Expression, Regulation, and Function of Atypical Receptor CCRL2 on Endothelial Cells

This information is current as Justin Monnier, Susanna Lewén, Edward O'Hara, Kexin of September 24, 2021. Huang, Hua Tu, Eugene C. Butcher and Brian A. Zabel J Immunol 2012; 189:956-967; Prepublished online 13 June 2012; doi: 10.4049/jimmunol.1102871

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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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Expression, Regulation, and Function of Atypical Chemerin Receptor CCRL2 on Endothelial Cells

Justin Monnier,*,†,‡ Susanna Lewe´n,‡ Edward O’Hara,*,† Kexin Huang,x Hua Tu,x Eugene C. Butcher,*,†,‡ and Brian A. Zabel‡

Chemokine (CC motif) receptor-like 2 (CCRL2) binds leukocyte chemoattractant chemerin and can regulate local levels of the at- tractant, but does not itself support cell migration. In this study, we show that CCRL2 and VCAM-1 are upregulated on cultured human and mouse vascular endothelial cells (EC) and cell lines by proinflammatory stimuli. CCRL2 induction is dependent on NF-kB and JAK/STAT signaling pathways, and activated endothelial cells specifically bind chemerin. In vivo, CCRL2 is constitutively expressed at high levels by lung endothelial cells and at lower levels by liver endothelium; and liver but not lung EC respond to systemic LPS injection by further upregulation of the receptor. Plasma levels of total chemerin are elevated in CCRL22/2 mice and 2 2 are significantly enhanced after systemic LPS treatment in CCRL2 / mice compared with wild-type mice. Following acute LPS- Downloaded from induced pulmonary inflammation in vivo, -like receptor 1 (CMKLR1)+ NK cell recruitment to the airways is significantly impaired in CCRL22/2 mice compared with wild-type mice. In vitro, chemerin binding to CCRL2 on endothelial cells triggers + robust adhesion of CMKLR1 lymphoid cells through an a4b1 integrin/VCAM-1–dependent mechanism. In conclusion, CCRL2 is expressed by EC in a tissue- and activation-dependent fashion, regulates circulating chemerin levels and its bioactivity, and enhances chemerin- and CMKLR1-dependent lymphocyte/EC adhesion in vitro and recruitment to inflamed airways in vivo. Its expression and/or induction on EC by proinflammatory stimuli provide a novel and specific mechanism for the local enrichment of chemerin at http://www.jimmunol.org/ inflammatory sites, regulating the recruitment of CMKLR1+ cells. The Journal of Immunology, 2012, 189: 956–967.

hemerin is a chemotactic for subsets, affinity chemerin receptor, G protein-coupled receptor 1 (GPR1), , and NK cells (1–3). Chemerin circulates has also been recently reported, although it also does not itself C in an inactive proform: activation of chemerin requires support chemerin-dependent cell migration (8). proteolytic processing of the carboxyl terminus and removal of Chemoattractants recruit leukocytes to inflamed tissues in part inhibitory amino acids (4–7). We and others identified chemerin by triggering integrin-dependent adhesion to activated vascular as a natural nonchemokine chemoattractant ligand for chemokine- endothelium. Several teams reported the colocalization of chemerin like receptor 1 (CMKLR1), and in a recent publication, we “de- with vascular endothelial cells (EC) in multiple inflammatory by guest on September 24, 2021 orphaned” an additional second receptor for chemerin, serpentine disorders, such as multiple sclerosis, lupus, and psoriasis, and in chemokine (CC motif) receptor-like 2 (CCRL2) (3, 5, 8–10). In- endothelial venules of secondary lymphoid tissues (11–14). Al- terestingly, although both CCRL2 and CMKLR1 bind chemerin though several human EC lines express CMKLR1 and can respond with high affinity, the downstream functional consequences of to chemerin in an angiogenesis assay, CCRL2 has not yet been ligand binding are quite different. Chemerin binding to CMKLR1 fully investigated in EC biology (15). Given the reported associ- triggers calcium mobilization, receptor and ligand internalization, ation of chemerin with vascular EC and the potential role of and cell migration. Alternatively, chemerin binding to CCRL2 nonclassical chemoattractant receptor CCRL2 in augmenting local does not induce intracellular calcium flux or ligand internalization, chemerin levels, we characterized the expression, regulation, and but can regulate chemerin bioavailability (3, 10). A third high- function of CCRL2 on human and murine vascular EC (10). In this study, we report that proinflammatory stimuli upregulate atypical chemerin receptor CCRL2 and VCAM-1 on EC via NF-kB *Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford † and JAK/STAT intracellular signaling pathways. Plasma chemerin University School of Medicine, Stanford, CA 94305; Center for Molecular Biology 2/2 and Medicine, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304; levels are significantly elevated in CCRL2 mice following ‡Palo Alto Institute for Research and Education, Veterans Affairs Palo Alto Health systemic LPS injection compared with wild-type (WT) mice and x Care System, Palo Alto, CA 94304; and LakePharma, Inc., Belmont, CA 94002 untreated controls, implicating CCRL2 in the regulation of cir- Received for publication October 4, 2011. Accepted for publication May 7, 2012. culating chemerin during inflammation. In an in vivo pulmonary This work was supported by National Institutes of Health Grant AI-079320 (to B.A.Z.), inflammation model, recruitment of CMKLR1+ NK cells into the by National Institutes of Health Grants R01 AI093981 and R37 AI047822 and a Merit 2/2 Award from the Department of Veterans Affairs (to E.C.B.), by National Institutes of airways is impaired in CCRL2 mice. In vitro, chemerin binding + + Health Training Grants T32-AI07290-25 and T32-AI07290-24 and American Cancer to CCRL2 EC triggers robust adhesion of CMKLR1 lymphoid Society Postdoctoral Fellowship PF-12-052-01-CSM (to J.M.), and by the FACS Core cells via a4b1 integrin/VCAM-1–mediated sticking. Thus, CCRL2 Facility of the Stanford Digestive Disease Center under Grant P30 DK056339. on EC acts in concert with CMKLR1 to coordinate chemerin- Address correspondence and reprint requests to Dr. Brian A. Zabel, Palo Alto Institute for Research and Education, Veterans Affairs Palo Alto Health Care System, dependent leukocyte adhesion in vitro and recruitment in vivo. m/c 154B, 3801 Miranda Avenue, Building 101, Room C4-121, Palo Alto, CA 94304. E-mail address: [email protected] Materials and Methods Abbreviations used in this article: BAL, bronchoalveolar lavage; CCRL2, chemokine Animals (CC motif) receptor-like 2; CMKLR1, chemokine-like receptor 1; EC, endothelial cell; 2/2 GAG, glycosaminoglycan; GPR1, G protein-coupled receptor 1; h, human; HDMEC, CCRL2 mice were obtained from Lexicon (The Woodlands, TX) and human dermal microvascular endothelial cell; m, mouse; poly(I:C), polyinosinic- backcrossed nine generations on the BALB/c background. WT BALB/c polycytidylic acid; RT-qPCR, reverse transcription quantitative PCR; WT, wild-type. mice were obtained from The Jackson Laboratory (Sacramento, CA). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102871 The Journal of Immunology 957

