Prostaglandin E2, an immunoactivator.

Abstract

Diseases caused by immune inflammation, such as rheumatoid arthritis, multiple sclerosis, and Crohn's disease, are intractable diseases to which novel therapeutics are highly demanded. Prostaglandin (PG) E(2) is the most ubiquitously produced PG with various actions. PGE(2) has been traditionally regarded as an immunosuppressant based on its inhibition of T cell activation in vitro. However, in vivo relevance of the immunosuppressant action of PGE(2) has remained obscure. Recently, several groups including ourselves have made unexpected findings that PGE(2) facilitates expansion of the Th17 subset of T helper cells of both human and mouse through elevation of cAMP via PGE receptors EP2 and EP4. We have further found that PGE(2) can induce and not suppress Th1 differentiation under certain conditions, again, through EP2 and EP4. Given the putative roles of these Th subsets in immune diseases such as the above, these findings suggest that, on the contrary to the traditional view, PGE(2) functions as a mediator of immune inflammation. Consistently, administration of an EP4 antagonist could suppress disease progression and development of antigen-specific Th17 cells in mice subjected to experimental allergic encephalomyelitis and contact hypersensitivity. In this perspective, we review these findings and discuss the prospect of EP4 antagonists as immunomodulatory drugs.

Full text

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1
J Pharmacol Sci 112, 1 – 5 (2010)Journal of Pharmacological Sciences
©2010 The Japanese Pharmacological Society
Helper T cell (Th) subsets and immune diseases
Immune diseases are caused by derangement of the
immune system. Normally, when foreign antigens such
as various pathogens invade into a host, they are taken
up by dendritic cells (DCs). DCs digest these antigens,
migrate to regional lymph nodes, and present processed
antigens to naïve T cells as a MHC-peptide complex.
There are two types of naïve T cells, CD4 + T cells (helper
T cells, Th) and CD8 + T cells (cytotoxic T cells, Tc). De-
pendent on types of cytokines to which they are exposed
upon antigen presentation, CD4 + T cells differentiate
into three distinct subsets of effector T cells, termed
Th1, Th2, and Th17 (1). Th1, Th2, and Th17 cells are
characterized by the cytokines they produce, interferon
(IFN)- γ , interleukin (IL)-4, and IL-17, respectively; and
they use these cytokines and facilitate pathogen elimi-
nation in a way specific to each subset (Fig. 1). IFN- γ
from Th1 cells induces activation of macrophages and
promotes elimination of pathogens. IL-4 from Th2 cells
instructs B cells to produce IgG1 and IgE type of anti-
bodies that bind to specific pathogens for elimination.
IL-17 from Th17 cells promotes tissue inflammation to
eliminate certain types of extracellular pathogens (2, 3).
Thus, these Th subsets primarily serve for host defense.
However, when activation of these Th cells is excessive
or aberrantly diverted to self-antigens, they attack host
tissues and induce various forms of immune diseases
(1) (Fig. 2). The close link between Th2 cells and atopy
such as atopic dermatitis and bronchial asthma is well
recognized, and involvement of Th17 cells or both Th1
and Th17 cells in immune diseases, such as rheumatoid
arthritis, multiple sclerosis, and Crohn’s disease, is indi-
cated by experiments in animal models of these diseases,
such as experimental autoimmune encephalomyelitis,
collagen-induced arthritis, and experimental colitis, as
well as accumulation of these Th cells and expression of
their signature cytokines in the lesion of the respective
*Corresponding author. snaru@mfour.med.kyoto-u.ac.jp
Published online in J-STAGE on January 6, 2010 (in advance)
doi: 10.1254/jphs.09R03CP

Invited article
Prostaglandin E 2 , an Immunoactivator
Daiji Sakata 1 , Chengcan Yao 1 , and Shuh Narumiya 1, *
1Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto 606-8501, Japan
Received October 3, 2009; Accepted November 16, 2009
Abstract. Diseases caused by immune inflammation, such as rheumatoid arthritis, multiple
sclerosis, and Crohn’s disease, are intractable diseases to which novel therapeutics are highly
demanded. Prostaglandin (PG) E 2 is the most ubiquitously produced PG with various actions.
PGE 2 has been traditionally regarded as an immunosuppressant based on its inhibition of T cell
activation in vitro. However, in vivo relevance of the immunosuppressant action of PGE 2 has
remained obscure. Recently, several groups including ourselves have made unexpected findings
that PGE 2 facilitates expansion of the Th17 subset of T helper cells of both human and mouse
through elevation of cAMP via PGE receptors EP2 and EP4. We have further found that PGE 2 can
induce and not suppress Th1 differentiation under certain conditions, again, through EP2 and EP4.
Given the putative roles of these Th subsets in immune diseases such as the above, these findings
suggest that, on the contrary to the traditional view, PGE 2 functions as a mediator of immune
inflammation. Consistently, administration of an EP4 antagonist could suppress disease progres-
sion and development of antigen-specific Th17 cells in mice subjected to experimental allergic
encephalomyelitis and contact hypersensitivity. In this perspective, we review these findings and
discuss the prospect of EP4 antagonists as immunomodulatory drugs.
Keywords: prostaglandin (PG) E 2 , PGE receptor subtype, helper T cell subset, dendritic cell,
immune inflammation

