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E2 Inhibits Trafficking through E- 2 Receptors

This information is current as Eva M. Sturm, Petra Schratl, Rufina Schuligoi, Viktoria of October 2, 2021. Konya, Gunter J. Sturm, Irmgard Th. Lippe, Bernhard A. Peskar and Akos Heinemann J Immunol 2008; 181:7273-7283; ; doi: 10.4049/jimmunol.181.10.7273 http://www.jimmunol.org/content/181/10/7273 Downloaded from

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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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Prostaglandin E2 Inhibits Eosinophil Trafficking through E-Prostanoid 2 Receptors1

Eva M. Sturm,2 Petra Schratl,2 Rufina Schuligoi, Viktoria Konya, Gunter J. Sturm, Irmgard Th. Lippe, Bernhard A. Peskar, and Akos Heinemann3

The accumulation of in lung tissue is a hallmark of , and it is believed that eosinophils play a crucial pathogenic role in allergic inflammation. Prostaglandin (PG) E2 exerts anti-inflammatory and bronchoprotective mecha- nisms in asthma, but the underlying mechanisms have remained unclear. In this study we show that PGE2 potently inhibits the of purified human eosinophils toward eotaxin, PGD2, and C5a. Activated similarly attenuated eosinophil migration, and this was reversed after pretreatment of the monocytes with a inhibitor. The selective E-prostanoid (EP) 2 agonist butaprost mimicked the inhibitory effect of PGE2 on eosinophil migration, whereas an EP2 antagonist completely prevented this effect. Butaprost, and also PGE2, inhibited the C5a-induced degran- Downloaded from ulation of eosinophils. Moreover, selective kinase inhibitors revealed that the inhibitory effect of PGE2 on eosinophil mi- gration depended upon activation of PI3K and protein kinase C, but not cAMP. In animal models, the EP2 agonist butaprost inhibited the rapid mobilization of eosinophils from bone marrow of the in situ perfused guinea pig hind limb and prevented the allergen-induced bronchial accumulation of eosinophils in OVA-sensitized mice. Immunostaining showed that human eosinophils express EP2 receptors and that EP2 receptor expression in the murine lungs is prominent in airway epithelium and, after allergen challenge, in peribronchial infiltrating leukocytes. In summary, these data show that EP2 receptor http://www.jimmunol.org/ agonists potently inhibit eosinophil trafficking and activation and might hence be a useful therapeutic option in eosinophilic diseases. The Journal of Immunology, 2008, 181: 7273–7283.

osinophils are important effector cells in allergic dis- vating factor are also potent chemoattractants for eosinophils eases, particularly in late-phase reactions, because they (9–11). Lately, a cyclooxygenase (COX)-derived4 mediator of release bronchoconstrictor mediators such as leukotri- activated mast cells, prostaglandin (PG) D , and several of its E 2 ene C4 and other chemoattractants that cause further influx of metabolites have been revealed to potently stimulate eosinophil inflammatory cells into the tissue (1). Eosinophils produce chemotaxis (12, 13). proinflammatory and growth factors, including im- by guest on October 2, 2021 PGE2 is the predominant cyclooxygenase product of airway munoregulatory Th2-type cytokines (2–4). Mucosal damage in , epithelial cells, and smooth muscle cells and is asthma is associated with cytotoxic mediators that are released regarded as a potent inflammatory mediator due to its effects on by activated eosinophils, and it has been shown that asthmatic vasodilatation, vascular permeability, and nociception; how- patients who received treatment based on eosinophil counts in ever, the role of PGE in allergic inflammation is unclear. In the sputum had significantly fewer severe asthma exacerbations 2 asthmatic lung, PGE affects both airway smooth muscle and than patients treated according to standard management therapy 2 the inflammatory process; PGE causes bronchial relaxation (5). Moreover, animal studies have also shown that genetically 2 (14) and inhibits allergen induced (15), but modified mice lacking eosinophils are protected against aller- it may also provoke bronchoconstrictor responses in some in- gen-induced lung injury and asthma (6, 7). A wide range of agonists can stimulate the accumulation of dividuals (16) because of activation of C fibers and reflex cho- eosinophils, in particular those of the CC family, linergic pathways (17). Moreover, PGE2 can induce cough di- which are acting through the CCR3 (8). In rectly and sensitize the cough receptor (18). In humans and rats, addition, activated complement factors such as C5a, leukotri- PGE2 reduces allergen-induced airway (19, 20), enes, 5-oxo-6,8,11,14-eicosatetraenoic acid, and -acti- abrogates eosinophil accumulation after passive cutaneous ana- phylaxis in the guinea pig (21), and protects against bleomycin- induced pulmonary fibrosis in mice (22). Conversely, eosino- Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Graz, Austria phil influx is exaggerated in COX-1 or COX-2 knockout mice Received for publication July 7, 2008. Accepted for publication September 18, 2008. (23, 24) and also in mice treated with selective COX-1 or The costs of publication of this article were defrayed in part by the payment of page COX-2 inhibitors (25). Decreased airway mucosal PGE2 pro- charges. This article must therefore be hereby marked advertisement in accordance duction during airway obstruction has been found in equine with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Jubila¨umsfonds of the Austrian National Bank Grants 10934 and 11967 and the Austrian Science Fund (FWF) Grant P19424-B05 V.K. is funded by the Ph.D. Program Molecular Medicine of the Medical University of Graz, Graz, Austria. 4 Abbreviations used in this paper: COX, cyclooxygenase; BAL, bronchoalveolar 2 lavage; CRTH2, chemoattractant receptor homologous molecules of Th2 cells; DM, E.M.S. and P.S. contributed equally to the study. demethylated; EP, E-prostanoid; EPO, eosinophil peroxidase; PG, prostaglandin; 3 Address correspondence and reprint requests to Dr. Akos Heinemann, Institute of PKA, protein kinase A; PKC, protein kinase C. Experimental and Clinical Pharmacology, Medical University of Graz, Universita¨- tsplatz 4, A-8010 Graz, Austria. E-mail address: [email protected] Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org 7274 EP2 RECEPTORS AND EOSINOPHIL TRAFFICKING

