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The Journal of Immunology, 2006, 176: 966-973.
Copyright © 2006 by The American Association of Immunologists

Prostaglandin E2 Is Generally Required for Human Dendritic Cell Migration and Exerts Its Effect via EP2 and EP4 Receptors1

Daniel F. Legler2,3,*,{dagger}, Petra Krause3,*,{dagger}, Elke Scandella{ddagger}, Eva Singer§ and Marcus Groettrup*,{dagger}

* Biotechnology Institute Thurgau, Tägerwilen, Switzerland; {dagger} Department of Biology, Division of Immunology, University of Konstanz, Konstanz, Germany; {ddagger} Cantonal Hospital, St. Gallen, Switzerland; and § Klinikum Konstanz, Konstanz, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The control of dendritic cell (DC) migration is pivotal for the initiation of cellular immune responses. In this study, we demonstrate that the migration of human monocyte-derived (Mo)DCs as well as of ex vivo peripheral blood DCs toward CCL21, CXCL12, and C5a is stringently dependent on the presence of the proinflammatory mediator PGE2, although DCs expressed CXCR4 and C5aR on their surface and DC maturation was accompanied by CCR7 up-regulation independently of PGE2. The necessity of exogenous PGE2 for DC migration is not due to the suppression of PGE2 synthesis by IL-4, which is used for MoDC differentiation, because maturation-induced endogenous production of PGE2 cannot promote DC migration. Surprisingly, PGE2 was absolutely required at early time points of maturation to enable MoDC chemotaxis, whereas PGE2 addition during terminal maturation events was ineffective. In contrast to mouse DCs, which exclusively rely on EP4 receptor triggering for migration, human MoDCs require a signal mediated by EP2 or EP4 either alone or in combination. Our results provide clear evidence that PGE2 is a general and mandatory factor for the development of a migratory phenotype of human MoDCs as well as for peripheral blood myeloid DCs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DCs)4 are professional APCs operating as sentinels in peripheral tissues and lymphoid organs. DCs have the unique ability to take up and process Ag, and to prime naive T cells, and are therefore critical for the induction of primary immune responses, for the induction of the immunological tolerance, as well as for the regulation of the T cell-mediated immune response (1, 2, 3). Due to these features, DCs loaded with specific Ags are currently being used in vaccinations against tumors and infectious agents in numerous clinical trials (4, 5, 6).

DC progenitors in the bone marrow give rise to circulating precursors that home to tissues, where they reside as immature cells. Thus, immature DCs are strategically located at portals of pathogen entry, such as the skin, the airways, or the gastrointestinal mucosa, and are particularly good at Ag ingestion through pinocytosis or receptor-mediated endocytosis and Ag processing (7). Exposure to pathogens triggers the maturation of DCs through recognition of the prototypic pathogen-derived macromolecules by TLRs (8, 9, 10). At the same time, DCs secrete large amounts of proinflammatory cytokines and chemokines, including CCL2, CCL3, CCL4, and CCL5, which in turn recruit other immature DCs, macrophages, and monocytes to the inflamed tissue (11). Along this line, immature DCs are also attracted by the complement component C5a and the bacterial peptide fMLP (12, 13, 14). DC maturation terminates the ability of Ag uptake, whereas the capacity to stimulate T cells is enhanced through the up-regulation of costimulatory molecules (such as CD80, CD86), MHC molecules, and T cell adhesion molecules (e.g., CD48 and CD58), and the enhanced production of cytokines (IL-12, IL-2, TNF-{alpha}) (2, 3). Importantly, maturing DCs lose their responsiveness to inflammatory chemokines by either down-regulation or desensitization of the chemokine receptors CCR1, CCR2, and CCR5 on monocyte-derived DCs and CCR6 on Langerhans cells (LCs) (14, 15, 16). Simultaneously, Ag-loaded DCs up-regulate surface expression of the homing chemokine receptor CCR7, and as a result acquire responsiveness to the chemokines CCL19 (EBV-induced gene 1 ligand chemokine, Exodus-3, MIP-3{beta}, CK{beta}11) and CCL21 (secondary lymphoid-tissue chemokine, Exodus-2, 6Ckine, TCA-4) (14, 15, 16, 17, 18). The fact that CCR7 and its ligands are mandatory for homing was demonstrated in CCR7-deficient and plt/plt mice lacking CCL19 and CCL21 (19, 20, 21, 22, 23, 24).

Recently, we and others found that maturation-induced up-regulation of CCR7 surface expression is not sufficient for monocyte-derived DCs (MoDCs) to migrate toward CCL19 and CCL21 (25, 26, 27). Indeed, MoDC migration toward CCL19 and CCL21 was readily observed upon maturation in the presence of the proinflammatory mediator PGE2, albeit PGE2 did not change the expression level of CCR7 on mature DCs (25, 26). CCR7 triggering in MoDCs matured in the presence of PGE2 induced an enhanced PI3K-mediated phosphorylation of protein kinase B/Akt (28). However, as PI3K inhibitors were not able to abrogate MoDC migration (28), the mechanism of how PGE2 permits DC migration remains largely unknown.

