Promotes Dendritic Cell Transmigration Derived Chemerin

Endothelial Cell−Derived Chemerin Promotes Dendritic Cell Transmigration Safiye Gonzalvo-Feo, Annalisa Del Prete, Monika Pruenster, Valentina Salvi, Li Wang, Marina Sironi, Susanne This information is current as Bierschenk, Markus Sperandio, Annunciata Vecchi and of September 28, 2021. Silvano Sozzani J Immunol published online 27 January 2014 http://www.jimmunol.org/content/early/2014/01/25/jimmun

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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 27, 2014, doi:10.4049/jimmunol.1302028 The Journal of Immunology

Endothelial Cell–Derived Chemerin Promotes Dendritic Cell Transmigration

Saﬁye Gonzalvo-Feo,*,1 Annalisa Del Prete,*,†,1 Monika Pruenster,‡ Valentina Salvi,† Li Wang,*,x Marina Sironi,* Susanne Bierschenk,‡ Markus Sperandio,‡ Annunciata Vecchi,* and Silvano Sozzani*,†

ChemR23 is a chemotactic receptor expressed by APCs, such as dendritic cells, macrophages, and NK cells. Chemerin, the ChemR23 ligand, was detected by immunohistochemistry, to be associated with inflamed endothelial cells in autoimmune diseases, such as lupus erythematosus, psoriasis, and rheumatoid arthritis. This study reports that blood and lymphatic murine endothelial cells produce chemerin following retinoic acid stimulation. Conversely, proinflammatory cytokines, such as TNF-a, IFN-g, and LPS, or calcitriol, are not effective. Retinoic acid–stimulated endothelial cells promoted dendritic cell adhesion under shear stress conditions and transmigration in a ChemR23-dependent manner. Activated endothelial cells upregulated the expression of the Downloaded from atypical chemotactic receptor CCRL2/ACKR5, a nonsignaling receptor able to bind and present chemerin to ChemR23+ dendritic cells. Accordingly, activated endothelial cells expressed chemerin on the plasma membrane and promoted in a more efficient manner chemerin-dependent transmigration of dendritic cells. Finally, chemerin stimulation of myeloid dendritic cells induced the high-affinity binding of VCAM-1/CD106 Fc chimeric protein and promoted VCAM-1–dependent arrest to immobilized ligands under shear stress conditions. In conclusion, this study reports that retinoic acid–activated endothelial cells can promote myeloid and plasmacytoid dendritic cell transmigration across endothelial cell monolayers through the endogenous production of http://www.jimmunol.org/

chemerin, the upregulation of CCRL2, and the activation of dendritic cell b1 integrin afﬁnity. The Journal of Immunology, 2014, 192: 000–000.

he correct localization of dendritic cells (DC) to peripheral the last 6 or 7 aa from the C terminus (7). Various proteases mediate tissues and secondary lymphoid organs is a crucial event for chemerin processing; these include neutrophil serine proteases and T optimal immune responses (1). Multiple chemotactic signals proteases from the coagulation and fibrinolytic cascades (8). Many regulate the trafficking of myeloid DC (mDC) and plasmacytoid DC tissues express chemerin in a constitutive manner, although the (pDC), the two main circulating DC subsets (2). Chemotactic ago- nature of the producing cells is still largely unknown (6). ChemR23, by guest on September 28, 2021 nists active on DC include chemokines, cytokines, formyl peptides, the functional chemerin receptor (also known as CMKLR1 in complement fragments, and bioactive lipid molecules (3, 4). humans and Dez in the mouse), is a G protein–coupled receptor ex- Chemerin was originally described as the product of the Tazarotene- pressed in various leukocyte populations, including mDC, pDC, induced gene 2 (Tig2) in stimulated skin cultures of psoriatic patients monocytes, macrophages, and NK cells (9–13). More recently, two (5) and subsequently purified from ascetic fluids from ovarian can- other high-affinity chemerin receptors were described, as follows: cer patients and synovial exudates from rheumatoid arthritis patients GPR1, a poor signaling receptor mainly expressed in the CNS, and (6). Chemerin is secreted as a poorly active precursor that is con- CCRL2/ACKR5, a member of the atypical chemokine receptor verted into a bioactive agonist following the proteolytic removal of family (also known as LCCR in the mouse) (8, 14). CCRL2 is a nonsignaling receptor that was proposed to bind and concentrate bioactive chemerin to ChemR23-positive cells (15, 16). *Humanitas Clinical and Research Center, Rozzano 20089, Italy; †Department of Chemerin is normally present in circulation of healthy subjects, Molecular and Translational Medicine, University of Brescia, Brescia 25123, Italy; and its levels are upregulated in many inflammatory conditions, ‡Walter Brendel Center for Experimental Medicine, Ludwig-Maximilians-Universi- x such as lupus erythematosus, psoriasis, rheumatoid arthritis, and ta¨t, Munchen€ 81377, Germany; and Department of Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China Crohn’s disease (17). We previously reported chemerin immuno- 1S.G.-F. and A.D.P. equally contributed to this work. staining in decidual cells during early pregnancy, with highest Received for publication July 31, 2013. Accepted for publication December 28, 2013. levels present in stromal and extravillous trophoblast cells (18). In addition, chemerin was found to be associated with tubular epi- This work was supported by the Italian Association for Cancer Research; Italian Ministry of Health (Progetto Giovani Ricercatori 2007); Ministero dell’Istruzione thelial cells and renal lymphatic endothelial cells in patients with Universita` e Ricerca; Fondazione Berlucchi; European Project Innovative Medicines lupus nephritis but not in normal kidneys (19). Chemerin expres- Initiative Joint Undertaking–funded project BeTheCure, Contract 115142-2; and Eurostars ChemExit 7306/8. sion also colocalized with high endothelial venules in lymph nodes and with inflamed endothelial cells in skin biopsies obtained from Address correspondence and reprint requests to Prof. Silvano Sozzani, Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, Bres- patients with autoimmune diseases, such as systemic lupus erythe- cia 25123, Italy. E-mail address: [email protected] matosus, lichen planus, and psoriasis (10, 11, 20). However, at The online version of this article contains supplemental material. present it is unknown whether endothelial cells produce chemerin or Abbreviations used in this article: DC, dendritic cell; KO, knockout; mDC, myeloid only bind and present on their surface the protein. Of note, in all DC; MELC, mouse lymphatic endothelial cell line; pDC, plasmacytoid DC; RA, cases, chemerin+ endothelial cells were surrounded by ChemR23+ retinoic acid. DC postulating a role for the ChemR23/chemerin axis in DC traf- Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 ficking across endothelial cell barriers.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302028 2 ENDOTHELIAL CELLS EXPRESS BIOLOGICAL ACTIVE CHEMERIN

