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Formyl Peptide Receptor-Like 2 Is Expressed and Functional in Plasmacytoid Dendritic Cells, Tissue-Specific Macrophage Subpopulations, and This information is current as of September 27, 2021. Thalie Devosse, Aude Guillabert, Nicky D'Haene, Alix Berton, Patricia De Nadai, Sophie Noel, Maryse Brait, Jean-Denis Franssen, Silvano Sozzani, Isabelle Salmon and Marc Parmentier

J Immunol 2009; 182:4974-4984; ; Downloaded from doi: 10.4049/jimmunol.0803128 http://www.jimmunol.org/content/182/8/4974 http://www.jimmunol.org/ References This article cites 62 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/182/8/4974.full#ref-list-1

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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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Formyl Peptide Receptor-Like 2 Is Expressed and Functional in Plasmacytoid Dendritic Cells, Tissue-Specific Macrophage Subpopulations, and Eosinophils1

Thalie Devosse,* Aude Guillabert,* Nicky D’Haene,† Alix Berton,† Patricia De Nadai,* Sophie Noel,‡ Maryse Brait,‡ Jean-Denis Franssen,‡ Silvano Sozzani,§ Isabelle Salmon,† and Marc Parmentier2*

The formyl peptide receptor (FPR) is a key player in innate and host defense mechanisms. In humans and other primates, a cluster of genes encodes two related receptors, FPR-like 1 and FPR-like 2 (FPRL1 and FPRL2). Despite their high sequence similarity, the three receptors respond to different sets of ligands and display a different expression pattern in leukocyte populations. Unlike FPR and FPRL1, FPRL2 is absent from , and two endogenous peptide agonists, F2L and humanin, Downloaded from were recently described. In the present work, we investigated the detailed functional distribution of FPRL2 in leukocytes by quantitative PCR, flow cytometry, , and assays, with the aim of raising hypotheses regarding its potential functions in the human body. We describe that FPRL2 is highly expressed and functional in plasmacytoid dendritic cells and up-regulated upon their maturation. FPRL2 is also expressed in eosinophils, which are recruited but do not degranulate in response to F2L. FPRL2 is expressed and functional in macrophages differentiated from in vitro in different conditions. However, in vivo, only specific subsets of macrophages express the receptor, particularly in the , colon, and , http://www.jimmunol.org/ three organs chronically exposed to and exogenous aggressions. This distribution and the demonstration of the pro- duction of the F2L peptide in mice underline the potential role of FPRL2 in innate immunity and possibly in immune regulation and allergic diseases. The Journal of Immunology, 2009, 182: 4974–4984.

receptor for formylated bacterial peptides was first de- FPR shares 69% amino acid identity with FPRL1 and 56% scribed in 1976 (1), and the human formyl peptide re- with FPRL2, while FPRL2 shares 83% identity with FPRL1 (5, 6). A ceptor (FPR)3 was cloned in 1990 (2, 3). FPR-like 1 FPR, and presumably the two other members of the family, are

(FPRL1) and FPR-like 2 (FPRL2) were subsequently cloned by involved in host defense mechanisms against invading pathogens by guest on September 27, 2021 low-stringency hybridization using the FPR cDNA as a probe (4– and in the sensing of internal signals of cellular disoperation. In- 6). The three genes are clustered on human chromosome 19q13.3. deed, N-formyl peptides such as fMLP, the prototypic agonist of FPR, derive either from bacterial proteins (7–9) or from endoge- nous mitochondrial proteins, released as a result of cell death or *Institut de Recherche Interdisciplinaire en Biologie Humaine et Mole´culaire, Uni- severe dysfunction (10). The three receptors display distinct dis- versite´ Libre de Bruxelles, Brussels, Belgium; †Service d’Anatomie Pathologique Erasme, Universite´Libre de Bruxelles, Brussels, Belgium; ‡Euroscreen SA, Gosse- tribution patterns. Neutrophils express FPR and FPRL1, but not lies, Belgium; and §Department of Biomedical Sciences and Biotechnologies, Uni- FPRL2 (11). Monocytes express the three receptors, and this ex- versity of Brescia, Brescia, Italy pression is retained following their maturation into macrophages. Received for publication September 23, 2008. Accepted for publication February 5, 2009. Differentiation of monocytes into immature dendritic cells (DC) leads to the loss of FPRL1 expression (12), whereas maturation of 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 DCs results in the further loss of FPR expression. FPRL2 is there- with 18 U.S.C. Section 1734 solely to indicate this fact. fore the only receptor expressed by both immature and mature 1 This work was supported by the Actions de Recherche Concerte´es of the Commu- DCs (13). naute´Franc¸aise de Belgique, the Interuniversity Attraction Poles Programme-Belgian State-Belgian Science Policy, the European Union (Grant LSHB-CT-2005-518167/ Formyl peptides display high affinity for FPR (1) and low af- INNOCHEM), the Fonds de la Recherche Scientifique Me´dicale of Belgium, the finity for FPRL1 (4) and are almost inactive on FPRL2 (11). In Walloon Region (Programme d’excellence CIBLES), the Fe´de´ration Belge contre le addition to formylated peptides, a number of structurally diverse Cancer, the Fonds Ithier and the Fondation Me´dicale Reine Elisabeth, the Fonds Yvonne Boe¨l, and grants from the Italian Ministero dell’istruzione, Universita`e agonists of FPR and/or FPRL1 have been described in recent years Ricerca (PRIN projects) and AIRC (Associazione Italiana per la Ricerca sul Cancro). (14). For FPRL2, on the other hand, few specific ligands have been T.D. is an Aspirant of the National Fund for Scientific Research of Belgium. identified. A newly discovered peptide, humanin, neuroprotective 2 Address correspondence and reprint requests to Dr. Marc Parmentier, Institut de in Alzheimer’s disease models, was shown to bind FPRL2 and Recherche Interdisciplinaire en Biologie Humaine et Mole´culaire, Universite´Libre de Bruxelles, Campus Erasme, Route de Lennik 808, B-1070, Brussels, Belgium. E-mail FPRL1 with high affinity (15, 16). So far, the most specific address: [email protected] ligand described for FPRL2 is the endogenous peptide F2L (17). 3 Abbreviations used in this paper: FPR, formyl peptide receptor; FPRL1, FPR-like 1; F2L is an acetylated 21-aa peptide derived from heme-binding FPRL2, FPR-like 2; DC, ; hF2L, human F2L; mF2L; mouse F2L; Fpr-rs, protein, a tetrapyrol-binding protein. It was isolated from porcine Fpr-related sequence; rh, recombinant human; HEBP1, heme-binding protein-1; SDF-1, -derived factor-1; pDC, plasmacytoid DC; ROS, reactive on the basis of its bioactivity. This highly conserved peptide species; TFA, trifluoroacetic acid; CHO, Chinese hamster ovary; MGC, multinucle- binds and activates FPRL2 in the low nanomolar range, is poorly ated ; TAM, tumor-associated macrophage. active on FPRL1, and is inactive on FPR. In FPRL2-expressing Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 cells, F2L triggers intracellular calcium release, inhibition of www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803128 The Journal of Immunology 4975

