The CD200 Receptor Regulation of Myeloid Cell Function Through

Regulation of Myeloid Cell Function through the CD200 Receptor Maria C. Jenmalm, Holly Cherwinski, Edward P. Bowman, Joseph H. Phillips and Jonathon D. Sedgwick This information is current as of September 27, 2021. J Immunol 2006; 176:191-199; ; doi: 10.4049/jimmunol.176.1.191 http://www.jimmunol.org/content/176/1/191 Downloaded from

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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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Regulation of Myeloid Cell Function through the CD200 Receptor1

Maria C. Jenmalm,2 Holly Cherwinski, Edward P. Bowman,3 Joseph H. Phillips, and Jonathon D. Sedgwick3

Myeloid cells play pivotal roles in chronic inflammatory diseases through their broad proinflammatory, destructive, and remod- eling capacities. CD200 is widely expressed on a variety of cell types, while the recently identified CD200R is expressed on myeloid cells and T cells. CD200 deletion in vivo results in myeloid cell dysregulation and enhanced susceptibility to autoimmune inflammation, suggesting that the CD200-CD200R interaction is involved in immune suppression. We demonstrate in this study that CD200R agonists suppress mouse and human myeloid cell function in vitro, and also define a dose relationship between receptor expression and cellular inhibition. IFN-␥- and IL-17-stimulated cytokine secretion from mouse peritoneal macrophages was inhibited by CD200R engagement. Inhibitory effects were not universal, as LPS-stimulated responses were unaffected. Inhibition Downloaded from of U937 cell cytokine production correlated with CD200R expression levels, and inhibition was only observed in low CD200R expressing cells, if the CD200R agonists were further cross-linked. Tetanus toxoid-induced human PBMC IL-5 and IL-13 secretion was inhibited by CD200R agonists. This inhibition was dependent upon cross-linking the CD200R on monocytes, but not on cross-linking the CD200R on CD4؉ T cells. In all, we provide direct evidence that the CD200-CD200R interaction controls monocyte/macrophage function in both murine and human systems, further supporting the potential clinical application of CD200R agonists for the treatment of chronic inflammatory diseases. The Journal of Immunology, 2006, 176: 191–199. http://www.jimmunol.org/

yeloid cells (i.e., macrophages, dendritic cells (DC),4 arthritis (CIA) (10), suggesting that CD200 normally induces im- neutrophils, mast cells, and eosinophils) play impor- mune suppression through CD200R. Consistent with this was the M tant roles in maintaining chronic inflammation (1–5). demonstration that a soluble form of CD200 (mouse (m) CD200Ig) They can be regulated through cell-cell interactions that trigger administered to mice blocks autoimmune inflammation in vivo matched sets of activating and inhibitory receptors, in addition to (11). In terms of mechanism of action, endogenous CD200 (and being regulated by secreted factors (6). The regulation of myeloid soluble CD200Ig) could send a signal that deviates the immune cell activity by direct cell-cell contact allows a more localized response away from a damaging effector pathway, as proposed by guest on September 27, 2021 control than that mediated by cytokines. The CD200-CD200R in- (12), and/or it could directly inhibit effector cell function (10). As teraction also provides a cell-cell contact regulatory interaction for in vivo studies cannot discriminate between these possibilities, a myeloid cells. The widely expressed glycoprotein CD200 is mast cell model was established to test whether CD200R directly closely related structurally to the T cell costimulatory