Table I. Compounds used in this study and their target selectivity , 3.33 mM CaCl2, 4.56 mM KCl, 1.5 mM NaH2PO4, 0.7 mM Na2HPO4, and 0.49 mM MgCl2. Reference Preparation of human leukocytes Name Selectivitya Activity No. Blood was sampled from healthy volunteers according to a protocol AH-6809 EP2 ϭ EP1 Ͼ Antagonist 57 approved by the Ethics Committee of the Medical University of Graz DP ϭ EP3 (Graz, Austria). All volunteers provided informed consent. Blood sam- Butaprost EP2 Agonist 57 ples were processed as described previously (29, 30). In brief, platelet- Chelerythrine PKC Inhibitor 58 rich plasma was removed by centrifugation of citrated whole blood, COX-1 ϭ COX-2 Inhibitor 59 after which erythrocytes and were removed by dextran sedi- H-89 PKA Inhibitor 60, 61 mentation. Preparations of polymorphonuclear leukocytes, containing LY-294002 PI3K Inhibitor 11 eosinophils and , and PBMC, including monocytes, ba- ONO-AE-248 EP3 Agonist 62 sophils, and , were made by Histopaque gradients (31). For ONO-AE1–329 EP4 Agonist 63 chemotaxis, eosinophils were further purified from polymorphonuclear ONO-AE3–208 EP4 Antagonist 64 populations by negative magnetic selection using an Ab mixture (CD2, PMA PKC Activator 48 CD14, CD16, CD19, CD56, and glycophorin A) and colloidal magnetic SB-202190 P38 MAPK Inhibitor 11 particles from StemCell Technologies. Resulting populations of eosin- SC-51089 EP1 Antagonist 57 ophils were typically Ͼ97%, with the majority of contaminating cells SQ-22536 Adenylyl cyclase Inhibitor 60 being neutrophils. Staurosporine Protein kinases Inhibitor 65 Culture of human peripheral blood monocytes a DP is D-prostanoid receptor. PBMC were resuspended in RPMI 1640 supplemented with penicillin (100

U/ml), streptomycin (100 ␮g/ml), glutamine (20 mM), nonessential amino Downloaded from acids, HEPES (50 mM) and sodium pyruvate (10 mM) at 6.7 ϫ 106/ml. asthma (26). At the cellular level, PGE2 has been found to at- Aliquots were placed into 6.5-mm Transwell inserts with 5-␮m polycar- tenuate Ig-dependent degranulation of eosinophils and leuko- bonate filters (Corning) in 24-well culture plates and human AB serum was triene biosynthesis. In contrast, PGE acts as a chemoattractant added. After 120 min of incubation at 37°C (5% CO2) nonadherent cells 2 were removed, which resulted in 105 monocytes per well and a purity of for immature murine mast cells derived from bone marrow (27). 95% (32). Moreover, PGE2 may favor Th2 responses over those of Th1 and promote the isotype switch to IgE (28). Chemotaxis http://www.jimmunol.org/ 6 Taken together, the role of PGE2 in allergic disease is not Purified eosinophils were suspended in assay buffer at 2 ϫ 10 /ml, and 50 unequivocal, but both animal and human studies suggest that ␮l of the suspension was placed onto the top of a 48-well micro-Boyden PGE has the potential of limiting the allergen-induced inflam- chemotaxis chamber with a 5-␮m pore-size polycarbonate filter (Neuro- 2 Probe) and 30 ␮l of agonists was placed in the bottom wells of the plate. matory response. However, the underlying mechanisms of this Baseline migration was determined in wells containing only assay buffer. effect and, hence, also the therapeutic usefulness of this ap- The plates were incubated at 37°C in a humidified incubator for 1 h, and proach still needs to be clarified. In this study we report that the membrane was carefully removed. Cells that had migrated to the lower chamber were enumerated by flow cytometric counting for 30 s, as de- PGE2 has a direct inhibitory effect on the migration of human scribed previously (30).

eosinophils and that this effect is mediated by E-prostanoid (EP) by guest on October 2, 2021 Transwell chemotaxis system (33) was used to assay the transmigration 2 receptors. We further show in animal models that EP2 ago- of eosinophils through layers of cultured adherent monocytes. Purified eo- nists potently inhibit the release of eosinophils from the bone sinophils were suspended in assay buffer at 2 ϫ 106/ml, and 100 ␮lofthe marrow and the allergen-induced accumulation of eosinophils suspension were placed onto the top of 5-␮m polyvinylpyrrolidone-free in the lung, thus pointing to EP2 receptors as therapeutic targets polycarbonate filters in 6.5-mm Transwell inserts containing adherent and overnight-cultured monocytes. The inserts were placed into 24-well culture in allergic diseases. plates containing 600 ␮l of buffer or chemoattractant in the bottom well. The plates were incubated at 37°C in a humidified incubator for 1 h, and Materials and Methods eosinophils that had migrated to the bottom wells were enumerated by flow Chemicals cytometry. Table I summarizes the pharmacological compounds used in this study Radioimmunoassay and shows their specificity. Reagents were obtained from Sigma-Al- Immunoreactive PGE in the supernatants of overnight-cultured monocytes drich unless specified. Dulbecco’s modified PBS (with or without 0.9 2 2ϩ 2ϩ was determined as described previously (34) using [5,6,8,11,12,14,15- mM Ca and 0.5 mM Mg ) was from Invitrogen. Assay buffer was 3 2ϩ 2ϩ H(N)]PGE2 as tracer and synthetic PGE2 as standard. Charcoal was used made from PBS with or without Ca /Mg supplemented with 0.1% to separate the free and Ab-bound fractions. The detection limit (defined as BSA, 10 mM HEPES, and 10 mM glucose (pH 7.4). CellFIX and 10% inhibition of binding of tracer to ) was 5.5 pg. FACSFlow were obtained from BD Immunocytometry Systems. Fixa- tive solution was prepared by diluting CellFIX 1/10 in distilled water Eosinophil degranulation and 1/4 in FACSFlow. Anti-mouse CCR3 (PE) was supplied by R&D Systems. Human eotaxin, IL-5, and IL-8 were from Peprotech. PGD , To determine the release of eosinophil peroxidase (EPO) from purified 2 ϫ 6 the CRTH2 (chemoattractant receptor homologous molecule of Th2 human eosinophils, cells were resuspended in assay buffer at 1 10 cells/ml, mixed with cytochalasin B (10 ␮g/ml), and 50-␮l aliquots were cells)-selective agonist 13,14 dihydro-15-keto-PGD2, PGE2, the EP2- selective agonists butaprost and its demethylated (DM) analog loaded into the wells of a 96-well microplate. Cells were stimulated with ␮ CAY10399 (butaprost-DM), and the EP/D-prostanoid receptor antago- 20 l of various concentrations of C5a for 20 min at 37°C. Thereafter, 60 ␮ nist AH-6809 were obtained from Cayman Chemical. The p38 MAPK lofH2O2 (1 mM) were added to each well to start the peroxidase reac- ␮ inhibitor SB-202190, the PI3K inhibitor LY-294002, the EP1 receptor tion. To detect the reaction, 70 l of 2.8 mM tetramethylbenzidine was antagonist SC-51089, the adenylyl cyclase inhibitor SQ-22536, the pro- used. Following incubation for 1 min at room , the peroxidase tein kinase A inhibitor H-89, and the protein kinase C (PKC) inhibitors reaction and the color development were terminated with 4 M acetic acid staurosporine and chelerythrine were supplied by BIOMOL. The rabbit (35). Microplates were analyzed on a bench reader at a wavelength of 630 polyclonal EP2 Ab and the corresponding blocking were ob- nm. Data were expressed as the percentage of the maximal control response tained from Cayman Chemical, and the Vectastain ABC kit and methyl (C5a at 300 nM). green were from Vector Laboratories. The EP3 receptor agonist ONO- In situ perfusion of the guinea pig hind limb AE-248, EP4 receptor agonist ONO-AE1–329, and the EP4 receptor antagonist ONO-AE3–208 were gifts from ONO Pharmaceutical. The guinea pig hind limb was perfused as previously described (29, 33). The composition of the Krebs-Ringer solution used for perfusion ex- The external iliac artery and vein were exposed and the caudal abdom-

periments was 0.1% BSA, 120 mM NaCl, 24 mM NaHCO3,10mMD- inal artery, superficial iliac circumflex artery, and pudendoepigastric The Journal of Immunology 7275