TABLE I. Summary of Abs and conditions of use

Ab Host Clone Dilution Source

FPRL2 Mouse IgG2a 2G3 1/100 Euroscreen FPRL2 Mouse IgG2a 2B9 1/100 Euroscreen PE-anti-mouse IgG Goat 1/100 Sigma-Aldrich FITC-CD206 Mouse IgG1 19.2 1/50 BD Pharmingen CD14 Mouse IgG2a M5E2 1/100 BD Pharmingen CD16 Mouse IgG1 3G8 1/50 BD Pharmingen FPRL2 Mouse IgG2a 145C 1/7500 Euroscreen CD68 Mouse IgG1 KP1 1/100 Dako CD209 Rabbit 1/50 eBioscience FITC-anti-mouse IgG2a Goat 1/200 Jackson ImmunoResearch Biotin-anti-mouse IgG2a Goat 1/200 Jackson ImmunoResearch StreptaNL557 1/3000 RnDSystems Cy2-anti-mouse IgG1 Goat 1/200 Jackson ImmunoResearch Cy3-anti-rabbit IgG Goat 1/500 Jackson ImmunoResearch

cAMP accumulation, and phosphorylation of ERK1/2 MAPKs Quantitative RT-PCR using TaqMan microfluidic cards Downloaded from through the Gi class of G proteins. When tested on monocytes and Gene expression was investigated by using custom TaqMan low-density -derived DCs, F2L promotes calcium mobilization and arrays (Applied Biosystems). The cards included TaqMan probes and chemotaxis (17). F2L inhibits the IL-12 production promoted by primer sets for the amplification of 354 G protein-coupled receptor LPS in DCs and appears therefore to be an inhibitor of DC mat- genes, 10 housekeeping genes, and 20 leukocyte markers. The cards uration (18). were assayed on an ABI Prism 7900 thermocycler (Applied Biosys- tems), using the TaqMan Universal PCR master mix kit. Each leukocyte In contrast to the human genome, the mouse genome encodes at population was tested in duplicate (independent cell preparations from http://www.jimmunol.org/ least eight FPR-related receptors: Fpr1 and Fpr-related sequences different donors). The relative expression of each gene was determined (Fpr-rs) 1–7 (19). Mouse Fpr1 is 76% identical with that of human by normalizing the Ct values against a reference housekeeping gene. FPR but displays lower affinity for fMLP (20). Fpr-rs1 was de- TATA box binding protein was selected as the most stable among the ⌬ 10 housekeeping genes present on the card. Ct values were further scribed as a murine FPRL1 ortholog, sharing 73% amino acid ⌬ ⌬ normalized against a calibrator to calculate the ( Ct) values. As a calibrator, identity, and responding to A4, one of the FPRL1 ligands a mixture of commercial RNA preparations was used, including the XpressRef (19, 21). Fpr-rs2 (or Fpr2) was shown to bind fMLP with low Human Universal Total RNA (SABiosciences), total RNA from human cell affinity (22), as well as other FPRL1 ligands. It appeared therefore lines (Universal Human Reference, Stratagene), and human testis total RNA as a second FPRL1 ortholog. Mouse Fpr2 is expressed by neutro- (Ambion) in the 20:10:1 proportions.

phils, DCs, and microglial cells, and its transcripts are detected at Semiquantitative RT-PCR by guest on September 27, 2021 high levels in spleen and lung (23, 24). After the testing of the Total RNA was extracted from ϳ3 ϫ 106 highly purified eosinophils, whole set of mouse FPR-related receptors, Gao et al. (25) identi- monocyte-derived DCs, or macrophages, using the TRIzol Reagent (Life fied Fpr2 as the only mouse receptor responding to F2L. Indeed, Technologies). Equal amounts of RNA were reverse transcribed, and PCR mouse neutrophils, which express Fpr2, respond to human F2L was conducted using a standard procedure. The sequence of the primers (hF2L) and mouse F2L (mF2L) in both calcium flux and chemo- were as follows: 5Ј-CGCACAGTCAACACCATCTG-3Ј as sense and 5Ј- Ј Ј taxis assays. Moreover, neutrophils from mice genetically deficient AGCTGTTAAAAAAGGCCAAG-3 ) as antisense for FPRL2; 5 -TCCTT CTCTCTTCCTATCAATC-3Ј as sense and 5Ј-GGCAATTTTCTGCAT for Fpr2 failed to respond to F2L. CTG-3Ј as antisense for CCR3; 5Ј-AATCTTCTTCATCATCCTCC-3Ј as In this study, we investigate the expression of FPRL2 in a broad sense and 5Ј-TCTCTGTCACCTGCATAGC-3Ј as antisense for CCR5; 5Ј- set of human leukocyte subpopulations by quantitative RT-PCR AGCCACATCGCTCAGAACAC-3Ј as sense and 5Ј-GAGGCATTGCTG Ј Ј and flow cytometry, using newly developed mAbs. Using tissue ATGATCTTG-3 as antisense for human GAPDH; 5 -CCTTGGTGTGC TGGGCAATGG-3Ј as sense and 5Ј-GCGGTCCAAGGCAATGAG microarrays and immunofluorescence microscopy, we detected AGC-3Ј as antisense for mouse Fpr-rs2; and 5Ј-ATGTCGTGGAGTCTAC FPRL2 expression only on tissue-specific macrophage subpopu- TGGT-3Ј as sense and 5Ј-GTAGGAACACGGAAGGCCAT-3Ј as lations, particularly in lung, colon, and skin. We demonstrate antisense for mouse GAPDH. The PCR conditions were: denaturation at also a strong and functional expression of FPRL2 on eosino- 94°C for 5 min, denaturation at 94°C for 1 min, annealing at 56°C for 1 min phils, contrasting with the previous description of FPRL2 being 30 s, extension at 72°C for 1 min 30 s, 25 cycles (35 cycles for eosino- phils); final extension step at 72°C for 6 min. The PCR products were expressed exclusively in the mononuclear lineage. Finally, loaded on 2% agarose gels. The gels were stained with 10 mg/ml ethidium FPRL2 is strongly expressed and functional in plasmacytoid bromide, and the PCR products were visualized with UV light and dendritic cells. These new cellular targets of the chemotactic photographed. factor F2L suggest that FPRL2 plays an important role in the Preparation of leukocyte populations positive or negative regulation of inflammatory reactions and in allergic diseases. FPRL2 could constitute therefore an appeal- Leukocytes were isolated from venous of healthy donors according to the manufacturer’s specification by immunomagnetic bead cell sorting ing pharmacological target. (MACS). Briefly, leukocytes were recovered on a Ficoll (Lymphoprep Ax- is-Schield) density gradient, and erythrocytes were lysed by ammonium Materials and Methods chloride. Eosinophils were isolated from the pellet by negative Reagents and Abs selection (CD16, CD2, CD14, CD19, CD56, and CD123 biotin-conjugated Abs and anti-biotin microbeads; Miltenyi Biotec), and the purity was es- Primers and reagents for RT-PCR were from Eurogentec and Qiagen, re- timated by FACS to be Ͼ95%. The population was eluted from spectively. RPMI 1640, DMEM, PBS, FBS, and FCS, and were the column with 99% purity. Monocytes and B and T subsets from Life Technologies. Recombinant human (rh) IL-4, IL-5, M-CSF, and were purified by positive selection, after Percoll density centrifugation, CCL5 were from R&D Systems. The characteristics and source of Abs are using, respectively, CD14, CD19, CD3, CD4, and CD8 microbeads (Milte- displayed in Table I. All chemicals were obtained from Sigma-Aldrich, nyi Biotec). Plasmacytoid DCs were magnetically sorted with blood DC unless otherwise specified. Ag (BDCA-4) cell isolation kits and matured by 24 h of incubation with 4976 FPRL2 EXPRESSION IN LEUKOCYTE POPULATIONS