molecules mediates cellular inhibition. IgE-mediated activation of mast cells CD80 and CD86 and is genetically linked to them on human chro- was inhibited by mCD200Ig or an agonist rat-anti-mCD200R mosome 3 and mouse chromosome 16 (7, 8). Structurally, CD200 (DX109) when the cells were transfected with mCD200R, and by contains two Ig superfamily (IgSF) domains in a typical V/C2 human (h) CD200Ig or agonist rat-anti-hCD200R (DX136 and arrangement (9). DX153) when the cells were transfected with hCD200R (13). Deletion of CD200 resulted in myeloid cell dysregulation and These agonists directly blocked mast cell degranulation as well as enhanced susceptibility to autoimmune inflammation such as ex- TNF and IL-13 secretion, with a typical IC50 of 0.2–1 nM. Thus, perimental autoimmune encephalitis (EAE) and collagen-induced CD200 directly triggered cellular inhibition through CD200R engagement. SP Biopharma, Palo Alto CA 94022 The CD200R is expressed at the surface of human and mouse Received for publication April 28, 2005. Accepted for publication October 7, 2005. myeloid cells, such as macrophages, DCs, neutrophils, and mast The costs of publication of this article were defrayed in part by the payment of page cells, and also on T cells (14, 15). CD200R is closely structurally charges. This article must therefore be hereby marked advertisement in accordance related to CD200, located on the same chromosome, the genes with 18 U.S.C. Section 1734 solely to indicate this fact. probably evolved by gene duplication (15). However, the receptor 1 SP Biopharma (previously DNAX Research) is supported by Schering Plough, New is distinct from CD200 by virtue of a longer cytoplasmic tail con- Jersey. taining three conserved tyrosine residues, one of which is con- 2 Current address: Department of Molecular and Clinical Medicine, Division of Pe- diatrics, and Clinical Research Centre, Faculty of Health Sciences, Linko¨ping Uni- tained within an NPXY motif (14, 15). Phosphorylated NPXY mo- versity, Sweden. tifs bind phosphotyrosine-binding domains that are present in 3 Address correspondence and reprint requests to Dr. Edward P. Bowman, SP Bio- signaling adaptor molecules such as Shc (16), suggesting that the pharma, 901 California Avenue, Palo Alto, CA 94022; E-mail address: CD200R can signal after ligation by CD200. CD200R lacks an [email protected] or Dr. Jonathon D. Sedgwick at the current address: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285; E-mail address: ITIM motif present in almost all immune inhibitory receptors (17). [email protected] Using the mast cell system, we have shown that the NPXY motif 4 Abbreviations used in this paper: DC, dendritic cell; IgSF, Ig superfamily; EAE, is critical for receptor activity, and that signaling and cellular in- experimental autoimmune encephalitis; CIA, collagen-induced arthritis; h, human; m, mouse; CBA, cytometric bead array; IP-10, interferon-inducible protein 10; MIG, hibition are linked to Dok1 and Dok2 phosphorylation and subse- monokine induced by IFN-␥; TT, tetanus toxoid. quent Ras/MAPK pathway inhibition (18).

The aim of the present study was to further characterize the at 37°C in a humidified incubator containing 5% CO2. After 5 days in Ϫ ϩ ϩ regulation of myeloid cell function through the CD200R in vitro, culture, cells were CD14 , CD11c , and HLA-DR . focusing especially on the effects of the CD200R on macrophage A total of 500,000 DCs were cultured in 0.5 ml of RPMI 1640 with 10% heat-inactivated FCS and 1% penicillin and streptomycin in Falcon 24-well and DC function in human and mouse. plates at 37°C in a humidified incubator containing 5% CO2. After a 30- min incubation with hCD200IgG, control