Current Perspective

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2 D Sakata et al
diseases (1 – 3). It is therefore believed that suppression
of excessive generation of these Th subsets might be
a means to treat various immune diseases. Currently,
a major focus in this area is elucidation of regulatory
mechanisms responsible for differentiation and expansion
of Th17 cells, because this Th subset is a new-comer, yet
is involved in many immune diseases.
Prostaglandin (PG) E 2 , an immunosuppressant: a
traditional view
PGE 2 is the most ubiquitously produced PG under
both physiological and pathophysiological conditions
(4, 5) and is long known as an immunosuppressant. This
is partly because it inhibits production of inflammatory
cytokines such as IL-1 β and TNF α by macrophages and
also because of its well-documented inhibitory action
on Th1 differentiation (6). It was already known in the
1980’s that PGE 2 is produced by APCs, inhibits produc-
tion of IL-2 and IFN- γ, and suppresses proliferation of
murine as well as human T cells in vitro (7, 8). Betz and
Fox (9) then examined the effect of PGE 2 on cytokine
production from Th1, Th2, and Th0 clones and found
that PGE 2 inhibited production of IL-2 and IFN- γ , two
Th1 cytokines, while it spared production of the Th2
cytokines, IL-4 and IL-5. This differential action of PGE 2
on Th1 and Th2 cells has been confirmed by many stud-
ies (10, 11). Because the best known action of PGE 2 at
that time was elevation of intracellular cAMP, and cAMP
exerted similar Th1-selective suppression (12, 13), most,
if not all, studies assigned PGE 2 as a modulator of T cells
raising the intracellular cAMP level. Among the four sub-
types of PGE receptor, EP2 and EP4 are coupled to a rise
in cAMP (14). Nataraj et al. (15) used T cells obtained
from mice deficient in each EP subtype individually
and found that immunosuppressive action of PGE 2 was
significantly attenuated in T cells obtained either from
EP2 −/− or EP4 −/− mice, suggesting that both EP2 and EP4
mediate suppression of PGE 2 on T cells. Thus there is
ample literature suggesting the Th1-suppressive action of
PGE 2 . Curiously, however, these T cell suppressive ef-
fects of PGE 2 have been shown mostly by in vitro studies
and are rarely seen in vivo, leaving the action of PGE 2 in
vivo in the immune system enigmatic.
PGE 2 action in Th1 differentiation revisited
As mentioned above, it has been known that cAMP
signaling induces suppression of T cell proliferation
(16). However, the mechanism of this action remained
obscure. T cell activation is primarily induced by
stimulation of T cell receptor (TCR) with the respective
antigen, and one of the mechanisms downstream of TCR
stimulation is activation of a Src-family kinase, Lck. It
was shown recently that cAMP in T cells activates PKA,
which then phosphorylates and activates C-terminal Src
kinase (CSK) that phosphorylates the C-terminal tyrosine
of Lck and inactivates it. cAMP and TCR thus compete
with each other in Lck activation (17). These results have
not only identified the suppression mechanism of T cell
proliferation by cAMP, but also indicate a possibility that
strong TCR stimulation may overcome the suppressive
action of cAMP (17). Indeed, experimentally, increased
TCR stimulation with higher amount of anti-CD3 and
anti-CD28 antibodies could overcome the cAMP-
mediated suppression of T cells (18). We exploited
these findings and re-examined the effects of PGE 2 on
differentiation of mouse naïve T cells to the T H 1 subset
under strengthened TCR stimulation, and we found that
under these conditions, PGE 2 facilitated IL-12–induced
Th1 differentiation significantly at nM concentrations
without affecting T cell proliferation (19). Furthermore,
we found that this PGE 2 effect was mimicked by EP2
and an EP4 agonists, and the action of these agonists
was not seen in T cells from mice lacking the respective
receptor. Interestingly, the inhibitor study suggested that
this effect of PGE 2 via EP2 and EP4 depended on the
PI3K pathway and not the cAMP pathway. Our results
were thus opposite to the previous findings and suggest a
possibility that PGE 2 can function as an immunoactivator
to facilitate Th1 differentiation via EP2 and EP4 under
some conditions (19).
PGE 2 action on Th17 cell expansion revealed
The Th17 subset is currently thought to be a principal
Th subset involved in immune inflammation (2, 3). Th17
cells are differentiated from naïve T cells by IL-6 and
TGF- β and become stabilized and expand in the presence
of IL-23 (3). IL-6 and TGF- β are provided, for example,
by DCs that have recognized apoptotic cells in tissue
injury, and IL-23 is also provided by DCs after certain
activation. We examined effects of PGE 2 on mouse Th17
development in vitro (19) and first found that PGE 2 po-
tently suppresses Th17 differentiation from naïve T cells
by IL-6 and TGF- β , which was consistent with the report
by Chen et al. (20). We then examined PGE 2 effects on
Th17 expansion and found that PGE 2 facilitated Th17
expansion in the presence of IL-23. This effect of PGE 2
was again mimicked by EP2 and EP4 agonists. However,
contrary to its effect on Th1 differentiation, this PGE 2 ef-
fect is elicited through the cAMP pathway and not through
the PI3K pathway. We further examined the effect of
PGE 2 on IL-23 production by DCs and found that treat-
ment with indomethacin almost completely suppressed
IL-23 production by DCs activated with anti-CD40