100 ng/ml LPS ( 055:B5; Sigma-Aldrich), or 20 ng of hemagglutinin per ml of inactivated influenza strain A/Moscow/10/99 (a gift from Dr. T. De Magistris, Instituto Superiore di Sanita`, Rome, Italy) (26). The purity of these cell preparations was evaluated by flow cytometry to exceed 95% for CD14ϩ, CD19ϩ, CD3ϩ, CD4ϩ, and CD8ϩ cells. Human monocytes were resuspended at the density of 106 cells/ml and seeded in six-well plates in RPMI 1640 supplemented with 10% heat- inactivated FBS. Immature monocyte-derived DCs were obtained by cul- turing monocytes in the presence of rhGM-CSF (800 U/ml; Schering- Plough) and rhIL-4 (500 U/ml) for 7 days. Macrophages were differentiated from monocytes in the presence of rhM-CSF (50 ng/ml,) for 6–11 days. The purity of the cell preparations was evaluated by flow cy- tometry to ϳ85% (CD11bϩCD11cϩ for DCs and CD11bϩCD206ϩ for macrophages). marrow-derived murine dendritic cells were prepared from C57BL/6 mice (Charles River Laboratories). was collected by flushing mouse femurs with RPMI 1640. The cells were washed twice with RPMI 1640, passed through a 70-␮m mesh pore size cell strainer to remove bone debris, and resuspended at a density of 2 ϫ 106 cells/ml in RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, 100 ␮g/ml Downloaded from streptomycin, and 20 ng/ml recombinant murine GM-CSF (Biosource). They were cultured for 10 days, one half of the medium being replaced every other day. The purity and viability of murine DCs were determined by flow cytometry (CD11cϩCD11bϩ population). Thioglycolate-elicited murine macrophages were obtained from the of 8-wk-old female C57BL/6 mice 4 days after i.p. in- jection of 4% thioglycolate broth. Peritoneal cells were resuspended in

DMEM supplemented with 10% FCS at a density of 2.5 ϫ 106 cells/ml, http://www.jimmunol.org/ FIGURE 1. Distribution of receptors of the FPR family in PBL popu- cultured for 4 h, washed twice, and further cultured overnight before col- A B C lection. The cell population was consistently composed of Ͼ95% macro- lations. Distribution of human FPR ( ), FPRL1 ( ), and FPRL2 ( ) was phages as determined by flow cytometry analysis (F4/80ϩCD11bϩ cells). analyzed by real-time quantitative RT-PCR on various leukocyte popula- tions, using microfluidic cards. For each population, two independent sam- FACS analysis ples prepared from healthy donors were used, and the data were normalized for the expression of an endogenous reference gene (TBP) used as an in- Mouse anti-FPRL2 mAbs (2G3 and 2B9) were used in FACS analysis. Briefly, cells were incubated for1hat4°CinPBScontaining 2% FBS and ternal control. D, Transcripts encoding human FPRL2 were amplified by 0.1% sodium azide (and 10% human serum for monocytes, macrophages, RT-PCR on purified peripheral blood monocytes, monocyte-derived mac- and DCs), washed, incubated with R-PE-labeled anti-mouse IgG for 30 rophages, monocyte-derived mDCs, and purified pDCs. GAPDH was used min at 4°C, and analyzed on a Cytomics FC 500 (Beckman Coulter). as control. E, Two anti-FPRL2 mouse (m.) mAbs (2G3 and 2B9) generated by guest on September 27, 2021 by genetic immunization were tested by flow cytometry on CHO-K1 cell Chemotaxis assays lines expressing FPRL2 (gray), FPRL1 (dashed line), ChemR23 (solid line) Leukocyte chemotaxis was assayed by a modification of the Boyden mi- or GPR23 (dotted line). F, FACS analysis of monocytes and neutrophils, cropore filter technique, as previously described (27, 28). Briefly, the assay using the 2G3 monoclonal, and an IgG2a control. G, Median shift was conducted in 48-well microchemotaxis chambers (Neuroprobe), using of fluorescence (relative to isotype control) obtained with the 2G3 mono- 5-␮m pore size Nuclepore track-etched polycarbonate membranes (What- clonal on various leukocyte populations prepared from peripheral blood or man). The cell suspension (50,000 cells/well for eosinophils and 10,000 derived ex vivo (macrophages). Data are the means of three to five inde- cells/well for human and murine DCs and macrophages) was loaded in the pendent experiments performed on cells from different donors. upper chamber and the chemoattractant solution or vehicle in the lower chamber. Positive controls of were 0.1 ␮M eotaxin for eo- sinophils, 1 ␮M fMLP for murine macrophages, and 0.5 ␮M CCL5 for murine DCs. Migration was conducted for 90 min at 37°C in humidified air degranulation assay containing 5% CO2. The filters were fixed and stained with Hoechst (di- lution 1/2000) for 2 min. Micrographs of the lower surface of the filters The production of reactive oxygen species (ROS) from eosinophils was were taken, and the number of cells was counted with ImageJ software analyzed as previously described by a luminol-dependent chemilumines- (version 1.36b). Migration assays for by plasmacytoid DCs (pDC) were cence assay (29). Purified eosinophils (3 ϫ 105 cells) were incubated in 25 performed using MultiScreen MIC plates (Millipore) with 5-␮m pore size ␮l of RPMI 1640 without phenol red in white 96-well plates for 30 min at 4 filters and 5 ϫ 10 cells/well. Stromal cell-derived factor-1 (SDF-1; 10 37°C in 5% CO2. The stimulatory agents (20 ng/ml IL-5, 10–500 nM nM) was used as a positive control. The number of migrating pDCs was hF2L) were then added to the eosinophil suspension in a volume of 25 ␮l, measured by an ATP assay (ATPlite; PerkinElmer). Results are expressed together with 50 ␮l of a luminol solution (7.5 ␮g/ml; Sigma-Aldrich), and as chemotactic index (ratio of cells in the lower chamber in the presence vs a kinetics was performed for 60 min at 37°C, by integrating the chemilu- the absence of chemoattractant). Statistical significance was determined minescence for periods of5sinaluminometer (Victor Wallac). using the t test for paired values, and p values of Ͻ0.05 were considered significant. Immunohistochemistry and tissue microarrays Air pouch assay Normal tissues obtained from 30 different human organs were selected using H&E-stained slides. Corresponding paraffin-embedded blocks were The air pouch assay was performed on 6- to 8-wk-old BALB/c mice then precisely aligned with the marked slides. To circumvent the problem (Charles River Laboratories). On days 1 and 4, 3 ml of sterile air were of tissue heterogeneity, three tissue cores (0.6 mm in diameter) for each injected under the back skin to create the pouch. On day 7, mice with a sample were punched using a precision instrument (Beecher) and arrayed well-formed pouch were randomized into groups and injected with 200 nM into a recipient block. Moreover, each organ was represented in triplicate. murine F2L in endotoxin-free PBS containing 0.1% DMSO or with vehicle Sections 5 ␮m thick of formalin-fixed and paraffin-embedded tissue only. Six hours later, mice were killed in a CO2 chamber, and the air samples and tissue microarrays were subjected to standard immunohisto- pouches were washed extensively with PBS. The collected cells were chemical procedures. Primary Abs were mouse monoclonal anti-FPRL2 counted, and the leukocyte populations were identified by flow cytometry. (clone 145C) and anti-CD68 Abs and rabbit anti-CD209 polyclonal Ab. , macrophages, and DCs were labeled by mAbs recognizing, Microwave Ag retrieval was performed twice in citrate buffer, pH 6.0, for respectively, CD11b, CD11c, and Gr1. 5 min. The immunohistochemical expression was visualized by means of The Journal of Immunology 4977

