Chemerin Contributes to Inflammation by Promoting Macrophage Adhesion to VCAM-1 and Fibronectin through Clustering of VLA-4 and VLA-5 This information is current as of October 1, 2021. Rosie Hart and David R. Greaves J Immunol 2010; 185:3728-3739; Prepublished online 18 August 2010; doi: 10.4049/jimmunol.0902154

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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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Chemerin Contributes to Inflammation by Promoting Macrophage Adhesion to VCAM-1 and Fibronectin through Clustering of VLA-4 and VLA-5

Rosie Hart and David R. Greaves

Chemerin is a potent macrophage chemoattractant . We used murine peritoneal exudate cells (PECs) in adhesion, flow cytom- etry, and confocal microscopy assays totestthehypothesisthat chemerin canalso contributetoinflammationby promoting macrophage adhesion. Chemerin stimulated the adhesion of PECs to the extracellular matrix protein fibronectin and to the adhesion molecule

VCAM-1 within a minute, with an EC50 of 322 and 196 pM, respectively. Experiments using pertussis toxin and PECs from ChemR232/2 mice demonstrated that chemerin stimulated the adhesion of macrophages via the Gi protein-coupled receptor ChemR23. Blocking Abs against subunits revealed that 89% of chemerin-stimulated adhesion to fibronectin was dependent on increased avidity of the integrin VLA-5 (a5b1) and that 88% of adhesion to VCAM-1 was dependent on increased avidity of VLA- Downloaded from 4(a4b1). Although chemerin was unable to induce an increase in integrin affinity as judged by the binding of soluble ligand, experiments using confocal microscopy revealed an increase in valency resulting from integrin clustering as the mechanism re- sponsible for chemerin-stimulated macrophage adhesion. PI3K, Akt, and p38 were identified as key signaling mediators in chemerin- stimulated adhesion. The finding that chemerin can rapidly stimulate macrophage adhesion to extracellular matrix and adhesion molecules, taken together with its ability to promote chemotaxis, suggests a novel role for chemerin in the recruitment and retention of macrophages at sites of inflammation. The Journal of Immunology, 2010, 185: 3728–3739. http://www.jimmunol.org/

he coordinated recruitment of leukocytes to sites of tissue In addition to mediating chemotaxis, many chemoattractants, injury is central to the generation of a successful in- including chemokines, are also able to induce the adhesion of T flammatory response (1). The directed migration of leu- leukocytes to adhesion molecules, such as VCAM-1 and ICAM-1, kocytes is regulated by a number of inflammatory molecules in- and to extracellular matrix proteins, such as fibronectin, laminin, cluding chemokines, cytokines and complement proteins (2). and collagen IV (15). They induce adhesion by stimulating an Chemerin was originally isolated from human inflammatory exu- increase in the affinity and/or the valency of integrin receptors dates as the protein ligand for the orphan Gi-linked G protein- expressed on the cell surface (16). Leukocyte adhesion plays a key coupled receptor (GPCR) ChemR23 (3). ChemR23 is structurally role in inflammation, contributing to leukocyte rolling and arrest by guest on October 1, 2021 related to chemokine receptors, and accordingly, chemerin was dis- on the vascular endothelium, migration through the extracellular covered to possess the chemoattractant abilities of a chemokine (3, matrix to the site of inflammation, retention of leukocytes within 4). Chemerin is chemotactic for ChemR23-expressing human mac- the inflammatory site, and leukocyte activation (17–22). rophages, plasmacytoid dendritic cells (pDCs), and NK cells (3, 5– In this study, we hypothesized that chemerin may contribute to 7). ChemR23 is also expressed on human monocytes (6). In the mo- inflammation not only by mediating macrophage chemotaxis, but use, macrophages express ChemR23 and migrate toward chemerin also by promoting the adhesion of macrophages to extracellular (8, 9). Recently, murine pDCs and NK cells were also shown to be matrix proteins and adhesion molecules. ChemR23+ (10). Unlike the majority of chemokines, chemerin is secreted in an inactive precursor form (3). It requires cleavage of the Materials and Methods C terminus by serine proteases of the coagulation, fibrinolytic, and Animals inflammatory cascades to become active as a chemoattractant for All animal experiments were performed with ethical approval from the Dunn cells expressing ChemR23 (11–13). Moreover, chemerin’s predicted School of Pathology Local Ethical Review Committee and in accordance structure, thought to be the reverse orientation of that of chemokines, with the U.K. Home Office regulations (Guidance on the Operation of distinguishes it from this family of proteins (14). Animals, Scientific Procedures Act, 1986). Reagents

Sir William Dunn School of Pathology, University of Oxford, Oxford, United King- Collagen IV, laminin and fibronectin were obtained Scientific Laboratory dom Supplies (Nottingham, U.K.). ICAM-1, VCAM-1, and VCAM-1-Fc were Received for publication July 6, 2009. Accepted for publication July 9, 2010. from R&D Systems (Abingdon, U.K.). The FITC-conjugated GRGDSP peptide was purchased from AnaSpec (Fremont, CA), as were all the blocking This work was supported by British Heart Foundation Grant FS/06/080. Abs and their isotype controls as follows: rat IgG1 (clone RTK2071), rat Address correspondence and reprint requests to Dr. David R. Greaves, Sir William IgG2a (clone RTK2758), rat IgG2b (clone RTK4530), Armenian hamster IgG Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, Oxon (clone HTK888), anti-mouse CD18 (b2; clone M18/2), anti-mouse/rat OX1 3RE, U.K. E-mail address: [email protected] CD29 (b1; clone HMb1-1), anti-mouse CD49d (a4; clone R1-2), anti-mouse The online version of this article contains supplemental material. CD49e (a5; clone 5H10-27), anti-mouse CD51 (av; clone RMV-7), and anti- Abbreviations used in this paper: GPCR, G protein-coupled receptor; i.c., isotype mouse integrin b7 (clone FIB27). Of the inhibitors, Akt inhibitor IV, cyto- controls; LPAM, lymphocyte Peyer’s patch adhesion molecule; pDC, plasmacytoid chalasin D, LY294002, and SB202190 were purchased from Sigma-Aldrich dendritic cell; PEC, peritoneal exudate cell; PTX, pertussis toxin; WT, wild-type. (Gillingham, U.K.), BAPTA-AM and pertussis toxin (PTX) were from Cal- biochem (Nottingham, U.K.), piceatannol and PP2 were from Tocris Bio- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 science (Bristol, U.K.), and U0126 was obtained from New England Biolabs www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902154 The Journal of Immunology 3729