PGE2 is a lipid mediator of the eicosanoid family of oxygenated arachidonic acid, and thus a potent modulator of immune responses in an autocrine and paracrine fashion. The production of PGs is initiated by the liberation of arachidonic acid from plasma membrane phospholipids by phospholipases, such as cytosolic phospholipase A2 (cPLA2), in a variety of cell types during inflammation. Arachidonic acid is then metabolized into PGH2 by the cyclooxygenases, i.e., the constitutively expressed cyclooxygenase-1 and the inducible cyclooxygenase-2 (29, 30, 31). Cell-specific PG synthases are responsible for the conversion of PGH2 into different PGs, including PGE2. The prime mode of PGE2 action is through signaling via four seven-transmembrane-domain, G protein-coupled receptors termed EP1-EP4 (32, 33). Interestingly enough, in mice, the importance of PGE2 for DC migration to draining lymph nodes in vivo has been demonstrated in Ptger4–/– animals lacking the PGE2 receptor EP4 (34). As MoDCs express the functional receptors EP2 and EP4 (25), it remains to be identified which of these PGE2 receptors is responsible for the development of a migratory DC phenotype in humans.

MoDCs, which are most frequently used for DC-based immunotherapies, are differentiated from peripheral blood monocytes in the presence of GM-CSF and IL-4. IL-4, however, was shown to inhibit cPLA2, thus limiting the endogenous production of PGE2 in MoDCs (35). As most MoDCs failed to leave the injection site after intradermal injection of patients undergoing an antitumor immunotherapy (36), Thurnher et al. (37) therefore suggested to replace IL-4 by IL-13 for the generation of MoDCs, as IL-13 enhances cPLA2.

In the present study, we investigate whether PGE2 is generally needed for immature and mature DCs to migrate toward chemokines and complement components. We also compare maturation and migratory capacities of human MoDCs generated in the presence of IL-4 or IL-13 in combination with GM-CSF. Furthermore, we assess the role of PGE2 on the migration of peripheral blood myeloid CD1c+ DCs, and we identify the PGE2 receptors responsible for facilitating human MoDC chemotaxis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Isolation of human peripheral blood myeloid DCs (PBDCs)

CD1c+ myeloid DCs from peripheral blood of healthy donors were purified using the CD1c (BDCA-1) Dendritic Cell Isolation Kit (Miltenyi Biotec), according to the manufacturer’s protocol. Briefly, PBMCs were separated by centrifugation over a density gradient of Ficoll-Paque (Amersham Biosciences), depleted of CD19+ cells, and positively selected for CD1c. After isolation, ex vivo PBDCs were directly subjected to FACS analysis and Transwell chemotaxis assays. Alternatively, PBDCs were cultured at 1 x 106 cells/ml in AIM-V medium (Invitrogen Life Technologies) supplemented with 50 ng/ml GM-CSF (Leukomax; Novartis Pharmaceuticals) and either 50 ng/ml IL-4 (Promo Cell) or 10 ng/ml IL-13 (Promo Cell) and matured with 20 µg/ml poly(I:C) (Sigma-Aldrich) in the presence or absence of 1 µg/ml PGE2 (Minprostin E2; Pharmacia). After 18–20 h, cells were harvested and analyzed for their migration capacity and maturation status by FACS.

Generation of human MoDCs

Monocytes were positively selected from PBMCs of healthy donors using anti-CD14-conjugated magnetic microbeads (Miltenyi Biotec), as previously described (28). CD14+ monocytes were cultured at 1 x 106 cells/ml in AIM-V medium supplemented with 50 ng/ml GM-CSF and IL-4 (1:50 of the supernatant of an IL-4-producing J558 cell line) or 10 ng/ml IL-13 (A. Minty, Sanofi-Synthelabo, Paris, France). After 5–6 days, immature DCs were matured for 2 days by adding 20 µg/ml poly(I:C), 0.5 µg/ml insoluble CD40L (Promo Cell), or 1 µg/ml LPS (Sigma-Aldrich), and, where indicated, 1 µg/ml PGE2, 1 µg/ml specific agonists for EP2 (Butaprost, Cayman Chemical; ONO-AE1-259-01, Ono Pharmaceutical), EP4 (PGE1-alcohol, Cayman Chemical; ONO-AE1-329, Ono Pharmaceutical), or 4 µg/ml specific EP4-antagonist ONO-AE3-208 (Ono Pharmaceutical).

Chemotaxis assay

DCs (2 x 105 cells in AIM-V medium) were placed on a polycarbonate filter (5-µm pore size) of a 24-well Transwell plate (Corning Costar) and allowed to migrate for 3 h at 37°C/5% CO2 to the lower chamber containing 250 ng/ml CCL21, 250 ng/ml CXCL12 (both Promo Cell), or 10 nM C5a (Sigma-Aldrich), respectively. Input and migrated cells were counted by flow cytometry acquiring events of the appropriate size for 60 s. The number of spontaneously migrated cells toward AIM-V medium without chemoattractants was subtracted.

Flow cytometry

For FACS analysis, MoDCs and PBDCs were stained at 4°C in PBS containing 0.5% FCS and 0.1% sodium azide using the following mouse anti-human mAb: anti-CD83 FITC (Beckman Coulter), anti-HLA-DR FITC, anti-CD86 FITC, anti-CD80 PE, anti-CD88 PE (clone D53-1473), anti-fMLPR PE (clone 5F1), anti-CXCR4 (mouse anti-human mAb 12G5) (all from BD Biosciences), anti-CD88 FITC (MCA1284F; Serotec), anti-CCR7 (rat anti-human mAb 3D12; provided by R. Förster, Hannover Medical School, Hannover, Germany), biotin-labeled anti-CD1c, anti-biotin PE (Miltenyi Biotec), anti-rat IgG F(ab')2 FITC (Jackson ImmunoResearch Laboratories), and anti-mouse IgG1 FITC (SILENUS Labs). Cell-associated fluorescence was measured using a FACScan II flow cytometer (BD Biosciences).