FIGURE 3. Detection of FPRL2 by immunohistochemistry and immu- Downloaded from nofluorescence. A, The anti-FPRL2 mAb 145C was validated for immu- nohistochemistry using a mixture of CHO-K1 cells expressing FPRL2 (10%) and wild-type CHO-K1 cells (90%). B, Double immunofluorescence staining on paraffin sections from duodenum showing partial colocalization (arrows) of FPRL2 (red) and the DC marker CD209 (DC-SIGN, green) in cells of the . Nuclei were stained with Hoechst (blue). Ab-

sence of nonspecific cross-staining was ascertained by replacing the pri- http://www.jimmunol.org/ mary Abs with isotype-matched control monoclonals. C, FPRL2 labeling (green) in the colonic crypts, corresponding in part to DCs probing the lumen by extending processes through the columnar epithelium (arrows).

Hoechst stain (Sigma-Aldrich; 1/2000 dilution), and sections were FIGURE 2. Expression of FPRL2 on monocyte-derived macrophages. mounted with the Glycergel mounting medium (Dako). For negative con- A, Monocytes differentiated into macrophages in the presence of 10% hu- trols, the primary Abs were replaced with control IgG2a or IgG1 isotypes man serum (HS), GM-CSF, M-CSF, M-CSF plus LPS plus INF-␥ (M1) or or rabbit Igs. by guest on September 27, 2021 M-CSF plus IL-4 (M2) were tested by FACS for polarization markers Identification of bioactive mF2L (CD206, CD14, and CD16). The percentage of cells displaying fluores- cence over the threshold in two independent experiments is displayed. B, Frozen mouse spleen tissue (500 mg) was homogenized in 4 volumes of FACS analysis of FPRL2 (2G3 monoclonal) on monocyte-derived macro- ice-cold 20% acetonitrile in water. The homogenate was centrifuged at ϫ phages and M1 and M2 polarized macrophages. C, Detection of FPRL2 10.000 g for 30 min at 4°C. The supernatant was diluted 4-fold in 0.1% ϫ transcripts by RT-PCR on total RNA from monocyte-derived macrophages trifluoroacetic acid (TFA) and loaded at 1 ml/min on a 4.6- 250-mm C18 column (Vydac) submitted to an acetonitrile gradient (0.25% acetonitrile and M1 and M2 polarized macrophages. The data are representative of per min) in 0.1% TFA. Collected fractions were tested for their bioactivity three independent experiments. D, The migration of macrophages toward a on FPRL2-expressing Chinese hamster ovary (CHO)-K1 cells in an ae- range of hF2L concentrations (0.1 to 1000 nM) was recorded in microche- quorin-based calcium mobilization assay, as described previously (17). All motaxis chambers. fMLP (10 nM) was used as a positive control. The active fractions were pooled, diluted to 20% acetonitrile, and loaded on a chemotactic index represents the number of migrated cells over that ob- second 4.6-mm C18 column as above. The active fractions were finally ϫ served in the absence of chemoattractant (buffer condition). Data are the loaded on a 2.1- 250-mm C4 column (Vydac) submitted to an acetoni- means Ϯ SEM of triplicate wells and are representative of five independent trile gradient at 0.2% acetonitrile per min in 0.1% TFA. The collected experiments. E, Chemotaxis of human pDCs in response to F2L in Multi- fractions were again tested for their activity on FPRL2-expressing cells. Screen MIC plates. SDF-1␣ (10 nM) was used as a positive control. Data The active fractions were analyzed by mass spectrometry in the absence of tryptic digestion, using a Q-TOF Ultima Global mass spectrometer are the mean Ϯ SEM of three independent experiments. Hi, High granu- (Micromass) equipped with a MALDI source. larity; Lo, low granularity; ns, non-stimulated. Statistical analysis t the Vectastain Elite ABC kit (Vector Laboratories) with 3,3Ј-diaminoben- Statistical analysis was performed with the unpaired Student test and the U zidine tetrahydrochloride (Dako) as the peroxidase substrate. The slides Mann-Whitney test (Instat; GraphPad Software). were subsequently counterstained with hematoxylin, dehydrated, and mounted in DPX mounting medium (Sigma-Aldrich). Negative controls Results were conducted by replacing primary Abs with nonimmune sera (Vector Distribution of human FPRL2 on PBL populations Laboratories). FPRL2 expression was evaluated by two independent ob- servers and assessed by scoring each spot: 0 for nonexpression; 1 for low The expression of receptors of the FPR family was previously expression; and 2 for high expression. reported in various leukocyte populations by RT-PCR, FACS, and functional assays (11–13, 17). In the present work, we performed Immunofluorescence studies a thorough analysis of the expression pattern of these (and other) Frozen and paraffin-embedded tissue sections were stained using standard receptors in a large panel of PBL populations, using real-time immunofluorescence methods. The monoclonal primary Abs (same as quantitative RT-PCR. Applied Biosystems microfluidic cards, con- above) were incubated overnight at 4°C. FPRL2 was further detected by a goat biotin-conjugated anti-mouse IgG2a-specific and streptavidin NL557, taining specific primers for the receptors of interest, were used for CD68 by goat Cy3-conjugated anti-mouse IgG1-specific, and CD209 by this purpose. Total RNA was prepared from highly purified pop- goat Cy3-conjugated anti-rabbit IgG. Nuclei were counterstained with ulations of neutrophils, monocytes, macrophages, B cells, naive T 4978 FPRL2 EXPRESSION IN LEUKOCYTE POPULATIONS

TABLE II. Expression of FPRL2 in various tissue-specific DC maturation (Fig. 1B). As reported earlier (12), FPRL2 tran- macrophages scripts were present in monocytes and myeloid DCs and absent from neutrophils. In addition, the highest FPRL2 expression levels Mean Score Mean Score were observed for monocyte-derived macrophages and mature Tissues of FPRL2 Tissues of FPRL2 pDCs (Fig. 1C). A low level of FPRL2 expression was also ob- Stomach 0.2 Prostate 0 served in NK cells. The results regarding FPRL2 were confirmed Duodenum 1 Testis 0 by classical RT-PCR, using RNA preparations from monocytes, Ileum 0.33 0 monocyte-derived-macrophages, myeloid DCs, and pDCs (Fig. Jejunum 0 Bladder 0 Appendix 0.5 1 1D). A much fainter band was also observed for NK cells (data not Colon 1.9 shown). 0 Spleen 0.5 The presence of FPRL2 at the protein level was further inves- Gallbladder Thyroid 0 tigated on purified blood leukocytes. For this purpose, a new set of Uterus Pancreas 0 Endometer 0 Brain 0 mAbs was generated against human FPRL2 by genetic immuni- Myometer 0 Amygdala 0 zation of mice. The monoclonals were tested by flow cytometry on Fallopian tube 0 Adrenal gland CHO-K1 cell lines expressing FPRL2, FPRL1 (closest relative), Skin 2 ChemR23, and GPR23 (negative controls). Two monoclonals Breast Esophagus 0 Ovary 0 Lung 2 (2G3 and 2B9) were selected for their labeling efficiency, their Seminal vesicle 0 high specificity for FPRL2, and their low background staining (Fig. 1, E and F). These two Abs were then tested on freshly Downloaded from prepared human PBL populations. For each population, three to cells, CD4ϩ T cells, CD8ϩ T cells, NK cells, mature and immature five independent experiments showed a similar expression profile myeloid DCs, and mature and immature pDCs. For each popula- for FPRL2, which was in agreement with the RT-PCR data. Clear tion, RNA samples were prepared from two independent donors. labeling was found for monocytes, macrophages, pDCs, and eo- As described previously, FPR was expressed at very high levels sinophils (Fig. 1G). For monocyte-derived DCs, no or weak label- in neutrophils and at lower levels in monocytes. We also detected ing was obtained on unpermeabilized cells, whereas staining was http://www.jimmunol.org/ expression in NK cells and immature myeloid DCs and pDCs. The consistently observed for all donors following permeabilization. maturation of DCs induced the loss of FPR expression (Fig. 1A). As previously described (17), FPRL2 appears therefore largely in- FPRL1 shared with FPR a similar expression pattern, with the tracellular in monocyte-derived DCs. No significant expression of exception of DCs, in which expression was maintained following FPRL2 was observed on T and B , NK cells, and by guest on September 27, 2021

