E-Prostanoid-3 Receptors Mediate the
Proinflammatory Actions of Prostaglandin E
2 in Acute Cutaneous Inflammation
This information is current as
of July 28, 2017.
Jennifer L. Goulet, Amy J. Pace, Mikelle L. Key, Robert S.
Byrum, MyTrang Nguyen, Stephen L. Tilley, Scott G.
Morham, Robert Langenbach, Jeffrey L. Stock, John D.
McNeish, Oliver Smithies, Thomas M. Coffman and Beverly
H. Koller
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2004 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2004; 173:1321-1326; ;
doi: 10.4049/jimmunol.173.2.1321
http://www.jimmunol.org/content/173/2/1321
The Journal of Immunology
E-Prostanoid-3 Receptors Mediate the Proinflammatory
Actions of Prostaglandin E2 in Acute Cutaneous Inflammation1
Jennifer L. Goulet,*† Amy J. Pace,† Mikelle L. Key,† Robert S. Byrum,† MyTrang Nguyen,†
Stephen L. Tilley,† Scott G. Morham,‡ Robert Langenbach,§ Jeffrey L. Stock,¶
John D. McNeish,¶ Oliver Smithies,‡ Thomas M. Coffman,* and Beverly H. Koller2†
I
nflammation is a complex physiologic process involving interactions among numerous mediators that results in erythema, edema, vasodilation, hyperemia, and cellular infiltration. An early event in this process is the release of arachidonic
acid (AA)3 from cell membranes, catalyzed by phospholipase enzymes, primarily phospholipase A2. Free AA can then be metabolized into a variety of biologically active lipids termed eicosanoids. Eicosanoids include the metabolites of the arachidonic
5-lipoxygenase (ALOX5) pathway, the proinflammatory leukotrienes (LTs), and the products of the cyclooxygenase (PG-endoperoxide synthase (PTGS)) pathways, the prostanoids (1–3).
The first step in the synthesis of prostanoids is the conversion of
AA into PGH2 by the enzyme PTGS, also known as cyclooxygenase (4). The two known PTGS isoforms are referred to as PTGS1
and PTGS2. The PTGS1 and PTGS2 enzymes are only 60% identical at the protein level and differ in their regulation at the transcriptional level (4). PTGS1 is a constitutive enzyme present in
almost all cell types, whereas PTGS2 is normally undetectable in
most tissues (4). However, high levels of PTGS2 can be induced
in a number of cells by proinflammatory and mitogenic stimuli (4),
*Division of Nephrology, Department of Medicine, Duke University and Durham
Veterans Affairs Medical Centers, Durham, NC 27705; Departments of †Medicine
and ‡Pathology, University of North Carolina, Chapel Hill, NC 27599; §Laboratory of
Experimental Carcinogenesis and Mutagenesis, National Institute of Environmental
Health Sciences, Research Triangle Park, NC 27709; and ¶Center for Experimental
Therapeutics, Pfizer Central Research, Groton, CT 06340
Received for publication January 21, 2004. Accepted for publication May 14, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants PO1-DK38108 (to
B.H.K. and T.M.C.) and R01-HL68141 (to B.H.K.), and the Duke Training Grant in
Nephrology 5-T32-DK07731.
2
Address correspondence and reprint requests to Dr. Beverly H. Koller, Department
of Medicine, University of North Carolina, 4341 Molecular Biology Research Building, Chapel Hill, NC 27599. E-mail address: [email protected]
3
Abbreviations used in this paper: AA, arachidonic acid; ALOX5, arachidonic 5-lipoxygenase; LT, leukotriene; PTGS, PG-endoperoxide synthase; EP, E-prostanoid;
INDO, indomethacin.
Copyright © 2004 by The American Association of Immunologists, Inc.
and these findings have led to the general belief that PTGS2 is the
major pathway for the synthesis of PGs in inflammatory responses.