FIGURE 1. PGE2 decreases the chemotactic respon- siveness of human eosinophils to chemoattractants but has no effect on migration. A–C, Purified hu- man eosinophils were mixed with vehicle or PGE2 (10 and 30 nM), loaded into the top wells of a micro-Boy- den chamber, and allowed to migrate toward PGD2 (A), eotaxin (B), and C5a (C). D, Purified human eosinophils were mixed with vehicle or increasing concentrations of Downloaded from PGE2 (1–100 nM) and allowed to migrate toward eotaxin (1 nM), C5a (1 nM) or PGD2 (10 nM) in the bottom wells. E and F, Neutrophils were mixed with vehicle or PGE2 (10 and 30 nM), and allowed to migrate toward IL-8 or C5a, respectively. Chemotaxis was ex- pressed as a percentage of the maximum response to the respective control stimulus (i.e., chemoattractant in the bottom well only). Data are shown as mean Ϯ S.E.; n ϭ http://www.jimmunol.org/ Ͻ ء 4–6. , p 0.05 PGE2 vs vehicle. by guest on October 2, 2021

trunk along with their satellite veins were ligated. Polyethylene cannu- cytometry as highly granular cells (high side scatter) expressing the las (0.8-mm outside diameter) were inserted into the external iliac ar- eotaxin receptor CCR3 (37). tery and vein. Modified Krebs-Ringer bicarbonate buffer (gassed with

95% O2 and 5% CO2 at 37°C) was infused at 4 ml/min via the arterial Immunostaining of EP2 receptors cannula and removed from the venous cannula using a peristaltic pump. After an equilibration period of 20 min, the perfusate fractions were Mice were deeply anesthetized with sodium pentobarbital (60 mg/kg; collected every 10 min and centrifuged at 300 ϫ g for 10 min. The i.p.), the lungs were perfused via the lung artery with 0.1 M PBS (pH pellet was resuspended in Kimura’s stain and nucleated leukocytes and 7.4) followed by 4% buffered paraformaldehyde. The lungs were dis- Kimura-positive eosinophils were counted in a Neubauer hemacytom- sected, postfixed for 48 h, and embedded in paraffin. Five-micrometer eter (36). sections were deparaffinized, Ag retrieval was performed in 10 mmol/L sodium citrate solution (pH 6.0) (10 min at 600 watts in a microwave Murine model of allergic lung inflammation oven), and endogenous peroxidase was blocked with 0.3% H2O2 in PBS containing 0.1% Triton X-100. To reduce nonspecific background, sec- The experimental procedure used in this study was approved by the tions were incubated in blocking solution (Vectastain ABC Kit). The Austrian Federal Ministry of Science. Male BALB/c mice, 6- to 8-wk samples were incubated overnight with a polyclonal EP2 Ab (diluted old, were immunized with i.p. injections of 20 ␮g of OVA adsorbed to 1/100) or, to test for specific staining, with preabsorbed Ab (Ab pre-

3.5 mg of Al(OH)3 on days 0 and 7. Three groups of mice were studied. absorbed with a 5-fold amount of the corresponding peptide Ag) at 4°C The control group did not receive any further treatment until the end of overnight. After washing, the bound Ab was detected using the Vec- the experiment. The second and third groups of mice were challenged tastain ABC kit with diaminobenzidine as the chromogen and counter- by an aerosol of 2% (w/v) of OVA in saline for 30 min on days 21, 23, stained with methyl green. Sections were visually examined with an and 25. Additionally, these groups received a daily s.c. injection from Olympus IX70 microscope (Olympus) and an Olympus U Plan Apo day 20 to day 26; Group 2 was treated with 100 ␮l of vehicle (5% (v/v) ϫ40/0.8 lens. Photographs were taken with an Olympus DP50 camera ethanol) and group 3 was treated with 50 ␮g of butaprost. On day 27, (2776 ϫ 2074 pixels) and further processed with CELLP software mice were sacrificed by an overdose of pentobarbital sodium for the (Olympus) for additional white balance, contrast, and brightness analysis of leukocytes in peripheral blood and bronchoalveolar lavage adjustments. (BAL) fluid. Blood was sampled by cardiac puncture. BAL was per- EP2 receptors on human peripheral blood eosinophils were quanti- formed with 3 ϫ 1mlofCa2ϩ- and Mg2ϩ-free PBS supplemented with fied by indirect immunofluorescence flow cytometry. Because the EP2 0.1 mM EDTA. Eosinophils in blood and BAL were recognized by flow Ab is directed against the C terminus of the EP2 receptor, aliquots of 7276 EP2 RECEPTORS AND EOSINOPHIL TRAFFICKING Downloaded from

FIGURE 2. Activated monocytes attenuate the migration of human eosinophils in a COX-dependent manner. Monocytes were made adherent to the top of the polycarbonate membranes of 24-well Transwell inserts and pretreated with the COX inhibitor diclofenac (1 ␮M) or vehicle before being stimulated http://www.jimmunol.org/ with LPS (1 ng/ml) or vehicle overnight. Purified eosinophils were loaded on top of the layer in the Transwell inserts and allowed to migrate toward eotaxin (1 nM) in the bottom well. A and B, Eosinophils that had migrated into the bottom wells were enumerated by flow cytometry. Eosinophils (Eo) and monocytes (Mo) were distinguished by their properties in cell shape (forward scatter) and granularity (side scatter). Baseline migration in the absence of a chemoattractant is shown in A and the chemotactic response of eosinophils toward eotaxin (1 nM) is shown in B. C, Monocytes that had been activated by LPS pretreatment markedly attenuated the transmigration of eosinophils as compared with control monocytes, and the pretreatment of

monocytes with diclofenac before LPS exposure restored the migration of eosinophils. D, Effects of LPS and diclofenac on PGE2 release by monocytes into the supernatants as measured by radioimmunoassay. Data were expressed as a percentage of the control response to eotaxin determined in the absence .p Ͻ 0.01, n ϭ 5 ,ء ;of LPS and diclofenac. Data are shown as mean ϩ SE by guest on October 2, 2021