FIGURE 4. Tissue microarray anal- ysis of FPRL2 expression. Two serial sections of tissue microarrays, includ- ing 30 human organs (3 samples from different subjects for each organ, 3 cores per sample) were stained for FPRL2 (left) and the macrophage marker CD68 (right). A score was as- signed for each spot (0 for no expres- sion, 1 for low expression, and 2 for high expression of FPRL2). Labeled macrophages were observed in lung (A), colon (B), and skin (C) with a score of 2. Low FPRL2 staining was ob- served on lymph node and spleen mac- rophages with a score of 1 (D and E), whereas no FPRL2 expression was de- tected in liver Kupffer cells (F) and in esophagus (G, score of zero). Other or- gans were negative as well (data not shown). Nuclei were counterstained with hematoxylin. Bars, 50 ␮m; origi- nal magnification, ϫ400. FPRL2 ex- pression on macrophages in the lung of a patient affected by (H, left) and in MGCs from a tuberculosis lesion (H, right). Original magnifica- tions, ϫ200 and ϫ400, respectively. Bars, 100 and 50 ␮m, respectively. The Journal of Immunology 4979 neutrophils (Fig. 1G), even after permeabilization (data not shown). Altogether, these results confirm that FPRL2 displays an expression profile clearly distinct from that of FPR and FPRL1, while identifying new cell types expressing FPRL2, namely pDCs and eosinophils. In the absence of FACS signal, the detection of low levels of FPRL2 transcripts in NK cells was considered as functionally irrelevant and was not investigated further. Macrophages are functionally very diverse in vivo, and at least two different polarities of macrophages in vitro (M1 and M2) have been described in the literature (30). We tested whether FPRL2 expression was affected by the polarity of macrophages generated in vitro from monocytes. Macrophages were generated in the pres- ence of M-CSF, GM-CSF, or human serum and further cultured in the presence of IL-4, LPS, or LPS and IFN-␥. The cells were tested by flow cytometry for polarization markers (HLA-DR, CD206, CD14, and CD16) as well as FPRL2 expression. M-CSF macro- phages and IL-4-stimulated M-CSF macrophages displayed a clear M2 polarity (HLA-DRhigh CD206high CD14high). GM-CSF mac- rophages and M-CSF macrophages activated by LPS and INF-␥ Downloaded from FIGURE 5. Immunofluorescence staining of FPRL2 in lung, colon, and showed a M1 polarity (HLA-DRlow CD206low CD14low). Macro- skin. A, Double immunofluorescence for FPRL2 (red) and the macrophage phages differentiated in the presence of human serum exhibited marker CD68 (green) was performed on paraffin sections from lung (A), mixed characters, and two populations were seen on the scatter colon (B), and skin (C). Most FPRL2-expressing cells coexpressed CD68 plot. A low-granularity population shared characteristics of M2 (thin arrows). CD68Ϫ cells stained with FPRL2 are presumably DCs (thick macrophages, whereas a high-granularity population showed M1 arrows). Nuclei were stained with Hoechst (blue). Negative controls were characteristics (Fig. 2A). No significant variation of FPRL2 ex- performed by replacing the primary Abs with isotype-matched control http://www.jimmunol.org/ pression was observed according to the macrophage polarity de- monoclonals. Original magnification, ϫ400. Bars, 50 ␮m. spite a trend toward higher expression in M1 and M2 subsets (Fig. 2B). RT-PCR did not show changes in FPRL2 transcript levels in the same conditions (Fig. 2C). FPRL2 is therefore highly ex- Tuberculosis, Crohn’s disease, , and sarcoid- pressed in vitro by monocyte-derived macrophages with a mild osis are known to share the presence of granulomatous lesions with up-regulation following activation. multinucleated giant cells (MGC), which arise from the fusion of To complement these observations in human tissues, we vali- macrophages. In sections from and tuberculosis dated one of our anti-FPRL2 monoclonals (1C4, 17) for immuno- histochemistry, using cytospin preparations of CHO-K1 cells ex- by guest on September 27, 2021 pressing or not FPRL2 (Fig. 3A). Provided the known expression of FPRL2 in DCs, we further validated this Ab by double immu- nofluorescence on duodenum and colon sections, using CD209 (DC-SIGN) as a DC marker. A number of cells labeled by the 145C monoclonal were detected in the stroma of the duodenum, in both the axis of the villi and the . A significant propor- tion of these cells expressed also CD209 (Fig. 3B). In the colon, FPRL2 labeling was also observed in cells within the columnar epithelium of the crypts (Fig. 3C), with a morphology previously described for DCs extending processes toward the lumen (31).

Expression of FPRL2 by macrophage subpopulations in human tissues The in vitro differentiation of monocytes into polarized macro- phages is not representative of the multiplicity of leukocyte sub- populations that are present in tissues in vivo. We therefore inves- tigated the expression of FPRL2 in a set of 30 human organs, using FIGURE 6. Functional expression of FPRL2 in human eosinophils. A, tissue microarrays. A score of expression was assigned for each Transcripts encoding human FPRL2 were amplified by RT-PCR from sample, and the results are displayed in Table II. High expression RNA prepared from human eosinophils (Ͼ95% purity) from six healthy of FPRL2 was observed in CD68ϩ macrophages from lung, colon, donors. CCR3, CCR5, and GAPDH were used as controls. Two represen- and skin (Fig. 4, A–C). In the lung, FPRL2 was expressed in both tative experiments are displayed. B, Cell surface expression of FPRL2 on alveolar and interstitial macrophages. In the skin, FPRL2 was also human purified eosinophils was analyzed by flow cytometry, using the 2G3 expressed in CD68Ϫ cells identified as DCs. Besides, very low monoclonal. C, hF2L (0.1–100 nM) promotes eosinophil migration in mi- crochemotaxis chambers with a maximum at 20 nM. Eotaxin (Eo, 100 nM) expression of FPRL2 was observed in lymph node and spleen mac- was used as a positive control and I309 (100 nM) as a negative control. D, rophages (Fig. 4, D and E), and no detectable expression of FPRL2 The production of ROS by purified eosinophils was tested with a luminol- was observed in Kupffer cells (Fig. 4F), macrophages from esoph- dependent chemiluminescence assay, in response to F2L (10–500 nM), agus (Fig. 4G), endocrine tissues, kidney, and microglial cells. IL-5 (20 ng/ml), and I309 (100 nM). The kinetics was performed for 60 Colabeling of FPRL2 and CD68 in lung, colon, and skin was also min at 37°C, and for each time point, chemiluminescence was recorded for demonstrated by double immunofluorescence (Fig. 5). 5 s. Data are representative of three independent experiments. 4980 FPRL2 EXPRESSION IN LEUKOCYTE POPULATIONS