The intermediate PGH2, generated from AA by the PTGS enzymes, is further metabolized by synthases and reductases into
PGs and thromboxanes. The type of prostanoid synthesized is dependent on the cell-specific expression of enzymes that metabolize
PGH2 into these biologically active lipids. In general, only one of
the major prostanoids is produced in abundance by a given cell
type. PGE2, generated from the intermediate PGH2 by the PGE
synthase enzyme, can be produced by both inflammatory cells and
tissue parenchyma, including keratinocytes. High levels of PGE2
have been measured in inflammatory exudates, and the injection of
PGE2 directly into tissue has been shown to induce a number of the
cardinal signs of inflammation (5). More importantly, a number of
studies suggest that PGE2 may play a synergistic role with other
mediators, such as histamine and bradykinin, especially in contributing to the pain and edema associated with the inflammatory
process (5). However, it has been difficult to determine whether
PGE2 plays an anti- or proinflammatory role in physiological responses because of the numerous, and often opposing, biological
actions of this lipid mediator.
The diverse effects of PGE2 are mediated by specific cell surface
receptors that belong to the large family of G protein-coupled receptors (6). Four classes of PGE2 E-prostanoid (EP) receptors,
designated EP1 through EP4, can be distinguished pharmacologically. EP receptors of each class have been cloned and sequenced,
and each is the product of a distinct gene (6). The tissue distribution and intracellular signaling pathways used by the subclasses of
EP receptors differ substantially (6). These differences may explain
the wide array of effects that are induced by PGE2.
Recently, the development of mouse lines deficient in the ability
to synthesize eicosanoids and/or their receptors has provided a
means by which the roles of these inflammatory mediators can be
examined in vivo. Mice deficient in LT biosynthesis were created
by the introduction of mutations into the alox5 gene (7, 8). Mice
deficient in the production of prostanoids were generated by inactivation of either the ptgs1 (9) or ptgs2 (10) gene, whereas mice
unable to express PGE2-specific receptors were created by introducing mutations in the Ep1, Ep2, Ep3, and Ep4 genes (11–13).
0022-1767/04/$02.00
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PGs are derived from arachidonic acid by PG-endoperoxide synthase (PTGS)-1 and PTGS2. Although enhanced levels of PGs are
present during acute and chronic inflammation, a functional role for prostanoids in inflammation has not been clearly defined.
Using a series of genetically engineered mice, we find that PTGS1 has the capacity to induce acute inflammation, but PTGS2 has
negligible effects on the initiation of this response. Furthermore, we show that the contribution of PTGS1 is mediated by PGE2
acting through the E-prostanoid (EP)3 receptor. Moreover, in the absence of EP3 receptors, inflammation is markedly attenuated,
and the addition of nonsteroidal anti-inflammatory agents does not further impair the response. These studies demonstrate that
PGE2 promotes acute inflammation by activating EP3 receptors and suggest that EP3 receptors may be useful targets for antiinflammatory therapy. The Journal of Immunology, 2004, 173: 1321–1326.
1322
EP3 MEDIATES PGE2 IN ACUTE CUTANEOUS INFLAMMATION
To isolate the role of lipid metabolites in acute inflammation, we
have used an experimental model, cutaneous inflammation in response to AA, that depends primarily on the synthesis and release
of eicosanoids. Using this model, and mice deficient in the ALOX5
enzyme, we and others have noted a significant reduction in edema
and leukocyte infiltration in the inflamed tissues of these animals,
thereby demonstrating the importance of LTs in acute inflammatory processes (7, 8, 14, 15). However, the inflammatory response
was not eliminated by the loss of ALOX5, and the remaining response was sensitive to the PTGS inhibitor indomethacin (INDO)
(7). These data suggested that prostanoids are also important proinflammatory mediators of cutaneous inflammation. In addition,
these findings indicated that the ALOX5-deficient mice would provide a genetic background that would allow us to isolate the contribution of prostanoids and, thus, provide a means for determining
the mechanism by which these lipid metabolites mediate acute
cutaneous inflammatory responses.