Quantitative real-time PCR

Total RNA of DCs was isolated using the NucleoSpin RNA II kit (Macherey Nagel) and transcribed into cDNA using the Reverse Transcription System Kit (Promega), according to the manufacturer’s protocols.

Real-time PCR was performed using a LightCycler together with the LightCycler FastStart DNA Master SYBR Green I kit (Roche), according to the manufacturer’s recommendations. Briefly, the cDNAs were initially denatured for 10 min at 95°C. Specific DNA fragments were amplified with steps of 15 s at 95°C, 5 s at 60°C, and 11 s at 72°C for 50 PCR cycles. The following oligonucleotide primers were used: for C5aR, 5'-CAG GAG ACC AGA ACA TGA ACT CC and 5'-TAC ATG TTG AGC AGG ATG AGG G; for CCR7, 5'-CCT GGG GAA ACC AAT GAA AAG C and 5'-GAG CAT GCC ACT GAA GAA GC; and for GAPDH, 5'-GAA GGT GAA GGT CGG AGT C and 5'-GAA GAT GGT GAT GGG ATT TC. Optimal MgCl2 concentrations were 3 mM for C5aR and CCR7, and 4 mM for GAPDH. The amount of amplified DNA fragments was normalized to GAPDH mRNA, and the specificity of the PCR products was verified by determining the melting profiles and analyzed by agarose gel electrophoresis.

Quantification of PGE2 by enzyme immunoassay

Culture supernatants of immature and mature MoDCs were collected after 2 days of stimulation, and the amount of secreted PGE2 was determined using the PGE2-enzyme immunoassay kit (Cayman Chemical), according to the manufacturer’s instructions.

Statistical evaluation

Differences between groups were assessed by Student’s paired t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
PGE2 is required for the ex vivo migration of human myeloid DCs

Human DCs are increasingly applied as vaccines for cancer patients. We and others (25, 26) have shown recently that PGE2 was required during maturation of MoDCs to permit migration in response to the lymph node-homing chemokines CCL19 and CCL21. To test whether the need for PGE2 for DC migration is a phenomenon that is confined to in vitro differentiated MoDCs, we investigated whether PGE2 has a similar effect on peripheral blood DCs. To this end, we isolated human PBDCs by positive selection of CD19– CD1c+ cells from fresh blood of healthy donors. Interestingly, ex vivo PBDCs under serum-free conditions either directly subjected to chemotaxis assays or cultured overnight in serum-free medium did not migrate in response to either CCL21 or CXCL12, which are known to attract mature DCs (Fig. 1, A and B). Ex vivo PBDCs kept in the presence of serum migrated in response to CXCL12, but barely to CCL21 (data not shown), in agreement with recent findings by Maraskovsky and colleagues (26, 27), which may indicate that serum contains substantial amount of PGE2 (data not shown). Indeed, overnight addition of only PGE2 in the absence of FCS facilitated PBDC migration (data not shown). Stimulation of PBMC with poly(I:C) alone permitted only a few PBDCs to chemotax to CCR7 and CXCR4 ligands, whereas the further addition of PGE2 to the culture medium induced a migratory phenotype (Fig. 1, A and B). Similarly, sCD40L-matured PBDCs efficiently migrated only in the presence of PGE2 (data not shown). The lack of responsiveness of ex vivo and poly(I:C)-stimulated PBDCs was not due to the lack of CCR7 and CXCR4 surface expression. All ex vivo PBDCs expressed CCR7 and CXCR4 as measured by FACS analysis (Fig. 1C). Stimulation with poly(I:C) led to an up-regulation of CCR7 and to a down-regulation of CXCR4, but PGE2 had no significant effect on the surface expression of these receptors on matured PBDCs (Fig. 1C). We further characterized the surface phenotype of PBDCs and found that freshly isolated CD1c+ DCs expressed high levels of HLA-DR and the costimulatory molecule CD86, but not CD80 and CD83 (Fig. 1C). Maturation of PBDCs by poly(I:C) led to a marked up-regulation of CD83, CD80, and CD86, independently of the addition of PGE2. Because IL-4 as well as IL-13 have been used to differentiate DCs in vitro (38, 39, 40), we compared the effect of these two cytokines on the maturation of PBDCs. However, surface expression of chemokine receptors and maturation markers was similar (Fig. 1C). Although the chemotactic responses to CCL21 and CXCL12 were higher in IL-4 PBDCs (Fig. 1A), compared with IL-13 PBDCs (Fig. 1B), in both cases PGE2 was mandatory for the efficient migration of PBDCs.



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  		FIGURE 1. PGE2 is required for human ex vivo myeloid DC migration in response to CCR7 and CXCR4 ligands. PBDCs were isolated from peripheral blood of healthy donors and directly analyzed for their migratory capacity toward the chemokines CCL21 and CXCL12 (ex vivo). Alternatively, PBDCs were cultured in serum-free medium containing GM-CSF and IL-4 (A) or GM-CSF and IL-13 (B) and stimulated or not with poly(I:C) and/or PGE2, as indicated. Mean values and SEM of five (A) or four (B) independent experiments are shown. Asterisks indicate statistical significance with p values <0.05 for * and <0.005 for **. C, The surface expression of CD1c, CD83, HLA-DR, CD80, CD86, CCR7, and CXCR4 on ex vivo and cultured PBDCs was analyzed by FACS. The solid line corresponds to PBDCs cultured in GM-CSF plus IL-4, whereas the dashed line represents PBDCs cultured in GM-CSF plus IL-13. Corresponding isotype controls are shown as thin gray line. Numbers indicated represent the mean fluorescent intensities for IL-4 (upper value) and for IL-13 (lower value).