FIGURE 7. Expression of Fpr2 by mouse DCs and macrophages and par- tial purification of bioactive mF2L from mouse spleen. A, Transcripts en- coding mouse Fpr2 were amplified from leukocyte populations by semi- quantitative RT-PCR. Mouse GAPDH was amplified in parallel as a house- keeping gene. Fpr-rs2 is expressed by monocyte-derived DCs and thioglyco- late-elicited peritoneal macrophages. LPS induces an up-regulation of Fpr2 expression in DCs. B–E, Mouse spleen homogenates from control animals (B) or animals receiving a nonlethal dose of LPS (600 ␮g i.p.) 24 h before sacrifice (C) were fractionated through three successive HPLC steps (optical density measured at 214 nM). The fractions were

tested in an aequorine-based lumines- Downloaded from cence assay (f) on a FPRL2 expressing CHO-K1 cell line. The measured activity was normalized to the response obtained for 20 ␮M ATP. No signal was observed on wild-type CHO-K1 cells (Œ). The fractions marked by arrows in C were processed further onto a 4.6-mm C18 col- http://www.jimmunol.org/ umn (D) and then a 2.1-mm C4 column (E). F and G, Mouse F2L was tested for its chemotactic activity on monocyte-de- rived DCs (F) and peritoneal macro- phages (G) in microchemotaxis cham- bers, using CCL5/RANTES (50 mM) and fMLP (fMLF; 1 ␮M) as positive controls, respectively, for DCs and macrophages. Maximal migration was observed for 10 ␮M in both cases, with by guest on September 27, 2021 efficiency comparable with that of the positive control. H and I, In vivo re- cruitment of leukocytes was tested by injecting mF2L or PBS in air pouches. Recruited leukocytes were counted with a hemocytometer. The collected cells were then characterized by flow cytometry using CD11b, CD11c, and GR-1 as markers (I). AU, Absorbance units; ns, non-stimulated; m. Macro, mouse macrophages. lesions, we observed high expression of FPRL2 on macrophages FPRL2 is expressed and functional in eosinophils H, left right and MGCs (Fig. 4 and ). We also investigated the expression and function of FPRL2 in Biological activity of F2L on monocyte-derived macrophages eosinophils. Indeed, high levels of FPR and FPRL1 transcripts and pDCs were previously reported in human eosinophils in addition to neu- trophils (32). The absence of FPRL2 in neutrophils is well docu- The hF2L peptide, derived from heme-binding protein, is presently mented (11), but no data were available regarding FPRL2 in eo- the only ligand displaying high specificity and high affinity for sinophils. Using total RNA prepared from highly purified FPRL2. As performed previously for monocytes and monocyte- preparations of human eosinophils (six donors), we detected by derived DCs (17), we investigated the biological activity of hF2L on monocyte-derived macrophages and native pDCs. In microche- RT-PCR transcripts for all three FPR family members. One rep- motaxis Boyden chambers, hF2L promoted chemotaxis of macro- resentative experiment performed with two donors is displayed in phages peaking for concentrations of 1–10 nM (Fig. 2D). Chemo- Fig. 6A. CCR3 and CCR5 were included as controls for highly and taxis of pDCs was also promoted by hF2L with a peak at 10 nM poorly expressed genes in eosinophils. The presence of FPRL2 at (Fig. 2E). The migration indexes obtained with hF2L for macro- the surface of eosinophils was confirmed by flow cytometry, using phages and pDCs were comparable with those of, respectively, the 2G3 and 2B9 mAbs (Fig. 6B). However, the labeling was fMLP (10 nM) and SDF-1 (10 nM), used as a positive controls. stronger on permeabilized cells, suggesting that as in monocyte- These data demonstrate the physiological role of hF2L on human derived DCs, a significant part of FPRL2 is kept in intracellular macrophages and pDC recruitment, through its action on FPRL2. structures. The presence of the receptor in eosinophils modifies The Journal of Immunology 4981 significantly the previously described expression pattern of derived DCs and peritoneal macrophages with a maximum of ϳ10 FPRL2, which was considered specific of the monocytic lineage. ␮M (Fig. 7, F and G), with an efficacy similar to that of CCL5/ On the basis of these results, we tested whether the hF2L peptide RANTES (50 mM) and fMLP (1 ␮M) used as positive controls, could promote chemotaxis of human eosinophils. As for macro- respectively, for DCs and macrophages. phages, hF2L promoted eosinophil migration (n ϭ 8 donors) in the We also investigated the chemoattractant activity of mF2L in low nanomolar concentration range, with a peak at 20 nM (Fig. vivo. In an air pouch assay, the cells recruited 6 h after injection of 6C). The efficacy of F2L (migration index, 3) was similar to that 200 nM mF2L (or buffer) were counted. In three independent ex- of eotaxin at 100 nM, used as positive control, whereas no migra- periments, a significantly larger number of cells was collected in tion over background was obtained for I309 at 100 nM, used as mF2L-injected pouches than in controls receiving PBS (Fig. 7H, negative control. Because some chemotactic factors for eosino- n ϭ 10). The cells were labeled with CD11b-PE, CD11c-FITC, phils promote also degranulation, resulting in the release of large and GR-1-PerCp to identify specific leukocyte populations. Neu- amounts of ROS, we tested whether F2L stimulated eosinophil trophils, DCs, and macrophages were detected, the last two pop- degranulation as well. Using a chemiluminescence assay for ROS, ulations being significantly overrepresented in the mF2L-injected we did not detect degranulation of eosinophils after stimulation by pouches (Fig. 7I). hF2L up to 500 nM, whereas 20 ng/ml IL-5, used as positive control, promoted strong degranulation (Fig. 6D). These data sug- gest that F2L is chemotactic for eosinophils through FPRL2 but Discussion does not promote the activation of cytotoxic properties of these FPR is well established as a key player in innate immunity and cells. host defense mechanisms. FPR shares with FPRL1 a number of Downloaded from characteristics, in terms of cell distribution, range of ligands, and Human FPRL2 shares with mouse Fpr2 some of its functional functional properties. The third member of the FPR family, properties FPRL2, displays more specific features, and its function is far less We have identified recently the mouse receptor Fpr2, encoded by established. Indeed, FPRL2 does not respond to fMLP and the Fpr-rs2, as a partial functional homolog of human FPRL2, and large variety of other FPR and FPRL1 ligands, unless at very high