isolated eosinophils were first permeabilized with Fix&Perm solution by PGE2 (10 and 30 nM; Fig. 1, E and F). In all experiments, (An Der Grub Bio Research) for 15 min at room temperature. Samples the treatment of eosinophils with PGE2 was without effect on were then treated with the following consecutive Abs for 30 min each spontaneous migration in the absence of a chemoattractant (data on ice with appropriate washing steps in between: 1 mg/ml human IgG to block Fc receptors; 20 ␮g/ml polyclonal EP2 Ab or preabsorbed Ab not shown). (EP2 Ab incubated with a 5-fold amount of the corresponding peptide In additional experiments we investigated the concentration-ef- ␮ immunogen); and 4 g/ml anti-rabbit IgG secondary Ab conjugated with fect relationship of the PGE2-induced inhibition of eosinophil mi- Alexa Fluor-488. After adding the fixative solution the cells were analyzed on gration. To this end, purified human eosinophils were mixed with a FACSCalibur flow cytometer (BD Biosciences). vehicle or serial dilutions of PGE2 (3–100 nM), and the cells were Statistical analysis allowed to migrate toward single concentrations of eotaxin, C5a, or PGD in the bottom wells for 60 min. The concentrations of Data are shown as the mean Ϯ S.E. Statistical comparisons of groups were 2 performed using two-way ANOVA for repeated measurements or Wil- eotaxin (1 nM), C5a (1 nM), and PGD2 (10 nM) used were se- coxon signed rank test. Probability values of p Ͻ 0.05 were considered as lected so that they induced chemotaxis of similar magnitude. In

statistically significant. keeping with the results from the first experiments, PGE2 concen- tration dependently depressed the chemotactic responses of eosin- Results ophils, with maximal inhibition being observed with 100 nM PGE inhibits the migration of human eosinophils 2 PGE2.

To assess the effects of PGE2 on chemoattractant-induced eo- sinophil chemotaxis, purified human eosinophils were mixed Activated monocytes attenuate eosinophil migration via the with vehicle or two dilutions of PGE2 (10 and 30 nM) for 5 min at room temperature and were allowed to migrate for 60 min in release of PGE2

a micro-Boyden chemotaxis chamber toward serial dilutions of These data suggested that PGE2 is a selective and potent inhibitor

PGD2 (1–300 nM), the chemokine eotaxin (0.01–10 nM), or of eosinophil migration and activation. We next investigated C5a (0.1 nM-30 nM) placed in the bottom wells. Migrated cells whether endogenous PGE2, released in a paracrine manner, was in the bottom wells were then enumerated by flow cytometry. involved in the regulation of eosinophil recruitment. In detail, we

Fig. 1 demonstrates that PGE2 potently inhibited the migration investigated how activated monocytes, which are a rich source of of human eosinophils toward eotaxin, PGD2, and C5a, while at PGE2 and are constituents of the inflammatory cell infiltrate in the same concentrations the migration of neutrophils toward asthma, could modulate eosinophil function. To this end, periph- serial dilutions (0.1–10 nM) of IL-8 or C5a was not attenuated eral blood monocytes were isolated and cultured overnight on The Journal of Immunology 7277

FIGURE 3. Inhibition of adenylyl cyclase or protein

kinase A does not prevent the PGE2-induced inhibition of eosinophil chemotaxis. Purified eosinophils were pre- treated with vehicle, the adenylyl cyclase inhibitor SQ- 22536 (10 ␮M), or the protein kinase inhibitor H-89 (1 ␮M) at 37°C for 30 min and then mixed with 30 nM

PGE2. Cells were loaded into the top wells of a micro- Boyden chamber and allowed to migrate toward eotaxin

(1 nM; A and B)orPGD2 (30 nM; C). The data shown in B are from the same experiments as in A, but were normalized to the response in the presence of the re-

spective inhibitor but in the absence of PGE2. Data are p Ͻ ,ء ;the mean Ϯ SE; n ϭ 5–12; n.d., not determined 0.05 as indicated. Downloaded from http://www.jimmunol.org/

Transwell filters in the presence or absence of 1 ng/ml LPS (Esch- adenylyl cyclase via EP2 and EP4 receptors coupled to Gs pro- erichia coli serotype 055:B5), and the migration of eosinophils teins. To investigate the involvement of the adenylyl cyclase/ through the monocyte layer toward eotaxin (1 nM) was deter- protein kinase A (PKA) pathway in the inhibitory action of

mined. As compared with untreated monocytes, the migration of PGE2 on eosinophil chemotaxis, purified human eosinophils eosinophils in the presence of activated monocytes was reduced by were pretreated with vehicle, the adenylyl cyclase inhibitor SQ- Ͼ50% (Fig. 2C). Pretreatment of monocytes with the COX inhib- 22536 (10 ␮M), or the PKA inhibitor H-89 (1 ␮M) at 37°C for ␮ itor diclofenac (1 M) 20 min before exposure to LPS completely 30 min, mixed with 30 nM PGE2 and then allowed to migrate restored eosinophil responsiveness to eotaxin (Fig. 2C). Similarly, toward eotaxin (1 nM) for 60 min. Interestingly, both inhibitors the low molecular fraction (Ͻ1 kDa) of the supernatants of LPS- significantly attenuated the migration of eosinophils to eotaxin by guest on October 2, 2021

activated monocytes, purified by centrifugation over 1-kDa filters in the absence of PGE2 (Fig. 3A). However, PGE2 was still able (Pall Microsep), significantly attenuated the chemotaxis of eosin- to further diminish the migratory response to eotaxin after pre- ophils toward eotaxin ( p Ͻ 0.05), whereas supernatants from treatment with SQ-22536 or H-89 (Fig. 3A). To account for the monocytes that had been treated with diclofenac plus LPS dis- inhibitory effects of the inhibitors that they caused in the ab- ϭ played no inhibitory effect (data not shown; n 6). These obser- sence of PGE2, we reanalyzed these data by normalizing the

vations were mirrored by increased amounts of PGE2 released into effects of PGE2 to the response in the presence of the respective

the supernatants in the presence of LPS and by diclofenac decreas- inhibitor but in the absence of PGE2. In fact, both cAMP in-

ing PGE2 production below baseline levels (Fig. 2D). This sug- hibitors tended to reduce the inhibitory effect of PGE2 on eo- gested that a COX product, most likely PGE2, was released by sinophil migration toward eotaxin (Fig. 3B). However, when

activated monocytes and accounted for the inhibition of eosinophil PGD2 was used as chemoattractant instead of eotaxin, inhibi- migration. tion of cAMP did not attenuate the migration of eosinophils

and, also, the inhibitory effect of PGE2 was left completely PGE2 inhibits eosinophil function via PI3K and PKC altered (Fig. 3C). Elevation of intracellular cAMP has been suggested to attenuate PI3K and p38 MAPK are known to take part in the regulation

leukocyte function and, in fact, PGE2 is an effective activator of of eosinophil responses to chemoattractants (11, 30). To this