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3Regulation of Immune System by PGE2
Fig. 1. Roles of Th cells in normal im-
mune response. Differentiation of naïve
T cells into each Th subsets is regulated
by specific cytokines in association with
antigen stimulation. IL-12, IL-4, or IL-6 and
transforming growth factor (TGF)- β are key
determinants for differentiation into Th1,
Th2, and Th17 cells, respectively. Th17
cells are then stabilized and expand in the
presence of IL-23. Th1 cells produce IFN- γ ,
which activate macrophages and promotes
elimination of pathogens. Th17 cells also
promote elimination of certain extracellular
pathogens by inducing the expression of
inflammatory cytokines and chemokines,
resulting in recruitment of inflammatory
cells such as neutrophils. On the other hand,
Th2 cells produce IL-4, and activate B cells
to facilitate production of antibodies.
Fig. 2. Roles of Th cells and their differen-
tiation in the disease condition. In diseases
such as autoimmune disease or allergy, Th
cells are excessively or abnormally activated
and/or react with self antigens, resulting in
tissue inflammation and destruction.
Fig. 3. Regulation of Th1 differentiation
and Th17 expansion by PGE 2. PGE 2 acts
on DCs and T cells to mobilize distinct
intracellular signaling pathways for Th1
differentiation and Th17 expansion. PGE 2
is produced by DCs and regulates IL-23
production from activated DCs through the
EP4–cAMP–Epac pathway. IL-23 then acts
on Th17 cells to stabilize and expand these
cells. PGE 2 promotes the IL-23–induced
Th17 cell expansion through the EP2/EP4–
cAMP–PKA pathway. PGE 2 , on the other
hand, also uses EP2 and EP4 to promote
IL-12–driven Th1 cell differentiation from
naïve T cells using PI3K signals.