The aim of the current study was to investigate the regulation cDNA reverse-transcription kit from Applied Biosystems, following man- of chemerin expression and production by vascular and lymphatic ufacturer’s instructions. RT-PCR was performed on cDNA samples using endothelial cells and to evaluate the role of the chemerin/ChemR23 a Power SYBR Green PCR master mix (Applied Biosystems, Warrington, U.K.) and speciﬁc primers. RT-PCRs were performed on a 7900HT Fast axis in the transmigration of DC subsets across endothelial cell Real-Time PCR System machine according to manufacturer’s guidelines barriers. (Applied Biosystems). The primer pairs used were as follows: mouse ChemR23 (forward: 59-CCATGTGCAAGATCAGCAAC-39, reverse: 59- GCAGGAAGACGCTGGTGTA-39), CCRL2 (forward: 59-TGTGTTTCCTG- Materials and Methods CTTCCCCTG-39,reverse:59-CGAGGAGTGGAGTCCGACAA-39), mCCR1 Reagents (forward: 59-CTGCCCCCCCTGTATTCTCT-39,reverse:59-GACATTGCCCA- CCACTCCA-39), mCCR2 (forward: 59-CTACGATGATGGTGAGCCTTGTC- DMEM (4.5 g/L glucose), RPMI 1640 medium, heat-inactivated FBS, 39,reverse:59-AGCTCCAATTTGCTTCACACTG-39), mCCR7 (forward: 59- penicillin/streptomycin, nonessential amino acids, and Na pyruvate were TGGTGGTGGCTCTCCTTGTC-39,reverse:59-CCTCATCTTGGCAGAAGC- from Lonza (Verviers, Belgium). The 1a,25 dihydroxyvitamin D3 (calci- ACA-39), mCCL3 (forward: 59-CATATGGAGCTGACACCCCG-39,reverse:59- triol), all trans retinoic acid (RA), BSA (A2934), gelatin type B from TCTTCCGGCTGTAGGAGAAGC-39), mCCL4 (forward: 59-GCCCTCTCTC- bovine skin, and heparin were from Sigma-Aldrich (St. Louis, MO). Re- TCCTCTTGCT-39,reverse:59-GAGGGTCAGAGCCCATTG-39), mCCL5 (for- combinant mouse chemerin (chemerin), TNF-a, and IFN-g were from ward: 59-TGCTCCAATCTTGCAGTCGT-39,reverse:59-ACACACTTGGCGG- R&D Systems (Minneapolis, MN). Endothelial cell growth supplement TTCCTTC-39), mCXCL10 (forward: 59-CGTCATTTTCTGCCTCATCCTG-39, was from Biomedical Technologies; 2-ME was from Life Technologies reverse: 59-CCGTCATCGATATGGATGCAGT-39), mCXCL12 (forward: 59- (Scotland, U.K.); CpG was from InvivoGen (San Diego, CA); and Flt3L, CTGTGCCCTTCAGATTGTTG-39,reverse:59-TAATTTCGGGTCAATGCA- GM-CSF, CCL3, CCL19, and CXCL12 were from PeproTech (Rocky Hill, CA-39), hb-actin (forward: 59-TCACCCACACTGTGCCCATCTACGA-39,re- NJ). LPS (Escherichia coli strain 055:B5) was from Difco Laboratories verse: 59-CAGCGGAACCGCTCATTGCCAATGG-39), and mGAPDH (forward:

(Detroit, MI). Sarcoma 180 (S180) cell line was from American Type 59-CGTGTTCCTACCCCCAATGT-39,reverse:59-TGTCATCATACTTGGCAG- Downloaded from Culture Collection. GTTTCT-39). Cell culture The primers/fluorogenic (FAM) probe sets Mm 00503581_gH and Hs 99999901_s1 from Applied Biosystems (Branchburg, NJ) were used to The mouse lung capillary endothelial cell line (1G11) was grown in com- amplify mouse chemerin and 18S, respectively. In this case, a TaqMan plete medium (DMEM, 20% FBS, 1% nonessential amino acids, 1 mM Na Universal PCR master mix was used (Applied Biosystems). pyruvate, 100 U/ml penicillin and streptomycin, freshly added heparin, and endothelial cell growth supplement at final concentration of 100 mg/ml), as Chemerin ELISA described (21). The mouse lymphatic endothelial cell line (MELC) was http://www.jimmunol.org/ cultured in complete medium supplemented with 10% supernatant from Supernatants of endothelial cells were collected at the indicated time points, 2 sarcoma 180 cells (growth medium), as described (22). Confluent cells centrifuged, and stored at 20˚C until ELISAs. The supernatants were were passed routinely at a split ratio of 1:3 after trypsin/EDTA digestion. concentrated 10 times using a vivaspin concentrator unit (3000 MWCO; Both endothelial cell lines were grown on flasks precoated with 1% gel- Sartorius Stedim Biotech) and examined for the presence of chemerin by atin. Mouse embryonic fibroblasts and 3T3L1 cells were prepared and ELISA, according to manufacturer’s instructions (Mouse Chemerin Quantikine cultured, as described (23, 24). ELISA kit; R&D Systems). Mouse bone marrow–derived DC and human DC from Immunofluorescence analysis peripheral blood The 1G11 cells (7.5 3 104) were seeded on glass coverslips precoated with by guest on September 28, 2021 Eight- to 12-wk-old C57BL/6J mice (Charles River Laboratories) and 1% gelatin in complete medium and then stimulated with RA (5 mM) for ChemR23 knockout (KO; provided by M. Parmentier [Universite´ Libre de 24 h. After 1% paraformaldehyde fixing, the slides were saturated with Bruxelles, Brussels, Belgium]) were used. CD34+ bone marrow–positive 1% BSA in PBS and incubated with an anti-chemerin Ab (Bioss, Woburn, cells were purified by positive immunoselection and cultured, as previ- MA) or an anti–VCAM-1 (clone M-K-2; Antibodies On-Line, Atlanta, ously described (25, 26), with murine GM-CSF (40 ng/ml) and Flt-3L (100 GA), followed by anti-rabbit Alexa 488 and anti-rat Alexa 594 (Invitrogen) ng/ml) to generate mDC, or with Flt3L (200 ng/ml) only to generate pDC. Abs, respectively. Nuclei were counterstained with DAPI. Analysis was per- Cells were split every 2–3 d, and used at day 9. DC were matured in the formed using a Zeiss Axio Observer Z1 microscope equipped with Apotome presence of TNF-a (20 ng/ml), LPS (100 ng/ml), or CpG (2 mg/ml) for the system and a Plan-Apochromat 1003/1.4 NA oil objective. indicated time points. PBMCs were isolated from buffy coats of normal donors by Ficoll Migration assays gradient (Ficoll-Paque Premium; GE Healthcare, Life Sciences) and were Endothelial cells were grown to confluence on 0.1% gelatin-coated transwell magnetically sorted with blood pDC Ag BDCA-4 cell isolation kits inserts in 24-well costar chambers (5 mm pore size; Corning). When indi- (Miltenyi Biotec), as previously described (27). cated, the endothelial cells were exposed to different stimuli in RPMI 1640 Flow cytometry medium containing 0.2% BSA (migration medium) for 18 h. For the transmigration assays, 100 mlDC(0.53 106 cells/ml 51Cr-labeled mouse DC were matured in the presence of TNF-a (20 ng/ml) or LPS (100 ng/ml) DC; 2 3 106 cells/ml human DC) in migration medium was placed in the for 24 h and, after blocking Fc-nonspecific binding with anti-CD16/32 upper chamber and 600 ml chemoattractant or control medium was added to (clone 2.4G2), incubated with an anti–ChemR23-PE (clone 477806) the lower chamber. DC were allowed to migrate for 90 min (mouse DC) or mAb or rat IgG2b-PE isotype-matched mAb (R&D Systems). The 1G11 4 h (human DC) at 37˚C in a 5% CO2 atmosphere. Mouse DC migration was cells were stimulated with RA (5 mM) or with IFN-g/TNF-a/LPS (50 ng/ evaluated as the percentage of radioactivity recovered in the lower com- ml, 20 ng/ml, 1 mg/ml, respectively) for 18 h. After Fc blocking, the cells partment relative to input. Values are expressed as net migration (% radio- were stained with an anti-CCRL2 mAb (28), washed with PBS (Life activity sample 2 % radioactivity control). Human DC were collected in the Technologies), and then incubated with an anti-mouse Alexa 488 mAb lower chamber and counted. The results were expressed as percentage of cell (Invitrogen, Life Technologies, Monza, Italy). Samples were read on a input in the upper chamber. To block ChemR23, human pDC were incubated Particle Analysing System cytofluorimeter (Partec, Muenster, Germany), (30 min at 4˚C) with an anti-ChemR23 mAb (clone 1H2, IgG2a, 3 mg/ml) and results were analyzed by FlowJo software (Tree Star). donated by M. Parmentier (6) and then added to the upper chamber. Chemerin was added to endothelial cells for 90 min at 37˚C in the upper Determination of mouse chemerin and ChemR23 expression by chamber and then removed before addition of DC to be tested. RT-PCR Flow chamber experiments Mouse endothelial cell lines were grown in six-well culture plates (BD Labware) till confluence. Then medium was replaced with medium with To analyze DC recruitment, we used an in vitro flow chamber assay. For this 0.2% BSAwithout serum and growth factors. Cells were stimulated with RA purpose, glass capillaries (2 3 0.2 mm; VitroCom, Mountain Lakes, NJ) (5 mM), 1a,25 dihydroxyvitamin D3 (calcitriol, 1 mM), and TNF-a (20 ng/ were assembled into microflow chambers, as described previously (29). ml) for reported times. DC and endothelial cell total RNA was isolated For adhesion studies, a combination of recombinant murine P-selectin using TRIzol reagent (Invitrogen), according to the manufacturer’s spec- (20 mg/ml), chemerin (10 mg/ml), and integrin-ligand murine rVCAM-1 ifications. Total RNA (1 mg) was reverse transcribed using a high-capacity (CD106, 15 mg/ml) and murine rCCL3 (10 mg/ml) from R&D Systems was The Journal of Immunology 3 used. After overnight incubation, the flow chambers were blocked with 5% DC, mRNA and protein levels were rapidly downregulated fol- casein (from bovine milk; Sigma-Aldrich, Munich, Germany) in PBS for 2 h lowing activation with LPS and TNF-a, or CpG, with LPS being 3 6 at room temperature and flushed with PBS. The DC suspension (2 10 /ml) already effective after 1-h incubation (Fig. 1A, 1B, Supplemental was flushed through the flow chamber using a high-precision syringe pump (Harvard Apparatus, Holiston, MA) at a flow rate of 1 ml/min, resulting in Fig. 1A, and data not shown). The expression of ChemR23 was func- a shear stress of ∼1 dyne/cm2. Results were evaluated after 10 min of per- tional, because chemerin was able to promote in a dose-dependent fusion. DC adhesion to 1G11 cells monolayer, grown on ibidi chambers, was manner the migration of DC subsets with a peak of activity observed investigated under shear stress conditions. The 1G11 cells were incubated at 100 pM chemerin. Conversely, chemerin was not active on acti- with RA (5 mM) overnight or with CCL3 (100 ng/ml) or chemerin (100 ng/ ml) for 90 min. The chambers were connected to an intramedic polyethylene vated DC nor in DC derived from ChemR23-deficient bone marrow tube (inner diameter 0.58 mm; outer diameter 0.965 mm; BD Biosciences) precursors (Fig. 1C, 1D, and data not shown). and perfused with mDC at the concentration of 0.5 3 106/ml and a flow rate of 0.28 ml/min, resulting in a shear stress of ∼0.5 dyne/cm2 based on Production of bioactive chemerin by vascular and lymphatic the manufacturer’s description (ibidi Application Note #11; www.ibidi. endothelial cells com) (30). Previous data have shown by immunohistochemistry that, in human Flow cytometry assessment of VCAM-1/CD106 Fc binding pathological conditions, chemerin expression is associated with activated blood and lymphatic endothelial cells (10, 11, 19), but no Measurement of the binding of mouse rVCAM-1/CD106 Fc chimera (R&D Systems) to DC was performed in the following manner: 3 3 105 DC in evidence is available on the nature of the signals involved in chem- HBSS, 1 mM HEPES, and 0.25% BSA (pH 7.4) were incubated with the erin regulation. Two mouse endothelial cell lines, one of lymphatic master mix containing murine rVCAM-1/CD106 Fc chimera (400 mg/ml), origin (MELC) (22) and the other of vascular derivation (1G11) anti-human IgG Fcg-specific biotin (eBioscience, San Diego, CA), PE-Cy5 (21), were used to better understand the possible role of chemerin Downloaded from streptavidin (BD Biosciences, San Jose, CA) plus murine CCL3 (100ng/ ml) or murine chemerin (100 ng/ml) for the indicated time points at 37˚C. production by endothelial cells. A constitutive basal expression of The binding reaction was stopped by the addition of FACS lysing solution chemerin was detected in both cell lines (Fig. 2A–D). Both vascular (BD Biosciences), and the cells were washed twice in ice-cold HBSS. and lymphatic endothelia were then stimulated with RA, calcitriol, Then the samples were acquired by FACSCalibur (BD Biosciences) and and a variety of proinflammatory stimuli, such as TNF-a,LPS, analyzed using CellQuest software. IFN-g, and IL-1b. Among the different agonists, only RA was able ∼

Statistical analysis to induce the upregulation of chemerin mRNA expression ( 4-fold http://www.jimmunol.org/ increase over control) in both MELC and 1G11 cells following Results are expressed as mean 6 SEM. Statistical significance was de- termined by Student t test or one-way ANOVA, as appropriate. Differences overnight stimulation with the optimal concentration of 5 mM were considered significant when p , 0.05. (Fig. 2A, 2B, and data not shown). This effect was paralleled by the concomitant secretion of chemerin, as evaluated by ELISA with 0.076 6 0.006 and 0.879 6 0.28 ng/106 cells (n = 5) released in Results 24 h by MELC and 1G11 cells, respectively (Fig. 2C, 2D). No Expression of functional ChemR23 by mouse bone marrow– statistically significant release of chemerin was observed with cal- derived mDC and pDC citriol and with any of the proinflammatory agonists tested. Thus,