FIGURE 4. Inhibition of PI3K, but not p38 MAPK,

prevents the PGE2-mediated inhibition of eosinophil chemotaxis. Purified eosinophils were pretreated with SB-202190 (2 ␮M; A), LY-294002 (20 ␮M; B)orthe respective vehicle at 37°C for 20 min and then mixed

with PGE2 (3–100 nM), and loaded into the upper wells of a 48-well micro-Boyden chemotaxis chamber. Cells were allowed to migrate toward eotaxin and responses were expressed as a percentage of the control response to eotaxin. Data are shown as mean Ϯ SE; n ϭ 4–5; .p Ͻ 0.05 vs vehicle ,ء 7278 EP2 RECEPTORS AND EOSINOPHIL TRAFFICKING

tration of 0.1 nM mimicked the inhibitory effect of PGE2 on eosinophil migration (Fig. 5B). As expected, the inhibitory ef- fect of PMA was prevented by both PKC inhibitors but not, however, by LY-294002 (Fig. 5B). These data suggested that

PGE2 negatively modulates eosinophil migration through PI3Kand PKC-dependent pathways.

EP2 and EP4 receptors mediate the inhibitory effect of PGE2 on eosinophils

The wide range of PGE2 effects is mediated by four - coupled receptors, i.e., EP1, EP2, EP3, and EP4. Pretreatment of eosinophils with the selective EP1/EP2/EP3 antagonist AH- 6809 (30 ␮M) completely prevented the inhibitory effect of

PGE2 on the migration of human eosinophils toward eotaxin, while the selective EP1 antagonist SC-51089 (3 ␮M) had no effect (Fig. 6A). The EP4 antagonist ONO-AE3–208 (30 nM) prevented the inhibitory effect of low concentrations (1–10

nM), but not higher concentrations (30–100 nM), of PGE2.In agreement with the inhibitory effect of the EP2 antagonist, eo- Downloaded from sinophil migration toward eotaxin was inhibited by the EP2- selective agonists butaprost (0.1–1000 nM) and its demethyl- ated analog butaprost-DM (0.01–100 nM) (38) with a potency

FIGURE 5. Protein kinase C inhibitors reverse the PGE2 inhibition of 10- to 30-fold higher than that of PGE2 (Fig. 6B). The selective eosinophil migration. Purified eosinophils were pretreated with vehicle, EP3 agonist ONO-AE-248 did not mimic the inhibitory effect of staurosporine (1 ␮M), chelerythrine (10 ␮M) or LY-294002 (20 ␮M; B)at PGE2, whereas the EP4 agonist ONO-AE1–329 was capable of http://www.jimmunol.org/ 37°C for 10 min and then mixed with PGE2 (10–100 nM; A) or PMA (0.1 blunting the migration of eosinophils toward eotaxin but was at nM; B). Cells were loaded into the top wells of a micro-Boyden chamber least 30 times less potent than butaprost (Fig. 6B). and allowed to migrate toward eotaxin (1 nM). Responses were expressed In additional experiments we addressed the role of EP2 recep- as a percentage of the control response to eotaxin. Data are shown as tors in eosinophil degranulation. In fact, we observed that the C5a- .p Ͻ 0.05 vs vehicle ,ء ;mean Ϯ S.E; n ϭ 4–8 induced release of EPO was significantly attenuated by both PGE2 and butaprost (30 nM; Fig. 7), although butaprost was less effec- end, eosinophils were pretreated with the respective vehicle, the tive than PGE2 in this respect. Moreover, we observed that the EP4 p38 MAPK inhibitor SB-202190 (2 ␮M) or the PI3K inhibitor agonist ONO-AE1–329 at equimolar concentrations was as effec-

LY-294002 (20 ␮M) at 37°C for 20 min and then mixed with tive as butaprost at inhibiting eosinophil degranulation. These data by guest on October 2, 2021 suggested that both EP2 and EP4 receptors can mediate the inhib- PGE2 (10–100 nM). As compared with its vehicle, SB-202190 itory effects of PGE on human eosinophils, with EP2 receptors was unable to prevent the PGE2-induced abrogation of eosino- 2 phil chemotaxis toward 1 nM eotaxin (Fig. 4A). In contrast, being more important for the negative regulation of eosinophil LY-294002 slightly reduced eosinophil migration toward migration. eotaxin by itself and almost completely prevented the inhibitory EP2 agonists inhibit eosinophil release from bone marrow effect of PGE2 (Fig. 4B). Similarly, pretreatment of eosinophils with the nonselective protein kinase inhibitor staurosporine (1 The rapid release of eosinophils from the bone marrow is an ␮M) or the selective PKC inhibitor chelerythrine (10 ␮M) at important prerequisite of the accumulation of eosinophils in the 37°C for 10 min completely prevented the inhibitory effect of tissue at sites of inflammation. The evasion of eosinophils from

PGE2 on eotaxin-induced migration (Fig. 5A) without having a the bone marrow sinuses is believed to be governed by che- profound effect on eosinophils in the absence of PGE2. Consis- moattractants such as eotaxin or PGD2 and by growth factors tent with this, the PKC activator PMA at the very low concen- such as IL-5 (39, 40). The possible inhibitory effect of PGE2

FIGURE 6. EP2 and in part also EP4 receptors me- diate the inhibitory effect of PGE2 on eosinophil che- motaxis. A, Purified human eosinophils were pretreated with vehicle, the EP1/EP2/EP3 antagonist AH-6809 (30 ␮M), the EP4 antagonist ONO-AE3–208 (30 nM), or the EP1 antagonist SC-51089 (3 ␮M) at room temper- ature for 10 min and mixed with PGE2 (10–100 nM). B, Purified eosinophils were mixed with vehicle or various concentrations of PGE2 (3–100 nM), the EP2 agonists butaprost (0.1–100 nM), or butaprost-DM (0.01–100 nM), the EP3 agonist ONO-AE-248 (3–100 nM), or the EP4 agonist ONO-AE1–329 (3–100 nM) and were al- lowed to migrate toward eotaxin (1 nM). Responses were expressed as percent of the eotaxin control re- sponse and are shown as the mean Ϯ SE; n ϭ 5–7; Ͻ ء , p 0.05 vs PGE2 alone (vehicle). The Journal of Immunology 7279