Reagents PCR (RT-qPCR) was performed using RT/Taq/SYBR Green qPCR reagents (Invitrogen) using a Stratagene Mx3000p thermocycler (Agilent Technol- Soluble mediators. IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-17, IL-23, ogies, Santa Clara, CA). Primers were validated using stringent criteria by CXCL12, GM-CSF, VEGF, Netrin-4, FLT3L, TGF-b, IFN-g,TNF-a, and verifying that the dissociation curve showed only one peak, and “no reverse chemerin were purchased from R&D Systems (Minneapolis, MN). Lipo- transcriptase” controls were used to confirm that qPCR results reflected teichoic acid (TLR2), flagellin (TLR5), R837 (TLR7), CpGa (TLR9), LPS RNA expression and not genomic DNA contamination. expression (TLR4), polyinosinic-polycytidylic acid (poly(I:C); TLR3) were purchased was normalized to CDC42 for mouse samples and b-actin for human from InvivoGen (San Diego, CA). Vitamin D , Vitamin D , and dexameth- 3 2 samples. The relative induction value of our of interest was calculated asone were obtained from Sigma-Aldrich (Saint Louis, MO, USA). IFN-a, 2 using the 2 DDCt method (19). All PCR reactions were done in duplicate. IFN-b were obtained from PBL InterferonSource (Piscataway, NJ, USA). Primary Abs. Anti-mouse (m) Abs included anti-mCCRL2 (clone BZ2E3, Flow cytometry generated in-house), rat IgG2a isotype control (clone 9B5, generated in- house), anti-mCMKLR1 (clone BZ194, generated in-house, rat IgG2a; A total of 0.5 million cells were used for each staining. For unconjugated clone BZ186, generated in-house, mIgG1), anti-mGPR1 (clone BZ48, Abs, cells were incubated with the indicated primary Abs at 4˚C for 30 min generated in-house, rat IgG2a), and anti-mVCAM-1 (clone MK2.7, in 100 ml PBS/2% FBS/2% mouse serum. Cells were then washed with generated in-house, rat IgG1). Anti–mCD31-PE-Cy7, anti–mCD146-FITC, PBS and centrifuged for 3 min at 2000 rpm. Following the washing step, anti–mVCAM-1-allophycocyanin (clone 429) were purchased from Bio- cells were incubated with secondary goat anti-rat PE (R&D Systems) in 50 Legend (San Diego, CA), and anti–CD3-PE-Cy7, anti–Ly6G-FITC, and ml PBS/2% FBS/2% goat serum. For directly conjugated Abs, cells are anti–DX5-PE were purchased from eBioscience (San Diego, CA). Anti- incubated with labeled Ab at 4˚C for 30 min in 100 ml PBS/2% FBS/2% human (h) Abs included anti–hCMKLR1 (clone BZ332, generated in- mouse serum. Cells were washed and centrifuged for 3 min at 2000 rpm, house), anti–hGPR1 (clone BZ1274, generated in-house, rat IgG2b); resuspended, and fixed in 200 ml PBS/1% paraformaldehyde and were anti–hVCAM-1-FITC (clone IE10), mIgG2b-FITC isotype control, and analyzed using a FACSCalibur (BD Biosciences, Franklin Lake, NJ). 125 mouse anti-hCCRL2 (clone 152211) were purchased from R&D Systems. [ I]chemerin binding assay Downloaded from Secondary Abs. Secondary Abs include goat anti-rat IgG-PE (R&D Sys- tems), goat anti–hIgG-PE, rat anti–mIgG-PE (Invitrogen, Carlsbad, CA), For radioligand binding assays, radioiodinated chemerin (residues 21–148; and goat anti-mouse IgG-Alexa 488 (Molecular Probes, Carlsbad, CA). R&D Systems; custom radiolabeling performed by PerkinElmer) was provided as a gift from J. Jaen (ChemoCentryx, Mountain View, CA). To Primers. Primers specific for mVCAM-1, hVCAM-1, and hCMKLR1 were assess the ability of chemerin to bind to bEND.3 cells treated with cyto- purchased from SABiosciences (Valencia, CA, USA). Primer sequences for kines, 5 3 104 cells per well were mixed with 4-fold dilutions of unlabeled mGPR1, hGPR1, and hCCRL2 are as follows: mGPR1 (forward, 59- chemerin competitor and ∼1 nM (0.025 mCi) [125I]chemerin tracer per

GGAGCTCAGCATTCATCACA-39, reverse, 59-GACAGGCTCTTGGTT- http://www.jimmunol.org/ well in a total volume of 200 ml, and agitated at 4˚C for 3 h. Levels of TCAGC-39), hGPR1 (forward, 59-AATGCCATCGTCATTTGGTT-39, re- cell-bound radioactivity were determined by harvesting the cells on verse, 59-CAACTGGGCAGTGAAGGAAT-39), and hCCRL2 (forward, 59- polyethyleneimine-treated GF/B glass filters (PerkinElmer, Waltham, MA) GAGGCAGAGCAATGTGACAA-39, reverse, 59-ATTTTTCCACGCGTT- using a cell harvester (PerkinElmer), washing the filters twice with buffer TGAGTC-39). Primers for mCCRL2, mCMKLR1, mouse chemerin, hu- (25 mM HEPES, 500 mM NaCl, 1 mM CaCl , and 5 mM MgCl , adjusted man chemerin, and human b-actin were used as previously described (10, 2 2 to pH 7.1) and measuring the amount of [125I]chemerin bound to each filter 16, 17). (cpm) with a TopCount scintillation counter (PerkinElmer). Inhibitors. Inhibitors included IKKb phopshorylation inhibitor BAY-11- 7082 (Calbiochem, Gibbstown, NJ) and JAK-1 inhibitor sc-204021 (Santa Fc-chemerin Cruz Biotechnology, Santa Cruz, CA). Recombinant Fc-chemerin protein (residues 23–156) was produced and