First, the expression level of ChemR23 in mouse bone marrow– these results indicate that RA is active in inducing chemerin pro- by guest on September 28, 2021 derived mDC and pDC was investigated by RT-PCR. Similarly to duction in both lymphatic and vascular endothelial cells in vitro, the data obtained with mouse tissue pDC (31) or with human DC although the levels of chemerin released were ∼1 log higher in (10), the expression of ChemR23 was easily detected in both mDC 1G11 cells. When compared with mouse embryonic ﬁbroblasts and and pDC. As expected, based on previous data obtained with human with differentiated 3T3L1 adipocytes, 1G11 cells were, respectively,

FIGURE 1. Expression of the functional chemerin receptor (ChemR23) by mouse bone marrow– derived mDC and pDC. mDC were stimulated with TNF-a (20 ng/ml) or LPS (100 ng/ml) up to 24 h; ChemR23 expression was evaluated by RT- PCR and FACS analysis. Levels of actin mRNA were used for normalization (A). ChemR23 membrane expression of one representative of four experiments is shown. For sake of clarity, only the staining of control cells with an isotype-matched mAb is shown (dotted line; B). Immature and TNF-a (20 ng/ml) or LPS (100 ng/ml)-activated mDC (C), or CpG (2 mg/ml)-activated pDC (D) for 18 h were tested for their ability to migrate in response to recombinant mouse chemerin across a 1G11 endothelial cell monolayer. 51Cr-labeled DC were allowed to migrate for 90 min in transwell inserts. Values are expressed as mean 6 SEM (n = 3) of the net migration (% radioactivity sample 2 % radioactivity control) of one experiment representative of four. *p , 0.05, TNF-a/LPS or CpG-activated DC versus medium. 4 ENDOTHELIAL CELLS EXPRESS BIOLOGICAL ACTIVE CHEMERIN

FIGURE 2. Expression of chemerin by RA-stimulated mouse lymphatic (MELC) and hematic (1G11) endothelial cells. Conﬂuent mouse endothelial cells were stimulated with 1a,25 dihydroxyvitamin D3 (calcitriol; 1 mM), RA (5 mM), or murine TNF-a (20 ng/ml) in serum-free medium, supplemented with 0.2% BSA for 18 h. Chemerin mRNA levels in MELC (A) and 1G11 (B) were evaluated by RT-PCR; 18S mRNA was used for normalization. Results are reported as fold of induction over unstimulated cells (medium). Secretion of chemerin in the supernatants of MELC (C) and1G11(D) was analyzed by ELISA. Ex- pression of chemokine mRNA (CCL3, CCL4, CCL5, CXCL10, and CXCL12) was evaluated in RA and TNF-a–stimulated MELC (E)and Downloaded from 1G11 (F) cells. Results are expressed as mean 6 SEM of triplicate determinations of one representative experiment [(A, B) n =3;(C–F) n = 2]. *p , 0.05, RA versus medium (A–D), or TNF-a versus medium (E, F). http://www.jimmunol.org/

3.1- and 4.2-fold more efficient in chemerin production (data not Endothelial cell–derived chemerin promotes DC shown). The action of RA on 1G11 cells was apparently selective for transmigration chemerin, because no mRNA induction of other DC bioactive CC Several reports suggested chemerin as a relevant chemotactic factor (CCL3, CCL4, and CCL5) and CXC (CXCL10 and CXCL12) for myeloid and plasmacytoid DC transmigration across endo- chemokines was detected under the same experimental conditions. thelial cells (32). To further explore this hypothesis, we tested the This selective regulation of chemerin RA was in contrast with the ability of endothelial cell–derived chemerin to induce DC trans- by guest on September 28, 2021 action of TNF-a, a prototypic proinflammatory cytokine, which was migration using MELC and 1G11 cells stimulated with RA in the able to induce chemokine production, but not chemerin, in both absence of any added chemotactic factor. Fig. 4A and 4B show endothelial cell types (Fig. 2E, 2F). that both vascular and lymphatic RA-stimulated endothelial cells Fig. 3 shows that chemerin could be detected by immunocy- were able to sustain the migration of mDC and pDC with efficacy tochemistry on the plasma membrane of RA-activated endothelial comparable to that of 100 pM recombinant chemerin. As ex- cells, with a distribution similar to that of VCAM-1. On the con- pected, TNF-a–, LPS-activated mDC, or CpG-activated pDC were trary, no immunostaining could be observed in resting 1G11 cells. unable to transmigrate across the RA-activated endothelial mono- This result demonstrates that at least part of the chemerin pro- layer but efficiently migrated in response to CCL19 (Fig. 4A, 4B). duced by activated endothelial cells remains associated to the Two different approaches were used to formally demonstrate the plasma membrane. role of membrane-bound chemerin in DC transmigration across RA-stimulated endothelial cells. First, Fig. 4C shows that ChemR23 KO DC did not migrate across activated 1G11 cells. Second, Fig. 4D shows that the transmigration of human pDC across RA-stimulated cells was completely blocked in the presence of a specific anti-ChemR23 blocking mAb (1H2) (10, 19, 20). Conversely, no effect was observed with an irrelevant isotype- matched control mAb. The action of the anti-ChemR23 mAb was specific because pDC transmigration in response to CXCL12 was not affected by the treatment. These results formally prove that membrane-bound endothelial cell–derived chemerin promote DC transmigration. Expression of CCRL2 by endothelial cells promotes chemerin-dependent DC transmigration FIGURE 3. Membrane expression of chemerin in RA-stimulated en- Recently, it was reported that the atypical chemotactic receptor dothelial cells. The 1G11 cells were stimulated with RA (5 mM) for 24 h. Chemerin expression was visualized by an anti-chemerin Ab (green). Cell CCRL2 binds and concentrates chemerin at the endothelial cell membrane was visualized by VCAM-1 staining (red), whereas the nucleus surface (33). Therefore, it was investigated whether a similar mech- was identified by DAPI staining (blue). One experiment representative of anism could function in mouse vascular 1G11 endothelial cells. To three is shown. Both resting and RA-stimulated endothelial cells did not this goal, vascular endothelial cell monolayers were treated with either show nonspecific binding of secondary Abs (Supplemental Fig. 1B). RA or a mixture of proinflammatory stimuli (IFN-g/TNF-a/LPS) The Journal of Immunology 5

