Interaction of eosinophils with endothelial cells is modulated by prostaglandin EP4 receptors

Interaction of eosinophils with endothelial cellsis modulated by prostaglandin EP4 receptors

Viktoria Konya1, Sonia Philipose1, Zoltan Balint2, Andrea Olschewski2,

Gunther Marsche1, Eva M. Sturm1, Rudolf Schicho1,

Bernhard A. Peskar1, Rufina Schuligoi1 and Akos Heinemann1

1 Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Graz, Austria2 Experimental Anesthesiology, Department of Anesthesia and Intensive Care Medicine, Medical

University of Graz, Graz, Austria

Eosinophil extravasation across the endothelium is a key feature of allergic inflammation.

Here, we investigated the role of PGE2 and its receptor, E-type prostanoid receptor (EP)-4, in

the regulation of eosinophil interaction with human pulmonary microvascular endothelial

cells. PGE2 and the EP4 receptor agonist ONO AE1-329 significantly reduced eotaxin-

induced eosinophil adhesion to fibronectin, and formation of filamentous actin and

gelsolin-rich adhesive structures. These inhibitory effects were reversed by a selective EP4

receptor antagonist, ONO AE3-208. PGE2 and the EP4 agonist prevented the activation and

cell-surface clustering of b2 integrins, and L-selectin shedding of eosinophils. Under

physiological flow conditions, eosinophils that were treated with the EP4 agonist showed

reduced adhesion to endothelial monolayers upon stimulation with eotaxin, as well as

after TNF-a-induced activation of the endothelial cells. Selective activation of EP1, EP2, and

EP3 receptors did not alter eosinophil adhesion to endothelial cells, whereas the EP4

antagonist prevented PGE2 from decreasing eosinophil adhesion. Finally, eosinophil

transmigration across thrombin- and TNF-a-activated endothelial cells was effectively

reduced by the EP4 agonist. These data suggest that PGE2–EP4 signaling might be

protective against allergic responses by inhibiting the interaction of eosinophils with the

endothelium and might hence be a useful therapeutic option for controlling inappropriate

eosinophil infiltration.

Keywords: Adhesion . Endothelium . Eosinophils . Migration . Prostaglandins

Supporting Information available online

Introduction

Eosinophil granulocytes are important effector cells in allergic

inflammation and are recruited to the tissue by chemotactic

signals [1–5]. The infiltrating eosinophils release several toxic

and proinflammatory mediators which induce epithelial injury,

airway hyper-responsiveness, and airway remodeling [6–8].

Therefore, eosinophils are regarded as potential therapeutic

targets in allergic diseases and asthma [9].

Prostaglandins (PGs) play diverse roles in inflammation,

depending on the cellular context, the type of PG being released,

and the differential expression of PG receptors [10, 11]. While

PGD2, which is the predominant PG formed in mast cells, exerts

proinflammatory effects by regulating the recruitment of eosino-Correspondence: Dr. Akos Heinemanne-mail: [email protected]

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

Eur. J. Immunol. 2011. 41: 2379–2389 DOI 10.1002/eji.201141460 Leukocyte signaling 2379

phils, basophils, and Th2 lymphocytes to the sites of allergic

inflammation [12], PGE2 seems to attenuate inflammatory

responses and reduce tissue injury in airways [13]. PGE2 was found

to exert bronchoprotective effects in patients with asthma [14]. In

rats, the ovalbumin-induced early and late phase airway responses

were inhibited by intratracheally administered PGE2 [15].

The activity of PGE2 is mediated by four subtypes of E-type

prostanoid receptors (EP), EP1, EP2, EP3, and EP4 [16]. These G

protein-coupled receptors have distinct biological imprints

depending on their affinity with synthetic agonists and the acti-

vated intracellular signaling pathways. Activation of the

Gq-coupled EP1 receptor leads to intracellular Ca21 increase and

protein kinase C activation. The EP2 and EP4 receptors are

G protein-coupled and elevate intracellular cyclic adenosine

monophosphate (cAMP) which activates protein kinase A. The

EP3 receptor is coupled to the Gi protein and its activation

increases the intracellular levels of Ca21. The EP3 receptor itself

has several splice variants exhibiting some constitutive activity

[16, 17]. At the cellular level and in murine allergic inflamma-

tion, PGE2 was found to inhibit eosinophil trafficking via EP2

receptor activation [18]. In mouse BM, PGE2 decreased the

number of eosinophil peroxidase-positive cells and reduced the

expression of a4 integrin in eosinophils [19].

The role of PGE2 in endothelial function has not been inves-

tigated in detail. Particularly, it is unknown how PGE2 modulates

the interaction of eosinophils with the endothelium, which is a

pivotal step in eosinophil extravasation to the sites of allergic

inflammation. In this study, we report that PGE2 potently

attenuates the adhesion of human peripheral blood eosinophils to

pulmonary microvascular endothelial cells and their trans-

endothelial migration by negatively regulating eosinophil polar-

ization and activation of adhesion molecules.

