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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. Koller^2,

^* 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

	Abstract

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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 anti-inflammatory therapy.

	Introduction

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Inflammation 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, 2, 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), 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, 12, 13).

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

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Mice

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, 10, 11, 12, 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 (Sigma-Aldrich, 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 µl of AA (100 µg/µl 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

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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 tissue (19, 20, 21, 22, 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 wild-type animals (Fig. 1A, p = 0.058). Serum protein extravasation was similar in wild-type and PTGS1-deficient animals (Fig. 1B, p = 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; _*, p = 3.3 x 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 ALOX5-deficient 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).

View larger version (29K): [in this window] [in a new window]   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. * and #, p < 0.005.

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; _*, p = 0.0006) and protein extravasation (B; _*, 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.

View larger version (40K): [in this window] [in a new window]   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; *, , and %, p < 0.001.

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, 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 in a significant reduction in AA-induced ear edema and serum protein extravasation compared with wild-type mice (Fig. 3; _*, p = 7.0 x 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.

View larger version (37K): [in this window] [in a new window]   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. *, p < 0.001.

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; _*, p = 1.2 x 10^–20 and 2.5 x 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).

View larger version (27K): [in this window] [in a new window]   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 *, p < 0.001.

	Discussion

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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.

View larger version (25K): [in this window] [in a new window]   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.

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 G_i (inhibition of intracellular cAMP formation) and G_q (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 G_i and G_q 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, 30, 31, 32, 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.

Inactivation of the PGE₂-specific EP3 receptor inhibited AA-induced 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, anti-inflammatory 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 AA-induced 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.

	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.

	Footnotes

 
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: treawouns{at}aol.com

³ Abbreviations used in this paper: AA, arachidonic acid; ALOX5, arachidonic 5-lipoxygenase; LT, leukotriene; PTGS, PG-endoperoxide synthase; EP, E-prostanoid; INDO, indomethacin.

Received for publication January 21, 2004. Accepted for publication May 14, 2004.

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