(Hitchin, U.K.). Anti-mouse CD16/CD32 (clone FCR4G8), FITC-conjugated Alexa Fluor 647-conjugated anti-mouse F4/80 and either 3 mg/ml PE- anti-mouse neutrophils (clone 7/4), Alexa Fluor 647-conjugated anti-mouse conjugated anti-mouse ChemR23 or 3 mg/ml PE-conjugated rat IgG2a F4/80 (clone CI:A3-1), FITC-conjugated anti-mouse CD49d (clone MFR5), isotype control for 30 min on ice, washed with assay buffer, and fixed with Pacific blue-conjugated anti-mouse F4/80 (clone CI:A3-1), and Alexa Fluor 2% formaldehyde (Sigma-Aldrich) in PBS. Cells were analyzed on a Cyan 647-conjugated anti-mouse CD206 (clone MR5D3) were from Serotec ADP flow cytometer (Dako, Ely, U.K.). Overnight-treated PECs were also (Oxford, U.K.). PE-conjugated anti-mouse ChemR23 (clone BZ194), PE- stained with 3 mg/ml FITC-conjugated anti-mouse CD80 or 1 mg/ml Alexa conjugated rat IgG2a isotype control (clone eBR2a), FITC-conjugated anti- Fluor 647-conjugated anti-mouse CD206 in combination with 2 mg/ml mouse CD49d (clone R1-2), FITC-conjugated rat IgG2a (clone eBR2a) and Pacific blue-conjugated anti-mouse F4/80. IgG2b (clone eB149/10H5), and FITC-conjugated anti-mouse CD80 (clone To measure the binding of a fibronectin peptide to macrophages, PECs 16-10A1) were purchased from eBioscience (Hatfield, U.K.). IFN-g and IL-4 from C57BL/6J mice were resuspended in assay buffer (HBSS [Invit- were from PeproTech (London, U.K.), and LPS was from Sigma-Aldrich. rogen] with CaCl2 and MgCl2 supplemented with 0.1% BSA and 20 mM Murine recombinant chemerin was obtained from R&D Systems, C15 was HEPES) to a concentration of 5 3 106 cells/ml, blocked with 10 mg/ml synthesized by Biosynthesis (Lewisville, TX), and C9 was a gift from anti-mouse CD16/CD32 for 15 min on ice, and stained with 1 mg/ml Alexa H. Wang and T. Schall (ChemoCentryx, Mountain View, CA). Fluor 647-conjugated anti-mouse F4/80 for 30 min on ice. FITC-labeled GRGDSP peptide (10 mM) and either assay buffer 10 mM MnCl2 (Sigma- Cells Aldrich) or 10 nM chemerin were added to 100 ml of the stained PECs and To obtain peritoneal exudate cells (PECs), Bio-Gel P100 polyacrylamide incubated at 37˚C for up to 5 min. Tubes were immediately placed on ice to beads (1 ml 2% w/v in PBS; Bio-Rad, Hemel Hempstead, U.K.) were stop the reaction and washed once with assay buffer. Cells were fixed using injected into the peritoneal cavities of 8- to 12-wk-old C57BL/6J mice, 2% paraformaldehyde (Sigma-Aldrich) in PBS on ice for 1 h. Samples were Sv129 mice, or ChemR232/2 mice on an Sv129 background. Four days analyzed by flow cytometry on a Cyan ADP flow cytometer. later, the mice were killed using a Schedule 1 method, and the peritoneal To measure the binding of VCAM-1-Fc to macrophages, PECs were 3 6 cavities were lavaged with 10 ml ice-cold PBS (Lonza, Slough, U.K.) plus 2 resuspended in assay buffer to a concentration of 5 10 cells/ml and mM EDTA (Sigma-Aldrich). The resulting cells were used immediately, 100 mg/ml VCAM-1-Fc plus either assay buffer 10 mM MnCl2 or 10 nM Downloaded from except for the polarized macrophage experiments in which 2 3 106 cells/ml chemerin were added to 10 ml of these cells and incubated at 37˚C for up to 2 PECs in RPMI 1640 (PAA Laboratories, Yeovil, U.K.) supplemented with min. Tubes were immediately placed on ice to stop the reaction and the cells 1% FBS (PAA Laboratories) 0.1% BSA (Sigma-Aldrich), 25 mM HEPES were fixed with 2% paraformaldehyde in PBS on ice for 1 h. Samples were (PAA Laboratories), 50 U/ml penicillin, and 50 mg/ml streptomycin (Invi- washed twice with assay buffer, blocked with 10 mg/ml anti-mouse trogen, Paisley, U.K.) were incubated overnight in polypropylene tubes CD16/CD32 for 15 min on ice, stained with 1 mg/ml Alexa Fluor 647-con- at 37˚C. Cells were polarized by the addition of 20 ng/ml IFN-g,20 jugated anti-mouse F4/80 and a 1/100 dilution of PE-conjugated anti-human ng/ml IFN-g plus 100 ng/ml LPS, or 20 ng/ml IL-4 to the media. The next IgG (Jackson ImmunoResearch Laboratories, Newmarket, U.K.) for 30 min day the cells were washed twice before use in further experiments. on ice and washed with assay buffer before resuspension in 2% para- http://www.jimmunol.org/ BaF3 cells stably transfected with the murine ChemR23 receptor formaldehyde in PBS and analysis by flow cytometry on Cyan ADP flow (BaF3-ChemR23) were a gift from H. Wang and T. Schall (ChemoCentryx). cytometer. ChemR23 expression was confirmed by flow cytometry using an anti-mouse ChemR23 Ab (data not shown). Wild-type (WT) BaF3 cells and BaF3- Confocal microscopy ChemR23 cells were cultured in RPMI 1640 supplemented with 10% FBS, 10% WEHI conditioned medium, 50 mM 2-ME (Sigma-Aldrich), 50 U/ml To visualize clustering of the a4 or a5 on macrophages, PECs from penicillin, and 50 mg/ml streptomycin. BaF3 cells were serum starved in C57BL/6J mice were resuspended in ice-cold RPMI 1640 supplemented 3 6 RPMI 1640 supplemented with 0.1% BSA and 25 mM HEPES for 24 h with 0.1% BSA and 25 mM HEPES to a concentration of 8 10 cells/ml. before their use in adhesion assays. After addition of either media, 100 ng/ml PMA or 10 nM chemerin, the cells were incubated at 37˚C for 1 min. Tubes were then immediately placed on