 
Influence of IL-4 and IL-13 on endogenous PGE2 production in DCs

To further characterize the role of PGE2 on mediating chemotaxis, we used monocyte-derived DCs, which are most frequently applied for immunotherapies. First, we investigated the endogenous production of PGE2 by MoDCs as well as by PBDCs. To this end, we collected culture supernatants of immature and poly(I:C)-matured MoDCs generated with IL-4 or IL-13 in the presence of GM-CSF. Immature MoDCs cultured for 2 days with IL-4 secreted on average 10.6 ng/ml PGE2, similar to poly(I:C)-matured MoDCs, which produced 9.7 ng/ml PGE2 (Fig. 2). Immature MoDCs differentiated with IL-13 and GM-CSF produced a comparable amount of PGE2, namely 8.9 ng/ml (Fig. 2). In contrast, a >7-fold increase of secreted PGE2 (67.9 ng/ml) was measured in the supernatant of mature IL-13/GM-CSF MoDCs (Fig. 2). Similarly, PBDCs cultured overnight in IL-13/GM-CSF produced on average 8 times more PGE2 than PBDCs cultured in IL-4/GM-CSF, namely 11.9 vs 1.5 ng/ml PGE2 (Fig. 2). This result is in agreement with the finding that IL-4 suppressed endogenous production of PGE2 in matured MoDCs by inhibiting the cytoplasmatic form of phospholipase A2 (35).



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  		FIGURE 2. Mature DCs generated with GM-CSF and IL-13 release high amounts of PGE2. Monocytes were differentiated into immature DCs with GM-CSF and IL-4 (IL-4) or GM-CSF and IL-13 (IL-13) and were either left immature (iDC) or matured by the addition of poly(I:C) (mDC). Ex vivo PBDCs were cultured in GM-CSF and IL-4 or GM-CSF and IL-13. The release of PGE2 into the supernatant of DC cultures was determined after 48 h for MoDCs, or 18–20 h for PBDCs by enzyme immunoassay. Mean values and SEM from supernatants derived from three to six donors are shown.

 
PGE2 is generally required for MoDC migration

Next, we intended to analyze in detail the two media used to generate MoDCs under serum-free conditions used for clinical applications. To this end, monocytes were cultured for 5–6 days in either IL-4/GM-CSF or IL-13/GM-CSF and matured by the addition of poly(I:C) for 2 days, both in the absence or presence of graded concentrations of PGE2. Similar levels of CD83, HLA-DR, CD80, and CD86 were expressed on immature MoDCs irrespective of the presence of IL-4 or IL-13 (Fig. 3). However, exogenous addition of PGE2 to immature MoDCs generated in the presence of IL-4, in contrast to IL-13, substantially up-regulated CD83 and CD80 surface expression (Fig. 3). The addition of 1 µg/ml PGE2 in conjunction with poly(I:C) for MoDC maturation had no effect on surface expression levels of the tested markers in both IL-4 and IL-13 culturing conditions (Fig. 3).



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  		FIGURE 3. Influence of PGE2 on phenotypic MoDC maturation. Human peripheral blood monocytes were cultured in serum-free medium containing either GM-CSF and IL-4 (solid line) or GM-CSF and IL-13 (dashed line) in the presence or absence of PGE2 to differentiate into immature DCs (iDC). DCs were matured (mDC) by the addition of poly(I:C) with or without PGE2 for 2 days. Surface expression of CD83, HLA-DR, CD80, and CD86 was measured by FACS. Corresponding isotype controls are depicted as thin dashed line. A representative experiment of 11 for IL-4 and 8 for IL-13, respectively, is shown. Numbers indicated represent mean fluorescent intensities for IL-4 (upper number) and for IL-13 (lower number).

 
Next, we investigated the chemotactic responses of MoDCs to various chemokines and chemoattractants. Therefore, we matured MoDCs generated in IL-4/GM-CSF with poly(I:C) in the presence of graded concentrations of PGE2. Mature MoDCs in the absence of PGE2 migrated neither in response to the chemokines CCL21 and CXCL12 nor to the chemoattractant C5a (Fig. 4A). In contrast, exogenous addition of as little as 10–50 ng/ml PGE2 during maturation was sufficient to allow MoDCs to chemotax in response to CCL21, CXCL12, as well as to C5a (Fig. 4A). The migration efficiency of mature MoDCs further increased with higher PGE2 concentration. To substantiate this finding, we subjected immature as well as mature MoDCs that were cultured in the presence or absence of PGE2 to migration assays. Indeed, MoDCs generated in IL-4/GM-CSF medium migrated efficiently only upon incubation with PGE2 (Fig. 4B). The migration efficiency was highest for all attractants in PGE2-treated mature MoDCs, but migration of immature MoDCs cultured in the presence of PGE2 was also observed for all three ligands. In addition, similar numbers of migrated cells to CCL21 were observed for sCD40L or LPS-matured MoDCs, and migration largely depended on the presence of PGE2 during maturation (Fig. 4C). Unexpectedly, under identical conditions, none of the MoDCs migrated in response to fMLP (data not shown). These results provide clear evidence that PGE2 is generally required to develop a migratory DC phenotype.