Materials and Methods
The generation of alox5⫺/⫺ (7), ptgs1⫺/⫺ (9), ptgs2⫺/⫺ (10), Ep2⫺/⫺ (11),
Ep3⫺/⫺ (12), and Ep4⫺/⫺ (13) mice has been described. The Ep1 null
mutation was introduced into the DBA/1Lac background by targeted disruption of this gene using an embryonic stem cell line derived directly from
the DBA/1Lac mouse strain (16). The alox5⫺/⫺ animals were bred to mice
deficient in either PTGS1, PTGS2, or EP3 to generate alox5⫺/⫺ptgs1⫺/⫺,
alox5⫺/⫺ptgs2⫺/⫺, and alox5⫺/⫺Ep3⫺/⫺ double-mutant mice. Mice were
screened for the appropriate targeted gene mutations by Southern blot or
PCR analysis of tail genomic DNA as previously described (7, 9 –13, 17).
The alox5⫺/⫺Ep3⫺/⫺ mice and their corresponding control animals are on
an inbred 129 genetic background. As mentioned above, the Ep1⫺/⫺ and
Ep1⫹/⫹ mice are on the DBA/1Lac strain. The other mouse lines are derived from a mixed genetic background, which is a combination of 129,
C57BL/6, and DBA/2 mouse strains. All mice studied were at least 8 wk
old and were bred and maintained in specific pathogen-free animal barrier
facilities at the University of North Carolina. All experiments were approved by the Institutional Animal Care and Use Committee.
Induction of inflammatory responses in mouse ear tissue
Animals were injected i.v. with either 0.5% Evans blue dye (SigmaAldrich, St. Louis, MO) dissolved in PBS (pH 7.5) (10 ml of dye solution/kg of body weight) or with INDO (1 mg/ml in 0.1 M Na2CO3 and 0.15
M Na2HPO4 (pH 7.4)) (Sigma-Aldrich) combined with 0.5% Evans blue
dye (10 mg of INDO/kg of body weight). The inner surface of the left ear
of each mouse was painted with 20 ␮l of AA (100 ␮g/␮l in acetone)
(Sigma-Aldrich), whereas the right ear was treated with an equal amount of
acetone alone. At 1 h after AA treatment, mice were sacrificed and an
8-mm-diameter disc of tissue was punched from the center of each ear.
Edema and vascular permeability measurements in mouse ear
tissue
Edema was assessed by determining the wet weight of each ear biopsy. To
extract extravasated Evans blue dye, ear biopsies were incubated in 1 ml of
formamide at 55°C for 48 h. Dye extravasation was quantified by measuring the absorbance of the formamide extracts at 610 nm (A610) with a
spectrophotometer (18).
Statistical analysis
Data are presented as mean ⫾ SEM. Statistical significance for comparisons between groups was determined using an unpaired two-sample t test.
Results
AA-induced inflammation is inhibited when both ALOX5 and
PTGS1 are absent
Topical application of AA to mouse ear tissue induces an inflammatory response characterized by immediate vasodilation, erythema, and edema formation. The onset of edema coincides with
the extravasation of plasma proteins and is followed by the accumulation of leukocytes, primarily neutrophils, in the inflamed tis-
FIGURE 1. AA-induced acute inflammatory responses in alox5⫺/⫺
ptgs1⫺/⫺ mice. Edema and vascular permeability changes were assessed for
wild-type, ptgs1⫺/⫺, alox5⫺/⫺, and alox5⫺/⫺ptgs1⫺/⫺ mice. Before the application of 2 mg of AA in acetone to the left ear and acetone alone to the right
ear, mice received an i.v. injection of 0.5% Evans blue dye solution. After 1 h,
mice were sacrificed, 8-mm-diameter discs of tissue were taken from each ear,
and the difference in weight between the right ear and left ear for each animal
was recorded (A). The extravasation of dye into the tissue was then quantitated
by extraction of dye with formamide and measurement of absorbance at a
wavelength of 610 nm (A610). For each animal, the A610 obtained for the right
ear was subtracted from that obtained for the left ear (B). Error bars indicate
SEM. n, Number of mice in each group. ⴱ and #, p ⬍ 0.005.
Downloaded from http://www.jimmunol.org/ by guest on July 28, 2017
Mice
sue (19 –23). Studies have also shown that high levels of LTs,
particularly cysteinyl-LTs, and PGs, specifically PGE2, are produced in mouse ear tissue after AA stimulation (20, 22).