Adhesion assay ice to stop the reaction and the cells were fixed with 2% paraformaldehyde in by guest on October 1, 2021 PBS on ice for 20 min. Samples were washed twice with assay buffer (HBSS Ninety-six-well cell culture plates (Corning, Amsterdam, The Netherlands) with CaCl2 and MgCl2 supplemented with 5% FCS, 0.5% BSA, and 20 mM were coated overnight at 4˚C with 30 mg/ml collagen IV, 30 mg/ml fibro- HEPES), blocked with 10 mg/ml anti-mouse CD16/CD32 for 30 min on ice, nectin, 30 mg/ml laminin, 2 mg/ml ICAM-1, or 2 mg/ml VCAM-1. The and stained overnight at 4˚C with 2.5 mg/ml FITC-conjugated anti-mouse plates were washed twice with PBS, and nonspecific binding sites were CD49d (a4) or CD49e (a5) or their isotype controls (FITC-conjugated rat blocked with 1% BSA in PBS for 1 h at 37˚C, before an additional three IgG2b or IgG2a, respectively) in combination with 1 mg/ml Alexa Fluor washes with assay buffer (RPMI 1640 supplemented with 0.1% BSA and 647-conjugated anti-mouse F4/80. The next day the cells were washed once 25 mM HEPES). PECs or BaF3 cells were resuspended in assay buffer, and in assay buffer then added to wells of a Lab-Tek chamber slide (Cole- 50 ml of cell suspension in the absence or presence of chemerin (10 nM Parmer, London, U.K.) that had previously been coated with 100 mg/ml unless otherwise stated), C9, or C15 peptide (0.001 nM to 1 mM) was added poly-D-lysine (Sigma-Aldrich) for 1 h at room temperature, washed two to each well of the plate (2 3 105 cells/well). Plates were incubated at 37˚C times with sterile dH2O, and left to air-dry. The cells were allowed to adhere for 1 min unless otherwise specified, before 20 s of shaking on an orbital for 1 h at room temperature before the wells were washed an additional three shaker, and three washes with assay buffer to remove nonadherent cells. times with assay buffer, and the slides were mounted in Dako fluorescent Adherent cells were quantified using CellTiter-Glo (Promega, South- mounting medium. The cells were analyzed using a Carl Zeiss LSM 510 ampton, U.K.) in a white luminometer plate (Greiner Bio-One, Stonehouse, confocal laser scanning system with a 633 Plan-Apochromat objective (NA U.K.), according to the manufacturer’s instructions and a SpectraMax M5 1.4). Macrophages were identified as those cells with an average fluores- microplate reader (Molecular Devices, Wokingham, U.K.). cence intensity for F4/80 exceeding a predefined lower limit. For adhesion assays using blocking Abs, PECs were blocked with 10 mg/ml anti-mouse CD16/CD32 for 15 min at 37˚C, followed by a 30-min incubation at 37˚C with 3 mg/ml blocking Ab or its isotype control. Cells RT-PCR analysis were then added to the coated plate as described previously. Total RNA was prepared from freshly obtained PECs using TRIzol (Invi- For adhesion assays using inhibitors, PECs were pretreated for 1 h at room trogen), isolated using an RNeasy (Qiagen, Crawley, U.K.), and reverse temperature with 0.015 mg/ml cytochalasin D, 1 mM Akt inhibitor IV, 5 mM transcribed using Moloney murine leukemia virus reverse transcriptase and BAPTA-AM, 5 mM LY294002, 20 mM piceatannol, 10 mM PP2, 20 mM randomprimers(Promega). RT-PCRanalysisforGAPDH,ChemR23, GPR1, SB202190, 20 mM U0126, or combinations of these inhibitors. For adhesion CCRL2, and BLT1 was performed using SYBRGreen (Qiagen) and the assays using PTX, PECs were pretreated with 200 ng/ml PTX for 1 h following primers: GAPDH, 59-TGTGTCCGTCGTGGATCTGA-39 (for- at 37˚C. To ensure that these concentrations of inhibitors were not cytotoxic, ward) and 59-ACCACCTTCTTGATGTCATCATACTT-39 (reverse); Chem- CellTiter-Glo was used according to the manufacturer’s instructions to R23, 59-ACACACTGTTCATCAGTAGCAA-39 (forward) and 59-CCTCC- compare the number of live untreated PECs with the number of live PECs ATGAGGAATGATGTCAA-39 (reverse); GPR1, 59-CTACGTGGCCTTG- that had been treated with inhibitors as described previously. AGTTTCCA-39 (forward) and 59-CAGTTGGGCAATGAAGGAATTAA- Flow cytometry 39 (reverse); CCRL2, 59-ACGGGCGTGTTCCTTTTG-39 (forward) and 59- TCCTGGAAAGCAGACAGGAAA-39 (reverse); and BLT1, 59-TTGCT- To assess ChemR23 expression on PECs, 5 3 105 PECs were resuspended CACTGCTCCCTTTTTC-39 (forward) and 59-GCGGCAACCCATCTCT- in assay buffer (PBS supplemented with 5% FBS, 25 mM HEPES, and CTAA-39 (reverse). The number of copies of receptor mRNA per copy of 5 mM EDTA), blocked with 10 mg/ml anti-mouse CD16/CD32 for 15 min GAPDH mRNA was quantified from a standard curve using mouse geno- on ice, stained with 0.8 mg/ml FITC-conjugated anti-mouse 7/4, 1 mg/ml mic DNA (Promega). 3730 CHEMERIN MEDIATES MACROPHAGE ADHESION

Statistical analysis mice (28). PECs from these three strains of mouse also expressed One-way ANOVA (with Dunnett’s or Bonferroni’s posthoc test) and leukotriene B4 receptor (BLT1) mRNA (28.4–40.3% of ChemR23 two-way ANOVA (with Bonferonni’s posthoc test) were performed as mRNA expression). appropriate using GraphPad Prism. Only macrophage and monocyte-macrophage PECs expressed ChemR23 as determined by flow cytometry (Fig. 2D). Therefore we Results concluded that the cells that adhere to fibronectin and VCAM-1 in a ChemR23-dependent manner in response to chemerin treatment Chemerin rapidly induces adhesion to fibronectin and VCAM-1 are macrophages and monocyte-macrophages. To investigate chemerin’s ability to induce macrophage adhesion to To test the effect of macrophage polarization on chemerin- extracellular matrix proteins and recombinant adhesion molecules, stimulated adhesion, we pretreated PECs with LPS and/or various we used Bio-Gel–elicited PECs, which are a good source of min- cytokines. Macrophages that were treated overnight with IFN-g imally activated macrophages, in a static adhesion assay. Bio-Gel– alone expressed increased levels of CD80, a marker of classically elicited PECs were composed of an average of 33% macrophages activated M1 macrophages (Supplemental Fig. 1A) and the same and 10% immature monocyte-macrophages as determined by their level of ChemR23 expression as untreated cells (Supplemental Fig. expression of 7/4 and F4/80 Ags (Fig. 1A). 1B). Overnight treatment of macrophages with IFN-g plus LPS PECs were allowed to adhere to plates coated with collagen IV, upregulated CD80 expression to a greater extent than IFN-g alone fibronectin, laminin, ICAM-1, or VCAM-1 in the presence or ab- but completely downregulated ChemR23 expression. Consistent sence of chemerin. Treatment of PECs with 10 nM chemerin resulted with the ChemR23 expression of these macrophage populations, in asignificant increase inthe number of adherentcells on fibronectin IFN-g–treated cells retained their ability to adhere to fibronectin (4.6-fold; p , 0.001), ICAM-1 (5.6-fold; p , 0.001), and VCAM-1 and VCAM-1 in response to chemerin stimulation, whereas IFN-g Downloaded from (4.5-fold; p , 0.001) (Fig. 1B). Chemerin was not able to induce plus LPS-stimulated cells lost this activity (Supplemental Fig. 2). PEC adhesion to collagen IV or laminin. Chemerin stimulated the Macrophages treated overnight with IL-4 displayed an upregulation adhesion of PECs to fibronectin (Fig. 1C) and VCAM-1 (Fig. 1D)in in expression of the , the classic marker of al- a dose-dependent manner with maximal effects achieved at 10 nM ternatively activated M2a macrophages (Supplemental Fig. 1A) and chemerin. This corresponds well with chemerin’s chemotactic ac- had the same level of ChemR23 expression as untreated cells tivity (8). Chemerin had an EC50 value of 322 and 196 pM for cell (Supplemental Fig. 1B). However, in the adhesion assay, IL-4– http://www.jimmunol.org/ adhesion to fibronectin and VCAM-1, respectively. treated cells did not adhere to fibronectin or VCAM-1 in response to Chemerin’s effects on adhesion were very rapid. The greatest chemerin stimulation (Supplemental Fig. 2). increase in the number of adherent cells compared with untreated To provide further evidence that chemerin is capable of promoting occurred at 1 min, the earliest time point that we could accurately cell adhesion to fibronectin and VCAM-1 solely through ChemR23, quantify, on both fibronectin (Fig. 1E) and VCAM-1 (Fig. 1F). we used BaF3 cells (a pro-B cell line) stably transfected with ChemR23 in the adhesion assay. Chemerin (10 nM) was capable of Chemerin stimulates the adhesion of macrophages through stimulating the adhesion of these cells to both fibronectin (Fig. 2E) ChemR23 and to VCAM-1 (Fig. 2F), but it was not able to induce the adhesion by guest on October 1, 2021 The effects of chemoattractants on adhesion are mediated through of WT BaF3 cells that did not express the ChemR23 receptor Gi-coupled GPCRs (23–26). To determine whether this was true for (Fig. 2E,2F). We also tested whether two peptides derived from chemerin’s effects, PECs from WT Sv129 mice were pretreated the C terminus of murine chemerin were able to promote adhesion with PTX. Chemerin induced a significant increase in the adhesion of the BaF3-ChemR23 cells to fibronectin. These peptides had of PECs from WT Sv129 mice to fibronectin (2.1-fold; p , 0.001) both been previously shown to induce ChemR23-dependent activ- (Fig. 2A) and VCAM-1 (3.7-fold; p , 0.001) (Fig. 2B). Chemerin’s ity (8, 10, 29). C9 (FLPGQFAFS) was a partial agonist, capable of effects on cell adhesion to fibronectin and VCAM-1 were com- producing 58.6% of the adhesive activity of full-length chemerin pletely inhibited by PTX pretreatment, demonstrating the require- (Fig. 2G). However, it was slightly more potent than chemerin ment for a Gi-coupled GPCR (Fig. 2A,2B). with an EC50 of 0.94 nM compared with 2.48 nM for chemerin. Bio-Gel–elicited peritoneal macrophages from ChemR232/2 Peptide C15 (AGEDPHGYFLPGQFA) was unable to promote the mice display no chemotaxis toward chemerin (8). To investigate adhesion of BaF3-ChemR23 cells to fibronectin at any concen- whether chemerin-stimulated adhesion is mediated via ChemR23, tration tested. we assessed the ability of chemerin to stimulate the adhesion of PECs from Sv129 ChemR232/2 mice (KO PECs) to both fibro- Chemerin-stimulated adhesion to fibronectin and VCAM-1 is a b a b nectin and VCAM-1. Chemerin was unable to promote the adhe- mediated through the VLA-5 ( 5 1) and VLA-4 ( 4 1) sion of PECs from ChemR232/2 mice to fibronectin (Fig. 2A). integrins, respectively Therefore, chemerin-stimulated adhesion to fibronectin is depen- Leukocytes bind to extracellular matrix proteins and adhesion dent on ChemR23 and its coupling to a Gi G protein. However, molecules, such as fibronectin and VCAM-1, through integrins a significant number of PECs from ChemR232/2 mice were still expressed on their surface (30). The adhesion of macrophages to capable of adhering to VCAM-1 (1.9-fold increase; p , 0.001). fibronectin is mediated via the integrins a4b1, a5b1, avb3, and These results suggest that although chemerin-stimulated adhesion a4b7, whereas a4b1, aDb2, aXb2, and a4b7 are involved in mac- to VCAM-1 is dependent on activation of a Gi-coupled GPCR, just rophage adhesion to VCAM-1 (31, 32). To identify which of these over half of this activity is independent of ChemR23. integrins mediates chemerin-induced adhesion to fibronectin and Quantitative RT-PCR analysis of PECs from C57BL/6J mice, VCAM-1, PECs were pretreated with blocking Abs against the Sv129 mice, and Sv129 ChemR232/2 mice showed minimal levels integrin subunits that constitute the candidate integrins. of mRNA for GPR1 (0.02–0.03% of ChemR23 mRNA expression), Chemerin-induced adhesion to fibronectin was significantly another GPCR shown to bind chemerin (Fig. 2C) (27). Low levels inhibited by Abs that blocked the a5 integrin subunit (92.5% in- of mRNA for CCRL2 (11.1–26.7% of ChemR23 mRNA expres- hibition; p , 0.001) and the b1 integrin subunit (86.4% inhibition; sion), a third nonsignaling receptor for chemerin, were expressed in p , 0.001) (Fig. 3A). This result demonstrates that chemerin stim- PECs from C57BL/6J mice, Sv129 mice, and Sv129 ChemR232/2 ulates the adhesion of macrophages to fibronectin through regulation The Journal of Immunology 3731