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  		FIGURE 4. PGE2 is generally required for MoDC migration. A, MoDCs were generated in GM-CSF and IL-4 and matured with poly(I:C) in the presence of graded concentrations of PGE2. After 2 days, the migration of mature DCs toward 250 ng/ml CCL21, 250 ng/ml CXCL12, and 10 nM C5a was measured in a Transwell chemotaxis assay. The number of migrated cells cultured with 1 µg/ml PGE2 was set to 100% for each chemoattractant. Absolute average values for the migration toward CCL21, CXCL12, and C5a are 32.5, 16.0, and 7.4%, respectively (n = 3). Basal migration in the absence of chemoattractants was always below 0.5%. Monocytes were differentiated into immature DCs (iDC) by culturing in GM-CSF and IL-4 (B and C) or GM-CSF and IL-13 (D) in the presence or absence of PGE2 and matured with poly(I:C) (A, B, and D) sCD40L or LPS (C) with or without PGE2 for 2 days. The migration of MoDCs was then analyzed by a Transwell chemotaxis assay in response to CCL21, CXCL12, and C5a. Mean values and SEM from four to six independent experiments of different donors are shown. Asterisks indicate statistical significance with p values <0.05 for *, <0.005 for **, and <0.0005 for ***.

 
As mature MoDCs generated in the presence of IL-13 and GM-CSF endogenously produced a similar amount of PGE2 (Fig. 2) as required for migration (Fig. 4A), we analyzed the migratory capacity of IL-13/GM-CSF MoDCs in Transwell assays. Surprisingly, endogenous production of PGE2 by IL-13/GM-CSF MoDCs was not sufficient to allow chemotaxis to any of the chemoattractants, but MoDCs did migrate when they were cultured in the presence of exogenous PGE2 (Fig. 4D). Strikingly, the PGE2 effect on migration was even more pronounced. CXCL12- and C5a-mediated migration was exclusively observed in poly(I:C)/PGE2-matured MoDCs. In agreement with IL-4/GM-CSF MoDCs, immature IL-13/GM-CSF MoDCs cultured in the presence of PGE2 barely migrated toward CCL21, whereas they did not respond to CXCL12 and C5a at all (Fig. 4D).

Taken together, we demonstrated that the necessity of PGE2 for the migration of DCs is not due to the inhibitory effect of IL-4 on PGE2 production. In fact, the maturation-induced endogenous production of PGE2 is not sufficient for migration of MoDCs, as previously suggested by Thurnher and coworkers (35, 37). Moreover, our results clearly indicate that PGE2 is a general and mandatory factor for the development of a migratory phenotype of human MoDCs as well as for PBDCs.

Role of PGE2 on CCR7, CXCR4, and C5aR expression on MoDCs

To exclude that the impaired DC migration in the absence of PGE2 was simply due to a lack of receptor expression, we subjected MoDCs to FACS analysis. As expected, immature IL-4/GM-CSF MoDCs did not express CCR7 (Fig. 5A), which is in agreement with previous observations (14, 15, 25). Addition of PGE2 led to a marked up-regulation of CCR7 on immature MoDCs, which was further increased upon maturation by poly(I:C) (Fig. 5A). PGE2 had no influence on CCR7 expression of mature MoDCs. CXCR4 was expressed on immature as well as on mature MoDCs, and PGE2 did not alter the expression level (Fig. 5A). We were unable to detect C5aR surface expression by FACS using two different commercially available Abs (data not shown). Therefore, we performed real-time PCR to quantify mRNA levels of C5aR under the various MoDC culturing conditions. C5aR mRNA was present in immature and mature MoDCs (Fig. 5B), and the amount of mRNA barely changed after maturation or after stimulation with PGE2. Compared with immature MoDCs, we found on average a 6-, 2-, and 4-fold increase in mRNA levels after treatment with PGE2, poly(I:C), and poly(I:C)/PGE2, respectively (Fig. 5B). For comparison, we also quantified mRNA expression of CCR7. There, the up-regulation of CCR7 mRNA increased by 41-, 300-, and 378-fold compared with immature MoDCs (Fig. 5B). In agreement with the unresponsiveness to fMLP, we found neither surface expression nor mRNA for fMLP receptor under these conditions (data not shown). However, the influence of PGE2 on the expression levels of CCR7, CXCR4, and C5aR was similar for MoDCs generated with IL-4 as compared with IL-13 (Fig. 5). We conclude from this data that the migratory inability of MoDCs without PGE2 is not due to the lack of CCR7, CXCR4, and C5aR expression; rather, PGE2 facilitated DC migration by a mechanism distinct from modulating the level of receptor expression.



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  		FIGURE 5. Impact of PGE2 on the expression of CCR7, CXCR4, and C5aR by MoDCs. The chemokine receptor expression on immature and poly(I:C)-matured MoDCs generated with GM-CSF and IL-4 (A, black solid line; B) or GM-CSF and IL-13 (A, gray dashed line; C) in the presence or absence of PGE2 was measured by FACS analysis using CCR7- and CXCR4-specific Abs (A). Numbers indicated represent mean fluorescent intensities for IL-4 (upper number) and for IL-13 (lower number). The mRNA expression of C5aR and CCR7 by MoDCs was examined by real-time PCR and normalized to the housekeeping gene GAPDH. Mean values and SEM from four independent experiments using different MoDC preparations are shown. Amplified transcripts (376 bp for C5aR, and 430 bp for CCR7) were visualized on agarose gel electrophoreses (inlet of B, lanes 2 and 3). The control PCR using H2O as template and C5aR or CCR7 primers, respectively, are shown in lanes 1 and 4 of the inlets.

