. We have detected the EP3 expression in bonemarrow–derived mast cells from wild-type mice and found thatadministration of the EP3 agonist indeed augmented the antigeninduced histamine release (data not shown). In addition, PGE2 andprostaglandin E analogs were shown to enhance mediator release by
an EP1- and/or EP3-dependent mechanism in the MC-9 mast cellline35. In contrast, another study showed that the EP2 and EP3 agonist
misoprostol inhibited mediator release from rat peritoneal mast cells
triggered by antibody to IgE36. Given the existence of several subclassesof mast cells that differ in their origin and functions37, these different
responses to PGE2 might reflect the differences in cell subclass. Bonemarrow–derived mast cells are considered the presumptive in vitro
counterpart of in vivo–differentiated mucosal mast cells and are
different from mast cells working in allergic reactions in the lung38.
Our findings suggest that a subclass of mast cells sensitive to EP3
stimulation has a chief function in OVA-induced allergic responses.
Alternatively, an EP3-dependent indirect mechanism may operate tosuppress mast cell activation in sensitized lungs in vivo.
The EP3 agonist is as potent as the glucocorticoid prednisolone in
suppressing allergic inflammation and is able to exert this inhibitory
effect at doses in the mg/kg range. Signaling by EP3 is mediated mainly
by coupling to the inhibitory G protein Gi and results in inhibition of
the generation of cyclic AMP. This signaling is elicited at PGE2 or EP3
agonist concentrations as low as 10 pM (ref. 39). The antiallergic effectof low doses of the EP3 agonist in the present study might therefore be
mediated by this signaling pathway. In addition inhibiting cyclic AMP
production, PGE2 acting at EP3 induces an increase in the intracellular
free calcium concentration at doses of 10 nM to 1 mM, an effectmediating such EP3 actions as uterine contraction. Given these facts
and also given that the proinflammatory action of PGE2 is exerted by
the EP2 and/or EP4 receptor, the EP3 agonist can circumvent theproinflammatory effects of PGE2 by its selectivity and also its own
adverse effects by administration of a low dose. Its unique property of
being effective when administered after antigen challenge, that is, after
appearance of early signs of allergy, may make an EP3 agonist acandidate for a new type of antiallergy drug.
METHODS
Animals and compounds. Ptger1/, Ptger2/, Ptger3/ and Ptger4/ mice
were generated as described15–17. Mice were maintained on a 12-hour–12-hour
light–dark cycle in specific pathogen–free conditions. All experimental procedures were approved by the Committee on Animal Research of Kyoto
University Faculty of Medicine (Kyoto, Japan). The agonists ONO-DI-004,
ONO-AE1-259, ONO-AE-248 and ONO-AE1-329 were supplied by ONO
Pharmaceutical; the ligand-binding specificities of these compounds for each529