Previous studies have shown a significant reduction in the levels
of edema and leukocyte infiltration in response to topical AA in
ALOX5-deficient mice. However, a response remaining in the
alox5⫺/⫺ mice was seen, and this response was sensitive to the
PTGS inhibitor INDO, suggesting that prostanoids are also important proinflammatory mediators of cutaneous inflammation. To
identify which PTGS pathway is involved in the synthesis of these
prostanoids, we generated mice that were deficient in both ALOX5
and each of the PTGS enzymes.
First, we examined the effect of topically applied AA on ear
tissue of wild-type, alox5⫺/⫺, ptgs1⫺/⫺, and alox5⫺/⫺ptgs1⫺/⫺
mice. Inflammation was measured by determining both the change
in weight of an ear biopsy and the extravasation of plasma protein.
Mice were injected with Evans blue, a dye that binds to serum proteins, before the administration of AA; protein extravasation was then
assayed by quantifying the amount of dye in the ear tissue. As shown
in Fig. 1, the loss of PTGS1 had little impact on the inflammatory
response elicited by AA. Only a slight decrease in ear weight in response to AA was observed in ptgs1⫺/⫺ mice compared with wildtype animals (Fig. 1A, p ⫽ 0.058). Serum protein extravasation was
similar in wild-type and PTGS1-deficient animals (Fig. 1B, p ⫽
The Journal of Immunology
1323
0.480). In contrast, a significant reduction in ear weight and protein
extravasation was seen in ALOX5-deficient mice compared with both
wild-type (Fig. 1; ⴱ, p ⫽ 3.3 ⫻ 10⫺5 and 0.0003, respectively) and
ptgs1⫺/⫺ mice (#, p ⫽ 0.0005 and 0.0029, respectively). However, a
very low level of inflammation in the AA-treated ears of ALOX5deficient mice was still apparent. When mice deficient in both
ALOX5 and PTGS1 were examined, AA-stimulated edema and vasopermeability were almost completely absent (Fig. 1, p ⬍ 0.05 compared with all other groups). Loss of both ALOX5 and PTGS1 enzymes strongly inhibited AA-induced ear inflammation (89 –94%)
compared with the effect of either the alox5 (74 –76%) or ptgs1 (1–
24%) mutations alone (Table I).
AA-induced ear inflammation in alox5⫺/⫺ptgs2⫺/⫺ mice is
inhibited by INDO
Table I. Inhibition of AA-induced ear inflammation
% Inhibitiona
Genotype
Ear weight
A610
Wild type
ptgs1⫺/⫺
ptgs2⫺/⫺
alox5⫺/⫺
alox5⫺/⫺ ptgs1⫺/⫺
alox5⫺/⫺ ptgs2⫺/⫺
alox5⫺/⫺ Ep3⫺/⫺
Ep3⫺/⫺
Ep1⫺/⫺
Ep2⫺/⫺
Ep4⫺/⫺
0
24
7
48–74b
94
30
86
45
⫺7
17
3
0
1
6
39–76b
89
20
88
50
⫺11
24
9
a
Percent inhibition was calculated from the mean values derived from analyses of
ear biopsies from mutant mice compared to wild-type mice.
b
Percent inhibition of AA-induced ear inflammation varies depending on the genetic background of the mice tested.
FIGURE 2. AA-induced inflammation in alox5⫺/⫺ptgs2⫺/⫺ mice.
Edema and vascular permeability changes were assessed for wild-type,
ptgs2⫺/⫺, alox5⫺/⫺, and alox5⫺/⫺ptgs2⫺/⫺ mice. For each mouse, the left
ear was treated with 2 mg of AA in acetone, and the right ear was treated with
vehicle alone. Mice received an i.v. injection of either 0.5% Evans blue dye
solution or 0.5% Evans blue plus INDO (10 mg/kg of body weight) before AA
treatment. After 1 h, mice were sacrificed, 8-mm-diameter discs of tissue were
taken from each ear, and the difference in weight between the right ear and left
ear for each animal was recorded (A). The extravasation of dye into the tissue
was then quantitated by extraction of dye with formamide and measurement of
absorbance at a wavelength of 610 nm (A610). For each animal, the A610
obtained for the right ear was subtracted from that obtained for the left ear (B).