 
PGE2 is required at early time points of MoDC maturation to permit migration

For a better understanding of how PGE2 permits DC chemotaxis, we incubated maturing MoDCs for different time periods with PGE2. As shown before, marginal or no migration in response to CCL21 and CXCL12, respectively, was measurable for MoDCs matured with poly(I:C) alone. However, the costimulation of MoDCs with PGE2 and poly(I:C) during the first 12 h of maturation, followed by a further incubation of 36 h in the presence of poly(I:C) alone, was almost as efficient as the stimulation with PGE2 and poly(I:C) throughout the whole maturation period with respect to chemotaxis of MoDCs toward CCL21 and CXCL12 (Fig. 6). Surprisingly, poly(I:C)-matured MoDCs that exclusively received PGE2 for the terminal 12 h of maturation were not attracted by the chemokines (Fig. 6). These data suggest that PGE2 may induce the expression of to date unidentified genes, which enable DCs to sense a chemokine gradient. Additional experiments are required to unravel such a putative mechanism.



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  		FIGURE 6. PGE2 is required at early time points of MoDC maturation to permit cell migration. Immature DCs differentiated with GM-CSF and IL-4 were matured with poly(I:C) for 48 h (poly(I:C)) and assessed for chemotaxis toward CCL21 (left) and CXCL12 (right). PGE2 was added either for the whole period of maturation (48 h), for the initial 12 h (12 h), or for the terminal 12 h (12 h day 2) of maturation, respectively. In the case of adding PGE2 at maturation initiation, MoDCs were incubated in medium containing 20 µg/ml poly(I:C) and 1 µg/ml PGE2 for 12 h, washed extensively, seeded in fresh IL-4/GM-CSF medium containing 20 µg/ml poly(I:C), and cultured for another 36 h before functional analysis. Mean values and SEM of two to three MoDC preparations are shown. Absolute average values for the migration toward CCL21 and CXCL12 are 31.1 and 24.2%, respectively.

 
In humans, the two PGE2 receptors, EP2 and EP4, can mediate MoDC migration

Gene-targeting experiments in mice revealed that exclusively PGE2 receptor EP4 was critical for LC migration to draining lymph nodes in vivo (34). Human MoDCs express two of the four described PGE2 receptors, namely EP2 and EP4 (25), and it remains to be determined which of these receptors can trigger human DC migration. To address this question, we made use of various specific EP2 and EP4 agonists. MoDCs were matured for 2 days with poly(I:C) and incubated either in the presence or absence of PGE2, or in the presence of an EP2 or an EP4 agonist, or a combination thereof, followed by testing the mobility of the DCs in a chemotaxis assay (Fig. 7). In contrast to mouse LC, human MoDCs migrated readily in response to CCL21 upon maturation in the presence of the EP2 agonists Butaprost and ONO-AE1-259-01 similar to MoDCs matured with poly(I:C) and EP4 agonists PGE1-alcohol and ONO-AE1-329 (Fig. 7A). Each agonist on its own and the combination of either Butaprost and PGE1-alcohol, or ONO-AE1-259-01 and ONO-AE1-329, were almost as potent as PGE2 in facilitating DC migration in response to CCL21 (Fig. 7A). Along this line, addition of a 4-fold excess of the EP4 antagonist ONO-AE3-208 over PGE2 was unable to inhibit migration (Fig. 7A). For CXCL12- and C5a-mediated chemotaxis, EP2 as well as EP4 agonists permitted MoDCs to migrate, but the EP2 agonists were less effective (Fig. 7, B and C). In agreement with these findings, the EP4 antagonist was able to partially antagonize the effect of PGE2. Thus, in contrast to mouse LCs, human MoDCs require a signal mediated by either EP2 or EP4 alone or the combination of both receptors to develop a migratory phenotype.