FIGURE 4. Transmigration of myeloid and plasmacytoid bone marrow–derived DC across RA-activated endothelial cells is ChemR23 dependent. Migration of mDC [left panel (A)] or pDC [left panel (B)], in the absence of added chemotactic factors in the lower chamber, across MELC and 1G11 monolayers stimulated with 5 mM RA for 18 h or in response to 100 pM recombinant mouse chemerin, added in the upper chamber. DC were activated, as described in the legend of Fig. 1. 51Cr-labeled DC were allowed to migrate for 90 min using transwell inserts. (A and B) Also shown is the migration of mDC and pDC to their positive controls, CCL3 and CCL19 (mDC), CXCL12 and CCL19 (pDC), respectively. Results represent mean 6 SEM of triplicate determinations of one representative experiment of four performed. Values are expressed as net migration (% radioactivity sample 2 % radioactivity control basal migration). *p , 0.05, TNF-a/LPS or CpG-activated DC versus medium. (C) Wild-type and ChemR23 KO DC transmigration across 1G11 monolayer. Experimental conditions are as in (A). (D) Blood human pDC Downloaded from were preincubated with 3 mg/ml 1H2 anti-ChemR23 mAb or with an irrelevant isotype-matched (IgG2a) mAb at 4˚C for 30 min before the transmigration assay across 1G11endothelial cells activated with 5 mM RA for 18 h. Migration across endothelial cells preincubated with 300 pM chemerin at 37˚C for 90 min and then washed is shown as positive control. Trans-

migration was evaluated after 4-h incubation; results are re- http://www.jimmunol.org/ ported as percentage of migrated cells relative to input, as detailed in Materials and Methods. Results are expressed as mean 6 SEM of one representative donor of four tested. *p , 0.05, ChemR23 KO versus wild-type DC (C); anti-ChemR23 mAb-treated human pDC versus irrelevant mAb (D).

known to induce CCRL2 expression (33). As shown in Fig. 5A, in cells rolling and undergoing adhesion at 10 min of perfusion and

both experimental conditions CCRL2 expression was induced at shows that chemerin caused a 3-fold increase in cell adhesion to by guest on September 28, 2021 the mRNA and protein levels in 1G11 cells. The mixture of proin- the immobilized substrate, an effect similar to that obtained in the flammatory stimuli (IFN-g/TNF-a/LPS) also increased ICAM-1 presence of CCL3. These results indicate that chemerin is able to expression but not the expression of VCAM-1 and several other induce the rapid activation of b1 integrin affinity. This conclusion chemokines (Fig. 5B). To functionally evaluate the contribution is further supported by the increased ability of chemerin-activated of CCRL2 expression on the ability of endothelial cells to support mDC to bind soluble VCAM-1/CD106 Fc chimera protein, as chemerin-induced DC transmigration, resting or proinflammatory evaluated by flow cytometry. Once again, the effect of chemerin agonist-activated 1G11 cell monolayers were preincubated with was comparable to that observed with an optimal (100 ng/ml) chemerin for 90 min, and then supernatant was removed and DC concentration of CCL3 (Fig. 6B). Finally, the ability of chem- was added to the transwells for the transmigration assay. As shown erin to induce adhesion of mDC was further exploited using flow in Fig. 5C, the basal net migration of DC across activated endo- chambers coated with 1G11 cells subsequently stimulated with thelial cells was not different from that across resting endothelial RA. Fig. 6C shows that also under dynamic conditions DC could cells, but the addition of chemerin to cytokine-activated endo- efficiently bind to activated endothelial cells in a chemerin- thelial cells induced DC transmigration more efficiently (2.3-fold dependent manner. Indeed, ChemR23 KO DC could perfectly increase) than to resting endothelial cells. These results strongly respond to CCL3 but failed to adhere to RA-activated endothelial support the concept that CCRL2 expressed on endothelial cell cells. membrane binds chemerin and promotes the transmigration of ChemR23+ DC. Discussion This study identifies vascular and lymphatic endothelial cells as an b Chemerin induces 1 integrin activation and DC arrest under important source of bioactive chemerin and provides evidence to shear stress conditions support the role of the chemerin/ChemR23 axis in DC transmi- Inside-out signaling generated by activated chemotactic receptors gration across endothelial cell barriers. promotes leukocyte transmigration through the induction of the Chemerin is a chemotactic agonist that becomes activated fol- high-affinity integrin conformation(34,35).Togainfurther lowing proteolytic processing. Active chemerin binds with high- insights on the possible role of chemerin in DC transmigration, affinity ChemR23, a heptahelic G protein–coupled chemotactic mDC rolling and adhesion were quantified under dynamic con- receptor involved in the recruitment of DC, macrophages, and NK ditions using a microflow chamber at a shear stress of 1 dyne/cm2, cells (3, 8). Although the chemerin inactive precursor is normally resembling the physiological shear stress found in postcapillary found in the plasma of healthy donors, increased levels of chemerin venules in vivo (36). Flow chambers were coated with a combi- are detected in many pathological conditions, including infectious nation of P-selectin, VCAM-1, and chemerin, or CCL3 as refer- and metabolic diseases (8, 37). The nature of the cells responsible ence chemokine acting on mDC (3). Fig. 6A reports the number of for chemerin production, as well as the signals involved in chemerin 6 ENDOTHELIAL CELLS EXPRESS BIOLOGICAL ACTIVE CHEMERIN Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021 FIGURE 5. Upregulation of CCRL2 expression by activated 1G11 endothelial cells. CCRL2 expression was evaluated by RT-PCR (left panel) FIGURE 6. Chemerin induces VCAM-1–mediated mDC adhesion under and FACS analysis (right panel) after overnight exposure to RA or dynamic conditions. (A) Recombinant P-selectin and VCAM-1 were im- proinflammatory stimuli (A). *p , 0.05, RA or IFN-g/TNF-a/LPS versus mobilized on glass capillaries together with 10 mg/ml chemerin or 10 6 medium. Expression of adhesion molecules and chemokines was investi- mg/ml CCL3. DC suspension (2 3 10 /ml) was flushed through the flow 2 gated after exposure to proinflammatory stimuli (B). Transmigration of chamber at the flow rate of 1 dyne/mm , using a high-precision syringe mDC across resting and activated (IFN-g + TNF-a + LPS for 18 h) 1G11 pump. The average numbers of cells rolling and adherent in at least five endothelial cell monolayers (C). For these experiments, 1G11 cells were fields of view (FOV) are shown. (B) Shown is the binding of VCAM-1/ preincubated with 100 pM chemerin for 90 min and then washed before CD106 Fc chimeric protein to DC stimulated with 100 ng/ml chemerin or the beginning of the assay. Transmigration was performed as described in 100 ng/ml CCL3, as assessed by flow cytometry. The results are expressed the legend of Fig. 1. Values are expressed as net migration (% radioactivity as fold of increase over medium; n =3.(C) Shown is adhesion of wild-type sample 2 % radioactivity control basal migration). Results represent mean 6 and ChemR23 KO DC to 1G11 cell monolayers in ibidi chambers under SEM of triplicates of one representative experiment of the two performed; shear stress conditions. Endothelial cells were stimulated overnight with *p , 0.05, activated versus resting endothelial cells (EC). RA (5 mM), or incubated with CCL3 (100 ng/ml) or chemerin (100 ng/ml) for 90 min. The number of adherent cells/FOV is expressed as mean 6 SEM; *p , 0.05, ChemR23 KO versus medium. regulation, is poorly understood. In previous reports, we described that chemerin immunoreactivity could be detected on the apical side and lymphatic endothelial cells produced chemerin in the super- of endothelial cells in lymph nodes and in the skin of patients with natant at low levels under basal conditions, and the production autoimmune diseases, such as systemic lupus erythematosus, lichen increased after an overnight stimulation, with 1G11 cells releasing planus, and psoriasis (10, 11, 19, 20). Both blood and lymphatic ∼10-fold higher levels than MELC cells. These results are in endothelium were positive by immunohistochemistry, but the nature agreement with the original description of chemerin as a Tazar- of the chemerin-producing cells is still elusive. otene (a RA-specific retinoid)-inducible gene (Tig2) in the skin This study was performed to directly address the question as to of psoriatic patients (5). Conversely, as previously reported with whether endothelial cells are capable of chemerin production and human endothelial cells (10), proinflammatory agonists, such as can support the transmigration of ChemR23+ DC. For this purpose, TNF-a, IL-1b, IFN-g, and LPS, were not active. Similarly, cal- two mouse endothelial cell lines were used to investigate chemerin citriol, the strongest agonist able to induce chemerin production in expression and regulation, namely MELC, of lymphatic origin human skin fibroblasts (20), was ineffective in both types of en- (22) and 1G11, of vascular derivation 1G11 (21), previously iso- dothelial cells, suggesting a cell-specific regulation of chemerin lated and characterized by our group. Among the different ago- production. RA-producing DC are localized in the lung, skin, and nists tested, RA was identified as the only agonist able to induce intestine and in their draining lymph nodes, and the expression of chemerin production both at the mRNA and protein level. Blood aldehyde dehydrogenase, the enzyme that controls RA production, The Journal of Immunology 7