demonstrated that mouse neutrophils expressing Fpr2 responded to concentrations which are likely physiologically irrelevant. The http://www.jimmunol.org/ mF2L in a chemotaxis assay (25). With the aim of comparing only significant exception is the neuroprotective peptide humanin, mouse Fpr2 expression with that of human FPRL2, we tested the which acts on both FPRL1 and FPRL2 with similar potency (15). presence of Fpr2 transcripts in various mouse leukocyte popula- FPRL2 is characterized by a specific high-affinity agonist, the pep- tions, using GAPDH as a housekeeping control gene. RT-PCR tide F2L, which is poorly active on FPRL1 and inactive on FPR showed expression of Fpr-rs2 on mouse bone marrow-derived DCs (17). Moreover, if FPR and FPRL1 are both highly expressed by and peritoneal macrophages (Fig. 7A). Activation of DCs with LPS neutrophils, FPRL2 is not detectable on these cells. It is rather induced up-regulation of Fpr2 transcript levels. To test the described with a specific expression pattern in the mononuclear physiological relevance of F2L activity on Fpr2, we attempted to lineage, suggesting a distinct role in the regulation of immune identify the production of endogenous mF2L in vivo. By analogy responses (11–13). by guest on September 27, 2021 with the initial identification of hF2L in humans (17), we fraction- In the present study, we have investigated more extensively the ated extracts from mouse spleen by reverse-phase HPLC and expression of FPRL2 in the various human leukocyte populations, tested the activity of the fractions on a cell line coexpressing hu- with the aim of raising more precise hypotheses regarding its po- man FPRL2, apoaequorin, and G␣16. For testing whether the pep- tential functions in the human body. By quantitative RT-PCR and tide is generated in inflammatory conditions, we used untreated flow cytometry, we confirmed the expression of FPRL2 in the mice, but also mice receiving a sublethal dose of LPS (600 ␮gin monocytic lineage, including monocytes, macrophages, and DCs 200 ␮l of PBS i.p.). Starting from ϳ0.5 g of spleen tissue, a bio- and its absence from lymphocytes. A low level of FPRL2 tran- logical activity was purified through three successive HPLC steps. scripts was detectable in NK cells, but, in the absence of FACS This activity was much stronger in LPS-stimulated conditions (Fig. signal, it was not considered as functionally relevant and was not 7, B–E). The partially purified material had no activity on control investigated further. FPRL2 was as expected absent from neutro- cell lines. The elution behavior of the bioactivity was identical phils, but present both at the transcript and protein levels in eo- with that of hF2L tested in the same conditions. This is consistent sinophils, which had not been described previously. Unlike its pre- with the conservation of the peptide between human and mouse viously described expression on monocyte-derived DCs, which which differ only by one amino acid (conservative lysine/ was found to be variable according to donors (17), FPRL2 was replacement at position 6). Mass spectrometry analysis of the par- consistently detected and functional on monocyte-derived macro- tially purified bioactive fraction resulting from the third HPLC phages, pDCs, and eosinophils from all donors tested. column revealed a mass corresponding to the predicted sequence The FPRL2 expression on in vitro monocyte-derived macro- for the mF2L peptide (data not shown). The peptide could not be phages was much more important than on DCs. Indeed, if the sequenced however. In conclusion, we have detected in mouse presence of the FPRL2 protein was previously described on DCs, spleen an activity specific for FPRL2, which is increased in in- it was essentially intracellular, suggesting a constant turnover of flammatory conditions and which behaves like F2L on several the receptor (17). Permeabilization of the cells was not required to HPLC columns. detect strong expression of FPRL2 on macrophages. Chemotaxis of macrophages was observed toward F2L, demonstrating that Biological activity of mF2L on murine DCs and macrophages FPRL2 is functional on these cells, and extending the chemotactic The ability of mF2L to promote chemotaxis of neutrophils through role of F2L, previously shown for monocytes and DCs (17). Hu- Fpr2 was demonstrated previously (25). We tested further the ca- man macrophages are present in almost all tissues of the body and pacity of mF2L to induce recruitment of DCs and macrophages, are key players in host defense mechanisms and responses to tissue which also express the receptor. mF2L was tested in 48-well mi- injuries. The activity of F2L on these cells suggests further the crochemotaxis chambers at concentrations ranging from 100 nM to involvement of FPRL2 and its ligand in the control of immune and 30 ␮M. The peptide promoted chemotaxis of mouse monocyte- inflammatory responses. 4982 FPRL2 EXPRESSION IN LEUKOCYTE POPULATIONS

However, the in vitro differentiation of monocytes into macro- proposed (30, 42). In vitro, INF-␥, GM-CSF, TNF-␣, and LPS phages is not representative of the multiple functional subtypes of induce the classically activated macrophages (M1) whereas IL-4, macrophages that are found in organs in vivo. Although all mac- IL-13, IL-10, and or secosteroid hormones lead to rophage populations exhibit in vivo mononuclear char- the alternative M2 activation. Each polarization is characterized by acteristics, it is clear today that a significant heterogeneity exists the up- and down-regulation of specific membrane and secreted within and between tissues (33, 34). Subpopulations of macro- proteins. M1 macrophages are described as proinflammatory cells phages, such as those present in intestine, skin, liver, kidney, with antimicrobial and antitumoral properties (43). M2 macro- spleen, and the nervous system, display functional, antigenic, and phages promote instead tumor proliferation and and morphological differences. Macrophages derive from pluripotent display immunoregulatory functions. We attempted to correlate the hemopoietic stem cells in bone marrow where they differentiate expression of FPRL2 with this simplified M1/M2 polarization into , , and monocytes. Peripheral blood scheme. On in vitro polarized macrophages, the profile of markers monocytes recruited to different organs then differentiate into tis- was consistent with previous reports (44), but we did not observe sue macrophages, in the presence of a combination of growth fac- significant differences in FPRL2 expression by flow cytometry or tors such as M-CSF (or CSF-1), GM-CSF, IL-6, IL-3, stem cell semiquantitative RT-PCR. FPRL2 was indeed highly expressed in factor, IL-1, LIF, and IFN-␥. The environment in which all situations of macrophage differentiation and activation. Never- monocytes differentiate in tissues dictates the polarization and theless, the M1/M2 polarization paradigm is essentially an in vitro functional properties of the resulting macrophages (30). concept that does not represent faithfully the in vivo situation. We wondered therefore whether FPRL2 is expressed by all mac- Recently, a set of up-regulated genes was associated with M2 po- rophage subpopulations, or rather restricted to some specialized larization of macrophages in vivo, but not in vitro (45). Moreover, Downloaded from populations. We investigated the distribution of FPRL2 in a set of pathogens and tumors can shape immune responses in a complex 30 human tissues by using tissue microarrays. This allowed us to way in vivo, by affecting the profile of released and the demonstrate the selective high expression of FPRL2 in macro- polarization of effector cells such as macrophages and DCs (46). phages from lung, colon, and skin, whereas low FPRL2 expression Finally, the M1/M2 concept is not appli- was seen in macrophages from spleen and lymph nodes, and no cable as such in oncogenesis. Indeed, the pro- or antitumoral prop-