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  		FIGURE 7. The PGE2 receptors EP2 and EP4 can both trigger MoDC migration in response to CCL21, CXCL12, and C5a. Immature MoDCs (GM-CSF and IL-4) were matured with poly(I:C), and the chemotactic responses to CCL21 (A), CXCL12 (B), and C5a (C) were measured in Transwell assays. To the maturation stimuli, either 1 µg/ml PGE2, 1 µg/ml specific EP2 agonists (Butaprost or ONO-AE1-259-01), 1 µg/ml specific EP4 agonists (PGE1-alcohol or ONO-AE1-329), or a combination of EP2 and EP4 agonists was added. In addition, 4 µg/ml EP4-specific antagonist ONO-AE3-208 was added to 1 µg/ml PGE2 for the whole maturation procedure. Migration relative to MoDCs matured in the presence of PGE2, which served as 100% value, is shown. Mean values and SEM of up to six independent experiments are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The maturation of DCs is a key event in the initiation of a T cell response. DC maturation results in the up-regulation of CCR7, which directs migration into the T cell zone of draining lymph nodes. There, DCs will meet naive T lymphocytes that were recruited by the same chemokine receptor. For the initiation of a T cell response, the up-regulation of CCR7 on DCs may represent a point of no return, and it is intriguing to observe that CCR7 surface expression is not enough to facilitate migration. Indeed, for a migratory phenotype, DCs require a second stimulus that permits chemokine receptor signaling and subsequent migratory response toward the chemokines. Recently, we and others have identified PGE2 as a permissive factor that couples CCR7 on human DCs differentiated from peripheral blood monocytes by IL-4 and GM-CSF to signaling, e.g., protein kinase B activation, and ultimately DCs migrated toward the chemokines CCL19 and CCL21 (25, 26, 28). In this study, we show that the effect of PGE2 on MoDC migration is not restricted to CCR7. Indeed, immature as well as mature MoDCs migrated exclusively to the constitutive chemokine CXCL12 via its cognate receptor CXCR4, when PGE2 was present in the culture condition (Fig. 4). The effect of PGE2 stimulation on CXCR4- and CCR7-mediated migration was very similar with respect to the required dose of PGE2 and the need for PGE2 early during maturation. Moreover, PGE2 did not affect the cell surface expression of either of the two receptors on matured MoDCs (Fig. 5). It is therefore likely that the same PGE2-triggered intracellular pathway determines the migratory capacity of CCR7 and CXCR4. In addition, we demonstrate that PGE2 is also a mandatory factor for the development of a migratory DC phenotype for the complement fragment C5a, although the migration in response to C5a was rather inefficient compared with CCL21 and CXCL12 (Fig. 4). Under our experimental setup, MoDCs did not migrate in response to the bacterial chemoattractant fMLP, although fMLP was fully active (data not shown). This was rather unexpected, as immature, but not mature, MoDC have been reported to migrate in response to fMLP and C5a (13, 14), whereas Yang et al. (12) showed chemotaxis in immature as well as mature MoDCs. However, at least the low portion of MoDCs that specifically migrated toward C5a was only able to do so when immature or maturing MoDCs were stimulated with PGE2. Nevertheless, we can conclude that PGE2 is a general permissive agent that controls MoDC migration in response to chemokines as well as complement chemoattractants.

Is the need for PGE2 for DC migration restricted to in vitro generated MoDCs? Maraskovsky and colleagues (26, 27) reported that freshly isolated CD1c+ PBDCs from human blood that were expanded in vivo with Flt-3 ligand did not require sensitization with PGE2, as the stimulation with CD40L in vitro sufficed to induce maturation and migration toward CCL19 under serum-containing conditions. In this study, we have reinvestigated this issue by magnetically isolating CD1c+ myeloid PBDCs and performing in vitro maturation and migration assays in serum-free conditions identical with MoDC preparations approved for clinical applications. Ex vivo PBDCs show a similar phenotype as immature MoDCs as they lack surface expression of CD83 and CD80, and express moderate levels of HLA-DR and CD86. Latter molecules are up-regulated upon maturation (Fig. 1C). In contrast to immature MoDCs, PBDCs express a substantial amount of CCR7. These data indicate that peripheral blood DCs are similar to MoDCs in respect of migration, but clearly represent two different DC populations. Under serum-free conditions, both CCR7 and CXCR4, however, are not functional, as the isolated PBDCs did not migrate to CCL21 and CXCL12 ex vivo. Strikingly, addition of 10% FCS to ex vivo PBDCs during the chemotaxis assay was sufficient for PBDCs to migrate in response to CXCL12 (data not shown), thus confirming previous findings (26, 27), but demonstrating that the presence of serum has a major effect on DC migration. Indeed, FCS can contain sufficient concentrations of PGE2 to trigger migration (P. Krause, unpublished observation). Maturation of PBDCs with poly(I:C) markedly up-regulated CCR7 expression and down-regulated CXCR4 expression, but facilitated only a minor population to migrate in response to CCL21, whereas no migration toward CXCL12 was observed (Fig. 1A). But, costimulation with PGE2 and poly(I:C) or sCD40L resulted in substantial migration to both chemokines. It hence appears that the permissive function of PGE2 for DC migration is not confined to MoDCs, but is also valid for PBDCs directly isolated from human blood under serum-free conditions. Taken together, our findings suggest that PGE2 is a general mandatory factor for DC migration. In this context, it is of interest that exogenous PGE2 seem also to enhance the activities of monocytes to certain chemokines (41, 42).