FIGURE 1. Chemerin induces a rapid and dose-dependent adhesion of PECs to fibronectin and VCAM-1. A, Bio-Gel–elicited PECs were stained with anti–7/4-FITC and anti–F4/80- Alexa Fluor 647 Abs and analyzed by flow cytometry. The graph is represen- tative of three separate experiments, and percent cell type is given beside each gate. B–F, PECs from C57BL6/J mice were added to wells that had been pre- coated with extracellular matrix proteins or adhesion molecules and incubated at 37˚C in the presence of media alone or chemerin. Plates were shaken for 20 s, and the wells were washed thoroughly with media to remove nonadherent cells. CellTiter-Glo was added to each well, and the luminescence was measured to quantify the remaining adherent cells. B, Wells were precoated with 30 mg/ml Downloaded from collagen IV, 30 mg/ml fibronectin, 30 mg/ml laminin, 2 mg/ml ICAM-1, or 2 mg/ml VCAM-1. PECs were allowed to adhere for 1 min at 37˚C in the presence of media alone or 10 nM chemerin; pppp , 0.001, two-way ANOVA with

Bonferroni’s posthoc test. C and D, http://www.jimmunol.org/ Wells were precoated with 30 mg/ml fi- bronectin (C)or2mg/ml VCAM-1 (D), and PECs were allowed to adhere for 1 min at 37˚C in the presence of media alone or 0.01 pM to 100 nM chemerin. E and F, Wells were precoated with 30 mg/ ml fibronectin (E)or2mg/ml VCAM-1 (F), and PECs were allowed to adhere for up to 2 h at 37˚C in the presence of media alone or 10 nM chemerin. Graphs by guest on October 1, 2021 show mean values 6 SEM from three independent experiments.