Primary EC isolation purified from Chinese hamster ovary cells via transient transfection and by guest on September 24, 2021 protein A purification. A DNA fragment corresponding to bioactive mouse 2 2 Mouse liver and lung EC were isolated from BALB/c WT and CCRL2 / chemerin isoform ending in residue 156 (serine) was amplified by PCR and mice. Briefly, livers and lungs were isolated from 8- to 10-wk-old mice and cloned in-frame downstream of human or mouse IgG1 Fc domain, which is digested in 5 mg/ml PBS/collagenase IV (Worthington, Lakewood, NJ) for downstream of a secretion signal peptide in mammalian expression vector 45 min at 37˚C. Digested tissue was passed over cell strainers of decreasing pLEV113 (LakePharma, Belmont, CA). There is a 9-aa glycine-rich linker size (100, 70, and 40 mM) and then centrifuged for 10 min at 300 3 g at 4˚C. between the Fc and chemerin domain. Plasmid DNA was transfected into Endothelial cells were enriched using 30% HistoDenz/RPMI 1640 solution Chinese hamster ovary cells using Lafectine transfection reagent (Lake- (Sigma-Aldrich); after centrifugation at 1500 3 g for 20 min at 4˚C, cells at Pharma), and cell culture supernatant was collected 3–5 d after transfec- the interface were collected and stained with CD31 and CD146 (both from tion. Fc fusion were purified with protein A resins (MabSelect BioLegend) to identify the purified endothelial cell population. SuRe; GE Healthcare), and final proteins were formulated in 100 mM Tris (pH 7.5), 150 mM NaCl, and 0.45% NaOAc. Cell culture EC adhesion assay Mouse EC line culture. bEND.3 cells were grown in DMEM media (Life Technologies, Carlsbad, CA) supplemented with pyruvate, nonessential To assess the ability of CCRL2 on bEND.3 cells to induce adhesion, bEND.3 amino acids, L-glutamine, penicillin-streptomycin, and 10% FBS. For in- cells were grown to confluence in 96-well petri dishes. After 24 h treatment hibitor experiments, bEND.3 cells were preincubated with the indicated with TNF-a/LPS/IFN-g (20 ng/ml, 1mg/ml, and 50 ng/ml, respectively), concentration of inhibitor for 1 h, after which fresh media with or without bEND.3 cells were loaded with 50 ml 200 nM chemerin in PBS/0.1% BSA inhibitor with the indicated cytokines were added to the cells and incu- and incubated at 37˚C for 30 min. This step serves to load CCRL2 with bated for an additional 24 h. HEK293 and L1.2 cells (pre-B cell mouse chemerin. The cells are then washed with PBS to remove unbound chemerin. lymphoma) were grown in RPMI 1640 supplemented with pyruvate, L1.2-CMKLR1+ cells (100 ml) at a concentration of 5 3 106 cells/ml, nonessential amino acids, L-glutamine, penicillin-streptomycin, and 10% prelabeled with calcein AM (BD Biosciences), were placed on top of the FBS and G418 (Life Technologies). bEND3 cells and coincubated for 30 min at 37˚C. The cells were washed Human EC culture. HUVEC and human dermal microvascular EC twice with PBS without calcium and magnesium. The number of cells that (HDMEC) were purchased from Lonza (Basel, Switzerland), and a novel adhered to the monolayer was then measured by a plate reader at an human brain microvascular endothelial cell line, hCMEC/D3, was a gift emission/excitation of 494/517 nm. Images of adherent cells were taken from Prof. P.O. Couraud (Universite´ Rene´ Descartes, Paris, France) (18). using a fluorescent microscope. Blocking Abs against VCAM-1 (MK2/7) Briefly, cells were seeded at a concentration of 10,000 cells/ml on 0.02% and a4b1 integrin (PS/2) were used at a concentration of 10 mg/ml. gelatin-coated plates. EBM media (Lonza) was changed every other day, ELISA and after 7 d confluent cells were ready for experimentation. Twenty- four hours prior to stimulation, cells were cultured in EBM base media Mice were injected i.p. with LPS (12 mg/kg), euthanized 12 h later, and containing reduced concentrations of supplemental growth factors. blood was collected by cardiac puncture. Plasma chemerin concentrations were measured by ELISA (R&D Systems). RNA isolation and reverse transcription quantitative PCR Chemerin internalization assay Total RNA was extracted from cells using an RNeasy (Qiagen, Valencia, CA), after which the total RNA concentration was measured using the HEK293 cells transfected with hCMKLR1 or hCCRL2, bEND.3 cells, and Nanodrop spectrophotometer ND-100. Reverse transcription quantitative HUVEC were used for chemerin internalization assays. One hundred 958 EXPRESSION, REGULATION, AND FUNCTION OF ENDOTHELIAL CCRL2 thousand cells per well were incubated with mFc-human chemerin for 30 induced by TNF-a alone (Fig. 1C, 1D); the latter observation is min at 4˚C and then washed with cold PBS to remove unbound chemerin. consistent with previous reports (20). Chemerin receptors CMKLR1 For the microscopy studies, HEK293 transfectants and bEND.3 cells were and GPR1 were not expressed under any condition, whether as- incubated with secondary Ab goat anti-mouse IgG Alexa 488 (Molecular Probes). After 20 min incubation at 4˚C the cells were washed in cold PBS. sessed by Ab staining or RNA analysis (data not shown). Subsequently, cells were either placed back at 4˚C or incubated at 37˚C to Kinetics of CCRL2 and VCAM-1 RNA and protein induction in allow for labeled Fc-chemerin to internalize. After a final wash in cold PBS, cells were fixed in PBS/1% paraformaldehyde and spun down on LPS-, IFN-g–, and TNF-a–treated bEND.3 cells microscope slides by cytospin. Fc-chemerin internalization was analyzed Consistent with the protein expression analysis, CCRL2 and by epifluorescence microscopy. For the flow cytometry studies, Fc-chem- erin–loaded HUVEC were incubated at 4˚C or 37˚C for 30 min, washed, VCAM-1 RNA were upregulated by proinflammatory stimuli (Fig. and then stained with secondary Ab goat anti-mouse PE. Fc-chemerin 2A, 2B). We next examined the RNA and protein induction ki- internalization was analyzed by flow cytometry. netics of CCRL2 and VCAM-1 following treatment with TNF-a, Acute LPS-induced lung inflammation LPS, and IFN-g (Fig. 2B, 2D). RNA expression for both CCRL2 and VCAM-1 occurred rapidly and peaked after just 2 h for each. WT and CCRL2 knockout mice were anesthetized and dosed with 1 mg LPS Despite similar RNA induction kinetics, CCRL2 protein expres- in 50 ml saline by intranasal injection. Twelve hours after LPS injection the mice were euthanized and the leukocytes that accumulated in the airways sion peaked at 24 h after treatment, whereas VCAM-1 surface were collected by bronchoalveolar lavage (BAL). expression peaked at 8 h (Fig. 2C, 2D). BAL fluid leukocyte isolation CCRL2 induction is controlled by NF-kB and JAK/STAT signaling pathways

After mice were euthanized, a blunt needle was inserted in the exposed Downloaded from trachea. The airway of the mice was washed three times with 1 ml PBS. The The robust and synergistic induction of CCRL2 by the combination recovered fluid was centrifuged, and the recovered leukocytes in the BAL of TNF-a, LPS, and IFN-g in bEND.3 cells prompted us to in- fluid were directly stained with surface markers for T cells (CD3), neu- trophils (Ly6G), and NK cells (DX5). vestigate the intracellular pathways involved. Previously, it was shown that TNF-a and LPS trigger the intracellular NF-kB pathway Blood leukocyte isolation in EC, whereas IFN-g activates the JAK-1/STAT-1 pathway; how- Blood was collected by cardiac puncture after euthanasia and directly mixed ever, more recent evidence indicates that TNF-a and LPS can also with 5 ml PBS without Ca2+/Mg2+ supplemented with 4 mM EDTA to activate the STAT-1 pathway, and, conversely, that IFN-g can in- http://www.jimmunol.org/ prevent clotting. An equal volume of 2% dextran T-500 in PBS was added, directly stimulate the NF-kB pathway (21–23). To test the role of and the solution was gently mixted by inversion and incubated at 37˚C for 45 min. The supernatant was collected and centrifuged and incubated with NF-kB and JAK-1/STAT-1 in the upregulation of CCRL2, we used 2 ml RBC lysis buffer (Sigma-Aldrich). The pelleted WBCs were then pharmacoinhibitors of the NF-kB pathway (BAY 11-7082) and of stained and analyzed by flow cytometry. the JAK-1/STAT-1 pathway (sc-204021). The NF-kB inhibitor al- In vitro transwell most completely prevented the induction of CCRL2 in bEND.3 cells by TNF-a, LPS, and poly(I:C), but only partially inhibited mCMKLR1/L1.2 cells were used to assess chemerin bioactivity by in vitro IFN-g–orIFN-b–dependent CCRL2 induction (Fig. 3). Alterna- transwell migration as previously described (3). For migration experiments,