Results

PGE2 reduces eosinophil adhesion to fibronectin viaEP4 receptor activation

The effect of PGE2 and the selective EP4 agonist ONO AE1-329 on

eosinophil adhesion was investigated using fibronectin-coated

plates (Fig. 1A). Eosinophils were preincubated with vehicle,

PGE2, or EP4 agonist (30 nM each) for 10 min at 371C. Eotaxin

induced concentration-dependent (0.3–3 nM) adhesion of eosi-

nophils to fibronectin which was significantly reduced by PGE2

and the EP4 agonist (Fig. 1A). The specific involvement of the

EP4 receptor was demonstrated by preincubating the cells with

an EP4-selective antagonist (ONO AE3-208; 300 nM) which

completely prevented the inhibitory effect of PGE2 on eosinophil

adhesion. Consistently, eosinophils showed membrane-associated

EP4 receptor expression as detected by laser-scanning confocal

microscopy (Fig. 1B and Supporting Information Fig. 1).

Formation of adhesive structures or protrusions is a key

element in eosinophil adhesion and migration. The effect of the

EP4 agonist and PGE2 on adhesive structure formation was

analyzed by preincubating eosinophils with vehicle, EP4 agonist,

or PGE2, 30 nM each (Fig. 2A and B). Adhesive structure

formation was induced by stimulation of eosinophils with eotaxin

(3 nM). Fluorescence microscopy revealed dramatic differences

in the cell shape and filamentous actin (F-actin) staining between

eosinophils stimulated in the presence of vehicle or EP4 agonist

and PGE2 respectively. The eotaxin-induced cellular polarization

and the development of lamellipodia/filopodia that contained

highly polymerized F-actin fibers were inhibited by the EP4

agonist and PGE2 (Fig. 2A). Gelsolin, an actin-severing protein,

was evenly distributed in vehicle-stimulated eosinophils but was

relocated to the cell periphery after eotaxin stimulation. In

eotaxin-stimulated eosinophils, gelsolin strongly colocalized with

F-actin as shown in the merged images (Fig. 2A). The colocali-

zation of gelsolin with F-actin was prevented by both the EP4

agonist and the PGE2. Importantly, the specific EP4 antagonist

largely reversed the inhibitory effect of PGE2 on adhesive struc-

ture formation and restored the colocalization of gelsolin with

F-actin (Fig. 2A). On average, eotaxin-stimulated eosinophils

exhibited 16 protrusions per cell; both the EP4 agonist and the

PGE2 reduced the number of adhesive structures to two to three

per cell (Fig. 2B) and the EP4 antagonist reversed the inhibitory

effect of PGE2 to 12 protrusions per cell (Fig. 2B).

Figure 1. The EP4 receptor is expressed on human eosinophils andinhibits the eotaxin-induced eosinophil adhesion to fibronectin.(A) Human-purified eosinophils were preincubated with the specificEP4 antagonist (antag) ONO AE3-208 or its vehicle, and then treatedwith vehicle, 30 nM of the EP4-selective agonist (ag) ONO AE1-329 orPGE2. In the presence of serial dilutions (0.3–3 nM) of eotaxin,eosinophils were allowed for 30 min to adhere to 96-well plates coatedwith 5mg/mL fibronectin. Results are mean1SEM percent of adherentcells relative to total eosinophils added. Experiments were performedfour times. �po0.05 versus vehicle for one-way ANOVA of repeatedmeasurements followed by Dunnett’s post-hoc test. (B) EP4 receptorexpression was determined by immunofluorescence microscopy. Thetop panel shows staining with the isotype control Ab, the bottom paneldisplays eosinophils stained with the EP4 Ab. The insert in the bottompanel is a laser-scanning micrograph showing cell-surface expressionof EP4 receptors on one single eosinophil (Z-stacks of the image areshown in Supporting Information). The secondary Ab was labeled withAlexa Fluor 647, and the nuclei are stained with DAPI. The micrographsare representative of eosinophil staining of three different donors.

Eur. J. Immunol. 2011. 41: 2379–2389Viktoria Konya et al.2380


These results showed that human eosinophils express EP4

receptors which negatively modulate the adhesion of eosinophils

to fibronectin, along with the morphological changes of eosino-

phils after eotaxin stimulation.

b2 integrin and L-selectin functions are altered by EP4agonist and PGE2

Since L-selectins and b2 integrins are highly involved in the

adhesion of eosinophils to endothelial cells [20] as well as to the

extracellular matrix [21], we investigated the regulatory function

of EP4 receptors and PGE2 on the activation and cell-surface

clustering of b2 integrins and on L-selectin shedding. Briefly, b2

integrin activation on eosinophils was detected by flow cytometry

using the conformation-dependent antibody mAb 24 which

specifically recognizes activated b2 integrin complexes. Stimula-

tion of eosinophils with eotaxin or PGD2 increased the

fluorescence intensity of cells stained with mAb 24, whereas the

signal of the isotype control Ab remained unchanged (Fig. 3A and

Supporting Information Fig. 2). The eotaxin- and PGD2-induced

b2 integrin activation was equally prevented by the EP4 agonist

ONO AE1-329 and PGE2 (30 nM each). Fluorescence microscopy

showed that b2 integrin cell-surface clustering was also inhibited

by both the EP4 agonist and the PGE2 (Fig. 3B). On eosinophils

stimulated with eotaxin (3 nM), mAb 24 staining was confined to

clusters of b2 integrin, whereas in vehicle-treated eosinophils

mAb 24 showed a diffuse staining. The EP4 agonist and PGE2

prevented the eotaxin-induced clustering of b2 integrin

complexes, which resulted in staining reminiscent of vehicle-

treated eosinophils (Fig. 3B).