ARTICLES© 2005 Nature Publishing Group http://www.nature.com/natureimmunology

prostaglandin E receptor subtype have been described16. We assessed thepharmacokinetic properties of AE-248 by monitoring the biological responseto this compound administered intravenously; injection of rats with a bolus of
AE-248 at a dose of 10 mg/kg resulted an increase in blood pressure of in
10 mmHg that persisted for 10 min (data not shown).
Analysis of asthmatic responses. Mice were immunized with OVA and werechallenged by OVA inhalation as described9. BALF was collected at 8 and 24 hfor cytokine measurement and cell analysis, respectively. The concentration ofcytokines was measured using enzyme-linked immunosorbent assay (ELISA)
kits (Endogen). The number of white blood cells was determined witha hemocytometer, and differential leukocyte counts were made using asmear stained with Diff-Quik solution (International Reagents). The serum
concentrations of total and OVA-specific IgE were measured by sandwich
ELISA as described40.
For examination of the effects of AE-248, dm-PGE2 (Sigma) and prednisolone (Sigma) on allergic inflammation in the lung, AE-248 (0.1–10 mg/kg) anddm-PGE2 (10 mg/kg) were administered subcutaneously to OVA-sensitizedmice twice a day beginning on day 21, the day before the first OVA challenge;
they were also administered in an additional dose 3 h after antigen challenge ondays 22, 26 and 30. Prednisolone (5 mg/kg, subcutaneously) was administeredonce a day from day 21 to day 30. For time course study, administration of the
AE-248 was limited to the days of antigen challenge only (days 22, 26 and 30)
and the following four treatment protocols were used: twice at 30 min before
and 3 h after OVA challenge; once at 30 min before challenge; once at 3 h after
the challenge; and once at 12 h after the challenge. BALF was collected 24 h after
the last challenge and the accumulation of inflammatory cells was assessed.
For measurement of airway resistance, mice treated with the EP3 agonist or
vehicle (saline) were anesthetized 24 h after the last antigen challenge and wereventilated at a tidal volume of 8 ml and a frequency of 2.5 Hz with a Flexivent
SAV system (Scientific Respiratory Equipment) as described41. Each mouse
was challenged by inhalation for 30 s of increasing doses of methacholine(1–100 mg/ml), with the doses administered at intervals of 5 min. Airway
resistance was monitored with a heated pneumotachograph, and the peak value
induced by each challenge was recorded using an oscillation technique.
Histological analysis. At 24 h after the last antigen challenge, lungs weredistended by injection through the trachea of 10% formalin in 0.1 M sodium
phosphate, pH 7.4, and then were excised. After immersion in fresh fixative for
24 h, the tissue was embedded in paraffin, sliced into sections 3 mm in thickness
and stained with hematoxylin and eosin or with PAS solution. Quantitative
analysis was done on microscopic images of three 100 magnification fields
per slide and three slides per mouse for each stain with the use of ImagePro
Plus software (Media Cybernetics). Perivascular inflammatory cell infiltration
was quantified as the total area of inflammatory cell accumulation relative tothe perimeter of the blood vessel. For quantification of mucus-producing cells,
the ratio of the PAS-positive area to the total area of the bronchial epithelium
was determined. Slides were analyzed by investigators ‘blinded’ to sample
identity and were presented in a random order for each examination.
PCA test. PCA was induced as described42. Mice were injected intradermally on
the back with 20 ml of antibody to dinitrophenyl IgE (0.3 mg/ml). After 24 h,
0.25 mg of dinitrophenyl-conjugated bovine serum albumin and 1.25 mg ofEvans blue in 0.25 ml of saline were injected intravenously to elicit PCA.
Vascular permeability was evaluated by measurement of the amount ofdye extravasated during the 30 min immediately after antigen challenge.
Control mice were injected with the antigen and Evans blue without priorinjection with IgE.
Histamine and cysteinyl leukotriene release in vitro. OVA-sensitized mice
were killed by exsanguination on day 19 and the lungs were immediately
removed and placed in Tyrode’s solution. After removal of bronchial tissue andblood vessels, the lungs were chopped into fragments 0.5 mm3 in size, and thefragments were washed and blotted dry. The tissue fragments (100 mg, wetweight) were suspended in Tyrode’s solution at a final volume of 2 ml, wereshaken for 10 min at 37 1C in the absence or presence of indomethacin(10 mM), PGE2 (10 mM) or prostaglandin E receptor agonist (10 mM for EP1,EP2 and EP3 agonists; 1 mM for the EP4 agonist), and then were challenged for530