expression was found in macrophages from kidney or endocrine erties of macrophages depend on their activation state, but also on http://www.jimmunol.org/ glands, or in Kupffer cells and . Colonic, alveolar, and the properties of the tumor cells and the resulting microenviron- skin macrophages are chronically exposed to pathogens, highly ment. Tumor-associated macrophages (TAM) release factors such phagocytic, and produce large amounts of ROS and reactive ni- as IFNs, angiostatin, factor-4, and thrombospondin that trogen species. Given their strategic localization allowing them to inhibit tumor growth, angiogenesis, and the metastatic process. On promote inflammatory responses to pathogens and tissue injury, the other hand, TAMs may favor tumor growth by releasing an- they constitute a macrophage subpopulation clearly involved in giogenic factors and molecules such as PGE2, which promotes an innate immunity. They display a quite distinctive pattern of surface immunosuppressive state (47, 48). It will therefore be interesting markers, as compared with other tissue macrophages. Remarkably, to investigate FPRL2 expression by TAMs in vivo, and determine colonic macrophages do not express all receptors mediating innate whether such expression is correlated with the pro- or antitumoral by guest on September 27, 2021 responses, particularly CD14, one of the most important pattern properties of macrophages. recognition molecules of the innate system. Such down-regulation We also demonstrated that FPRL2 is expressed on pDCs, more ensures the control of potentially harmful inflammation in the rich- abundantly than by myeloid DCs. FPRL2 is significantly up-reg- est site in proinflammatory signals (35, 36). Moreover, intestinal ulated during maturation of pDCs, whereas expression is essen- macrophages do not produce proinflammatory cytokines in re- tially unchanged during maturation of murine DCs (17). pDCs, sponse to several inflammatory stimuli (37) and release instead named according to their morphology, constitute one anti-inflammatory molecules such as IL-10 (38). Macrophages can of two main subsets of human DCs. Discovered as professional also contribute to adaptive immunity by uptake and processing of INF-␣/␤-producing cells in response to the activation of a set of Ags, thus triggering T lymphocyte responses. In the lung, alveolar TLRs (TLR7, -8, and -9) activated by viral products (49, 50), macrophages play a crucial role of scavengers, keeping the alveoli pDCs can also activate adaptive immunity through their Ag-pre- clean and sterile (39). These properties distinguish them from other senting properties and elicit Th1 polarization (51). In several tissue macrophages, mainly involved in the processing and transfer chronic viral diseases, including HIV and hepatitis B and C infec- of Ags to lymphoid receptors. There is presently increasing evi- tions, pDC number and function are impaired (52, 53). Besides this dence for alveolar macrophages contributing to the pathophysiol- pivotal role in innate and adaptive immunity, a number of studies ogy of important lung diseases such as emphysema and interstitial have implicated pDCs in the development of tolerance. pDCs can fibrosis. In granulomatous diseases, the fusion of macrophages shape the repertoire, inducing Ag-specific CD4ϩ CD25ϩ- leads to the formation of MGCs, which are observed in tubercu- regulatory T cells (54, 55) and suppressing the inflammatory cell losis, rheumatoid arthritis, and sarcoidosis (40, 41). We have infiltrate in some autoimmune diseases such as lupus (56). Pres- shown that FRPL2 expression is maintained in MGCs from tuber- ently, only a few pDC chemotactic agents have been identified, culosis disease lesions. The restricted localization of FPRL2 on including SDF-1, CXCR3 ligands, adenosine, and (26, macrophage subsets suggests a contribution of the receptor in the 57). Here, we have demonstrated that the F2L peptide is chemo- phenotypic specificity of these cells in lung, intestine, and skin. tactic for highly purified human pDCs. This observation and the Whether F2L contributes to the recruitment of macrophages to high amount of FPRL2 in pDCs suggest a possible implication of inflammatory sites in these organs, or rather modulates the func- FPRL2 in Th1 immune responses against or in the devel- tional properties of these cells remain to be determined. opment of tolerance. Moreover, the diversity of this cell population extends beyond Another new expression site of FPRL2 is eosinophils, which the tissue-specific differentiation of monocytes into functionally constitutes a significant exception to the thus far restricted expres- different macrophage subsets. Indeed, macrophage activation is sion pattern of the receptor in the monocytic lineage. It was pre- also polarized according to the local microenvironment. A simpli- viously reported that eosinophils express FPR and FPRL1, but not fied dichotomy of macrophage polarization (M1 and M2) has been FPRL2 (58). We demonstrate, however, a solid expression of The Journal of Immunology 4983

FPRL2 on highly purified eosinophils by semiquantitative RT- macrophages following mF2L injection was observed. These ele- PCR and flow cytometry, and F2L promoted chemotaxis of eosin- ments demonstrate that F2L is generated and functional in vivo in ophils with a potency incompatible with an action through other mice. Therefore, mouse can be used as a model to study some receptors (FPRL1 or FPR). As macrophages, eosinophils are pleio- aspects of the F2L pathway, despite the absence of a strictly or- tropic multifunctional leukocytes involved in the initiation and thologous receptor for FPRL2. propagation of diverse inflammatory responses in parasitic infec- As a conclusion, we have described in this study the detailed tions and allergic diseases. They are also considered as modulators distribution of FPRL2 in human leukocytes, demonstrated its func- of innate and adaptative immunity (reviewed in Ref. 59). Eosino- tionality in macrophages and uncovered new cell targets of F2L, phils can function as APCs and promote T cell proliferation and namely plasmacytoid DCs and eosinophils. Moreover, in vivo, polarization toward Th1 or Th2 pathways. The trafficking of eo- FPRL2 appears to be expressed only in some tissue-specific mac- sinophils to inflammatory sites requires the coordination of numer- rophage populations, including in lung, colon, and skin, three or- ous cytokines, , and molecules. Platelet-acti- gans chronically subjected to pathogens and exogenous aggres- vating factor, leukotriene B4, and C5a were among the first sions. This distribution underlines the potential role of FPRL2 in eosinophil chemoattractants identified, but many others have been innate immunity and possibly in immune regulation. Further stud- described. In the lung, alveolar macrophages, which constitute the ies will be undertaken to elucidate whether F2L has pro- or anti- first line of defense, can, upon activation, release chemotactic fac- inflammatory properties, and whether FPRL2 expression can be tors for eosinophils (60). Once recruited to inflammatory sites, correlated with pro- or antitumoral properties of macrophages. In activated eosinophils secrete, by regulated exocytosis and degran- this context, understanding the pathway leading to the proteolytic ulation, an arsenal of capable of inducing tissue damage cleavage of the precursor HEBP1 and the release of F2L will cer- Downloaded from and cell dysfunction. Unlike IL-5, F2L did not promote degranu- tainly facilitate future investigations. lation of eosinophils. These data suggest therefore a role of FPRL2 in innate immunity against helminths or in allergic diseases, by Acknowledgments promoting the recruitment but not the activation of eosinophils. A We thank Jean-Marie Vanderwinden for his help with confocal similar behavior was described for PGD2 (61), in contrast to other microscopy. mediators (eotaxin, C5a), which promote both chemotaxis and de- Disclosures http://www.jimmunol.org/ granulation. F2L, by recruiting both macrophages and eosinophils The authors have no financial conflict of interest. via FPRL2, could lead to a coordinated action of these effector cells and affect the inflammatory process in lung allergic diseases References such as . Other members of the FPR family genes have been 1. Showell, H. J., R. J. Freer, S. H. Zigmond, E. Schiffmann, S. Aswanikumar, implicated in the activation of eosinophils by exogenous B. Corcoran, and E. L. Becker. 1976. The structure-activity relations of synthetic peptides as chemotactic factors and inducers of lysosomal secretion for neutro- (62). phils. J. Exp. Med. 143: 1154–1169. To investigate further the role of the FPRL2/F2L system, animal 2. Boulay, F., M. Tardif, L. Brouchon, and P. Vignais. 1990. The human models would be helpful. The FPR gene family has, however, N-formylpeptide receptor: characterization of two cDNA isolates and evidence

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