Error bars indicate SEM. n, Number of mice in each group. # and ⫹, p ⬍ 0.05;
ⴱ, ⌬, and %, p ⬍ 0.001.
tively; Table II). Treatment with INDO also significantly reduced
edema (81%) and protein extravasation (82%) elicited by AA in
the ears of alox5⫺/⫺ptgs2⫺/⫺ mice (Fig. 2; %, p ⫽ 1.7 ⫻ 10⫺5 and
0.0002, respectively; Table II). Moreover, the magnitude of the
responses of the INDO-treated alox5⫺/⫺ and alox5⫺/⫺ptgs2⫺/⫺
mice were virtually identical (Fig. 2, p ⫽ 0.244 and 0.455, respectively; Table II). With respect to the PTGS pathways, these data
demonstrate that the PTGS1 enzyme provides the major contribution to AA-induced cutaneous acute inflammation in the mouse.
EP3 receptors mediate the actions of PGE2 in AA-induced ear
inflammation
To determine the specific PTGS metabolites involved in AA-induced cutaneous inflammation, we examined this response in mice
deficient in each of the four PGE2-specific receptors, EP1, EP2,
EP3, and EP4. As seen in Fig. 3 and Table I, ear inflammation
elicited by topical AA, as measured by changes in ear weight and
dye extravasation, was unaffected by the absence of either the EP1
( p ⫽ 0.371 and 0.344, respectively), EP2 ( p ⫽ 0.223 and 0.170,
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To confirm that the PTGS1 pathway, and not the PTGS2 pathway,
contributes to this inflammatory response, the ears of wild-type,
alox5⫺/⫺, ptgs2⫺/⫺, and alox5⫺/⫺ptgs2⫺/⫺ mice were treated
with AA. As can be seen in Fig. 2 and Table I, the loss of PTGS2
did not result in decreased AA-induced ear edema and serum protein extravasation compared with wild-type animals ( p ⫽ 0.337
and 0.365, respectively). In contrast, and consistent with previous
data (7, 14, 15), a significant reduction in both ear weight (Fig. 2A;
ⴱ, p ⫽ 0.0006) and protein extravasation (B; ⴱ, p ⫽ 0.012) was
seen in ALOX5-deficient mice compared with wild-type mice (Table I). The AA-induced increase in ear weight and serum protein
extravasation was somewhat lower in alox5⫺/⫺ptgs2⫺/⫺ mice than
in wild-type animals (Fig. 2; #, p ⫽ 0.025 and 0.150, respectively;
Table I). Unlike the effect of the ptgs1 mutation, the ptgs2 mutation did not reduce or eliminate the inflammatory responses to AA
seen in the alox5⫺/⫺ mice.
To examine whether the inflammatory responses of the alox5⫺/⫺
ptgs2⫺/⫺ mice are due to PTGS1 activity, we treated mice of each
genotype with the PTGS inhibitor INDO. As can be seen in Fig. 2
and Table II, INDO significantly reduced AA-induced edema and
vasopermeability, as determined by changes in ear weight and protein extravasation, respectively, in wild-type mice by 43–53% (⫹,
p ⫽ 0.0005 and 0.005, respectively). INDO treatment had no effect
on responses to topical AA in PTGS2-deficient animals (Fig. 2,
p ⫽ 0.235 and 0.497, respectively; Table II). In contrast, INDO
treatment had dramatic effects on inflammation in alox5⫺/⫺ and
alox5⫺/⫺ptgs2⫺/⫺ groups. The AA-induced inflammatory responses remaining in the alox5⫺/⫺ mice were inhibited by 78 –
81% by INDO (Fig. 2; ⌬, p ⫽ 8.6 ⫻ 10⫺5 and 0.0004, respec-
1324
EP3 MEDIATES PGE2 IN ACUTE CUTANEOUS INFLAMMATION
Table II. Inhibition of AA-induced ear inflammation by genetic
mutations and INDO treatment
% Inhibition by INDOa
% Inhibition by
Combination of Genetic
Mutations and INDOb
Genotype
Ear weight
A610
Ear weight
A610
Wild type
ptgs2⫺/⫺
alox5⫺/⫺
alox5⫺/⫺ ptgs2⫺/⫺
alox5⫺/⫺ Ep3⫺/⫺
Ep3⫺/⫺
19–53c
19
78–81c
81
55
⫺16
(⫺8)–43c
0.4
76–78c
82
11
⫺49
19–53c
25
90–92c
86
94
36
(⫺8)–43c
6
86–93c
86
89
25
a
Percent inhibition was calculated from the mean values derived from analyses of
ear biopsies from INDO-treated mice compared with untreated mice of the same
genotype.