It has been speculated that the permissive role of PGE2 for MoDC migration may be a consequence of the in vitro differentiation procedure. IL-4 has been reported to suppress the endogenous PGE2 production of MoDCs (35, 37) by down-regulating enzymes required for PGE2 biosynthesis, such as phospholipase A2 or cyclooxygenase-2 (43). For the differentiation and maturation of MoDCs, IL-4 may be replaced by IL-13 (15, 39, 44), which rather enhances phospholipase A2 expression at least in macrophages (45). Thurnher et al. (37) thus hypothesized that MoDCs generated with IL-13 may be able to produce PGE2, which could potentially allow MoDCs to migrate. To investigate whether IL-13 MoDCs indeed produce PGE2 and whether endogenous production of PGE2 by IL-13 MoDCs would facilitate migration, we generated MoDCs with IL-4 or IL-13 in the presence of GM-CSF. Indeed, we found that the concentration of endogenously produced PGE2 in the supernatant of IL-4-treated mature MoDCs was very low (Fig. 2). In contrast, mature MoDCs raised in IL-13 and GM-CSF secreted >70 ng/ml PGE2 into the growth medium, which should suffice to trigger DC migration. Nevertheless, IL-13-treated MoDCs also required the addition of exogenous PGE2 to permit migration (Fig. 4D). Similarly, PBDCs cultured in the presence of IL-13, compared with IL-4, secreted more PGE2. Although IL-13 DCs endogenously produced PGE2, they migrated for an unknown reason less efficiently than IL-4 DCs. For a better understanding of the permissive role of PGE2 for MoDC migration, we investigated whether PGE2 may act on the level of gene transcription or whether PGE2 may trigger a signal transduction module mandatory for chemotaxis. As shown in Fig. 6, PGE2 was required during the first 12 h of maturation and could not facilitate migration when added during the last 12 h of the maturation period. In several, but not all, experiments, it was even sufficient to add PGE2 during the first 2 h of maturation (data not shown). We are therefore in favor of the hypothesis that PGE2 may regulate chemotaxis by turning on or shutting off yet unknown genes required for migration. This may also explain why the endogenous secretion of PGE2 even by IL-13-treated MoDCs was insufficient to accumulate enough PGE2 during the initial period of maturation to facilitate migration. Although the conditions in vitro and in vivo are difficult to compare, it may well be that in vivo the endogenous PGE2 production by DCs is insufficient to trigger DC migration. Alternatively, it may be that blood DCs have had contact with PGE2 during maturation at sites of inflammation. This may also explain why ex vivo PBDCs require a shorter period of PGE2 contact to acquire a migratory phenotype, or why they migrated in response to CXCL12 in the presence of serum without PGE2 supplementation (26). Noteworthily, inflammation, which promotes DC migration, is associated with the rapid induction of arachidonic acid metabolism and PGE2 production, thus leading to a local coproduction of cytokines (such as TNF-{alpha} and IL-1{beta}) and PGE2 in inflamed lesions. The exogenous supply of PGE2 may derive from IL-1-, TNF-{alpha}-, or LPS-stimulated macrophages or fibroblasts at sites of inflammation.

The essential role of PGE2 for DC migration has been highlighted by Kabashima et al. (34) in gene-targeted mice lacking the PGE2 receptor EP4. The morphology and density of class II-positive LC in epidermal sheets were similar in ptger4–/– and ptger4+/+ mice, indicating that EP4 deficiency does not affect LC generation or LC recruitment into the tissue. However, LC emigration from ptger4–/– skin explants and migration into the draining lymph node after FITC application in ptger4–/– mice were significantly reduced (34). These experiments imply that PGE2 cannot be replaced by other agents that up-regulate cAMP levels in DCs. Moreover, they open an attractive therapeutic option for the pharmacological control of DC migration by the inhibition of the EP4 receptor. In humans, we have recently shown that two of the four PGE2 receptors, EP2 and EP4, are expressed on MoDCs (25). This is in accordance with the study by Luft et al. (26) reporting that an agonist specific for EP2 and EP4 can trigger MoDC migration, but not an agonist specific for EP1 and EP3. To explore potential therapeutic options for the control of MoDC migration in humans, we used two specific agonists each for EP2 and EP4 as well as a specific antagonist of the EP4 receptor to dissect the role of these two PGE2 receptors for MoDC migration in vitro (Fig. 7). To our surprise, the EP2 and EP4 agonists were both equally competent in facilitating MoDC migration via CCR7. This finding was substantiated by the fact that the specific EP4 antagonist could not inhibit the effect of PGE2 in enabling DC migration. Interestingly, for the migration toward CXCL12 and, even more pronounced, for C5a, EP4 agonists seemed to be more potent than EP2 agonists, which correlated with a stronger inhibition of MoDC migration by the EP4 antagonist, although we observed substantial donor to donor variations. Nevertheless, there seems to be a clear difference between murine LCs and human MoDCs in the usage of PGE2 receptors that can mediate migration. This result is pharmacologically relevant as we surmise that the treatment with EP2 and EP4 agonists or antagonists will most likely be required to interfere with DC migration in humans.

In summary, we show that PGE2 is a general and mandatory factor for human MoDCs and PBDCs to migrate in response to the chemokines CCL21 and CXCL12 as well as to the chemoattractant C5a. Thereby, endogenous production of PGE2 by DCs was not sufficient for the development of a migratory phenotype. Furthermore and in contrast to mouse DCs, which exclusively rely on EP4 receptor triggering for migration, human MoDCs require a signal mediated by EP2 or EP4 either alone or in combination.


   Acknowledgments
 
We thank Ono Pharmaceutical for the generous gift of the EP2 and EP4 agonists and the EP4 antagonist, Dr. Adrian Minty from Sanofi-Synthelabo Research for rIL-13, and Dr. Reinhold Förster from the Institute of Immunology, Hannover Medical School, for the anti-CCR7 Ab.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft, TR-SFB 11), the Swiss Cancer League OncoSuisse (to M.G. and D.F.L.), and the Vontobel Stiftung (to D.F.L.). D.F.L. is a recipient of a career development award from the Prof. Dr. Max Cloëtta Foundation. Back

2 Address correspondence and reprint requests to Dr. Daniel F. Legler, Biotechnology Institute Thurgau, Konstanzerstrasse 19, CH-8274 Tägerwilen, Switzerland. E-mail address: daniel.legler{at}bitg.ch Back

3 D.F.L. and P.K. are both considered first authors. Back

4 Abbreviations used in this paper: DC, dendritic cell; cPLA2, cytosolic phospholipase A2; LC, Langerhans cell; MoDC, monocyte-derived DC; PBDC, peripheral blood myeloid DC; sCD40L, soluble CD40L. Back

Received for publication March 31, 2005. Accepted for publication November 1, 2005.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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