is increased following infections and stimulation of TLRs (38). previously documented ability to induce b1 integrin clustering (13), Thus, these results suggest that chemerin production by endo- can also promote in DC the high-affinity conformational state of b1 thelial cells stimulated by RA released by aldehyde dehydroge- integrin and induce VCAM-1–dependent cell arrest. nase–positive DC may represent a positive feedback mechanism of In conclusion, this study reports that chemerin is produced by DC to promote the recruitment of ChemR23+ cells. RA-stimulated activated vascular and lymphatic endothelial cells and becomes endothelial cells promoted DC transmigration in the absence of any associated with the endothelial cell membrane through the possible added chemotactic agonist. The migration was comparable to that interaction with membrane proteoglycans and CCRL2. Endothelial induced after the preincubation of the endothelial cell monolayer cell–derived chemerin promotes b1 integrin activation and direc- with recombinant chemerin and was completely blocked in the tional migration of DC across activated endothelial cell mono- presence of a specific anti-ChemR23 mAb. These results imply two layer. This mechanism is apparently tightly regulated. First, the different concepts, as follows: first, chemerin released by activated activation of TLRs by infectious or autoimmune stimuli is respon- endothelial cells undergoes proteolytic activation by endothelial sible for the upregulation of inflammatory cytokines that will in- cell–derived proteases; second, at least some of the released protein duce the production of RA by aldehyde dehydrogenase–expressing becomes membrane associated and available for the recognition by DC and the upregulation of CCRL2 in endothelial cells (33, 38). ChemR23+ DC. Second, RA will further upregulate the expression of CCRL2 and the Prochemerin C-terminal processing was shown to occur in the production of prochemerin by activated endothelial cells. Finally, presence of different proteases, such as those released by activated endothelial cell–associated proteases will proteolytically generate neutrophils, or generated during the coagulation and fibrinolytic biologically active chemerin. Taken together, these results show that cascades (39, 40), or by circulating carboxypeptidases (41). The the ChemR23/chemerin axis represents a new pathway for activated Downloaded from production of bioactive chemerin was reported in different cell endothelial cells to promote DC extravasation and provides functional lines transfected with the chemerin full-length cDNA, including implications to the previously described observation of chemerin im- CHO-K1, COS-7, and HEK293 cells, suggesting the ability of munoreactivity of endothelial cells in pathological tissues (32). membrane-associated proteases to process prochemerin (6). The finding that DC can undergo ChemR23-dependent transmigration Disclosures across RA-activated endothelial cells implies that both MELC and The authors have no financial conflicts of interest. http://www.jimmunol.org/ 1G11 cells are able to process and secrete bioactive chemerin in the absence of the contributions of other inflammatory cells. Immobilization of chemotactic agonists on the surface of en- References dothelial layers is crucial for maintaining the chemotactic gradient 1. von Andrian, U. H., and T. R. Mempel. 2003. Homing and cellular traffic in lymph nodes. Nat. Rev. Immunol. 3: 867–878. under shear stress conditions and for leukocyte extravasation (42, 2. Penna, G., M. Vulcano, S. Sozzani, and L. Adorini. 2002. Differential migration 43). Chemerin is positively charged, and it is likely to bind neg- behavior and chemokine production by myeloid and plasmacytoid dendritic atively charged heparin or sulfated glycosaminoglycans expressed cells. Hum. Immunol. 63: 1164–1171. 3. Sozzani, S. 2005. Dendritic cell trafficking: more than just chemokines. Cytokine on the endothelial cell membranes. In addition, chemerin was re- Growth Factor Rev. 16: 581–592. ported to bind the atypical chemotactic receptor CCRL2/ACKR5 as 4. Del Prete, A., W. H. Shao, S. Mitola, G. Santoro, S. Sozzani, and B. Haribabu. by guest on September 28, 2021 2007. Regulation of dendritic cell migration and adaptive immune response by a mechanism to concentrate bioactive chemerin on the endothelial leukotriene B4 receptors: a role for LTB4 in up-regulation of CCR7 expression cell surface (16, 33). CCRL2 is constitutively expressed by MELC and function. Blood 109: 626–631. and 1G11 cells and can be further upregulated by RA and by a 5. Nagpal, S., S. Patel, H. Jacobe, D. DiSepio, C. Ghosn, M. Malhotra, M. Teng, M. Duvic, and R. A. Chandraratna. 1997. Tazarotene-induced gene 2 (TIG2), mixture of TNF-a,IFN-g, and LPS acting in a synergistic manner a novel retinoid-responsive gene in skin. J. Invest. Dermatol. 109: 91–95. (16, 33). Conceivable with CCRL2 induction, activated 1G11 en- 6. Wittamer, V., J. D. Franssen, M. Vulcano, J. F. Mirjolet, E. Le Poul, I. Migeotte, dothelial cells support more efficiently than resting cells chemerin- S. Bre´zillon, R. Tyldesley, C. Blanpain, M. Detheux, et al. 2003. Specific recruitment of antigen-presenting cells by chemerin, a novel processed ligand from induced DC transmigration. Because activation of 1G11 cells did human inflammatory fluids. J. Exp. Med. 198: 977–985. not result in the upregulation of DC active chemokines (e.g., CCL3, 7. Wittamer, V., F. Gre´goire, P. Robberecht, G. Vassart, D. Communi, and CCL4, CCL5, CXCL10, and CXCL12) or in a general upregulation M. Parmentier. 2004. The C-terminal nonapeptide of mature chemerin activates the chemerin receptor with low nanomolar potency. J. Biol. Chem. 279: 9956–9962. of integrin expression, it is tempting to speculate that the increased 8. Bondue, B., V. Wittamer, and M. Parmentier. 2011. Chemerin and its receptors in transmigration of DC is due to the improved binding of chemerin to leukocyte trafficking, inflammation and metabolism. Cytokine Growth Factor Rev. 22: 331–338. CCRL2 expressed by activated endothelial cells. 9. Samson, M., A. L. Edinger, P. Stordeur, J. Rucker, V. Verhasselt, M. Sharron, In the multistep process of leukocyte extravasation, chemotactic C. Govaerts, C. Mollereau, G. Vassart, R. W. Doms, and M. Parmentier. 1998. factors play a crucial role in promoting firm adhesion of leukocyte ChemR23, a putative chemoattractant receptor, is expressed in monocyte-derived dendritic cells and macrophages and is a coreceptor for SIV and some primary to endothelial cells through the activation of integrin adhesive HIV-1 strains. Eur. J. Immunol. 28: 1689–1700. properties. The engagement of chemotactic receptors generates an 10. Vermi, W., E. Riboldi, V. Wittamer, F. Gentili, W. Luini, S. Marrelli, A. Vecchi, inside-out signaling leading to the increase integrin affinity and J. D. Franssen, D. Communi, L. Massardi, et al. 2005. Role of ChemR23 in directing the migration of myeloid and plasmacytoid dendritic cells to lymphoid clustering (35, 44). In our experimental conditions, chemerin was organs and inflamed skin. J. Exp. Med. 201: 509–515. able to induce a rapid increase of the binding of soluble VCAM-1/ 11. Parolini, S., A. Santoro, E. Marcenaro, W. Luini, L. Massardi, F. Facchetti, CD106 Fc chimeric protein in DC, a sign of the increase of b D. Communi, M. Parmentier, A. Majorana, M. Sironi, et al. 2007. The role of 1 chemerin in the colocalization of NK and dendritic cell subsets into inflamed integrin-binding affinity. In addition, to better understand the po- tissues. Blood 109: 3625–3632. tential role of chemerin in DC extravasation, we performed ad- 12. Zabel, B. A., T. Ohyama, L. Zuniga, J. Y. Kim, B. Johnston, S. J. Allen, hesion assays under dynamic conditions. Flow chambers assays D. G. Guido, T. M. Handel, and E. C. Butcher. 2006. Chemokine-like receptor 1 expression by macrophages in vivo: regulation by TGF-beta and TLR ligands. were performed in the presence of immobilized P-selectin, VCAM-1, Exp. Hematol. 34: 1106–1114. and chemerin at the shear stress of 1 dyne/cm2. This experimental 13. Hart, R., and D. R. Greaves. 2010. Chemerin contributes to inflammation by promoting macrophage adhesion to VCAM-1 and fibronectin through clustering approach revealed that chemerin was as effective as CCL3, a refer- of VLA-4 and VLA-5. J. Immunol. 185: 3728–3739. ence chemokine, in inducing DC arrest on coated glass capillary 14. Bachelerie, F., A. Ben-Baruch, A. M. Burkhardt, C. Combadiere, J. M. Farber, surfaces, and similar results were obtained using flow chambers G. J. Graham, R. Horuk, A. H. Sparre-Ulrich, M. Locati, A. D. Luster, et al. 2014. International Union of Pharmacology. LXXXIX. Update on the extended coated with RA-activated endothelial cells. Taken together, these two family of chemokine receptors and introducing a new nomenclature for atypical experimental approaches indicate that chemerin, in addition to the chemokine receptors. Pharmacol. Rev. 66: 1–79. 8 ENDOTHELIAL CELLS EXPRESS BIOLOGICAL ACTIVE CHEMERIN