b
Percent inhibition was calculated from the mean values derived from analyses of
ear biopsies from INDO-treated mice compared with untreated wild-type mice.
c
Percent inhibition of AA-induced ear inflammation varies depending on the genetic background of the mice tested.
in a significant reduction in AA-induced ear edema and serum
protein extravasation compared with wild-type mice (Fig. 3; ⴱ, p ⫽
7.0 ⫻ 10⫺5 and 0.0004, respectively; Table I). These data demonstrate that the EP3 receptor mediates the proinflammatory actions of PGE2 in this experimental model.
AA-induced inflammation is inhibited when both ALOX5 and
EP3 receptors are absent
To confirm that the EP3 receptor mediates the actions of PGE2 in
this acute inflammatory model, we tested mice deficient in both
EP3 and ALOX5. These results are shown in Fig. 4. When both
ALOX5 and EP3 receptors are absent, the AA-induced inflammatory responses were almost completely eliminated (Fig. 4; ⴱ, p ⫽
1.2 ⫻ 10⫺20 and 2.5 ⫻ 10⫺15, respectively). The level of inhibition resulting from the combination of alox5 and Ep3 mutations
(86 – 88%) was similar to that seen in alox⫺/⫺ptgs1⫺/⫺ mice (89 –
94%), INDO-treated alox5⫺/⫺mice (86 –93%), and INDO-treated
alox5⫺/⫺ptgs2⫺/⫺ animals (86%) (Tables I and II).
FIGURE 3. AA-induced inflammatory responses in PGE2 receptor-deficient mice. Edema and vascular permeability changes were assessed for
wild-type, Ep1⫺/⫺, Ep2⫺/⫺, Ep3⫺/⫺, and Ep4⫺/⫺ mice. Before the application of 2 mg of AA in acetone to the left ear and acetone alone to the right
ear, mice received an i.v. injection of 0.5% Evans blue dye solution. After
1 h, mice were sacrificed, 8-mm-diameter discs of tissue were taken from
each ear, and the difference in weight between the right ear and left ear for each
animal was recorded (A). The extravasation of dye into the tissue was then
quantitated by extraction of dye with formamide and measurement of absorbance at a wavelength of 610 nm (A610). For each animal, the A610 obtained
for the right ear was subtracted from that obtained for the left ear (B). Error
bars indicate SEM. n, Number of mice in each group. ⴱ, p ⬍ 0.001.
Downloaded from http://www.jimmunol.org/ by guest on July 28, 2017
respectively), or EP4 receptor ( p ⫽ 0.447 and 0.442, respectively)
compared with the responses of corresponding wild-type animals.
In contrast, a deficiency in expression of the EP3 receptor resulted
FIGURE 4. AA-induced inflammation in alox5⫺/⫺Ep3⫺/⫺ mice.
Edema and vascular permeability changes were assessed for wild-type,
Ep3⫺/⫺, alox5⫺/⫺, and alox5⫺/⫺Ep3⫺/⫺ mice. For each mouse, the left ear
was treated with 2 mg of AA in acetone, and the right ear was treated with
vehicle alone. Mice received an i.v. injection of either 0.5% Evans blue dye
solution or 0.5% Evans blue plus INDO (10 mg/kg of body weight) before
AA treatment. After 1 h, mice were sacrificed, 8-mm-diameter discs of
tissue were taken from each ear, and the difference in weight between the
right ear and left ear for each animal was recorded (A). The extravasation
of dye into the tissue was then quantitated by extraction of dye with formamide and measurement of absorbance at a wavelength of 610 nm (A610).