of the VLA-5 (a5b1) integrin. Blocking Abs against the av, a4, and investigate the hypothesis that chemerin promotes adhesion by b7 subunits had no significant effect in this assay, and therefore, increasing integrin affinity, we measured the binding of soluble the avb3, a4b1, and a4b7 integrins do not play a role in chemerin- fluorescently conjugated VLA-4 and VLA-5 ligands to chemerin- stimulated adhesion to fibronectin. treated macrophages by flow cytometry. MnCl2 binds to the ex- Chemerin-stimulated adhesion of PECs to VCAM-1 was signif- tracellular domain of integrins, inducing a conformational change icantly reduced by blocking Abs against the a4 integrin subunit that increases integrin affinity and was therefore used as a positive , (100% inhibition; p 0.001) and the b1 integrin subunit (87.9% control (33). , inhibition; p 0.001) (Fig. 3B). This result indicates that chemer- GRGDSP is a peptide derived from fibronectin that contains the in-stimulated adhesion to VCAM-1 occurs through regulation of the key RGD sequence required for binding to integrins (34). In our VLA-4 (a4b1) integrin. As adhesion to VCAM-1 is almost com- experiments, over a period of 5 min, MnCl2 significantly increased pletely blocked by these Abs, it suggests that regulation of VLA-4 is the amount of GRGDSP-FITC peptide bound to F4/80+ PECs critical for both the ChemR23-dependent and the ChemR23-in- compared with untreated F4/80+ PECs (Fig. 4A). An increase in the dependent adhesion to VCAM-1 in response to chemerin treatment. amount of peptide bound to F4/80+ PECs was seen as early as 10 s A blocking Ab against the b7 subunit also significantly reduced and became significant after 2 min (1.69-fold increase; p , 0.05). chemerin-induced adhesion to VCAM-1 (43.1% inhibition; p , This indicates that MnCl2 is capable of inducing an increase in the 0.05), although to a lesser extent, suggesting a role for lymphocyte affinity of fibronectin-binding integrins on macrophages. In con- Peyer’s patch adhesion molecule (LPAM)-1 (a4b7) in chemerin- trast, chemerin (10 nM) was unable to stimulate an increase in the stimulated adhesion to VCAM-1. A blocking Ab against the b2 amount of GRGDSP-FITC peptide bound to F4/80+ PECs com- subunit excluded the aDb2 and aXb2 integrins from playing a role in pared with untreated F4/80+ PECs at any time point. Therefore, chemerin-stimulated adhesion to VCAM-1 as no significant in- chemerin does not induce macrophage adhesion to fibronectin by hibition of adhesion was observed following preincubation with increasing VLA-5 affinity. this Ab. MnCl2 also stimulated a rapid and sustained increase in the amount of VCAM-1-Fc bound to F4/80+ cells compared with un- Chemerin does not stimulate an increase in integrin affinity treated F4/80+ PECs, demonstrating its ability to increase the affinity Many chemoattractants stimulate leukocyte adhesion by promoting of VCAM-1–binding integrins on macrophages (Fig. 4B). A signif- an increase in the affinity of an integrin for its ligand (16, 25). To icant increase in the amount of VCAM-1-Fc was observed after just 3732 CHEMERIN MEDIATES MACROPHAGE ADHESION Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 2. Chemerin’s effects on adhesion are mediated largely through ChemR23 expressed on macrophages. A and B, PECs from Sv129 mice (WT PECs) 6 1 h of pretreatment with 200 ng/ml PTX at 37˚C or PECs from ChemR232/2 mice (KO PECs) were added to wells that had been precoated with 30 mg/ml fibronectin (A)or2mg/ml VCAM-1 (B) and incubated for 1 min at 37˚C in the presence of media alone or 10 nM chemerin. Plates were shaken for 20 s, and the wells washed thoroughly with media to remove nonadherent cells. CellTiter-Glo was added to each well, and the luminescence was measured to quantify the remaining adherent cells. Graphs show mean values 6 SEM from three independent experiments; pppp , 0.001, two-way ANOVA with Bonferroni’s posthoc test. C, GPCR mRNAwas quantified using SYBRGreen RT-PCR. Data presented as the number of copies of receptor mRNA per copy of GAPDH mRNA as quantified from a standard curve. D, PECs were stained with anti–7/4-FITC, anti–F4/80-Alexa Fluor 647, and either rat IgG2a-PE or rat anti-mouse ChemR23-PE Abs and analyzed by flow cytometry. Graphs representative of three separate experiments. E and F, WT-BaF3 cells or BaF3- ChemR23 cells were added to plates that had been precoated with 30 mg/ml fibronectin (E)or2mg/ml VCAM-1 (F) and incubated for 1 min at 37˚C in the presence of media alone or 10 nM chemerin. Plates were washed and analyzed as described above. Graphs show mean values 6 SEM from three independent experiments. G, BaF3-ChemR23 cells were added to plates precoated with 30 mg/ml fibronectin and incubated for 1 min at 37˚C in the presence of media alone, 0.01–100 nM chemerin, or 0.001–1 mM C9 or C15 peptide. Plates were washed and analyzed as described above. Graphs show mean values 6 SEM from three independent experiments.

30 s (2.83-fold increase; p , 0.001). Again, chemerin had no effect does not induce macrophage adhesion to VCAM-1 by increasing on the amount of VCAM-1-Fc bound to F4/80+ PECs compared with the affinity of VLA-4 (or LPAM-1). Representative histograms are untreated F4/80+ PECs over a 2-min period, indicating that chemerin shown in Fig. 4C and 4D. The Journal of Immunology 3733 Downloaded from http://www.jimmunol.org/

FIGURE 3. Chemerin-stimulated adhesion to fibronectin and VCAM-1 is mediated through the regulation of VLA-5 (a5b1) and VLA-4 (a4b1), by guest on October 1, 2021 respectively. A and B, PECs from C57BL6/J mice were pretreated with 3 mg/ml integrin blocking Abs or their i.c. for 30 min at 37˚C then added to wells that had been precoated with 30 mg/ml fibronectin (A)or2mg/ml VCAM-1 (B) and incubated for 1 min at 37˚C in the presence of media alone or 10 nM chemerin. Plates were shaken for 20 s, and the wells washed thoroughly with media to remove nonadherent cells. CellTiter-Glo was added to each well, and the luminescence was measured to quantify the remaining adherent cells. Graphs show mean values 6 SEM from three independent experiments; pp , 0.05; pppp , 0.001, one-way ANOVA with Bonferroni’s posthoc test. i.c., isotype controls. FIGURE 4. Chemerin does not stimulate an increase in integrin affinity. A, Chemerin stimulates integrin clustering PECs from C57BL/6J mice were stained with anti–F4/80-Alexa Fluor 647 Ab. A total of 10 mM GRGDSP-FITC–conjugated peptide and either media,

Some chemokines have been shown to promote leukocyte adhesion 10 mM MnCl2, or 10 nM chemerin were added to the F4/80-stained PECs and by stimulating the clustering of integrins on the cell surface (16, 35). incubated at 37˚C for up to 5 min. The PECs were immediately washed with To test whether chemerin could induce the clustering of VLA-4 and/or ice-cold buffer and fixed in 2% PFA. The quantity of peptide remaining bound VLA-5 integrins, we used confocal microscopy to visualize localized to the F4/80+ cells was measured by flow cytometry. B, A total of 100 mg/ml VCAM-1-Fc and either media, 10 mM MnCl , or 10 nM chemerin were changes in the distribution of the a4 integrin subunit and the a5 in- 2 tegrin subunit, respectively, on the surface of F4/80+ macrophages. added to PECs from C57BL/6J mice and incubated at 37˚C for up to 2 min. PECs were immediately fixed in 2% PFA on ice, washed with ice-cold buffer, The phorbol ester PMA was used as a positive control (36, 37). and stained with PE-conjugated anti-human IgG and anti–F4/80-Alexa Fluor Treatment of the PECs in suspension with PMA induced the 647 Abs. The quantity of VCAM-1-Fc remaining bound to the F4/80+ cells clustering of both the a5 (Fig. 5A) and the a4 (Fig. 5B) integrin was measured by flow cytometry. Graphs show mean values 6 SEM from + subunits on the surface of F4/80 macrophages. This was shown three independent experiments; pp , 0.05; pppp , 0.001, two-way ANOVA by an increase in fluorescence intensity on certain parts of the cell with Bonferroni’s posthoc test. C and D, Representative plots showing the compared with the low fluorescence intensity of untreated cells in amount of GRGDSP-FITC (C) or VCAM-1-Fc (D) bound to F4/80+ PECs which the integrins were more evenly distributed. Treatment of the after 300 or 120 s, respectively. PECs for 1 min with 10 nM chemerin had a very similar effect to that of PMA. It increased both a5 and a4 fluorescent intensity on + patches of the macrophage surface, suggesting that chemerin can the average a5 fluorescence intensity of F4/80 macrophages (Fig. induce rapid clustering of both VLA-5 and VLA-4 integrins, re- 5C) and a 6-fold (p , 0.01) increase in the average a4 fluores- spectively. Chemerin stimulated a 2.6-fold (p , 0.01) increase in cence intensity of F4/80+ macrophages (Fig. 5D) (compared with 3734 CHEMERIN MEDIATES MACROPHAGE ADHESION Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 5. Chemerin stimulates integrin clustering. A–D, PECs from C57BL/6J mice were stimulated for 1 min at 37˚C with media, 100 ng/ml PMA or