15. Zabel, B. A., S. Nakae, L. Zuń˜iga, J. Y. Kim, T. Ohyama, C. Alt, J. Pan, H. Suto, 29. Smith, M. L., M. Sperandio, E. V. Galkina, and K. Ley. 2004. Autoperfused D. Soler, S. J. Allen, et al. 2008. Mast cell-expressed orphan receptor CCRL2 mouse flow chamber reveals synergistic neutrophil accumulation through binds chemerin and is required for optimal induction of IgE-mediated passive P-selectin and E-selectin. J. Leukoc. Biol. 76: 985–993. cutaneous anaphylaxis. J. Exp. Med. 205: 2207–2220. 30. Nussbaum, C., A. Gloning, M. Pruenster, D. Frommhold, S. Bierschenk, 16. Del Prete, A., R. Bonecchi, A. Vecchi, A. Mantovani, and S. Sozzani. 2013. O. Genzel-Boroviczeńy, U. H. von Andrian, E. Quackenbush, and M. Sperandio. CCRL2, a fringe member of the atypical chemoattractant receptor family. Eur. J. 2013. Neutrophil and endothelial adhesive function during human fetal ontog- Immunol. 43: 1418–1422. eny. J. Leukoc. Biol. 93: 175–184. 17. Weigert, J., F. Obermeier, M. Neumeier, J. Wanninger, M. Filarsky, S. Bauer, 31. Luangsay, S., V. Wittamer, B. Bondue, O. De Henau, L. Rouger, M. Brait, C. Aslanidis, G. Rogler, C. Ott, A. Scha¨ffler, et al. 2010. Circulating levels of J. D. Franssen, P. de Nadai, F. Huaux, and M. Parmentier. 2009. Mouse chemerin and adiponectin are higher in ulcerative colitis and chemerin is ele- vated in Crohn’s disease. Inflamm. Bowel Dis. 16: 630–637. ChemR23 is expressed in dendritic cell subsets and macrophages, and mediates 18. Carlino, C., E. Trotta, H. Stabile, S. Morrone, R. Bulla, A. Soriani, an anti-inflammatory activity of chemerin in a lung disease model. J. Immunol. M. L. Iannitto, C. Agostinis, C. Mocci, M. Minozzi, et al. 2012. Chemerin 183: 6489–6499. regulates NK cell accumulation and endothelial cell morphogenesis in the de- 32. Sozzani, S., W. Vermi, A. Del Prete, and F. Facchetti. 2010. Trafficking prop- cidua during early pregnancy. J. Clin. Endocrinol. Metab. 97: 3603–3612. erties of plasmacytoid dendritic cells in health and disease. Trends Immunol. 31: 19. De Palma, G., G. Castellano, A. Del Prete, S. Sozzani, N. Fiore, A. Loverre, 270–277. M. Parmentier, L. Gesualdo, G. Grandaliano, and F. P. Schena. 2011. The pos- 33. Monnier, J., S. Leweń, E. O’Hara, K. Huang, H. Tu, E. C. Butcher, and sible role of ChemR23/chemerin axis in the recruitment of dendritic cells in B. A. Zabel. 2012. Expression, regulation, and function of atypical chemerin lupus nephritis. Kidney Int. 79: 1228–1235. receptor CCRL2 on endothelial cells. J. Immunol. 189: 956–967. 20. Albanesi, C., C. Scarponi, S. Pallotta, R. Daniele, D. Bosisio, S. Madonna, 34. Ley, K., C. Laudanna, M. I. Cybulsky, and S. Nourshargh. 2007. Getting to the P. Fortugno, S. Gonzalvo-Feo, J. D. Franssen, M. Parmentier, et al. 2009. site of inflammation: the leukocyte adhesion cascade updated. Nat. Rev. Immu- Chemerin expression marks early psoriatic skin lesions and correlates with nol. 7: 678–689. plasmacytoid dendritic cell recruitment. J. Exp. Med. 206: 249–258. 35. Sanz, M. J., and P. Kubes. 2012. Neutrophil-active chemokines in in vivo im- 21. Dong, Q. G., S. Bernasconi, S. Lostaglio, R. W. De Calmanovici, I. Martin- aging of neutrophil trafficking. Eur. J. Immunol. 42: 278–283. Padura, F. Breviario, C. Garlanda, S. Ramponi, A. Mantovani, and A. Vecchi. 36. Kim, M. B., and I. H. Sarelius. 2003. Distributions of wall shear stress in venular 1997. A general strategy for isolation of endothelial cells from murine tissues: Downloaded from convergences of mouse cremaster muscle. Microcirculation 10: 167–178. characterization of two endothelial cell lines from the murine lung and subcu- 37. Yoshimura, T., and J. J. Oppenheim. 2011. Chemokine-like receptor 1 (CMKLR1) taneous sponge implants. Arterioscler. Thromb. Vasc. Biol. 17: 1599–1604. and chemokine (C-C motif) receptor-like 2 (CCRL2); two multifunctional recep- 22. Sironi, M., A. Conti, S. Bernasconi, A. M. Fra, F. Pasqualini, M. Nebuloni, E. Lauri, M. De Bortoli, A. Mantovani, E. Dejana, and A. Vecchi. 2006. Gen- tors with unusual properties. Exp. Cell Res. 317: 674–684. eration and characterization of a mouse lymphatic endothelial cell line. Cell 38. Guilliams, M., K. Crozat, S. Henri, S. Tamoutounour, P. Grenot, E. Devilard, Tissue Res. 325: 91–100. B. de Bovis, L. Alexopoulou, M. Dalod, and B. Malissen. 2010. Skin-draining 23. Doni, A., G. Mantovani, C. Porta, J. Tuckermann, H. M. Reichardt, A. Kleiman, lymph nodes contain dermis-derived CD103(-) dendritic cells that constitutively produce retinoic acid and induce Foxp3(+) regulatory T cells. Blood 115: 1958– M. Sironi, L. Rubino, F. Pasqualini, M. Nebuloni, et al. 2008. Cell-specific http://www.jimmunol.org/ regulation of PTX3 by glucocorticoid hormones in hematopoietic and non- 1968. hematopoietic cells. J. Biol. Chem. 283: 29983–29992. 39. Wittamer, V., B. Bondue, A. Guillabert, G. Vassart, M. Parmentier, and D. Communi. 24. Patel, R., A. Apostolatos, G. Carter, J. Ajmo, M. Gali, D. R. Cooper, M. You, 2005. Neutrophil-mediated maturation of chemerin: a link between innate and K. S. Bisht, and N. A. Patel. 2013. Protein kinase C d (PKCd) splice variants adaptive immunity. J. Immunol. 175: 487–493. modulate apoptosis pathway in 3T3L1 cells during adipogenesis: identification 40. Zabel, B. A., S. J. Allen, P. Kulig, J. A. Allen, J. Cichy, T. M. Handel, and of PKCdII inhibitor. J. Biol. Chem. 288: 26834–26846. E. C. Butcher. 2005. Chemerin activation by serine proteases of the coagulation, 25. Vecchi, A., L. Massimiliano, S. Ramponi, W. Luini, S. Bernasconi, R. Bonecchi, fibrinolytic, and inflammatory cascades. J. Biol. Chem. 280: 34661–34666. P. Allavena, M. Parmentier, A. Mantovani, and S. Sozzani. 1999. Differential 41. Du, X. Y., B. A. Zabel, T. Myles, S. J. Allen, T. M. Handel, P. P. Lee, responsiveness to constitutive vs. inducible chemokines of immature and mature E. C. Butcher, and L. L. Leung. 2009. Regulation of chemerin bioactivity by mouse dendritic cells. J. Leukoc. Biol. 66: 489–494. plasma carboxypeptidase N, carboxypeptidase B (activated thrombin-activable 26. Del Prete, A., W. Vermi, E. Dander, K. Otero, L. Barberis, W. Luini, fibrinolysis inhibitor), and platelets. J. Biol. Chem. 284: 751–758.