15 min with OVA (10 mg/ml). The tissue and medium were separated byfiltration and histamine was extracted from the tissue and the medium andassayed as described43. Histamine release is expressed as a percentage of total
tissue histamine. Cysteinyl leukotrienes were extracted from the incubation
medium with the use of a Sep-Pak C18 cartridge44 and were assayed with an
ELISA kit for LTC4, LTD4 and LTE4 (Amersham Pharmacia Biotech).
DNA microarray analysis. At 3 h after each antigen challenge, OVA-challenged
mice were injected with either vehicle or AE-248; saline-challenged control
mice were injected with vehicle instead of the EP3 agonist. Lung tissue wasisolated from all mice 24 h after the last inhalation. Polyadenylated RNA wasprepared from lung tissue as described8 and was subjected to microarray
analysis with the MG-U74Av2 gene chip (Affymetrix). Expression of each genein each of the three control mice was then compared with that in each of thethree vehicle-treated, OVA-challenged mice using Affymetrix GeneChip Expression Analysis software (Suite, version 4.0, 3  3 ¼ 9 comparison pairs). Achange in the expression of a gene was considered relevant if it differed in morethan six of the nine pairs. Comparison of gene expression in the EP3 agonist–
treated, OVA-challenged mice with that in the vehicle-treated, OVA-challenged
mice was done in a similar way.
RT-PCR. A GeneAmp PCR system 9700 (Perkin Elmer Applied Biosystems) wasused for PCR. Primers (sense and antisense, respectively) were 5¢-ATCCTCGTG
TACCTGTCACAGCGACGCTGG-3¢ and 5¢-TGCTCAACCGACATCTGATTGAAGATCATT-3¢ for Ptger3 (EP3); 5¢-CAGGAAGTTGGTGAGCTGGTATA-3¢
and 5¢-TTGTGTTCGCCTGTAGTGCATA-3¢ for Ccl17 (TARC); 5¢-AGAGCTCCACAGCGCTTCTATT-3¢ and 5¢-GGTGCATCTGTTGTTGGTGATT-3¢ for
Ccl11 (eotaxin); 5¢-CACAACACTAAGGTGGCCTACCTCCAAG-3¢ and 5¢-AC
TTGGAGTGAGGTGTTGAAGTGGTCCCT-3¢ for Clca3 (Gob-5); and 5¢-TGA
AGGTCGGTGTGAACGGATTTGGC-3¢ and 5¢-CATGTAGGCCATGAGGTCCA
CCAC-3¢ for Gapd (glyceraldehyde-3-phosphodehydrogenase).
Cell preparation. AutoMACS (Miltenyi Biotec) was used for preparation of the
subpopulations of inflammatory cells in BALF. BALF was recovered 24 h after
last challenge from OVA-challenged mice. Samples were enriched for CD11b+
and CD4+ cells by auto-MACS with antibody to CD11b or to CD4 MicroBeads,
respectively (Miltenyi Biotec). Cells in each subpopulation were examined bysmear staining using Diff-Quik solution.
Histochemistry. For staining to assess b-galactosidase activity, freshly isolated
lung tissue was embedded in Tissue-Tec optimum cutting temperature compound (Sakura Finetechnicals), was frozen at 80 1C and was cut into sections
14 mm in thickness using a cryostat. The sections were fixed for 30 min at 4 1C
with 2% paraformaldehyde in PBS, were washed with PBS and were incubatedfor 48 h at 30 1C with a solution containing 5-bromo-4-chloro-3-indolyl-b-Dgalactopyranoside (X-gal; 1 mg/ml), 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6,
2 mM MgCl2, 0.01% SDS and 0.02% Nonidet P-40. For immunohistochemistry, the lungs were first fixed by systemic perfusion with 4% paraformaldehyde
in PBS and then were dissected. The dissected tissue was fixed further for 4 h
and then was incubated overnight at 4 1C with 15% sucrose. Sections with athickness of 14 mm were then prepared and, after inhibition of endogenous
peroxidase activity with Peroxo-Block (Zymed), were incubated overnightat 4 1C with goat anti–mouse TARC (AF529; R&D Systems). Immune
complexes were detected by incubation for 1 h at 25 1C with horseradish
peroxidase–conjugated secondary antibodies (Zymed) followed by exposure
to 3,3¢-diaminobenzidine.
Statistical analysis. Data are presented as means 7 s.e.m. and were analyzed by
the unpaired t-test, Mann-Whitney test, or one-way or two-way analysis ofvariance followed by Dunnett’s test with the use of Prism 3.0 and SPSS 10.1
software. A P value of less than 0.05 was considered statistically significant.
GEO accession number. Microarray data, GSE2276.
Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTS
We thank ONO Pharmaceutical for prostaglandin E receptor–selective
compounds; T. Fujiwara for animal care; T. Arai and Y. Kitagawa for help in

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preparation of the manuscript; and S. Matsui for help in statistical analyses.
Supported in part by the Program for the Promotion of Fundamental Studies in
Health Sciences of the Pharmaceuticals and Medical Devices Agency of Japan;
Grants-in-Aid for Scientific Research from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan; and ONO Research Foundation.

© 2005 Nature Publishing Group http://www.nature.com/natureimmunology

COMPETING INTERESTS STATEMENT
The authors declare competing financial interests (see the Nature Immunology
website for details).
Received 29 November 2004; accepted 25 February 2005
Published online at http://www.nature.com/natureimmunology/
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