2.5 3 105 mCMKLR1/L1.2 cells in 100 ml chemotaxis media (either tively, the JAK-1 inhibitor marginally inhibited TNF-a–, LPS-, IL- by guest on September 24, 2021 RPMI 1640 plus 10% FCS for prochemerin activation experiments, or 1b–, or poly(I:C)-induced induction of CCRL2, but almost com- RPMI 1640 plus 0.5% BSA for detection of bioactive chemerin in the pletely blocked IFN-g–orIFN-b–dependent CCRL2 induction absence of proteases) were added to the top wells of 5-mm pore transwell (Fig. 3). Cells treated with NF-kB or JAK inhibitors only partially inserts (Corning Costar, Lowell, MA), and 25 ml plasma samples in 600 ml blocked CCRL2 induction when TNF-a, LPS, and IFN-g were media were added to the bottom wells. After incubating the transwell plates for 2 h at 37˚C, the bottom well cells were harvested and flow combined together. However, the combination of both NF-kBand cytometry was used to assess migration. To test the amount of prochemerin JAK-1 inhibitors almost completely blocked CCRL2 induction by in the plasma samples, 25 ml plasma was incubated with 5 ml plasmin (1 combined TNF-a, LPS, and IFN-g, suggesting that both pathways mg/ml, reconstituted in PBS) for 5 min at 37˚C, and then immediately are involved and that they can independently and synergistically diluted in 600 ml cold chemotaxis media (RPMI 1640 plus 10% FBS). upregulate endothelial CCRL2 (Fig. 3). Statistical analysis Activated bEND.3 cells bind chemerin Evaluation of significance was performed using a Student t test or ANOVA 125 followed by a Bonferroni posttest. Statistical tests were calculated using Excess unlabeled chemerin inhibited the binding of either [ I] the Instat statistical program (GraphPad Software, La Jolla, CA), and chemerin (IC50, 3 nM; Fig. 4A) or Fc-chemerin (IC50, 8 nM; Fig. graphs were plotted using Prism graphing software (GraphPad Software). 4B) to bEND.3 cells treated with proinflammatory stimuli. 6 Data are expressed as means SD or SEM as indicated, and a p value of Chemerin did not bind to unstimulated bEND.3 cells (Fig. 4). ,0.05 was considered to be significant. Expression and regulation of CCRL2 in human EC Results Primary human umbilical vein and dermal microvascular EC CCRL2 and VCAM-1 are upregulated on mouse brain vascular (HUVEC and HDMEC, respectively), as well as a human brain endothelioma cells by proinflammatory cytokines and certain endothelial cell line (hCMEC/D3), significantly upregulated TLR ligands CCRL2 RNA following exposure to TNF-a, LPS, and IFN-g (Fig. Given the reported colocalization of chemerin with activated EC in 5A). VCAM-1 RNA was also significantly upregulated as antici- multiple inflammatory diseases, we tested a panel of cytokines and pated (data not shown). Furthermore, unstimulated HUVEC TLR ligands for CCRL2 induction in bEND.3 endothelioma cells, expressed CCRL2 protein and bound Fc-chemerin, and stimula- a model cell line of mouse brain vascular EC (11–14, 20). A subset tion with TNF-a, LPS, and IFN-g slightly increased CCRL2 of proinflammatory cytokines and TLR ligands [TNF-a,LPS,IFN- protein expression and Fc-chemerin binding (Fig. 5B). g,IL-1b,IFN-b, and poly(I:C)] induced CCRL2 protein expression (Fig. 1A, 1B). The cytokines and factors that upregulated CCRL2 Expression and regulation of CCRL2 on mouse primary EC were similar to those that induced VCAM-1, although optimal We next asked whether CCRL2 was expressed on freshly isolated upregulation of CCRL2 required synergistic activity of TNF-a with mouse vascular lung and liver EC, as these organs provided an other stimuli (e.g., IFN-g and LPS), whereas VCAM-1 was highly adequate quantity of primary EC for analysis. Interestingly, CD31+ The Journal of Immunology 959 Downloaded from http://www.jimmunol.org/

FIGURE 1. Selective upregulation of CCRL2 and VCAM-1 in endothelioma cells by proinflammatory stimuli. CCRL2 (A) and VCAM-1 (B) protein expression was measured by flow cytometry on mouse brain endothelioma cells (bEND.3) treated for 24 h with the indicated soluble inflammatory mediators. The induction of CCRL2 and VCAM-1 is represented as a heat map. Histogram plots of CCRL2 (C) and VCAM-1 (D) are representative of the induction of CCRL2 and VCAM-1. Results are representative of three independent experiments. by guest on September 24, 2021

CD146+ mouse lung EC expressed high levels of CCRL2 in the lung or liver EC from CCRL2-deficient mice, confirming the absence of experimental exogenous activation, and mouse liver specificity of the Ab staining. We did not detect any genotype- EC were moderately positive. Abs against CCRL2 failed to stain dependent differences in VCAM-1, CD31, or CD146 expression

FIGURE 2. Kinetic analysis of CCRL2 and VCAM-1 induction. CCRL2 (A) and VCAM-1 (B) mRNA and protein induction was measured in bEND.3 cells treated with the indicated cytokines for 24 h. The means of three different experiments 6 SD are shown. TNF-a plus LPS plus IFN-g were incubated with bEND.3 cells for the indicated times. At each time point CCRL2 (C) and VCAM-1 (D) induction was measured by either RT-qPCR (normalized to CDC42) or by mAb staining and flow cytometry. Datum points represent the mean of two to three experiments. *p , 0.05 by t test. 960 EXPRESSION, REGULATION, AND FUNCTION OF ENDOTHELIAL CCRL2

FIGURE 3. NF-kB and JAK/STAT signaling pathways regulate CCRL2 induction in EC. Mouse bEND.3 cells were preincubated with IKKb in- hibitor BAY (11-7082) and/or with JAK-1 inhibitor (sc-204021) followed by incubation with the indicated cytokines and TLR ligands. CCRL2 protein expression was measured by flow cytometry. Blocking of CCRL2 induction by specific inhibitors is displayed as a percentage inhibition. The means of fivetoeightdifferentexperiments6 SD are shown. *p , 0.05 by t test. Downloaded from on lung or liver EC, suggesting that overall the endothelial cell phenotype is not altered in the CCRL2-deficient animals (Fig. 6). In vivo injection of LPS upregulates CCRL2 on liver EC FIGURE 5. Human endothelial cells upregulate CCRL2. HUVEC, HDMEC, and a human brain endothelial cell line (hCMEC/d3) were in- LPS injection activates vascular EC in vivo (24, 25). To determine A