10 nM chemerin. The cells were immediately fixed in 2% PFA on ice, washed, and stained with either anti-CD49e (a5)-FITC (A, C) or anti-CD49d (a4)- + FITC (B, D) Abs and anti–F4/80-Alexa Fluor 647 Ab. The cells were adhered to poly-D-lysine–coated slides and integrin clustering on F4/80 macrophages analyzed by confocal microscopy. F4/80+ cells were identified as those cells with average fluorescence intensity for F4/80 exceeding a predefined lower limit. Images representative of confocal photomicrographs taken from three independent experiments. Magnification 31260. Graphs show mean values 6 SEM from three independent experiments; pp , 0.05; ppp , 0.01, one-way ANOVAwith Dunnett’s posthoc test compared with “untreated.” E, PECs were pretreated for 1 h at room temperature with cytochalasin D (0.0015–0.5 mg/ml), CellTiter-Glo was added to each well, and the luminescence was measured to quantify the number of live cells. Graph shows mean values 6 SD. F and G, PECs were pretreated for 1 h at room temperature with 0.015 mg/ml cytochalasin D, then added to wells that had been precoated with 30 mg/ml fibronectin (F)or2mg/ml VCAM-1 (G) and incubated for 1 min at 37˚C in the presence of media alone or 10 nM chemerin. Plates were shaken for 20 s, and the wells washed thoroughly with media to remove nonadherent cells. CellTiter-Glo was added to each well, and the luminescence was measured to quantify the remaining adherent cells. Graph shows mean values 6 SEM from three independent experiments; pp , 0.05; ppp , 0.01, two-way ANOVA with Bonferroni’s posthoc test. the respective 2.5-fold [p , 0.05] and 5.2-fold [p , 0.05] in- toxic effects on the PECs (determined using Promega’s CellTiter-Glo creases following PMA stimulation). The greater capacity of assay) (Fig. 5E). Pretreatment with 0.015 mg/ml cytochalasin D had no chemerin to stimulate VLA-4 clustering compared with VLA-5 effect on untreated PEC or chemerin-stimulated PEC adhesion to clustering is in accordance with the adhesion data in Fig. 1C and fibronectin (Fig. 5F). However, cytochalasin D pretreatment increased 1D in which chemerin stimulates a greater fold change in the both the number of untreated cells (64.0% increase; p , 0.05) and number of adherent cells on VCAM-1 than on fibronectin. chemerin-treated cells (75.9% increase; p , 0.01) adhering to Low doses of cytochalasin D (0.05–5 mg/ml), an inhibitor of actin VCAM-1 (Fig. 5G). These results suggest that actin polymerization polymerization, have been shown to both inhibit and promote integrin is not required for chemerin-stimulated integrin clustering as no clustering and leukocyte adhesion (36–38). To investigate the role of inhibitory effect of cytochalasin D was observed. Cytochalasin D the actin cytoskeleton in chemerin-stimulated adhesion, we looked has been shown to increase the lateral mobility of integrins by re- at the effect of pretreating PECs with cytochalasin D for 1 h in the leasing them from their cytoskeletal restraints, leading to integrin adhesion assay. We used 0.015 mg/ml cytochalasin D, because this clustering and adhesion. This would explain the results of Fig. 5G was the highest concentration of the inhibitor that exhibited no cyto- and implies that chemerin may act via a similar mechanism. The Journal of Immunology 3735

Akt, PI3K, and p38 play key roles in chemerin-stimulated pathway. A combination of LY294002 and SB202190 produced adhesion to fibronectin and VCAM-1 a significantly greater inhibition (83.6% reduction) than the two To investigate which intracellular signaling pathways are important inhibitors alone but did not completely block chemerin-stimulated in chemerin-mediated macrophage adhesion, we pretreated PECs macrophage adhesion to fibronectin. This suggests that activation of for 1 h with various cell signaling inhibitors at concentrations Akt is not completely dependent upon p38 activation. A putative demonstrated not to have cytotoxic effects (data not shown) and signaling flow diagram based on our results is presented in Fig. 7. used these cells in the adhesion assay. Chemerin-induced adhesion In contrast with the results obtained in the fibronectin adhesion to fibronectin was significantly reduced by pretreatment of PECs assays, none of the inhibitor combinations could completely block with Akt inhibitor IV (54.6% reduction; p , 0.001), LY294002 chemerin-stimulated adhesion to VCAM-1, and they had only a (54.3% reduction; p , 0.001), and SB202190 (49.6% reduction; slightly greater inhibitory effect than Akt inhibitor IVor SB202190 p , 0.001), indicating a role for Akt, PI3K, and p38, respectively alone (Akt inhibitor IV plus LY294002,77.4%; Akt inhibitor IV plus (Fig. 6A). Inhibitors of calcium flux (BAPTA-AM), Syk (picea- SB202190, 74.3%; and LY294002 plus SB202190, 84.1% re- tannol), Src (PP2), and ERK1/2 (U1026) had no effect on chemerin- duction) (Fig. 6D). stimulated adhesion to fibronectin. Inhibitors of Akt, PI3K and p38 also significantly reduced ad- Discussion hesion to VCAM-1 in response to chemerin treatment (Akt inhibitor Chemerin functions as a potent macrophage chemoattractant via the IV,57.3%; LY294002, 78.6%; and SB202190, 65.6% reduction; p , GPCR ChemR23 (3, 8). In this study, we provide the first evidence 0.001), suggesting the involvement of Akt, PI3K, and p38 in that chemerin can induce the adhesion of ChemR23-expressing

chemerin-mediated adhesion to VCAM-1 as well as to fibronectin macrophages to recombinant adhesion molecules and extracellular Downloaded from (Fig. 6B). In contrast to the adhesion to fibronectin, BAPTA-AM matrix proteins through an increase in integrin clustering. Chemerin (34.6% reduction; p , 0.001), piceatannol (36.8% reduction; p , stimulates the adhesion of PECs to fibronectin and VCAM-1 in 0.001), PP2 (39.0% reduction; p , 0.001), and U0126 (42.0% re- a dose-dependent, PTX-sensitive manner within a minute. The use duction; p , 0.001) all significantly inhibited PEC adhesion to of PECs from ChemR232/2 mice revealed that chemerin-stimulated VCAM-1 resulting from chemerin treatment. This suggests that adhesion to fibronectin is dependent on the expression of the Gi- calcium flux, Syk, Src, and ERK1/2, respectively, play a role in coupled receptor ChemR23, as is nearly half of chemerin-stimulated http://www.jimmunol.org/ chemerin-stimulated adhesion to VCAM-1 but not to fibronectin. adhesion to VCAM-1. Flow cytometry demonstrated that ChemR23 Pretreatment of PECs with a combination of Akt inhibitor IV and is expressed only on macrophages and immature monocyte-mac- LY294002 almost completely blocked (96.5% reduction) chemer- rophages within this mixed cell population. Therefore, we conclude in-stimulated adhesion to fibronectin, suggesting that Akt and PI3K that chemerin promotes the adhesion of ChemR23-expressing mac- are involvedinseparate pathways downstreamof ChemR23(Fig.6C). rophages to fibronectin and VCAM-1. A combination of Akt inhibitor IV and SB202190 did not further A peptide derived from the last nine amino acids of murine inhibit adhesion to fibronectin (67.5% reduction) than either of these chemerin, C9, has previously been shown to bind to ChemR23 and to inhibitors alone; therefore, p38 and Akt appear to reside in the same inducecalcium flux through this receptor (10). In agreement with this, by guest on October 1, 2021