PGD2 (30 nM) and eotaxin (3 nM) induced L-selectin shedding

on eosinophils (Fig. 3C), as determined by flow cytometry using

fluorescently labeled Ab against L-selectin (gating strategies are

shown in Supporting Information Fig. 3). The EP4 agonist ONO

AE1-329 and PGE2 (30 nM each) reversed the eotaxin- and PGD2-

induced L-selectin shedding (Fig. 3C). These data suggest that

activation and cell-surface clustering of eosinophil b2 integrins,

as well as L-selectin shedding, are markedly inhibited by EP4

receptor activation in eosinophils.

EP4 agonist attenuates eosinophil–endothelial inter-action

In further experiments, we addressed the impact of EP4

activation on eosinophil adhesion to pulmonary microvascular

endothelial cells under physiological flow conditions. For this

purpose, we grew endothelial monolayers on Cellix VenaEC

biochip substrates for 2 days; confluence of the cell layers was

determined visually by an inverted phase-contrast microscope

(Supporting Information Fig. 4). VenaEC biochips containing the

endothelial monolayers were superfused with isolated eosino-

phils and images were recorded for 140 s. EP4 agonist pretreat-

ment of eosinophils significantly reduced both the basal adhesion

Figure 2. An EP4 agonist and PGE2 abrogate the formation of adhesivestructures in eosinophils. Samples of purified eosinophils were treatedwith vehicle, EP4 agonist, PGE2 (30 nM each), or first with EP4 antagonist(300 nM) followed by PGE2. Eotaxin (3 nM) was used for stimulatingeosinophil adhesion to cover slips coated with 5mg/mL fibronectin.F-actin was detected with Phalloidin-Texas Red, whereas gelsolin wasstained with a monoclonal mouse Ab followed by an appropriatesecondary Ab labeled with Alexa Fluor 488. (A) Representative micro-graphs of the formation of adhesive structures in differentially treatedadherent eosinophils. (B) Filopodia containing gelsolin as markers ofadhesive structures were enumerated by microscopy. Data are mean1

SEM, experiments were repeated three times. �po0.001 EP4 or PGE2 pluseotaxin versus eotaxin alone for one-way ANOVA of repeated measure-ments followed by Dunnett’s post-hoc test.

Eur. J. Immunol. 2011. 41: 2379–2389 Leukocyte signaling 2381


(Fig. 4A, top) and the eotaxin-induced (3 nM) adhesion to the

endothelial layer (Fig. 4A, middle). Preactivation of the

endothelial layers with TNF-a (10 pM) for 4 h greatly enhanced

the number of adherent eosinophils. Similarly, eosinophil

adhesion to TNF-a-activated endothelial cells was reduced by

more than 50% in the presence of the EP4 agonist (Fig. 4A,

bottom). Corresponding quantitative data were obtained from

computerized image analysis of adherent eosinophils (Fig. 4B).

Representative video footage can be viewed on the journal

homepage. On the contrary, the EP1/EP3 agonist (17-phenyl

trinor PGE2, 30 nM), EP2 agonist (butaprost, 30 nM), or EP3

agonist (sulprostone, 30 nM) were found to have no effect on

eosinophil adhesion to TNF-a-stimulated endothelial monolayers

(Fig. 4C). Treatment with PGE2 mimicked the action of the EP4

agonist, and the inhibitory effect of PGE2 was prevented by the

selective EP4 receptor antagonist (Fig. 4C). Collectively, these

data demonstrated that the activation of EP4 receptors negatively

regulates the adhesion of eosinophils to endothelial cells.

Next, we tested the possibility that EP4 receptor activation

was capable of reversing adhesion, resulting in detachment of

eosinophils from endothelial cells. In Fig. 4D, eosinophils were

allowed to adhere to TNF-a-stimulated endothelial layers for

140 s and were then superfused with vehicle or EP4 agonist

(30 nM) for further 10 min. Images were acquired before and at

5–10 min after the start of the vehicle/EP4 agonist treatment.

While the number of adherent eosinophils remained stable when

superfused with vehicle, the EP4 agonist led to a time-dependent

detachment of adherent eosinophils from the endothelial mono-

layer (Fig. 4D).

Finally, we investigated the effect of the EP4 agonist on

eosinophil transmigration across monolayers of pulmonary

microvascular endothelial cells (Fig. 5). Endothelial cells were

grown in transwell inserts until confluence, as confirmed by

measuring transendothelial electrical resistance. Transendothe-

lial migration was calculated as the percent of total eosinophils

added to each well. Eosinophil migration was more than six-fold

enhanced when eotaxin (3 nM) was present in the bottom well.

After pretreatment of the endothelial layer with thrombin (0.5 U/

mL) for 15 min, eosinophil migration was further increased to

almost 15-fold of baseline (Fig. 5A). Pretreatment of eosinophils

with 30 nM of the EP4 agonist reduced the eotaxin-triggered

transendothelial migration of eosinophils from approximately 35

to 12% (Fig. 5A). Similar observations were made when endo-

thelial monolayers were activated with TNF-a for 4 h which

resulted in a 20-fold increase of eosinophil migration (Fig. 5B).