cubated for 24 h with TNF-a plus LPS plus IFN-g.( ) Induction of http://www.jimmunol.org/ whether endothelial CCRL2 is induced by LPS in vivo, we injected CCRL2 RNA was measured in all samples by RT-qPCR normalized to mice systemically with endotoxin, isolated vascular EC from liver b-actin. The means of three different experiments 6 SD are shown. (B) and lung, and assessed CCRL2 and VCAM-1 expression and HUVECs were either untreated or treated with TNF-a plus LPS plus chemerin binding by flow cytometry. CD31+CD146+ liver EC IFN-g for 24 h and stained for CCRL2 expression and Fc-chemerin from LPS-injected WT mice significantly upregulated CCRL2 and binding. Results are representative of three individual experiments with bound to Fc-chemerin, whereas similar cells from saline-injected similar results. *p , 0.05, **p , 0.01 by t test. WT mice were CCRL2low (Fig. 7). LPS injection had no effect on CCRL2 expression or Fc-chemerin binding to WT lung EC rela- CMKLR1+ HEK293 cells efficiently internalized bound Fc-

tive to isotype control staining (Fig. 8). Furthermore, neither chemerin when incubated at 37˚C, as evidenced by the cytoplas- by guest on September 24, 2021 CCRL2 Ab nor Fc-chemerin stained liver or lung EC from LPS- mic puncti and lack of membrane staining (Fig. 9B). To determine injected or control CCRL22/2 mice (Figs. 7, 8). Consistent with whether these results extend to primary human EC, Fc-chemerin– previous reports, LPS injection upregulated VCAM-1 on liver and loaded HUVEC (stimulated with TNF-a, LPS, and IFN-g) were lung EC in both genotypes (Figs. 7, 8) (24, 26–29). incubated at 4˚C or 37˚C, washed, and then stained for surface chemerin. The staining intensity of surface Fc-chemerin on EC CCRL2 captures and concentrates chemerin on the cell HUVEC incubated at 37˚C was similar to the staining intensity at surface 4˚C, indicating that HUVEC did not internalize bound chemerin + Given our previous data that CCRL2 lymphoid cells do not in- (Fig. 9C). ternalize bound chemerin, we next asked whether CCRL2+ vas- cular EC also concentrated chemerin on the cell surface. CCRL2+ CCRL2 regulates circulating chemerin levels in vivo bEND.3 cells (treated with TNF-a, LPS, and IFN-g) bound to Fc- Given the constitutive expression of CCRL2 lung vascular EC and, chemerin, whereas untreated cells were negative for chemerin to a lesser extent, liver vascular EC, we hypothesized that circu- binding (Fig. 9A). Upon shifting the chemerin-loaded cells to an lating chemerin levels may be altered in CCRL22/2 mice owing to internalization-permissive temperature (37˚C), the bEND.3 cells the lack of chemerin sequestration in the vasculature. Indeed, did not internalize bound ligand (Fig. 9A). CCRL2+ HEK293 plasma levels of total chemerin (measured by ELISA, which transfectants also did not internalize bound Fc-chemerin; however, detects bioactive chemerin, prochemerin, and larger fragments of

FIGURE 4. Chemerin binds to activated bEND.3 cells. bEND.3 cells were activated with TNF-a plus LPS plus IFN-g for 24 h and incubated with either [125I]chemerin (A) or Fc-chemerin (B) in the presence of various concentrations of unlabeled chemerin competitor. The mean of triplicate wells 6 SD is shown for each experiment. Data shown are representative of three individual experiments with similar results. The Journal of Immunology 961

FIGURE 6. CCRL2 protein expression in mouse primary EC. After collagenase digestion and endothelial enrichment by density gradient, lung (A) and liver (B) EC from WT and CCRL22/2 mice were evaluated for CCRL2 expression. CD31 and CD146 were used to identify EC by flow cytometry. Data shown are representative of three different experiments with similar results. chemotactically inert chemerin) were slightly but significantly Downloaded from elevated in CCRL22/2 mice compared with WT mice (Fig. 10A). There was no significant difference in the level of bioactive plasma chemerin between WT and CCRL22/2 (Fig. 10B), and http://www.jimmunol.org/

FIGURE 8. LPS injection does not alter CCRL2 expression on mouse lung endothelial cells in vivo. WT or CCRL22/2 mice were injected i.p. with LPS. EC from the lung were isolated 12 h later, enriched, and identified by costaining for CD31 and CD146. (A) The surface expression

of CCRL2 (mAb staining and Fc-chemerin binding) and VCAM-1 (mAb by guest on September 24, 2021 staining) were determined by flow cytometry. The dataset shown is rep- resentative of three to six individual experiments with similar results. (B) Induction of CCRL2, Fc-chemerin, and VCAM-1 in LPS-treated mice was quantified by normalizing the specific Ab mean fluorescence intensity value to its isotype mean fluorescence intensity value. The mean 6 SEM is shown. Each graph represents three to six independent experiments pooled together (n = 1–2 mice/group/experiment).*p , 0.05 by t test.

there was a slight but nonsignificant increase in prochemerin (detected by ex vivo proteolytic [plasmin] activation) in CCRL22/2 plasma compared with WT (Fig. 10C), as measured by in vitro CMKLR1+ cell migration. Interestingly, in mice dosed with endotoxin to induce systemic inflammation and vascular CCRL2 expression, total chemerin plasma levels were 2-fold higher in CCRL22/2 mice versus WT mice, and 2-fold higher than un- treated CCRL22/2controls (Fig. 10A). Although there was no difference in bioactive plasma chemerin levels between LPS- treated WT and CCRL22/2 mice (Fig. 10B), prochemerin levels in CCRL22/2 plasma were significantly elevated compared FIGURE 7. CCRL2 upregulation on mouse liver EC following LPS with those in WT mice (Fig. 10C). Taken together, these data 2 2 injection in vivo. WT or CCRL2 / mice were injected i.p. with LPS. EC indicate that the increase in total circulating chemerin in LPS- from the liver were isolated 12 h later, enriched, and identified by cos- treated CCRL22/2 mice is due to an increase in prochemerin taining for CD31 and CD146. (A) The surface expression of CCRL2 (mAb and possibly inactive chemerin fragments. Interestingly, plasma staining and Fc-chemerin binding) and VCAM-1 (mAb staining) were levels of bioactive chemerin and prochemerin were significantly determined by flow cytometry. The dataset shown is representative of three B reduced in LPS-treated WT mice compared with untreated con- to six individual experiments with similar results. ( ) Induction of CCRL2, 2/2 Fc-chemerin, and VCAM-1 in LPS-treated mice was quantified by nor- trols (Fig. 10B, 10C). Although plasma from CCRL2 mice malizing the specific Ab mean fluorescence intensity value to its isotype showed a similar trend, the differences did not reach significance mean fluorescence intensity value. The mean 6 SEM is shown. Each graph (Fig. 10B, 10C). Thus, CCRL2 regulates circulating chemerin represents three to six independent experiments pooled together (n = 1–2 levels and its proteolytic processing in vivo during systemic in- mice/group/experiment). *p , 0.05 by t test. flammation. 962 EXPRESSION, REGULATION, AND FUNCTION OF ENDOTHELIAL CCRL2