Ig, rat-anti-hCD200R (DX153), or rat IgG1 isotype control, the cells were stimulated with LPS, rCD40L Materials and Methods (R&D Systems) and rIFN-␥, rIFN-␥ alone or rTNF (R&D Systems), and CD200R-binding reagents rIL-1␤ (R&D Systems) for 24 and 48 h. In some experiments, cells were washed after the 30-min incubation, and cell-bound hCD200IgG or rat- Rat mAbs that were agonistic for mouse and human CD200R were ␮ Ј anti-hCD200R were cross-linked with 5 g/ml endotoxin-depleted F(ab )2 generated in rats as previously described (15). Abs used were DX109 goat anti-mouse/rat IgG, and incubated for another 30 min before addition (ratIgG1-anti-mCD200R), DX136 (ratIgG2a-anti-hCD200R), and DX153 of stimuli. Supernatants were collected and IL-1␣, IL-1␤, IL-6, IL-8/ (ratIgG1-anti-hCD200R). DX109 does not bind the activating DNAX activat- CXCL 8 (CXCL8), IL-10, IL-12p70, IFN-inducible protein 10 (IP-10)/ ing protein 12 (DAP-12)-paired mCD200RLa and mCD200RLb (15). CXCL10, MCP-1/CCL2, monokine induced by IFN-␥ (MIG)/CXCL9, hCD200RLa is not expressed and is probably nonfunctional (15). MIP-1␣/CCL3, RANTES/CCL5, and TNF secretion was analyzed by Ligand (CD200)-Ig fusion proteins were generated as described (13) by ELISA, CBA, or the LINCOplex Simultaneous Multianalyte Detection fusion of the extracellular domain of human or mouse CD200 to the Fc System as described below. Expression of the cell surface activation mark- domain of mIgG1 mutated in the CH2 domain (D265A) to inhibit binding ers CD1a, CD32, CD80, CD83, CD86, and HLA-DR was analyzed by flow to FcRs (19). mCD200Ig does not bind the activating DAP-12-paired cytometry as described below. mCD200RLa and mCD200RLb (15, 20). Human monocyte cell line. All mAb and protein preparations used contained Ͻ0.1 ng of endotox- Human U937 monocytic cells (American Type Culture Collection) were transfected with hCD200R. cDNA encoding in/mg protein, as determined by the Limulus Amebocyte Lysate Pyrogen Downloaded from Testing kit, QCL-1000 (BioWhittaker). full-length hCD200R was subcloned into the pMX-pie retrovirus expression vector (21) containing a puromycin resistance gene, an internal ribo- somal entry site (IRES) element, and an enhanced GFP gene. Plasmid Cells and cell culture DNA was then transfected into the Phoenix ecotropic virus packaging cell Mouse peritoneal cells. Resident peritoneal cells were obtained by peri- line (a gift from G. Nolan, Stanford University, Palo Alto, CA). The U937 toneal lavage of 8- to 12-wk-old C57BL/6 mice (The Jackson Laboratory) cells were infected by coculture with the transfected packaging cell line. with 5 ml of DMEM (Mediatech). A total of 1 ϫ 106 cells were cultured After drug selection, the resulting hCD200R or control vector transfectants in 1 ml of DMEM with 10% heat-inactivated FCS (HyClone) and 1% were isolated by FACS on a FACSVantage System (BD Immunocytometry http://www.jimmunol.org/ penicillin and streptomycin (Sigma-Aldrich) in Falcon 24-well plates (BD Systems) based on GFP expression. The hCD200R transfectant was further Biosciences Discovery Labware) at 37°C in a humidified incubator con- sorted into very low, low, medium, and high hCD200R-expressing cells after staining with PE-labeled anti-hCD200R (DX136). Stable expression taining 5% CO2. In some experiments, nonadherent cells were removed after2htoobtain a more pure macrophage population. After a 30-min of the hCD200R was seen over several months of culture. The cell lines incubation with mCD200Ig, control Ig, rat-anti-mCD200R (DX109), or rat were grown in RPMI 1640 (Mediatech) with 10% heat-inactivated FCS, IgG1 isotype control (BD Pharmingen), cells were stimulated with recom- 1% penicillin and streptomycin, 20 mM HEPES buffer (Fisher Chemical), binant murine IFN-␥ (R&D Systems), recombinant murine IL-17 (DNAX), 1 mM sodium pyruvate (BioWhittaker), and 0.1 mM nonessential amino or Salmonella typhimurium LPS (Sigma-Aldrich) for 18 h. Cell superna- acids (Invitrogen Life Technologies) at 37°C in a humidified incubator tants were collected and analyzed for IL-6, IL-10, IL-12p70, MCP-1/ containing 5% CO2, and split every 3–4 days. ϫ 6 CCL2, IFN-␥, and TNF secretion by ELISA or cytometric bead array A total of 1 10 hCD200R or vector control-transfected U937 cells by guest on September 27, 2021 (CBA) as described below. were cultured in 1 ml of RPMI 1640 with the supplements described above in Falcon 24-well plates at 37°C in a humidified incubator containing 5% Human peripheral blood cells. PBMC were isolated from heparinized CO . After a 30-min incubation with hCD200Ig, control Ig, rat-anti- blood of recently tetanus toxoid (TT)-vaccinated healthy donors, using 2 ϫ 6 hCD200R (DX153), or rat IgG1 isotype control, hCD200R or vector con- Ficoll-Paque density gradient (Amersham Biosciences). A total of 1 10 trol-transfected U937 cells were stimulated with 100 U/ml recombinant PBMC were cultured in 1 ml of RPMI 1640 with 10% heat-inactivated human IFN-␥ (R&D Systems) for 18 h. In some experiments, cells were FCS and 1% penicillin and streptomycin in Falcon 24-well plates at 37°C washed after the 30-min incubation and the cell-bound hCD200Ig or rat- in a humidified incubator containing 5% CO . After a 30-min incubation 2 anti-hCD200R were cross-linked with 5 ␮g/ml endotoxin-depleted F(abЈ) with hCD200IgG, control-Ig, rat-anti-hCD200R (DX153), or rat IgG1 iso- 2 goat anti-mouse/rat IgG (Jackson ImmunoResearch Laboratories), and in- type control, the cells were stimulated with inactivated Clostridium tetani cubated for another 30 min before addition of IFN-␥. Cell supernatants TT (Calbiochem) for 6 days. In some experiments, cells were washed after were collected and analyzed for IL-1␤, IL-6, IL-8/CXCL8, IL-10, TNF, the 30-min incubation, and cell-bound hCD200IgG or rat-anti-hCD200R IL-12p70, IP-10/CXCL10, MCP-1/CCL2, MIG/CXCL9, and RANTES/ were cross-linked with 5 ␮g/ml endotoxin-depleted F(abЈ) goat anti- 2 CCL5 secretion by ELISA or CBA as described below. mouse/rat IgG, and incubated for another 30 min before addition of TT. Supernatants were collected and IL-2, IL-4, IL-5, IL-10, IL-12p70, IL-13, IFN-␥, and TNF secretion was analyzed by ELISA, CBA (BD Pharmin- gen), or the LINCOplex Simultaneous Multianalyte Detection Systems Flow cytometry (Linco Research) as described below. ϩ Cells were stained and washed in PBS containing 0.1% NaN3 (Sigma- Both human blood monocytes and CD4 T cells express the CD200R, Aldrich) and either 1% heat-inactivated FCS for mouse cells or 1% heat- and Ag-induced cytokine responses require cooperation between Ag-pre- inactivated normal human serum (Jackson ImmunoResearch Laboratories) senting monocytes and T cells (15). To study the relative contribution of for human cells. To block Fc␥Rs, 1 ϫ 106 cells/tube were incubated with cross-linking the CD200R on monocytes or CD4ϩ T cells before TT stim- ␮ ϩ 0.1 g of Fc block (anti-CD16/CD32 (clone 2.4G2); BD Pharmingen) for ulation, CD14 monocytes were positively selected from PBMC on an mouse cells or 10% heat-inactivated normal human serum for human cells. autoMACS (Miltenyi Biotec) using CD14 microbeads (Miltenyi Biotec), The cells were then stained with FITC-, PE-, PerCP-, allophycocyanin-, or according to the manufacturer’s protocol. CD4ϩ T cells were negatively Ϫ ϩ biotin-conjugated Abs (BD Pharmingen, except as indicated) for 30 min selected on an autoMACS from the CD14 fraction using the CD4 T cell 4°C in the