The EP4 agonist reduced eosinophil transmigration across

TNF-a-stimulated endothelial layers from 57 to 29% of input

eosinophils (Fig. 5B). These data suggested that EP4 agonists are

able to control transendothelial migration of eosinophils.

Discussion

In the present study, we show that PGE2 and EP4 receptors

potently regulate eosinophil interaction with pulmonary endothe-

lial cells, as PGE2 and the EP4 agonist ONO AE1-329 significantly

reduced adhesion of eosinophils to endothelial cells under

physiological flow conditions and blocked eosinophil trans-

endothelial migration. Visualization of eosinophil adhesion to

fibronectin revealed that PGE2 and the EP4 agonist deranged

the polarization and formation of cellular adhesive structures in

eosinophils. Furthermore, activation and cell-surface clustering of

b2 integrins and L-selectin shedding were inhibited by PGE2 and

the EP4 agonist of that ilk. Eosinophil EP4 receptor expression on

the cell membrane was confirmed by laser-scanning confocal

Figure 3. Eosinophil b2 integrin activation and cell-surface clusteringand L-selectin shedding are inhibited by an EP4 agonist and PGE2.(A) Purified human eosinophils were treated with the EP4 agonist (ag)ONO AE1-329 or PGE2 (30 nM each) and stimulated with eotaxin (3 nM)or PGD2 (30 nM). The activation of b2 integrins was thereafterdetermined by indirect immunofluorescence staining using mAb 24and flow cytometry. Data are mean1SEM fluorescence intensity,experiments were performed four times. �po0.001 EP4 agonist orPGE2 versus vehicle for one-way ANOVA of repeated measurementsfollowed by Dunnett’s post-hoc test. (B) Clustering of activatedb2 integrins was detected by staining with mAb 24 and immuno-fluorescence microscopy. Micrographs are representative of threeexperiments. (C) Polymorphonuclear leukocyte preparations wereincubated with vehicle, EP4 agonist (ONO AE1-329) or PGE2 (30 nMeach). L-selectin shedding was induced by eotaxin (3 nM) or PGD2

(30 nM) and was determined using flow cytometry. Eosinophils weredistinguished from neutrophils as CD16neg cells. Data are presented asmean1SEM fluorescence intensity, experiments were repeated fourtimes. �po0.001 EP4 agonist or PGE2 versus vehicle for one-way ANOVAof repeated measurements followed by Dunnett’s post-hoc test.



microscopy. Therefore, EP4-mediated attenuation of eosinophil

–endothelial interaction might afford a novel therapeutic

approach to preventing eosinophil recruitment at the sites of

allergic inflammation.

In detail, we found that selective activation of the EP4

receptor significantly inhibited the chemokine-induced adhesion

of eosinophils to fibronectin. One of the initial steps in the

extravasation process is the capture and adhesion of eosinophils

to the endothelium, via the molecular interaction of eosinophil

selectins and integrins with endothelial adhesion molecules.

Integrins also mediate adhesion to extracellular matrix compo-

nents, such as fibronectin, vitronectin, laminin, or collagen

[21, 22]. In the latter case, integrins recognize the RGD (Arg-Gly-

Asp) amino acid sequence of the matrix macromolecules which is

the recognition and binding site for the a5b1 and aMb2 integrins

[23]. Addition of PGE2 elicited the same inhibitory effect on

eosinophil adhesion to fibronectin as the EP4 agonist. Morpho-

logical analysis of eosinophils adhering to fibronectin showed

that the actin cytoskeleton was reorganized upon eotaxin

stimulation, an event that was coupled to cellular polarization

and formation of lamellipodia/filopodia and F-actin/gelsolin-rich

adhesive structures. Treatment with PGE2 and the EP4 selective

agonist prevented these morphological changes. The highly

dynamic F-actin-rich protrusions contain large amounts of

integrins, and actin-binding and actin-severing proteins such as

gelsolin. The major function of gelsolin is to rapidly depolymerize

Figure 4. EP4 receptor activation suppresses eosinophil adhesion to endothelial cells under flow conditions. Eosinophils treated with vehicle orthe EP4 agonist ONO AE1-329 (30 nM) were flowed over human pulmonary microvascular endothelial cells grown on VenaEC biochips (Cellix).Eosinophil adhesion was induced by stimulating eosinophils with eotaxin (3 nM) or by activating the endothelial monolayer with TNF-a (10 pM).(A) Representative images taken 2 min after the start of the superfusion with eosinophils. (B) Images were quantified by computerized imageanalysis. Data are shown as mean1SEM of four experiments with different donors. �po0.001, EP4 agonist versus vehicle for one-way ANOVA ofrepeated measurements followed by Dunnett’s post-hoc test. (C) Eosinophils were treated with EP1/EP3 agonist (EP1, 17-phenyl trinor PGE2, 30 nM),EP2 agonist (butaprost, 30 nM), EP3 agonist (sulprostone, 30 nM), EP4 agonist (ONO AE1-329, 30 nM), or PGE2 (30 nM). Alternatively, eosinophils werefirst pretreated with EP4 antagonist (ONO AE3-208, 300 nM) followed by PGE2 (30 nM). Cells were then flowed over TNF-a-stimulated endothelial cellsand their adherence was recorded 2 min later. Results are shown as mean1SEM of four experiments with different donors. �po0.001, EP4 agonist andPGE2 versus vehicle, or EP4 antagonist plus PGE2 versus PGE2 alone for one-way ANOVA of repeated measurements followed by Dunnett’s post-hoctest. (D) Vehicle-treated eosinophils were flowed over TNF-a-stimulated endothelial cells and the attached eosinophils were then superfused withvehicle or EP4 agonist (ONO AE1-329, 30 nM) for an additional 5 or 10 min. Data are presented as mean1SEM of four experiments with differentdonors. �po0.05, EP4 agonist after 5 or 10 min versus before for one-way ANOVA of repeated measurements followed by Dunnett’s post-hoc test.