EP2 agonist inhibits allergen-induced accumulation of eosinophils in lung

Our data suggested that PGE2 acting through EP2 receptors is a potent inhibitor of the in vitro migration of eosinophils and the rapid mobilization of eosinophils from the bone marrow. To investigate whether an EP2 agonist, butaprost, was also able to prevent the allergen-induced accumulation of eosinophils in the airways in vivo, mice were sensitized to OVA, injected daily with 50 ␮g of butaprost or vehicle s.c., and challenged with OVA by aerosol. An additional group of mice that were sensi- tized to OVA but received no aerosol challenge served as con- FIGURE 7. EP2 and EP4 receptors mediate the inhibitory effect of trol. Thereafter, BAL was performed and eosinophil numbers in PGE on eosinophil degranulation. Purified human eosinophils were pre- 2 the fluid were analyzed by flow cytometry. In additional groups treated with vehicle, the EP2 agonist butaprost, the EP4 agonist ONO- of mice (n ϭ 3 each), lungs were not subjected to lavage but AE1–329, or PGE2 (30 nM each) for 5 min and then stimulated with C5a for 30 min at 37°C. The release of EPO activity into the supernatant was were processed for histology. Immunochemistry revealed that determined by photometry. Data were expressed as a percentage of the EP2 receptors were predominantly expressed in bronchial epi- maximal control response (300 nM C5a) and are shown as the mean Ϯ SE; thelial cells and in some of the few inflammatory cells present ,p Ͻ 0.005 vs C5a alone (vehicle). in control lungs (Fig. 9, A and B). In addition to epithelial cells ,ء ;n ϭ 5–7 we observed profound EP2 staining in the infiltrating leuko- Downloaded from cytes (most likely eosinophil) in the lungs of allergen-chal- and EP2 receptors on the release of eosinophils from bone mar- lenged mice (Fig. 9, C and D). Similarly, human eosinophils row was investigated in the in situ perfused hind limb of the stained positive for EP2 when analyzed by flow cytometry (Fig. guinea pig. Infusion of the selective CRTH2 receptor agonist 9G). Preabsorption of the Ab with EP2-blocking peptide com- pletely prevented the staining of lungs of allergen-challenged 13,14-dihydro-15-keto-PGD2 (30 nM) or IL-5 (50 nM) into the hind limb caused a rapid increase of eosinophils in the effluent mice (Fig. 9, D and E) and also that of human eosinophils (Fig. http://www.jimmunol.org/ perfusate (Fig. 8, A and C) without having an effect on the 9G), hence proving specificity of the EP2 staining. In the BAL release of other nucleated cells (Fig. 8, B and D). Concomitant fluid, allergen challenge caused an ϳ3-fold increase in total infusion of PGE2 (30 nM), starting 10 min before the infusion leukocyte numbers as compared with control animals (Fig. 10). of 13,14-dihydro-15-keto-PGD2 completely prevented the mo- Excess leukocytes in the BAL fluid were identified as being bilization of eosinophils from bone marrow (Fig. 8A). Simi- eosinophils, while the numbers of other leukocytes did not larly, the mobilization of bone marrow eosinophils by IL-5 was change to a significant extent. Consequently, the frequency of Ͼ Ϯ significantly attenuated by PGE2 and even more profoundly by eosinophils in the BAL fluid increased 10-fold from 2.4

Ϯ Ͻ ϭ by guest on October 2, 2021 the EP2-selective PGE2 analog butaprost-DM (Fig. 8C). 1.0% to 27.9 7.3% ( p 0.05, n 8) in control vs challenged

FIGURE 8. PGE2 and the EP2-selective agonist butaprost-DM attenuate the release of eosinophils from guinea pig femoral bone marrow. The isolated hind limb preparation was perfused with Krebs-Ringer solution, the effluent was collected in 10-min fractions and eo- sinophils and other nucleated cells in the effluent were enumerated. Infusion of 13,14-dihydro-15-keto-PGD2 7 (DK-PGD2; 30 nM) or IL-5 (50 pM) for 30 min ( ) caused a rapid increase in eosinophil numbers in the effluent (A and C) while the number of other nucleated cells in the perfusate remained largely unchanged (B and D). Infusion of PGE2 or butaprost-DM (30 nM), which was started 10 min before that of DK-PGD2 or IL-5, respectively, markedly depressed the eosinophil mobilization. Data are shown as mean Ϯ SE; n ϭ 8–9; .p Ͻ 0.05 vs control ,ء 7280 EP2 RECEPTORS AND EOSINOPHIL TRAFFICKING

FIGURE 10. Butaprost attenuates the allergen-induced accumulation of murine eosinophils in BAL fluid in vivo. Male BALB/c mice were immu- nized i.p. with OVA and then divided into three groups, of which one received no further treatment (, A). The second group of mice was challenged with OVA by aerosol on days 21, 23, and 25 and treated with the vehicle of butaprost (allergy plus challenge, AC). The third group ad- ditionally received a daily s.c. injection of 50 ␮g of butaprost from day 20 to day 26 (allergy plus challenge plus butaprost, ACB). On day 27 BAL was performed and cells in the fluid were analyzed by flow cytometry. Data are shown as the cell counts per lung and as a percentage of total cell count. p Ͻ 0.05. Downloaded from ,ء ;Data are the mean Ϯ SE; n ϭ 8 each group

Discussion In this study we describe that the chemotactic responsiveness of isolated human eosinophils is restrained by low concentrations of http://www.jimmunol.org/ PGE2 in vitro. The PGE2-induced inhibition of migration is brought about mainly by EP2 receptors and involves PI3K and PKC. Consistent with this notion, we show in animal models that EP2 agonists are capable of ameliorating the mobilization of eo- sinophils from bone marrow and eosinophil recruitment to sites of allergic inflammation. Therefore, our data provide a novel mech- anistic concept to substantiate previous observations that exoge-

nous PGE2 negatively controls eosinophil recruitment to sites of allergic inflammation (19–21), while inhibition of endogenous by guest on October 2, 2021