For each animal the A610 obtained for the right ear was subtracted from that
obtained for the left ear (B). Error bars indicate SEM. n, Number of mice
in each group. #, p ⬍ 0.05; ⌬, p ⬍ 0.005; ⫹ and ⴱ, p ⬍ 0.001.
The Journal of Immunology
1325
FIGURE 5. Eicosanoid pathways
involved in AA-induced acute cutaneous inflammation. In this experimental
model, AA is metabolized by ALOX5
to generate LTB4 and LTC4 and by
PTGS1 to produce PGE2. Each inflammatory mediator then activates specific
cell surface receptors, which results in
certain characteristic signs of acute inflammation. B-LTR, LTB4 receptor;
CYS-LTR, cysteinyl-LT receptors;
EP3-R, EP3 receptor.
Discussion
Previous studies using mice deficient in ALOX5 have established
the importance of LTs in AA-induced ear inflammation in the mouse
(7, 8, 14, 15). In this report, using mice deficient in both ALOX5 and
PTGS1, we show that PTGS1 metabolites of AA also contribute to
edema formation in this model of cutaneous inflammation (Fig. 5).
This observation is consistent with the demonstration of high levels of
PTGS1 expression in skin (4). In contrast, the loss of PTGS2 has no
impact on the inflammatory response to topical AA. It is likely that,
because of the time required for the induction of PTGS2 expression,
this enzyme is not present at substantial levels during the initiating
events of an inflammatory response (24, 25). Therefore, PTGS2 does
not make a significant contribution to the early rise in prostanoid
levels in the inflamed tissue.
Previously, it has been difficult to definitively assign a specific
prostanoid and prostanoid receptor to a given physiologic response
due to the incomplete selectivity of pharmacological receptor agonists and antagonists. However, gene targeting and the generation
of mice deficient in specific prostanoid receptors has allowed us to
identify the prostanoid and, with respect to PGE2, the correspond-
ing receptor that contribute to various inflammatory processes. In
this study, we examined AA-induced cutaneous inflammation in
mice deficient in each of the four PGE2-specific receptors and
found that only inactivation of the EP3 receptor resulted in a significant decrease in edema formation and plasma protein extravasation in response to topical AA. These data show that PGE2 activation of the EP3 receptor mediates the major proinflammatory
actions of prostanoids in this model.
Initially, these results appear to be contrary to the current understanding of the mechanism by which PGE2 contributes to inflammatory processes. It has been widely assumed that PGE2 potentiates acute inflammatory responses primarily by its
vasodilatory actions on arterioles. However, EP3 receptors are unlikely candidates for mediating such actions. EP3 receptors have
been shown to couple to different signaling pathways, including Gi
(inhibition of intracellular cAMP formation) and Gq (stimulation
of intracellular Ca2⫹ release), in most cell lines and tissues examined to date (6). These secondary messenger systems would be
expected to result in vasoconstriction, not vasodilation (26). Additional evidence supporting these data has come from our recent
demonstration that EP3 receptors do not contribute to PGE2-induced vasodilation, and may promote vasoconstriction (27).
Activation of both Gi and Gq signaling pathways might also
stimulate and/or enhance activation of leukocytes, such as mast
cells and Langerhans cells (28). Previous studies have demonstrated that pharmacological agents that preferentially activate EP3
receptors potentiate bradykinin-induced inflammation in rabbit
skin by enhancing plasma extravasation without altering blood
flow (29 –33). It has also been shown that EP3 receptor agonists
induce chemotaxis in neutrophils and the release of LTB4 from
these cells in vitro (34, 35). The results of our present study,
together with these pharmacological data, suggest that the EP3
receptor contributes to cutaneous inflammatory responses by a leukocyte-dependent mechanism. Therefore, PGE2, acting through EP3
receptors, may contribute to AA-induced acute inflammation by exerting proinflammatory effects on leukocytes present in the tissue.