FIGURE 6. Akt, PI 3K and p38 play key roles in chemerin-stimulated adhesion to fibronectin and VCAM-1. A–D, PECs from C57BL6/J mice were pretreated for 1 h at room temperature with 1 mM Akt inhibitor IV, 5 mM BAPTA-AM, 5 mM LY294002, 20 mM piceatannol, 10 mM PP2, 20 mM SB202190, 20 mM U0126, or combinations of these inhibitors as indicated in the graphs. The pretreated PECs were added to wells that had been precoated with 30 mg/ ml fibronectin (A, C)or2mg/ml VCAM-1 (B, D) and incubated for 1 min at 37˚C in the presence of media alone or 10 nM chemerin. Plates were shaken for 20 s, and the wells washed thoroughly with media to remove nonadherent cells. CellTiter-Glo was added to each well, and the luminescence was measured to quantify the remaining adherent cells. Graphs show mean values 6 SEM from three independent experiments; ppp , 0.01; pppp , 0.001, one-way ANOVA with Dun- nett’s posthoc test compared with “no in- hibitor” (A, B) or Bonferroni’s posthoc test (C, D). 3736 CHEMERIN MEDIATES MACROPHAGE ADHESION

macrophages resulting from IFN-g stimulation alone retain their ChemR23 expression and their ability to adhere to fibronectin and VCAM-1 in response to chemerin.) This suggests that chemerin- stimulated adhesion may only be relevant in the very early initiating stages of inflammation. However, M1 and M2a macro- phages represent the extreme phenotypes and in inflammatory environments, where a number of stimulatory ligands are present, macrophages exhibit plasticity and may display a spectrum of func- tionalities for which it is hard to predict their responsiveness to chemerin (46). Although we have focused on macrophages, ChemR23 is addi- FIGURE 7. A diagram to illustrate the putative signaling pathway tionally expressed on monocytes, pDCs, and NK cells in humans and downstream of ChemR23 that leads to adhesion to fibronectin. was also recently shown to be expressed by pDCs and NK cells in the mouse (5–7, 10). Because we have shown that chemerin rapidly increases murine macrophage adhesion to VCAM-1, it is possible we found that C9 is capable of stimulating the adhesion of that chemerin can induce the rapid adhesion of monocytes, NK cells, ChemR23-transfected cells to fibronectin and is in fact slightly more or pDCs to VCAM-1 presented on activated vascular endothelium potent than chemerin in this assay, although it is a partial agonist that as well as to fibronectin, thereby recruiting these cells to sites of is unable to achieve the maximal cell adhesion induced by chemerin. inflammation. Chemerin has an overall positive charge that would C15, another peptide derived from the C terminus of chemerin, has allow it to be presented on negatively charged heparin or sulfated Downloaded from been shown to have anti-inflammatory and prophagocytic effects on glycosaminoglycans, making monocyte, NK cell, and pDC re- macrophages that are dependent on expression of the ChemR23 cruitment from the bloodstream a feasible hypothesis (14). Indeed, receptor (8, 29). We were unable to see an effect of C15 on the ad- chemerin immunoreactivity has been detected on endothelial cells hesion of ChemR23-transfected cells to fibronectin. This also agrees lining blood vessels in inflamed tissues and chemerin expression has with the data of Luangsay et al. (10) who were unable to detect been shown to correlate with ChemR23+ pDC recruitment in pso- calcium release in ChemR23-transfected cells in response to C15 riatic skin biopsies (6, 47–49). http://www.jimmunol.org/ treatment. This does not, however, rule out the possibility that C15 activates alternative signaling pathways downstream of ChemR23 Chemerin-stimulated integrin regulation on macrophages by acting as an allosteric modulator or by binding to Integrins are the receptors expressed on leukocytes that bind to ex- a heterodimer of ChemR23 and another unknown GPCR. tracellular matrix proteins and adhesion molecules and are conse- quently responsible for leukocyte adhesion (30). The binding of many Chemerin, macrophages, and inflammation chemoattractants to their GPCRs can initiate a process termed insi- Leukocyte adhesion and chemotaxis are important for maintaining de-out signaling that results in an increased capacity for the integrins tissue homeostasis as well as in generating a successful inflammatory to bind their ligands (16, 50). Although the regulation of integrin by guest on October 1, 2021 response (2). Given that chemerin is activated by inflammatory activity has been well studied in many leukocyte subsets, such as proteases and that ChemR23 is predominantly expressed by mac- neutrophils, monocytes and lymphocytes, this process in primary rophages in the mouse, chemerin is most likely to contribute to the macrophages remains relatively unexplored. We used blocking Abs to development of inflammatory diseases in which macrophages play identify VLA-5 and VLA-4 as the integrins responsible for chemerin- a central role (e.g., atherosclerosis or arthritis [9, 11]). Taking ath- stimulated macrophage adhesion to fibronectin and VCAM-1, re- erosclerosis as an example, the migration of tissue and newly dif- spectively. LPAM-1 was also partially responsible for chemerin- ferentiated macrophages from the surrounding intima and adventitia stimulated adhesion to VCAM-1. to the atherosclerotic lesion requires adhesion via integrins to ex- Chemerin’s effects on adhesion are extremely rapid and so we tracellular matrix proteins, such as fibronectin (22, 39, 40). The ad- considered it unlikely that an upregulation in integrin expression on hesion of macrophages to the extracellular matrix and to VCAM-1 the surface of macrophages was responsible for the increase in expressed on smooth muscle cells that have migrated into the sub- adhesion to fibronectin and VCAM-1. GPCR agonist-stimulated endothelial space, are thought to contribute to macrophage retention inside-out signaling increases integrin avidity and subsequently within the atherosclerotic plaque, thereby potentiating the pathogenic leukocyte adhesion (16). An increase in integrin avidity can result process (41, 42). Macrophage adhesion to fibronectin has been shown from either an increase in integrin affinity and/or an increase in to increase the expression of proinflammatory molecules, such as integrin valency (51). Many chemoattractants are capable of al- matrix metalloproteinases, which have a deleterious effect on plaque tering the conformational state of integrins, thereby increasing their stability (21, 43, 44). Our findings suggest that chemerin could affinity. For example, CXCL12 promotes a rapid increase in the promote any one of the above processes, thereby contributing to the affinity of VLA-4 on monocytes for VCAM-1 (25). However, our progression of an inflammatory disease, such as atherosclerosis. experiments using soluble ligands revealed that chemerin is unable Macrophages that have been activated by a combination of IFN-g to increase the affinity of either VLA-4 or VLA-5 (or LPAM-1) on plus TNF-a (LPS) are known as classically activated M1 macro- macrophages. phages, whereas macrophages activated by IL-4 are known as al- Increased integrin valency results from cytoskeletal rearrange- ternatively activated M2a macrophages (45). These two proinflam- ments that result in the redistribution of integrins on the cell surface matory macrophage types have distinct functional properties. Our (51, 52). This increases the cooperative binding of the integrins, results suggest that polarized macrophages no longer adhere to fi- thereby increasing the overall bond strength and allowing leukocyte bronectin or VCAM-1 in response to chemerin stimulation, either adhesion (51, 53). Using confocal microscopy, we demonstrated that because of a complete downregulation in ChemR23 expression chemerin stimulates the ligand-independent clustering of VLA-4 in the case of M1 macrophages or, in the case of M2a macrophages and VLA-5, thereby providing a mechanism for chemerin-stimu- that retain their ChemR23 expression, for