PGE2 biosynthesis in COX-1 or COX-2 knockout mice or after FIGURE 9. Infiltrating inflammatory cells in a mouse model of aller- treatment with selective COX-1 or COX-2 inhibitors exaggerates gen-challenged lungs and human eosinophils express EP2 receptors. A–F, the eosinophil infiltration in response to allergen (23–25). As determined by immunohistochemistry, EP2 receptors (stained brown) The inhibitory effect of PGE on eosinophil recruitment seems were detected in sparse inflammatory cells and bronchial epithelial cells 2 to occur directly at the subcellular level; PGE2 very potently (IC50 (indicated by arrows) in lung sections from control mice (A and B). The ϳ dense infiltrates of inflammatory cells in allergen-challenged lungs were 3 nM) inhibited the migratory responsiveness of eosinophils, as also EP2 positive (C). Epithelial cells (arrows) from allergen-challenged chemotaxis toward three different classes of chemoattractants, i.e., mice can be seen in D. Preabsorption of the Ab resulted in the loss of eotaxin, PGD2, and C5a, was abrogated alike. Eosinophil migra- specific staining in allergen-challenged lungs (E and F). The photomicro- tion was also markedly inhibited in the presence of activated ϭ graphs show typical examples of each group (n 3). Scale bars are 100 monocytes, which are important sources of PGE2 production in ␮m. G, Histogram of indirect flow cytometric staining of isolated human vivo. In fact, LPS treatment of monocytes resulted in concentra- eosinophils with rabbit polyclonal EP2 Ab and preabsorbed EP2 Ab fol- ϳ tions of 3nMPGE2 in the supernatants, which corresponded to lowed by anti-rabbit secondary Ab conjugated with Alexa Fluor 488. Un- the IC50 of exogenously added PGE2. Inhibition of COX in mono- stained eosinophils and cells incubated with secondary Ab alone are also cytes abrogated both the biosynthesis of PGE and the inhibition of shown for comparison. The histogram shows one representative staining of 2 eosinophil migration. These observations, hence, demonstrate that four with different donors. the inhibitory action of PGE2 might also bear physiological relevance. mice, respectively. Treatment of mice with butaprost com- The effects of PGE2 are mediated via four rhodopsin-type G pletely prevented the allergen challenge-induced increase in to- protein-coupled receptors termed EP1, EP2, EP3, and EP4 (41). tal leukocyte numbers and eosinophil counts in the BAL fluid Expression of EP2 and EP4 receptors in eosinophils has been dem- (Fig. 10). This observation was reflected by a decrease of the onstrated at mRNA and protein levels, whereas data on EP1 and frequency of eosinophils after allergen challenge form 27.9 Ϯ EP3 receptor expression have remained inconclusive (42, 43). 7.3% to 9.8 Ϯ 2.3% ( p Ͻ 0.05, n ϭ 8) in animals treated with Functionally, stimulation of EP4 but not EP2 receptors inhibits the vehicle or butaprost, respectively (Fig. 10). The absolute C4 biosynthesis in human eosinophils (42). Our data numbers of other leukocytes was unchanged by butaprost treat- show that PGE2 inhibition of eosinophil responsiveness toward ment. The percentage of peripheral blood eosinophils did not chemoattractants is mediated mainly by EP2 receptors, as this was significantly differ in the three treatment groups (3.4 Ϯ 0.9% in mimicked by the EP2-selective agonists butaprost and its demeth- Ϯ sensitized animals, 8.7 3.2% in challenged and vehicle- ylated analog with considerably higher potency than with PGE2 treated mice, and 7.9 Ϯ 2.4% in challenged and butaprost- itself. This notion was corroborated by the observation that the treated mice; n ϭ 8, p ϭ 0.25). nonselective EP1/EP2/EP3 antagonist AH6809, which has very The Journal of Immunology 7281 low affinity for EP4 receptors, completely blocked the inhibitory chelerythrine. In contrast, LY-294002 did not prevent the inhibi- effect of PGE2 on eosinophil migration, while the EP4 antagonist tory effect of PMA on eosinophil migration. These data demon- was only partially effective. The involvement of EP1 and EP3 strated that PI3K acting up-stream of PKC might be involved in receptors could be ruled out, as the selective EP1 antagonist SC- the inhibitory action of PGE2 (49). Different PKC subtypes have 51089 was without effect on PGE2 inhibition of eosinophil migra- been shown to modulate eosinophil functions, such as chemotaxis, tion, and an EP3-selective agonist did not mimic the inhibitory degranulation, or adhesion, and increased expression and activa- effect of PGE2. However, EP4 receptors also seem to be capable of tion of eosinophil PKC␨ was recorded after allergen challenge conferring an inhibitory signal upon eosinophil migration, because (50). an EP4 agonist likewise decreased the migratory responsiveness of Recently, p38 MAPK has been hypothesized to be essential for eosinophils in chemotaxis assays. Interestingly, PGE2 and both the neutrophil orientation in opposing gradients of chemoattractants EP2 and EP4 agonists inhibited eosinophil degranulation as mea- (51). Furthermore, we described that the MAPK pathway deter- sured by C5a-induced EPO release, which might add to the anti- mines the hierarchy of eosinophil chemoattractants (11). In that inflammatory effects of PGE2 in vivo. study we observed that pretreatment of eosinophils with the che- Stimulation of EP2 or EP4 receptors usually increases intracel- moattractants eotaxin or 5-oxo-6,8,11,14-eicosatetraenoic acid in- lular cAMP levels. Unexpectedly, the inhibitory effect of PGE on 2 hibited the subsequent migration of the eosinophils toward PGD2 eosinophil migration does not seem to be integrally dependent on and that inhibition of p38 MAPK prevented this inhibitory effect activation of the adenylyl cyclase pathway, because neither the and even converted it to enhancement of eosinophil migration. adenylyl cyclase inhibitor SQ-22536 nor the PKA inhibitor H-89 This pathway, however, does not seem to be involved in PGE2 could prevent the inhibitory effect of PGE2. In detail, both cAMP

inhibition of eosinophil migration because SB-202190, a highly Downloaded from inhibitors slightly reduced the inhibitory effect of PGE2 on eosin- selective inhibitor of the ␣- and ␤-isoforms of p38 MAPK (52), ophil migration toward eotaxin, but this was not the case when failed to reverse the PGE2-induced inhibition of eosinophil PGD2 was used as the chemoattractant. Therefore, it seems that migration. cAMP plays a major role in eosinophil migration toward eotaxin It has been suggested previously that PGE2 also inhibits neu- per se in agreement with the differential roles that cAMP plays in trophil chemotaxis with an IC of 90 nM (53). In agreement with the complex process of leukocyte migration, depending on the sub- 50