S. Bernasconi, M. Sironi, A. Santoro, C. Garlanda, et al. 2004. Defective den- by guest on September 28, 2021 dritic cell migration and activation of adaptive immunity in PI3Kgamma- 42. Rot, A. 2010. Chemokine patterning by glycosaminoglycans and interceptors. deﬁcient mice. EMBO J. 23: 3505–3515. Front. Biosci. (Landmark Ed.) 15: 645–660. 27. Penna, G., S. Sozzani, and L. Adorini. 2001. Cutting edge: selective usage of 43. Proudfoot, A. E., T. M. Handel, Z. Johnson, E. K. Lau, P. LiWang, I. Clark- chemokine receptors by plasmacytoid dendritic cells. J. Immunol. 167: 1862– Lewis, F. Borlat, T. N. Wells, and M. H. Kosco-Vilbois. 2003. Glycosamino- 1866. glycan binding and oligomerization are essential for the in vivo activity of 28. Otero, K., A. Vecchi, E. Hirsch, J. Kearley, W. Vermi, A. Del Prete, S. Gonzalvo- certain chemokines. Proc. Natl. Acad. Sci. USA 100: 1885–1890. Feo, C. Garlanda, O. Azzolino, L. Salogni, et al. 2010. Nonredundant role of 44. Laudanna, C., J. Y. Kim, G. Constantin, and E. Butcher. 2002. Rapid leukocyte CCRL2 in lung dendritic cell trafﬁcking. Blood 116: 2942–2949. integrin activation by chemokines. Immunol. Rev. 186: 37–46.