FIGURE 9. CCRL2+ EC bind but do not in- ternalize chemerin. HEK293 cells transfected with hCCRL2 or hCMKLR1 (A)aswellas bEND.3 cells treated with or without TNF-a plus LPS plus IFN-g (B) were preincubated with Fc- chemerin and goat anti-mouse IgG-Alexa 488 at 4˚C, and then shifted to 4˚C or 37˚C. Internali- zation of fluorescently labeled Fc-chemerin was monitored by fluorescence microscopy. The dataset shown is representative of three individ- ual experiments with similar results. Scale bars, 25 mm. (C) HUVEC pretreated for 24 h with TNF-a plus LPS plus IFN-g were incubated with Fc-chemerin at 4˚C for 30 min. Cells were washed to remove unbound chemerin and then shifted to either 4˚C or 37˚C for 30 min. Surface- bound Fc-chemerin was detected using rat anti- mouse IgG1-PE secondary Ab. Results are rep- resentative of three individual experiments with similar results. Downloaded from

To isolate the role of vascular endothelium-expressed CCRL2 Impaired CMKLR1+ NK cell trafficking into inflamed airways in regulating circulating chemerin levels, mice were injected i.v. in CCRL22/2 mice with Fc-chemerin and the levels of plasma Fc-chemerin (as op- Intranasal injection of LPS causes acute lung inflammation and the posed to total chemerin) were measured over time. Plasma Fc- 2/2 accumulation of leukocytes into the broncheoalveolar space (30). chemerin levels were significantly higher in CCRL2 mice Given the high level of CCRL2 expression and chemerin binding http://www.jimmunol.org/ compared with WT controls (area under the curve for CCRL2 by lung EC, we used this pulmonary inflammation model to de- 6 mice, 40,540 2,630 ng h/ml versus area under the curve for WT termine whether CCRL2 deficiency altered the accumulation of 6 , mice, 29,550 1,240 ng h/ml; *p 0.05 by t test; Fig. 10D). CMKLR1+ NK cells into inflamed airways. Although there were no genotype-dependent differences in the total number of BAL infiltrating leukocytes, T cells, or neutrophils, significantly fewer NK cells (measured as absolute number and as a percentage of total BAL leukocytes) accumulated in the airways of CCRL22/2 mice (Fig. 11A–C). Blood NK cells from WT and CCRL22/2 mice expressed similar levels of CMKLR1 (Fig. 11D) and Fc- by guest on September 24, 2021 chemerin binding (data not shown), ruling out differential CMKLR1 receptor expression as a contributing factor in impaired airway NK cell trafficking in CCRL22/2 mice. NK cells themselves are CCRL22 (data not shown). Additionally, there were no differences in total numbers (or percentages) of circulating NK cells (or other major leukocyte subsets, such as neutrophils) between CCRL22/2 and WT mice (Fig. 11E, 11F, and data not shown). Thus, CCRL2 deficiency selectively impairs the recruitment of CMKLR1+ NK cells in an in vivo model of airway inflammation. Chemerin bound to CCRL2+ EC triggers CMKLR1+ cell adhesion CCRL2 binds chemerin such that the critical cell-signaling car- boxyl terminus remains exposed at the cell surface (10), and FIGURE 10. CCRL2 regulates circulating chemerin levels and its bio- + 2 2 chemerin triggers CMKLR1 adhesion by inducing activity in vivo. (A) WT and CCRL2 / mice were either untreated or injected with 12 mg/kg LPS for 12 h, after which chemerin levels in a4b1 integrin clustering and binding to VCAM-1–coated plates plasma were measured by ELISA. For untreated mice, the means of n = (31). Because activated bEND.3 cells express high levels of both 100 (WT) and n = 92 (CCRL22/2) individual mice 6 SEM are shown. For VCAM-1 and CCRL2 (Fig. 1), and L1.2 lymphoid cells express LPS-treated mice, the mean of six individual mice 6 SEM is shown. *p , endogenous a4b1 integrin (32), we hypothesized that CCRL2 on 0.05, ***p , 0.001 by t test. (B) The chemotactic bioactivity of plasma bEND.3 cells could bind chemerin and trigger CMKLR1+ L1.2 2 2 chemerin from WT and CCRL2 / mice with or without LPS treatment cell adhesion (33). Using a static endothelial adhesion assay, we was evaluated by in vitro mCMKLR1/L1.2 cell migration. **p , 0.01 by compared the ability of WT or CMKLR1+ L1.2 cells to adhere to t test. (C) Plasma prochemerin levels were evaluated by preincubating untreated or activated CCRL2+ bEND.3 cells in the presence or plasma samples with plasmin to proteolytically activate chemerin and then absence of chemerin. Activated CCRL2+ EC loaded with chem- assessing mCMKLR1/L1.2 cell migration. For (B) and (C), the mean of n = erin triggered significant and robust adhesion of CMKLR1+ L1.2 9–10 mice 6 SD is shown. *p , 0.05, **p , 0.01 by t test. Results are 2 representative of two independent experiments. (D) Fc-chemerin (3 mg) cells compared with unstimulated CCRL2 EC (Fig. 12A, 12B). was injected i.v. in WT or CCRL22/2 mice, and Fc-chemerin plasma WT L1.2 cells did not adhere to the endothelial monolayer under levels were quantified at the indicated time points. The mean of n =3 any condition tested, and chemerin was required for adhesion mice 6 SD is shown. Statistical significance (*p , 0.05) was determined triggering (Fig. 12A). Blocking Abs against a4 integrin or VCAM- by ANOVA followed by a Bonferroni post hoc test. 1 abolished chemerin-dependent CMKLR1+ cell adhesion to The Journal of Immunology 963 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 11. Impaired in vivo recruitment of CMKLR1+ NK cells into inflamed airways in CCRL22/2 mice. Following intranasal LPS administration, BAL fluid from WT and CCRL22/2 mice was collected and cells were stained for neutrophils (Ly6G), T cells (CD3), and NK cells (DX5) and plotted either as absolute number of infiltrating cells (A) or as a percentage of total BAL leukocytes (B). Each graph represents three to four independent experiments pooled together (n = 15–29 mice per group; each symbol represents one mouse). (C) Representative flow cytometry dot plots of CD3 versus DX5 staining of BAL cells from WT and CCRL22/2 mice. (D) Blood NK cells (DX5+CD32) from WT or CCRL22/2mice were stained for CMKLR1. Representative histogram of n = 3 independent experiments. WBC were collected from WT and CCRL22/2 mice, stained for neutrophils (Ly6G) and NK cells (CD32 DX5+), and plotted either as absolute number (E) or as a percentage of total leukocytes (F)(n = 3 mice/group). **p , 0.01, ***p , 0.001 by t test.