that, PGE at concentrations up to 30 nM did not attenuate neu- http://www.jimmunol.org/ cellular localization of cAMP production and the relative expres- 2 trophil migration toward IL-8 and C5a in the present study, sion level of different cAMP effectors such as PKA or Epac-1 whereas the inhibition of eosinophil migration was near maximal (exchange protein directly activated by cAMP 1). As recently re- at that concentration. In contrast to its inhibitory effects in eosin- viewed by Lorenowicz et al. (44), activation of PKA results in ophils and neutrophils, recent studies suggested that PGE en- inhibition of cell surface expression of selectins and integrins and 2 hances the chemotactic responsiveness of human monocytes and integrin activation, thereby resulting in reduced adhesion; but PKA dendritic cells through activation of the EP2 and EP4 receptors is necessary for the clustering of integrins, which is a prerequisite (54, 55). It should be noted, however, that these effects were ob- for stable, sustained adhesion. Furthermore, PKA inhibits RhoA, a served only at concentrations of PGE at least 10-fold higher than critical regulator of -based contractility. In contrast, cAMP 2 those in the present study. by guest on October 2, 2021 also activates Epac-1, which mediates the activation of integrins The numbers of eosinophils at sites of inflammation depend on locally at the leading edge and therefore facilitates leukocyte ad- the rate of their release into the circulation from the bone marrow hesion and . These findings hence might explain how both stimulation and inhibition of cAMP biosynthesis might have a neg- and the rate of their extravasation into the tissue. Only a few in- ative impact on leukocyte migration. However, cAMP seems to flammatory mediators have been described to regulate eosinophil release from the bone marrow, including IL-5, eotaxin and PGD2 play a smaller role in PGD2-induced migration, because inhibition of the cAMP pathway did not attenuate eosinophil chemotaxis to- (33, 39, 40). In the present study we found that PGE2 via EP2 receptors also controls agonist-induced rapid mobilization of eo- ward PGD2 in the absence of PGE2. One explanation for this ap- parent discrepancy might be the fact that the eotaxin receptor, sinophils from bone marrow. In the in situ perfused guinea pig hind limb, 30 nM PGE2 completely prevented the mobilization of CCR3, signals through Gi proteins, leading to inhibition of adeny- eosinophils in response to infusion of the selective CRTH2 agonist lyl cyclase, whereas the PGD2 receptor, CRTH2, signals through 13,14-dihydro-15-keto-PGD2 and significantly attenuated the IL-5 both Gi and Gq in eosinophils (30). Therefore, disruption of a major signaling pathway by lowering cAMP levels at baseline induced eosinophil release. The EP2-selective agonist butap- might cause more profound alterations in the well-coordinated rost-DM showed even higher efficacy at the same concentration, subcellular events that govern the migration of the eosinophil in suggesting that the EP2 receptor was involved. Administration of PGE to humans and experimental animals the case of eotaxin, as compared with PGD2. 2 Apart from increasing cAMP biosynthesis, a downstream event has been shown to inhibit allergen-induced bronchoconstriction following stimulation of EP2 receptors is activation of PI3K (45, and airway eosinophilia (19, 20). Using immunohistochemistry of 46). Moreover, activation of PI3K can eventually lead to PKC murine lung tissue we observed that EP2 receptor expression was activation (47, 48). Therefore, we investigated the effect of the confined to the epithelium in control lungs. After exposure to al- PI3K inhibitor LY-294002 on eosinophil migration in the presence lergen, infiltrating leukocytes, presumably eosinophils, were also of PGE2. In agreement with previous findings, LY-294002 slightly EP2 positive. EP2 expression was also confirmed in human pe- inhibited eosinophil migration to eotaxin in the absence of PGE2, ripheral blood eosinophils by using flow cytometry. Because stim- but almost completely prevented the inhibitory effect of PGE2. ulation of EP2 receptors attenuates the mobilization of eosinophils Moreover, exposure of eosinophils to the broad-spectrum protein from bone marrow and eosinophil migration, we assessed the ef- kinase inhibitor staurosporine and the selective PKC inhibitor fect of the EP2 agonist butaprost on allergen-induced airway eo- chelerythrine likewise prevented the PGE2-induced inhibition of sinophilia in mice sensitized to OVA. Butaprost almost completely eosinophil chemotaxis. The PKC activator PMA very potently (0.1 abolished the allergen-induced increase in eosinophils in the BAL nM) mimicked the inhibitory effect of PGE2 on eosinophil che- fluid, although it had no significant effect on blood eosinophilia motaxis, and this effect was also prevented by staurosporine or after repeated allergen challenge. Nevertheless, this observation 7282 EP2 RECEPTORS AND EOSINOPHIL TRAFFICKING does not preclude that butaprost inhibits the allergen-induced mo- 14. Smith, A. P., M. F. Cuthbert, and L. S. Dunlop. 1975. Effects of inhaled pros- bilization of eosinophils from bone marrow, but its concomitant taglandins E1,E2, and F2␣ on the airway resistance of healthy and asthmatic man. Clin. Sci. Mol. Med. 48: 421–430. inhibitory effect on eosinophil extravasation seems to compensate 15. Pavord, I. D., C. S. Wong, J. Williams, and A. E. Tattersfield. 1993. Effect of for the reduced rate of the bone marrow supply of eosinophils. inhaled prostaglandin E2 on allergen-induced asthma. Am. Rev. Respir. Dis. 148: Taken together, in the present study we have described for the 87–90. 16. Mathe, A. A., and P. Hedqvist. 1975. Effect of F2␣ and E2 on first time that PGE2 directly controls eosinophil migration on the airway conductance in healthy subjects and asthmatic patients. Am. Rev. Respir. cellular level and is highly potent and efficacious in this respect. Dis. 111: 313–320. 17. Roberts, A. M., H. D. Schultz, J. F. Green, D. J. Armstrong, M. P. Kaufman, We have dissected the receptors, the cellular effector mechanisms, H. M. Coleridge, and J. C. Coleridge. 1985. Reflex tracheal contraction evoked and the biochemical pathways behind these PGE2 effects. In detail, in dogs by bronchodilator prostaglandins E2 and I2. J. Appl. Physiol. 58: we have shown in vitro that eosinophil locomotion is attenuated 1823–1831. 18. Costello, J. F., L. S. Dunlop, and P. J. Gardiner. 1985. Characteristics of pros- mainly via EP2 receptors involving PI3K- and PKC-dependent taglandin induced cough in man. Br. J. Clin. Pharmacol. 20: 355–359. pathways. Furthermore, using in vivo models we could show that 19. Gauvreau, G. M., R. M. Watson, and P. M. O’Byrne. 1999. Protective effects of EP2 agonists can attenuate the release of eosinophils from bone inhaled PGE2 on allergen-induced airway responses and airway inflammation. Am. J. Respir. Crit. Care Med. 159: 31–36. marrow and prevent the allergen-induced recruitment of eosino- 20. Martin, J. G., M. Suzuki, K. Maghni, R. Pantano, D. Ramos-Barbon, D. Ihaku, phils into the lungs. Previous studies have shown that PGE2 has F. Nantel, D. Denis, Q. Hamid, and W. S. Powell. 2002. The immunomodulatory bronchoprotective effects and also acts as an anti-inflammatory actions of prostaglandin E2 on allergic airway responses in the rat. J. Immunol. 169: 3963–3969. mediator in human airways (15, 19). However, the use of PGE2 as 21. Teixeira, M. M., T. J. Williams, and P. G. Hellewell. 1993. E-type prostaglandins a therapeutic agent in asthma is hampered by major side effects enhance local oedema formation and neutrophil accumulation but suppress eo- such as acute bronchoconstriction, retrosternal soreness, transient sinophil accumulation in guinea-pig skin. Br. J. Pharmacol. 110: 416–422. 22. Moore, B. B., M. N. Ballinger, E. S. White, M. E. Green, A. B. Herrygers, Downloaded from cough, and flu-like symptoms (15, 16, 19). Because these effects C. A. Wilke, G. B. Toews, and M. Peters-Golden. 2005. Bleomycin-induced E are thought to arise from EP1 or EP3 receptor stimulation, selec- prostanoid receptor changes alter fibroblast responses to prostaglandin E2. J. Im- tive EP2 agonists might have a more favorable pharmacological munol. 174: 5644–5649. 23. Gavett, S. H., S. L. Madison, P. C. Chulada, P. E. Scarborough, W. Qu, profile as therapeutic options for allergic diseases. 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