Downloaded from http://www.jimmunol.org/ by guest on July 28, 2017
Fig. 4 and Table II also show the effect of INDO on the responses of ALOX5- and EP3-deficient mice. AA-induced changes
in ear weight were slightly reduced (19%) in INDO-treated wildtype animals (Fig. 4A; #, p ⫽ 0.035). However, in wild-type controls, serum protein extravasation, as determined by the amount of
dye in the ear tissue, was not affected by INDO treatment (Fig. 4B,
p ⫽ 0.287). INDO treatment also had no effect on the remaining
AA-induced inflammatory responses in EP3-deficient animals.
Once again, the inflammatory responses to topical AA in the
alox5⫺/⫺ mice were inhibited by 76 –78% by treatment with INDO
(Fig. 4; ⫹, p ⫽ 4.9 ⫻ 10⫺6 and 4.9 ⫻ 10⫺5, respectively). Edema
was slightly lower in INDO-treated alox5⫺/⫺Ep3⫺/⫺ mice (Fig.
4A; ⌬, p ⫽ 0.0014; Table II), however, the responses of both
INDO-treated and untreated mice of this genotype were very low.
In addition, INDO had little effect on protein extravasation (11%)
in alox5⫺/⫺Ep3⫺/⫺ mice (Fig. 4B, p ⫽ 0.354).
1326
EP3 MEDIATES PGE2 IN ACUTE CUTANEOUS INFLAMMATION
Acknowledgments
We thank M. Wade for his critical review of the manuscript. We also thank
V. A. Wagoner and B. Hawkins for assistance with animal husbandry, genotyping, and experiments; and J. B. Garges and T. Mason for help with
genotyping.
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Inactivation of the PGE2-specific EP3 receptor inhibited AAinduced cutaneous inflammation to a greater degree than the lack
of PTGS1 expression. A number of explanations for this observation are possible. First, PGE2, synthesized by PTGS1, has both
anti- and proinflammatory properties. In EP3-deficient mice, antiinflammatory pathways would remain intact, because our data
demonstrate that EP3 receptors mediate the proinflammatory actions of PGE2 in this experimental model. The anti-inflammatory
activities of PGE2 would dampen the inflammatory response, especially in the absence of the proinflammatory EP3 receptors. In
contrast, all prostanoid production is eliminated in PTGS1-deficient animals, and, as a result, both anti- and proinflammatory
PGE2 pathways are inhibited. However, this interpretation of the
data is not supported by our observation that the inflammatory
responses of mice deficient in the other PGE2-specific receptors,
EP1, EP2, and EP4, were not enhanced or significantly different
from their wild-type controls.
Second, our findings do not exclude the possibility that other
prostanoids, with anti-inflammatory actions, are produced in the
EP3-deficient mice, but not in the ptgs1⫺/⫺ mice, and contribute to
the differences seen in the inflammatory responses of these animals. Again, this hypothesis is not supported by data presented in
this report that show that INDO treatment has no impact on AAinduced cutaneous inflammation in EP3-deficient mice.
Finally, an alternate explanation for the differences in the level
of inhibition resulting from the loss of the EP3 receptor, compared
with the absence of PTGS1, is suggested by the observation that the
Ep3 and ptgs1 mutations had similar effects on ear inflammation when
examined on the ALOX5-deficient background. These data suggest
that enhanced synthesis and secretion of ALOX5 metabolites in the
PTGS1-deficient mice compensates for and minimizes the effect of
the ptgs1 mutation on AA-induced acute inflammation. These data,
taken together with our previous findings that the loss of the ALOX5
pathway might lead to an increased role for prostanoids (7), suggest
that these eicosanoid pathways are interrelated.
In summary, we have used a series of genetically engineered animals to identify the pathway, mediator, and receptor that contribute to
the prostanoid component of acute cutaneous inflammation. Our data
support a model in which AA is metabolized by the PTGS1 enzyme
to produce PGE2, which then activates EP3 receptors present on tissue
leukocytes. This action stimulates the release of additional inflammatory mediators from resident and infiltrating cells in the skin, and
promotes the early phases of the acute inflammatory process. These
findings may have significant clinical relevance, in that EP3 receptor
antagonists may prove to be effective, highly specific treatments for
acute inflammatory conditions.