F-actin, and thus gelsolin sustains the dynamics of protrusions

and withdrawal which is essential for directed locomotion

[24, 25].

Adhesion sites contain clustered integrins that transmit

mechanical forces in the migration process and are involved in

signaling cascades essential for cell survival and morphogenesis

[26]. Eotaxin- and PGD2-induced b2 integrin activation and

clustering was markedly reduced by the EP4 agonist and PGE2.

Therefore, it is very likely that the PGE2/EP4-induced blockade of

b2 integrin activation and cell-surface clustering might take a

profound effect on eosinophil recruitment and function at the

sites of inflammation. A potential limitation of our clustering

experiments may be that we used the activation-sensitive mAb 24

for detecting b2 integrin complexes. Since a recent report showed

that integrin activation and clustering can occur independently

[26], it is possible that we missed detecting the lateral clustering

of nonactivated b2 integrins. L-selectin mediates the capture and

rolling of eosinophils on the endothelium. Chemoattractant-

induced activation of eosinophils can lead to shedding of

L-selectin due to metalloproteinase activity. The EP4 agonist and

PGE2 prevented the eotaxin- and PGD2-stimulated L-selectin

shedding, which seems consistent with the ability of EP4 recep-

tors to reverse eosinophils activation. Since these changes in

eosinophil function might also translate to compromised eosi-

nophil–endothelial interaction, we examined the impact of EP4

receptor activation on eosinophil adhesion to human pulmonary

microvascular endothelial cells under flow conditions. In fact,

eosinophils that had been pretreated with EP4 agonist showed

reduced adhesion to resting as well as TNF-a-activated endo-

thelial cells. On the contrary, other PGE2 receptors, i.e. EP1, EP2,

and EP3, do not seem to play a role since agonists of these

receptors did not mimic the inhibitory effects of the EP4 agonist

or PGE2. Interestingly, the activation of EP4 receptors was suffi-

cient to deactivate eosinophils that had previously become

adherent, leading to detachment from endothelial cells.

TNF-a, a predominant proinflammatory cytokine, has been

shown to be a chemoattractant for both neutrophils and eosino-

phils [27]. Furthermore, TNF-a has been suggested to play an

important role in severe refractory asthma, a rare type of asthma

which affects 5–10% of all asthmatic patients. Being largely

unresponsive to inhaled corticosteroids, this subgroup of

asthmatics urgently requires novel therapies [28–30]. Anti-TNF-atherapy has been proved to improve lung function, airway hyper-

responsiveness, and exacerbation rates in some but not all

refractory asthma patients [31–34]. However, a potential risk of

anti-TNF-a therapy is the development of malignancies and

increased susceptibility for infections which can limit the clinical

use of TNF-a blockers [35]. Importantly, the present study

suggests a possible application of EP4-selective agonists in

treating severe refractory asthma, as an EP4 agonist markedly

reduced the adhesion of eosinophils to TNF-a-treated pulmonary

microvascular endothelial cells.

In additional interaction studies, we investigated eosinophil

transmigration across endothelial monolayers activated with

TNF-a or thrombin. Eosinophil chemotaxis was triggered by

eotaxin. In further support of the inhibitory role of the EP4

receptor in eosinophil trafficking, we observed that the EP4

agonist markedly reduced the transendothelial migration of

eosinophils through thrombin-activated as well as TNF-a-stimu-

lated endothelial monolayers. The involvement of thrombin in

bronchial asthma is supported by several reports; increased levels

of thrombin were found in asthmatic airways [36–38]. Moreover,

thrombin was shown to exert chemotactic activity on eosinophils

via activation of protease-activated receptor-1 [39]. In our assay,

TNF-a and thrombin were present in the experimental setup

throughout the 4 h migration period and could hence exert their

effects both on endothelial cells and on eosinophils.

Importantly, the reduced responsiveness of eosinophils after

EP4 receptor activation was not due to downregulation of the

respective C–C chemoattractant receptors (CCR3 and chemo-

attractant receptor homologous molecule expressed on Th2

lymphocytes) or reduced viability or a proapoptotic effect of the

EP4 agonist (Supporting Information Figs. 5 and 6). Moreover,

PGE2 showed the same potency in all assays tested at inhibiting

eosinophil responses as the EP4 agonist, and the effects of PGE2

were completely prevented by EP4 antagonist. This included

eosinophil adhesion to fibronectin, adhesion to endothelial

monolayers, polarization of eosinophils, and formation of adhe-

sive structures, which suggests that PGE2 might exert inhibitory

effects on eosinophil–endothelial interaction also physiologically

through EP4 receptors.

Collectively, the results of the current study are in agreement

with our previous findings that PGE2 decreases the migration of

eosinophils through uncoated filters toward PGD2 and eotaxin.