CCRL2+-activated endothelium, confirming that the adhesion experimental pulmonary inflammation. Thus, our data suggest that molecules that mediate cell sticking in this model are a4b1 CCRL2 on EC functions to increase local concentrations of integrin and VCAM-1 (Fig. 12C). chemerin and recruit CMKLR1+ cells to sites of inflammation. Although we tested an array of proinflammatory and immune Discussion suppressive cytokines, ILs, growth factors, and TLR ligands, Chemerin is associated with vascular endothelium in the affected only proinflammatory stimuli [TNF-a,IFN-g,IFN-b, LPS, and tissues of multiple inflammatory disorders, such as multiple poly(I:C)] induced CCRL2 on the mouse brain endothelial model sclerosis, lupus, and psoriasis (11–14), but little is known re- cell line bEND3. Additionally, proinflammatory factors induced garding the regulation and role of its receptors on EC. In this CCRL2 in three human endothelial model cell lines (HUVEC, study, we show that in a variety of EC, CCRL2, a high-affinity HDMEC, and hCMEC/d3). We and others reported similar results chemerin receptor, is either constitutively expressed (lung EC, for CCRL2 induction by mouse peritoneal macrophages and liver EC, and HUVEC) and/or induced (bEND.3, hCMEC/D3, dendritic cells (8, 27), suggesting the involvement of shared HDMEC, HUVEC, and liver EC) by proinflammatory stimuli. pathways for CCRL2 regulation across cell types. Endothelial As with lymphoid cell-expressed receptor, CCRL2 on EC binds cells express TNF-aR, IFN-gR, IFN-bR, TLR4, and TLR3, con- chemerin but does not internalize the ligand. Chemerin bound to sistent with responsiveness (as measured by CCRL2 or VCAM-1 CCRL2+ EC triggered robust adhesion of CMKLR1+ lymphoid induction) to their respective ligands (34–37). Combinations of cells via a4b1 integrin/VCAM-1 interactions (illustrated in Fig. proinflammatory mediators were significantly more robust in 12D). In vivo, CCRL2 deficiency resulted in selective impairment triggering CCRL2 induction than any individual stimuli (maximal of CMKLR1+ NK cell accumulation into the airways following response with TNF-a/LPS/IFN-g), consistent with enhanced in- 964 EXPRESSION, REGULATION, AND FUNCTION OF ENDOTHELIAL CCRL2 Downloaded from http://www.jimmunol.org/

FIGURE 12. CCRL2+-activated EC bind chemerin and trigger CMKLR1+ cell adhesion. (A) Mouse bEND.3 cells were untreated or treated with TNF-a plus LPS plus IFN-g, loaded with chemerin, and then coincubated with WT or CMKLR1+ L1.2 lymphoid cells prelabeled with calcein AM. The mean of quintuplet wells 6 SD of n = 3 independent experiments with similar results is shown. (B) Fluorescence microscopy of adherent CMKLR1+ cells. Panels show adhesion of L1.2-CMKLR1+ cells to bEND.3 cells treated with or without TNF-a plus LPS plus IFN-g and with or without chemerin. Representative images of n = 3 different experiments are shown. Scale bars, 25 mm. (C) Left panel, Inhibition of L.12-CMKLR1+ cell adhesion to bEND.3 cells was + evaluated by preincubating L1.2-CMKLR1 cells either with PBS, rat IgG isotype control, or with rat anti-mouse a4 integrin. Right panel, Inhibition of L.12-CMKLR1+ cell adhesion to bEND.3 cells was evaluated by preincubating bEND.3 cells with either PBS, rat IgG isotype control, or with rat anti- by guest on September 24, 2021 mouse VCAM-1. The mean of quintuplet wells 6 SD of n = 3 independent experiments with similar results is shown. (D) Model summarizing the components involved in chemerin-dependent CMKLR1+ cell adhesion to CCRL2+ EC. ***p , 0.001 by t test. duction of CCRL2 on human neutrophils by cotreatment with higher levels in mouse lung EC compared with liver EC, al- TNF-a and IFN-g (38), implying that multiple intracellular sig- though it is well documented that EC isolated from anatomically naling pathways (NF-kB, JAK/STAT, and possibly others) work different vascular beds are phenotypically and functionally dis- synergistically to regulate CCRL2 expression. Indeed, treating tinct in leukocyte adhesion and trafficking mechanisms (20, 40, cells with pharmacoinhibitors targeting both NF-kB and JAK/ 41). STAT pathways significantly reduced CCRL2 induction by TNF-a/ Given prior reports indicating CMKLR1 expression and function LPS/IFN-g. Furthermore, the addition of immune suppressive fac- in cultured EC in vitro (15, 42), we monitored CMKLR1 and tors such as dexamethasone, TGF-b, or IL-10 failed to inhibit the GPR1 protein (and in most cases RNA) expression in bEND.3, TNF-a/LPS–stimulated induction of CCRL2 or VCAM-1, indi- hCMEC/D3, HUVEC, HDMEC, and primary mouse lung and cating that the proinflammatory signals are dominant (Fig. 1). liver EC. In all conditions tested, EC did not express CMKLR1 or To confirm that the endothelioma cell lines accurately reflected GPR1 at the protein or RNA level (data not shown). Part of the primary EC biology, we evaluated CCRL2 expression on freshly discrepancy may be due to different culture conditions, which isolated lung and liver EC from mice dosed with endotoxin to could affect gene regulation (15). However, liver and lung EC induce systemic inflammation and vasculitis (24). Systemic ad- from LPS-dosed CCRL2-deficient mice did not bind to Fc- ministration of endotoxin has been reported to increase circu- chemerin, thus indicating that CCRL2 is the primary receptor lating levels of TNF-a and IFN-g, mimicking to an extent the for chemerin on liver and lung EC in vivo. in vitro stimulation of CCRL2 on EC (24, 25). Indeed, liver EC With its lack of classical signaling responses (i.e., cell migration, upregulated CCRL2 in response to LPS challenge in vivo. In- intracellular calcium mobilization) and absence of a “DRY” motif terestingly, EC isolated from the lung of normal WT mice con- in the second intracellular loop (thought to enable coupling to stitutively expressed CCRL2 and bound Fc-chemerin, but LPS G proteins; see Ref. (43), CCRL2 may be considered a member treatment did not alter lung CCRL2 expression. Primary human of the family of atypical chemoattractant receptors that include EC (HUVEC and HDMEC) treated in vitro with proinflammatory DARC, D6, CCX-CKR, and CXCR7 (reviewed in Ref. 44). These stimuli upregulated CCRL2 and bound Fc-chemerin, indicating receptors modulate immune responses by regulating the bio- conserved regulation in primary EC across species. Liver and availability of chemoattractants, usually through specialized and lung EC from LPS-dosed mice of both genotypes upregulated efficient ligand internalization and degradation (44, 45). Mice VCAM- 1, which is consistent with previous reports (24, 26, 39). deficient in D6 or DARC, for example, show increased inflam- It is not yet clear why CCRL2 is expressed endogenously at mation in models of skin inflammation and endotoxemia, re- The Journal of Immunology 965 spectively, owing to impaired chemokine clearance (46, 47). In chemerin receptors to leukocyte recruitment during pulmonary line with their biological function to intercept excess circulating inflammation, we investigated the role of CCRL2 in CMKLR1+ , D6, DARC, and CXCR7 are widely expressed on NK cell recruitment to the airways in response to intranasal numerous endothelial cell types (48–50). CCRL2 is also expressed LPS challenge. We hypothesized that EC CCRL2-dependent on a variety of EC from different tissues (brain, skin, lung, and anchoring/accumulation of bioactive chemerin contributes to the liver), suggesting a role for CCRL2 in regulating the bioavail- recruitment of CMKLR1+ NK cells to inflamed airways, an effect ability of circulating chemerin. Indeed, the intravascular time- that should be attenuated in CCRL2-deficient mice. Indeed, sig- integrated chemerin levels during 2 wk following a single i.v. nificantly fewer CMKLR1+ NK cells accumulated in the airways injection of Fc-chemerin was significantly greater (by 40%) in of CCRL22/2 mice compared with WT mice (Fig. 11). There CCRL22/2 mice compared with WT mice. This is also likely were no differences in the recruitment of CMKLR12 neutrophils reflected in the small but significantly elevated plasma chemerin or CD3+ cells. Additionally, there were similar numbers of cir- levels in unchallenged CCRL22/2 mice. Furthermore, treatment culating NK cells and other major WBC subsets in CCRL22/2 of mice with endotoxin, which upregulates vascular EC CCRL2 and WT mice, a similar expression of CMKLR1 on NK cells from and enhances chemerin binding in WT mice, produced a robust 2- both genotypes, and a lack of expression of CCRL2 on NK cells. to 3-fold greater than WT increase in circulating chemerin levels Taken together, these results indicate that CCRL2 selectively in CCRL22/2 mice. Other examples of increased chemoattractant coordinates the recruitment of CMKLR1+ NK cells in a manner levels in mice deficient for their cognate receptor include CCL2/ consistent with our model of EC CCRL2-dependent chemerin CCR2 and chemerin/CMKLR1 in a model of pneumonia (51, 52). anchoring.