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4D Sakata et al
antibody, which was restored by the addition of PGE 2 or
an EP4 agonist, whereas an EP2 agonist showed variable
effects. In addition, an EP4-specific antagonist, ONO-
AE3-208, also completely suppressed IL-23 production.
Consistent with our findings, Ganea and collaborators
(21, 22) found that exogenously added PGE 2 enhanced
lipopolysaccharide-induced IL-23 production by bone
marrow-derived DCs and that DCs differentiated with
GM-CSF in the presence of exogenously added PGE 2
acquired a Th17-inducing phenotype, producing higher
amounts of IL-1 β , IL-6, TNF- α , IL-10, and IL-23.
Thus, PGE 2 can facilitate both Th1 differentiation and
Th17 expansion of mouse T cells in vitro through the
same receptors EP2 and EP4 (Fig. 3).
PGE 2 in human Th17 differentiation and expansion
The above studies have thus revealed novel actions
of PGE 2 -EP2/EP4 signaling on expansion of mouse
Th17 cells, which are exerted both on primed T cells and
activated DCs. The next question is whether the same
mechanism operates in human cells. Indeed, concomitant
with our report on mouse cells, several groups have used
human peripheral blood mononuclear cells and reported
the actions of PGE 2 on human Th17 differentiation.
Boniface et al. (23) reported that PGE 2 in the combina-
tion with IL-1 β and IL-23 promote production of IL-17
from differentiating Th17 cells by up-regulating the IL-
1 β R and IL-23R expression through the EP2/EP4–cAMP
pathway. In addition, they also found that the combination
of PGE 2 with IL-1 β and IL-23 induces CCR6 expression,
a chemokine receptor preferentially expressed on Th17
cells. Chizzolini et al. (24) reported that PGE 2 synergizes
with IL-23 and increases the number of Th17 cells from
human CD4 + CD45RO + (memory) T cells but not from
CD4 + CD45RO − (naïve) T cells, consistent with our
finding in mouse T cells that PGE 2 cannot enhance Th17
differentiation but facilitates the action of IL-23 on Th17
expansion. Again, IL-1 β is required for this PGE 2 action.
They also observed that PGE 2 itself facilitates the CCR6
expression on CD4 + T cells. Napolitani et al. (25) used
human peripheral memory T cells and obtained results
similar to Boniface et al. and Chizzolini et al.
Role of PGE 2 –EP4 signaling in vivo in immune
inflammation
Thus, evidence now accumulates that PGE 2 facilitates
Th17 expansion, and, in some instances, Th1 differ-
entiation. Given the involvement of these Th subsets,
particularly Th17, in immune inflammation (3), this
suggests that PGE 2 acting on EP2 and EP4 amplifies
immune pathology. To test this hypothesis, we subjected
wild-type and EP2-deficient mice to two models of
immune inflammation, 2,4-dinitro-1-fluorobenzene–
induced contact hypersensitivity (CHS) and experi-
mental allergic encephalomyelitis (EAE), and assessed
effects of an EP4 antagonist on disease progression
and development of Th1 and Th17 subsets (19). We
found that treatment with the EP4 antagonist suppressed
disease severity and decreased accumulation of antigen-
specific Th1 and Th17 cells in regional lymph nodes
in both models. Deficiency of EP2 alone did not affect
significantly the disease progression, and no augmented
suppression was found with administration of the EP4
antagonist to EP2-deficient animals in the CHS model.
These findings indicate that the PGE 2 –EP4 signaling in-
deed positively regulates development of Th1 and Th17
subsets to specific antigens and determine the extent of
immune inflammation. The fact that the EP2-deficiency
alone was unable to lessen the disease and did not exhibit
additive suppression in combination the EP4 antagonism
in CHS indicates that the EP4 signaling is a dominant, if
not the sole, mechanism operating in the body. Converse
to the findings by us, Sheibanie et al. (26, 27) adminis-
tered various PGE analogs to animals subjected to either
TNBS-induced colitis or collagen-induced arthritis
and found that administration of misoprostol, an EP3/
EP4 agonist, aggravated the disease in both models and
induced higher expressions of IL-23 and IL-17.
Conclusion: potential of EP4 antagonist as an im-
munomodulatory drug
In this perspective, we have reviewed recent progress
in research on the role of PGE 2 in immune inflammation.
On the contrary to the traditional belief, PGE 2 has now
emerged as an immuno-activator that acts on the EP4
receptor and facilitates Th1 differentiation and Th17
expansion, two Th subsets involved in immune inflam-
mation. Given a variety of evidence for involvement of
these Th subsets in various immune diseases of humans,
an EP4 antagonist can be a good therapeutic drug target
for diseases such as rheumatoid arthritis, multiple sclero-
sis, psoriasis, and Crohn’s disease. One finding support-
ing this assumption is the genomic analysis identifying
EP4 as a causative gene of Crohn’s disease (28). On the
other hand, Kabashima et al. previously reported that the
PGE 2 –EP4 signaling protects mice from dextran sodium
sulfate–induced mouse colitis (29), and a recent clinical
trial of an EP4 agonist, ONO-4819CD, in ulcerative
colitis patients showed some beneficial effects (30). We
assume that the discrepancy may come from the differ-
ence in pathophysiology between ulcerative colitis and
Crohn’s disease. While the two diseases are categorized

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5 Regulation of Immune System by PGE2
together as inflammatory bowel diseases, ulcerative
colitis could be a disease of impaired barrier function
of intestinal epithelium, which the PGE 2 –EP4 signaling
improves as shown in the DSS model, while Crohn’s
disease is primarily caused by a disorder of the immune
system. Thus, analysis of the role of the PGE 2 signal-
ing may provide deeper insight into the pathological
mechanisms underlying each disease, which should be
fully taken into account in developing an EP4 antagonist
as a therapeutic agent and its clinical application.
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