This effect was at least partially reversed by the EP4 antagonist

[18], although EP2 receptors were also found to be involved in

this setup. In further experiments, we could substantiate the

Figure 5. EP4 agonist inhibits eosinophil transendothelial migrationacross thrombin- and TNF-a-activated endothelial monolayers.Endothelial cells were grown in Transwell inserts and were activatedby (A) 0.5 U/mL thrombin or (B) 10 pM TNF-a. Eosinophils werepretreated with vehicle or the EP4 agonist ONO AE1-329 (30 nM), andtransmigration was stimulated with eotaxin (3 nM) or vehicle for 4 h.Results are shown as the mean1SEM percentage of transmigratedeosinophils relative to total eosinophils added. Experiments wereperformed four times, �po0.05 EP4 agonist versus vehicle for one-wayANOVA of repeated measurements followed by Dunnett’s post-hoctest.



inhibitory role of EP4 receptors in eosinophils in assays of CD11b

upregulation, production of reactive oxygen species and Ca21

mobilization [40]. These findings have now fundamentally been

extended by the current study as we found that the regulatory

role of EP4 receptors translates to preventing the interaction of

eosinophils with the endothelium, which is probably the most

crucial step in eosinophil recruitment to the sites of inflamma-

tion. Interestingly, we recently identified the EP4 receptor as a

potent anti-aggregatory regulator. Inhibition of platelet aggre-

gation by a selective EP4 agonist was characterized by attenuated

Ca21 flux, inhibition of glycoprotein IIb/IIIa, and downregulation

of P-selectin. Moreover, adhesion of platelets to fibrinogen under

flow conditions and in vitro thrombus formation were similarly

prevented by the EP4 agonist [41]. Other anti-inflammatory

actions of EP4 receptors reported previously include inhibition of

cytokine production in monocytes and macrophages [42–44],

and mucus secretion in airway epithelial cells [45]. From our

current data, we cannot exclude the possibility that parts of the

inhibitory effects of PGE2 and the EP4 agonist also involve EP4

receptors expressed on the endothelial cells. Therefore, the role

of EP4 receptors in endothelial function awaits further investi-

gation.

In summary, increased levels and protective effects of PGE2 in

pulmonary inflammatory diseases have been described previously

[14, 46]. The accumulation of eosinophils in asthmatic airways

greatly contributes to the outcome of the disease by releasing

cytotoxic mediators, leading to airway remodeling and angiogen-

esis in the chronically inflamed pulmonary tissue [7]. The present

study provides an explanation for the protective role of PGE2 in

allergic inflammation, by (i) identifying the EP4 subtype as key

binding site for PGE2 and (ii) revealing the cellular mechanisms by

which the EP4 receptor is involved in controlling eosinophil

interaction with the endothelium. Furthermore, our findings

suggest that EP4-selective agonists might be useful therapeutic

agents for treating otherwise uncontrolled bronchial asthma.

Materials and methods

Chemicals

Laboratory reagents were purchased from Sigma-Aldrich

(Vienna, Austria), unless specified. Human eotaxin/CCL11 and

TNF-a were from Peprotech (London, UK). PGE2, PGD2, and the

selective agonists for EP1/EP3, 17-phenyl trinor PGE2, EP2,

butaprost, and EP3, sulprostone were purchased from Cayman

(Ann Arbor, MI, USA). The monoclonal mouse anti-EP4 receptor

Ab was purchased from SantaCruz (Heidelberg, Germany). ONO

AE1-329 and ONO AE3-208 were kind gifts from ONO

Pharmaceutical (Osaka, Japan). The EP4 agonist ONO AE1-329

(2-[[2-[2-(2-methylnaphthalen-1-yl) propanoylamino] phenyl]-

methyl]benzoic acid) has been shown to selectively bind to EP4

receptors (Ki 5 35 nM) relative to the EP1, EP2, and EP3

receptors (Ki 5 3000, 2000, and 43000 nM respectively) [47].

The Ki values of the EP4 antagonist ONO AE3-208

(2-[[2-[2-(2-methylnaphthalen-1-yl)propanoylamino]phenyl]-

methyl]benzoic acid) are 1.3, 30, 790, 2400 nM for EP4, EP3, FP,

and TP, respectively, and more than 10 000 nM for other

prostanoid receptors [48]. Anti-rabbit and anti-mouse IgG

secondary Abs conjugated with Alexa Fluor 488 and 647 were

supplied by Invitrogen. mAb 24 (activation-sensitive b2 integrin

Ab) was from Hycult Biotechnology (Uden, The Netherlands).

Phalloidin-Texas Red was purchased from Molecular Probes

(Eugene, OR, USA). Vectashield/DAPI mounting medium was

from Vector Laboratories (Burlingame, CA, USA). Ultra V Blocking

solution was purchased from Lab Vision (Fremont, CA, USA) and

Ab diluent from Dako (Carpinteria, CA, USA). Anti-CD62L (FITC),

anti-CD1 (PE), CellFix, and FACS-Flow were obtained from BD

(Vienna, Austria). Assay buffer was prepared from Dulbecco-

modified PBS (with 0.9 mmol/L Ca21 and 0.5 mmol/L Mg21;

Invitrogen, Vienna, Austria), 0.1% BSA, 10 mmol/L HEPES, and

10 mmol/L glucose, pH 7.4 [49]. Fixative solution was made by

adding 9 mL of distilled water and 30 mL of FACS-Flow to 1 mL of

CellFix [50]. Drugs were dissolved in distilled water, ethanol, or

dimethyl sulfoxide and further diluted in assay buffer to have a

final concentration of the solvents ofo0.1%.