another reason that lated adhesion to VCAM-1 and fibronectin, respectively. Some may involve the functionality of the integrins themselves. (M1 chemokines have also been shown to stimulate integrin clustering The Journal of Immunology 3737 without stimulating an increase in integrin affinity. For example, Many studies have found a role for PI3K in integrin activation. For CXCL12 promotes the rapid clustering of VLA-4 on lymphocytes to example, clustering of LFA-1 in lymphocytes was shown to be achieve adhesion to VCAM-1 while having no effect on VLA-4 dependent on PI3K activity, but PI3K was not involved in increases affinity (35). Integrin clustering alone is sufficient to support cell in LFA-1 affinity in these cells (60). It was suggested that PI3K, adhesion under the flow conditions that model the environment through its ability to promote cytoskeletal remodeling, might re- found within blood vessels (54). lease LFA-1 from its cytoskeletal restraints, resulting in integrin It is generally thought that release of integrins from cytoskeletal clustering and lymphocyte adhesion. The calcium-dependent pro- restraints increases integrin lateral mobility and allows integrin tease calpain has also been implicated in the cleavage of a cyto- clustering and leukocyte adhesion to take place (52). Evidence for this skeletal component, triggering the release of LFA-1 from the cyto- comes from the stimulatory effect of cytochalasin D, an inhibitor of skeleton and integrin clustering (61). A similar mechanism may be actin polymerization on integrin clustering and the inhibitory effect partly responsible for chemerin-stimulated VLA-4 clustering, be- of integrin mutations on integrin clustering arising from an increase cause inhibition of calcium flux partially inhibited cell adhesion to in the cytoskeletal anchoring of the integrin tail (37, 38, 55). In VCAM-1 agreement with this, cytochalasin D pretreatment increased un- treated and chemerin-stimulated adhesion to VCAM-1. It did not ChemR23-independent adhesion inhibit chemerin-stimulated adhesion to VCAM-1 or fibronectin, Chemerin-stimulated adhesion of macrophages to fibronectin is arguing against an active recruitment of integrins into clusters completelydependenton theexpression of ChemR23.Unexpectedly, through actin polymerization (36). This implies that chemerin may chemerin is still capable of promoting the adhesion of PECs function through a similar mechanism, acting to release cytoskeletal from ChemR232/2 mice to VCAM-1, revealing that just under half restraints holding VLA-4 and VLA-5 in place, thereby allowing of chemerin-stimulated adhesion to VCAM-1 is dependent on Downloaded from them to passively diffuse into clusters. There is some evidence to ChemR23 expression. Chemerin was originally thought to bind only suggest that movement of integrins into lipid rafts allows focused to ChemR23, thereby forming a highly specific receptor-ligand pair, clustering to take place and is required for cell adhesion (56). but recent studies have identified two additional high-affinity re- The lack of effect of cytochalasin D on cell adhesion to fibronectin ceptors for chemerin. CCRL2 is a nonsignaling GPCR expressed may be explained by a low expression of VLA-5 on the macrophage on mast cells and macrophages that is proposed to bind chemerin, surface. The number of untreated PECs adhering to fibronectin was increasing its local concentration and presenting it for interaction http://www.jimmunol.org/ 3.4-fold lower than the binding of these cells to VCAM-1 (data not with ChemR23 expressed on adjacent cells (28). Low levels of shown). This may result from a lower expression of VLA-5 on the CCRL2 mRNA are expressed by the PECs used in our experiments; surface, and together with the low dose used of this inhibitor to avoid however, CCRL2’s reported inability to initiate intracellular sig- cytotoxic effects, cytochalasin D may not be able to sufficiently re- naling would seem to rule it out as the additional receptor mediating lease enough VLA-5 integrins from the cytoskeleton to cause clus- chemerin-stimulated adhesion to VCAM-1 (28). Chemerin also tering and adhesion, unlike 10 nM chemerin. binds to the GPCR GPR1, initiating an increase in intracellular calcium that is also seen in cells expressing ChemR23 (27). How- Signaling pathways mediating chemerin-stimulated adhesion ever, negligible levels of GPR1 mRNA are expressed by PECs, by guest on October 1, 2021 In common with other chemoattractants, chemerin’s ability to in- suggesting the existence of a fourth unknown chemerin receptor. duce adhesion to fibronectin and VCAM-1 is dependent on the Pretreatment with PTX completely ablates chemerin-stimulated activation of a Gi G protein (23–26). Many studies have looked at adhesion to VCAM-1, implying that the unknown receptor is cou- the signaling requirements downstream of the Gi G protein that are pled to a Gi G protein. A possible candidate for the unknown re- required for stimulation of leukocyte adhesion (16, 57). However, it ceptor is the leukotriene B4 receptor (BLT1), which is expressed at has become clear that no single pathway is responsible, rather the the mRNA level by PECs. The lipid resolvin E1 has been reported to signaling pathway depends on the specific leukocyte subset, the be a ligand for both ChemR23 and BLT1 (62, 63). agonist, GPCR, and specific integrin involved. The inside-out In summary, we have shown for the first time that the macrophage pathways responsible for increased integrin avidity in macro- chemoattractant protein chemerin, which is generated at sites of in- phages, in particular, have not been studied in detail, and the same is flammation, rapidly stimulates the adhesion of macrophages to fi- true of the pathways responsible for increases in integrin valency in bronectin and VCAM-1 in a ChemR23-dependent manner, by pro- other leukocyte subsets. Agonists at Gi-coupled GPCRs are capable moting clustering of the integrins VLA-5and VLA-4,respectively.We of activating a multitude of signaling pathways involving PI3K, have also identified some of the key signaling components involved in phospholipase C, tyrosine kinases, MAPKs, and small GTPases the regulation of VLA-5 and VLA-4 valency on macrophages in re- (58, 59). Chemerin was previously demonstrated to induce calcium sponse to chemerin treatment. Chemerin-stimulated adhesion may flux and activate ERK through ChemR23 (3). play an important role in recruiting and retaining macrophages at sites In this study, we have clearly demonstrated that PI3K, Akt, and of inflammation and in their subsequent activation. p38 are involved in chemerin-stimulated adhesion to fibronectin and therefore in the increase in VLA-5 clustering on the macrophage Acknowledgments surface. Combinations of inhibitors revealed that Akt and PI3K are We thank Linda Randall, Denise Jelfs, Jenna Cash, Thomas Tan, and involved in separate pathways downstream of ChemR23, whereas Gemma White for advice and support. p38 and Akt reside in the same pathway (Fig. 7). Similarly, PI3K, Akt, and p38 play a key role in chemerin-stimulated adhesion to Disclosures VCAM-1 and therefore in the increase in the clustering of VLA-4. The authors have no financial conflicts of interest. However, combinations of inhibitors against these signaling com- ponents are unable to completely block chemerin-stimulated ad- hesion to VCAM-1, and instead, calcium flux, Syk, Src, and ERK References play a role. Taken together, our data suggest that chemerin activates 1. Majno, G., and I. Joris. 2004. Cells, Tissues, and Disease: Principles of General Pathology. Oxford University Press, New York, Oxford. at least one additional separate signaling pathway that leads to 2. Sallusto, F., and C. R. Mackay. 2004. Chemoattractants and their receptors in VLA-4 clustering and adhesion to VCAM-1. homeostasis and inflammation. Curr. Opin. Immunol. 16: 724–731. 3738 CHEMERIN MEDIATES MACROPHAGE ADHESION

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