Although a contribution of extravascular CCRL2 is not formally When bound to CCRL2, the carboxyl terminus of chemerin Downloaded from excluded by our studies, CCRL2 control of circulating chemerin important for CMKLR1 signaling remains exposed at the cell levels most likely occurs at the level of vascular EC. Thus, in surface (10). Recently, Hart and Greaves (31) demonstrated that regulating the bioavailability of leukocyte attractant chemerin, chemerin is a potent inducer of CMKLR1+ peritoneal macrophage CCRL2 exhibits another trait in common with the other atypical adhesion to VCAM-1 by inducing a4b1 integrin clustering. Thus, chemoattractant “interceptors.” However, one critical difference we hypothesized that CCRL2+ EC could bind and effectively + that sets CCRL2 apart from the other atypical receptors is that present chemerin to CMKLR1 lymphoid cells to trigger cell http://www.jimmunol.org/ CCRL2 does not internalize bound chemerin, demonstrated in this adhesion. Adhesion of L1.2 lymphoid cells to EC required the study with EC and previously with CCRL2+ lymphoid cells (10). following components: 1) CCRL2+ activated EC, 2) CMKLR1+ Chemerin circulates in an inactive proform and requires pro- L1.2 cells, and 3) chemerin. Furthermore, adhesion of CMKLR1+ teolytic processing to increase its biological activity. It is tempting cells was completely dependent on a4b1 integrin and VCAM-1. to speculate that CCRL2 could bind and present prochemerin on the Thus, we propose the following possible mechanisms to describe surface of EC to circulating serine proteases widely present during the concerted actions of CCRL2, CMKLR1, and chemerin: 1) Di- endotoxemia, thus removing the inhibitory peptides and presenting rect mechanism: CCRL2 directly presents chemerin to CMKLR1+ the active chemerin to circulating CMKRL1+ cells (reviewed in cells and thus creates a trio with CCRL2 binding the N terminus, Ref. 53). Other surface-bound receptors such as endothelial pro- whereas CMKLR1 interacts with the critical signaling C terminus by guest on September 24, 2021 tein C receptor are known to bind and concentrate their soluble of chemerin. 2) Indirect mechanism: Chemerin binds to CCRL2 ligand on the surface of EC, allowing for more efficient proteolytic with an affinity typical for a chemoattactant/receptor pair (low activation by their cognate enzyme. Indeed, endothelial protein C nanomolar KD) (10); coupled with the lack of ligand internaliza- receptor is widely expressed on vessels and binds and concentrates tion, it is likely that chemerin is released from the cell surface at a protein C, accelerating by 20-fold the activation of protein C by certain rate dependent on, for example, receptor density, temper- thrombin (54, 55). If EC CCRL2 captures circulating prochemerin ature, pH, and salt concentration, and at some rate is reacquired and enhances its proteolytic activation during inflammation, we by CCRL2. Elevated local concentrations of soluble chemerin in would predict 1) a reduction in circulating prochemerin levels in the media near the CCRL2 cells, however, may trigger CMKLR1 LPS-treated WT mice versus untreated WT controls, and 2) an activation and, subsequently, a4b1 integrin avidity upregulation, increase in circulating prochemerin in LPS-treated CCRL22/2 without requiring a trio complex. mice compared with WT mice. Indeed, there was significantly less In conclusion, our results provide a novel mechanism by which circulating prochemerin in WT LPS-treated mice compare with the chemoattractant chemerin is presented by CCRL2+ EC to untreated WT controls, likely reflecting sequestration by EC trigger CMKLR1+ cell adhesion. Extracellular matrix glyco- CCRL2 during systemic inflammation (Fig. 10B). Additionally, saminoglycans (GAGs) on the luminal side of the endothelium there was significantly more plasma prochemerin in LPS-treated and are thought to immobilize and present chemokines to rolling CCRL22/2 mice than in WT mice (Fig. 10C), along with a sig- leukocytes, which triggers integrin activation and leukocyte ex- nificant 2- to 3-fold increase in total circulating chemerin levels travasation (58). In several human inflammatory disorders (mul- (Fig. 10A). Thus, our results are consistent with the hypothesis tiple sclerosis, lupus, and psoriasis) in which chemerin is asso- that EC CCRL2 binds plasma prochemerin for enhanced proteo- ciated with inflamed endothelium, CMKLR1+ leukocytes (NK lytic activation during inflammation. Additional work is necessary cells, plasmacytoid dendritic cells) are found to infiltrate into the to characterize protease-specific effects of CCRL2-dependent affected tissues (11–14). Furthermore, in two separate in vivo anchoring of chemerin in its proteolytic activation. inflammatory models, CCRL22/2 mice displayed less severe al- Depending on the model, chemerin and its receptors CCRL2 and lergic inflammation (10) and less severe OVA-induced airway CMKLR1 can play a pathogenic or protective role in pulmonary inflammation than in WT counterparts (57); however, it is not inflammation: CMKLR1 plays a pathogenic role in cigarette clear whether this protective effect is linked with a decrease in smoke-induced lung inflammation (56) and CCRL2 plays a path- CMKLR1+ cell recruitment. Although GAGs likely play a role in ogenic role in an OVA model of lung inflammation (57), whereas chemerin binding (chemerin binds to heparin, a type of GAG), CMKLR1 plays a protective role in viral pneumonia and an LPS we hypothesize that CCRL2 expressed on inflamed endothelium airway challenge model (30, 51). Given the robust expression of provides a novel specific and selective mechanism to bind and CCRL2 on lung EC and the recent reported contributions of the concentrate chemerin (3). A recent report indicates that CCL19 966 EXPRESSION, REGULATION, AND FUNCTION OF ENDOTHELIAL CCRL2 may be an alternate chemoattractant ligand for CCRL2, thus 17. Huss, R. S., J. I. Huddleston, S. B. Goodman, E. C. Butcher, and B. A. Zabel. 2010. Synovial tissue-infiltrating natural killer cells in osteoarthritis and peri- widening the biological spectrum of action for CCRL2 (59). prosthetic inflammation. Arthritis Rheum. 62: 3799–3805. Nevertheless, selective inhibition of CCRL2 binding to chemerin, 18. Weksler, B. B., E. A. Subileau, N. Perrie`re, P. Charneau, K. Holloway, rather than inhibition of GAGs, which bind all chemokines, could M. Leveque, H. Tricoire-Leignel, A. Nicotra, S. Bourdoulous, P. Turowski, et al. 2005. 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