Preparation of human peripheral blood eosinophils

Blood was drawn from healthy nonatopic volunteers, according

to a protocol approved by the Ethics Committee of the Medical

University of Graz. Polymorphonuclear leukocytes (PMNLs,

containing eosinophils and neutrophils) were isolated by dextran

sedimentation of erythrocytes then by centrifugation on Histo-

paque gradients as described previously [51, 52]. Eosinophils

were purified from polymorphonuclear leukocyte using negative

magnetic selection with an Ab cocktail (CD2, CD14, CD16, CD19,

CD56, and glycophorin A) and colloidal magnetic particles from

StemCell Technologies (Vancouver, Canada) [53]. All separation

steps were performed at room temperature. The eosinophil

preparations showed more than 95% purity and viability.

Culture of endothelial cells

Human lung microvascular endothelial cells (HMVEC-L, Lonza,

Verviers, Belgium) were cultivated in EGM-2 MV Bullet kit

medium (Lonza) with 5% FCS. Endothelial cell attachment and

growth was promoted by precoating with 1% gelatin for 1 h at

371C. Fresh medium was added every 2 days and cells were

passaged after reaching 90% confluence (5–6 days); the cells

were used between 5 and 10 passages [54, 55].

Eosinophil adhesion to fibronectin

Adhesion of purified eosinophils to fibronectin was determined by

incubating the eosinophils (6� 104/well) for 30 min at 371C in flat-



bottom 96-well plates that had been precoated with 5mg/mL

fibronectin for 2 h [55]. Eosinophils were treated with vehicle, EP4

agonist (ONO AE1-329, 30 nM), or PGE2 (30 nM) or first

preincubated with EP4-selective antagonist (ONO AE3-208,

100 nM) for 15 min at 371C and then treated with PGE2 (30 nM).

Adhesion of eosinophils was induced by increasing concentrations

of eotaxin (0.3–1.3 nM). After 30 min incubation, first the non-

adherent cells were collected, and thereafter the adherent eosino-

phils were harvested with 100mL trypsin/EDTA. Both nonadherent

and adherent cells were enumerated by means of flow cytometry.

Eosinophil adhesion was calculated as the percentage of adherent

eosinophils with respect to the total cells added.

L-selectin shedding

Polymorphonuclear leukocyte preparations were incubated with

vehicle or EP4 agonist (ONO AE1-329, 30 and 300 nM) for 5 min

at 371C. L-selectin shedding was induced by eotaxin (3 nM) or

PGD2 (30 nM) for 10 min at 371C. Cells were then stained with

anti-CD62L (FITC) and anti-CD16 (PE) Abs for 30 min at 41C.

CD62L expression on CD16neg eosinophils was quantified by flow

cytometry [56].

Immunofluorescence staining of adherent eosinophils

For double staining of F-actin and gelsolin in eosinophils, chamber

slides were precoated with 5mg/mL fibronectin for 2 h, and

purified eosinophils were incubated for 12 min at 371C. Eosino-

phils were pretreated in Eppendorf tubes with vehicle, ONO AE1-

329 (30 nM), or PGE2 (30 nM) or first with EP4 antagonist

(300 nM) or its vehicle for 15 min and subsequently with PGE2

(30 nM). Eosinophil adhesion was stimulated with eotaxin (3 nM).

After the removal of nonadherent cells, adherent eosinophils were

fixed with 3.7% formaldehyde for 10 min, permeabilized with

0.1% Triton X-100 for 10 min, and nonspecific binding sites were

blocked with 1% BSA for 20 min. Eosinophils were first incubated

with anti-gelsolin primary Ab (1:100) for 1 h, then with a mixture

of phalloidin-Texas Red (5 U/mL) and the second Ab labeled with

AF-488 (1:500) for 30 min in the dark. All steps were performed at

room temperature [57]. The slides were then mounted with

Vectashield mounting medium.

Activation and clustering of b2 integrins

Activation of b2 integrins in eosinophils was determined by

means of indirect immunofluorescence flow cytometry with

mAb 24, which recognizes only the activated conformation of

b2 integrins (CD18) [58]. Purified eosinophils were treated with

vehicle or 30 nM ONO AE1-329 for 10 min, activated with vehicle,

eotaxin (3 nM), or PGD2 (30 nM) for 12 min at 371C in the

presence of mAb 24 (1:50) or isotype control Ab followed by

staining with Alexa Fluor 488-labeled secondary Ab (1:500). The

stained samples were either directly measured by flow cytometry

[59] or were cytospun onto microscope slides (600 rpm 3 min,

Cytospin3, Shandon) and observed with a Olympus IX70 fluor-

escence microscope and UPlanFI – 60� /1.20 lens; images were

acquired using a Hamamatsu ORCA-ER digital camera [60].

Immunofluorescence staining of EP4 receptor oneosinophils

EP4 expression of purified human eosinophils was detected by

immunofluorescence and laser-scanning microscopy. The mono-

clonal mouse Ab used targets the C-terminal intracellular tail of the

EP4 receptor, and thus the cells were fixed and permeabilized, and

nonspecific binding sites were blocked with Ultra V Blocking

solution for 30 min at room temperature. Samples were then

incubated with the specific mouse EP4 primary Ab or an irrelevant

mouse IgG control Ab (4mg/mL each) for 1 h at room temperature.

Finally, 4mg/mL goat anti-mouse IgG secondary Ab conjugated

with Alexa Fluor 647 was added, cells were fixed, cytospun

(600 rpm 3 min, Cytospin3, Shandon) to microscope slides and

analyzed on a laser-scanning confocal microscope, LSM 510 Meta

(Zeiss), with the following Ex/Em settings: 405/BP420-480

(DAPI); 633/679-754 (Alexa Fluo 647). The images were taken

with a Zeiss 100� oil immersion objective and 0.6mm Z-slice

thickness over the whole cell. Typically, 12–15 slices were taken

from one cell with 1024� 1024 resolution; these images were

reconstituted to a 3-D image with the software of the instrument.

Eosinophil adhesion to endothelial cells under flowconditions

Endothelial cells (4� 105/substrate) were grown on VenaEC

biochips (Cellix, Dublin, Ireland). After reaching confluence

(usually within 2 days), endothelial layers were incubated with

vehicle or TNF-a (10 pM) for 4 h. Endothelial monolayers were

superfused with suspensions of 3� 106/mL eosinophils at

0.5 dyne/cm2 for 2 min and 20 s at 371C in a OKOLAB H201-

T1-heated cage. Eosinophils were treated with vehicle or ONO

AE1-329 (30 nM) for 10 min at 371C. In some cases, eosinophil

adhesion was stimulated with eotaxin treatment (3 nM for 5 min

at 371C). Eosinophils were treated with 17-phenyl trinor PGE2,

butaprost, sulprostone, ONO AE1-329, or PGE2 (each 30 nM) for

10 min at 371C. Alternatively, eosinophils were first incubated

with the EP4-selective antagonist (ONO AE3-208, 300 nM for

15 min) followed by PGE2. In other cases, the EP4 agonist

(30 nM) or its vehicle was superfused over eosinophils already

attached to TNF-a-stimulated endothelial cells as post-treatment.

Cell adhesion was monitored by phase contrast on a Zeiss

Axiovert 40 CFL microscope and Zeiss A-Plan 10� /0.25 Ph1

lens, using a Hamamatsu ORCA-03G digital camera and Cellix

VenaFlux software. Computerized image analysis was performed

by DucoCell analysis software (Cellix, Dublin), where adherent

eosinophils were quantified on each single image [41, 61].



Eosinophil transendothelial migration

Endothelial cells (3.5� 104/insert) were grown to confluence on

5mm filters in 6.5 mm Transwell inserts (Corning, NY, USA).

Confluence of the monolayers (usually 3 days after seeding) was

confirmed by measuring transendothelial electrical resistance

with an Endohm device (WPI, Sarasota, Fla) [54, 55]. The

monolayers grown in the inserts were activated by 0.5 U/mL

thrombin for 15 min or by 10 pM TNF-a for 4 h at 371C and placed

into 24-well plates containing eotaxin (3 nM) or vehicle in the

bottom wells. Eosinophils were preincubated with vehicle or the

EP4 agonist ONO AE1-329 for 10 min at 371C prior to being

added to the top wells (2� 105/well). The plates were incubated

at 371C in a humidified incubator for 4 h, and the nonmigrated

and transmigrated eosinophils were collected separately and

were enumerated by flow cytometry. Transendothelial migration

was calculated as the percentage of transmigrated eosinophils

with respect to total cells added to the filters [55, 62].

Statistical analyses

Data are shown as means1SEM for n observations. Statistical

analysis was performed with SigmaPlot 11.0 software using one-

way ANOVA or two-way ANOVA for repeated measurements

followed by Dunnett’s post-hoc test. p-Values of o0.05 were

considered as statistically significant.

Acknowledgements: V. Konya and S. Philipose were funded by

the Ph.D. Program Molecular Medicine of the Medical University

of Graz. This work was supported by the Start Funding Program

of the Medical University of Graz (ASO109000101 to V. Konya),

Jubilaumsfonds of the Austrian National Bank (12552 to

A. Heinemann and 13487 to R. Schuligoi), the Austrian Science

Fund FWF (Grants P19424 and P22521 to A. Heinemann, P21004

to G. Marsche, and P22771 to R. Schicho). The expert technical

help of Wolfgang Platzer, Martina Ofner and Gerald Parzmair is

highly appreciated.

Conflict of interest: The authors declare financial or commercial

conflict of interest.

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Abbreviations: EP: E-type prostanoid receptor � F-actin: filamentous

actin � PG: prostaglandin

Full correspondence: Dr. Akos Heinemann, Institute of Experimental

and Clinical Pharmacology, Medical University of Graz,

Universitaetsplatz 4. A-8010 Graz, Austria

Fax: 143-316-380-9645

e-mail: [email protected]

Received: 28/1/2011

Revised: 18/4/2011

Accepted: 18/5/2011

Accepted article online: 17/6/2011



Interaction of eosinophils with endothelial cells is modulated by prostaglandin EP4 receptors

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