dark. Biotinylated Abs were secondarily labeled with strepta- Isolation kit II (Miltenyi Biotec), according to the manufacturer’s protocol. vidin-allophycocyanin (BD Pharmingen). Cells were stained with Abs to Cells were Ͼ95% pure after isolation, as analyzed by flow cytometry as ϩ mouse CD11b (clone M1/70), mouse CD200R (DX109; DNAX), mouse described below. A total of 250,000 monocytes/well and 750,000 CD4 T F4/80 (clone CI:A3-1; Caltag Laboratories), mouse Gr-1 (Ly-6G/Ly-6C, cells/well were incubated separately, in the presence of rat-anti-hCD200R clone RB6-8C5), human CD1a (clone HI149), human CD3 (clone HIT3a), (DX153) and cross-linking Ab as above. After washing, the monocytes and human CD4 (clone RPA-T4), human CD8 (clone HIT8a), human CD11c T cells were then mixed and stimulated with TT. (clone B-ly6), human CD14 (clone M5E2), human CD19 (clone HIB19), Human DCs. Monocytes were negatively selected from PBMC on an au- human CD20 (clone L27), human CD32 (clone FLI8.26), human CD80 toMACS using the Monocyte Isolation kit II (Miltenyi Biotec), according (clone L307.4), human CD83 (clone HB15e), human CD86 (clone FUN-1), to the manufacturer’s protocol. To generate immature DCs, 2 ϫ 106 mono- human CD200R (DX136; DNAX), human HLA-DR (clone L243), lin 1 cytes were cultured in 100 ng/ml GM-CSF (Schering-Plough) and 5 ng/ml (mixture of CD3 (clone SK7), CD14 (clone M␾P9), CD16 (clone 3G8), IL-4 (Schering-Plough) for 5 days in 4 ml of RPMI 1640 with 10% heat- CD19 (clone SJ25C1), CD20 (clone L27), and CD56 (clone NCAM16.2)) inactivated FCS and 1% penicillin and streptomycin in Falcon 6-well plates and corresponding mouse IgG1, mouse IgG2a, mouse IgG2b, rat IgG1, and The Journal of Immunology 193 rat IgG2a isotype controls. Flow cytometry was performed on a FACS- Human cytokines and chemokines (IL-1␣, IL-1␤, IL-2, IL-4, IL-5, IL-6, Calibur flow cytometer (BD Biosciences) or an LSR II flow cytometer (BD IL-8/CXCL8, IL-10, IL-12p70, IL-13, IP-10/CXCL10, MCP-1/CCL2, Biosciences). Data was analyzed using CellQuest Pro Software (BD MIP-1␣/CCL3, RANTES/CCL5, TNF, and IFN-␥) were also analyzed us- Biosciences). ing the LINCOplex Simultaneous Multianalyte Detection System (Linco Research), according to the manufacturer’s instructions. Cytokine levels Cytokine and chemokine assays were quantitated using the Luminex 100 system. Values were expressed as ␥ picograms per milliliter deduced from the mean fluorescence intensity of The levels of mouse TNF and IL-6, and human IFN- , IL-5, IL-8/CXCL8, the standard curves after subtracting the blanks, using a 5-parameter lo- IL-13, and TNF were determined by ELISA, as described in (22), except Ј Ј gistic algorithm with MasterPlex QT Quantitation Software version 2.0 that Ultra-3,3 ,5,5 -tetramethylbenzidine substrate (Pierce) was used as (MiraiBio). substrate. The following coating Abs were used: polyclonal goat anti- mouse TNF (R&D Systems), rat anti-mouse IL-6 (clone MP5-20F3; BD Gene expression analysis using TaqMan real-time quantitative Pharmingen), mouse-anti-human IFN-␥ (clone NIB42; BD Pharmingen), mouse anti-human IL-5 (clone TRFK 5; BD Pharmingen), mouse anti- PCR human IL-8 (clone 3IL8-H10; Endogen), mouse anti-human IL-13 (clone Methods were essentially as described in Ref. 23. JES10-5A2; BD Pharmingen), and mouse anti-human TNF (clone 28401; R&D Systems). Recombinant mouse TNF and IL-6 and human IL-5 (BD Statistical methods Pharmingen), recombinant human IFN-␥, IL-13, and TNF (R&D Systems), and recombinant human IL-8 (Endogen) were used for standards. Biotin- Comparisons between groups were assessed with the two-tailed unpaired t ylated polyclonal goat anti-mouse TNF (R&D Systems), rat anti-mouse test. A p value of Ͻ0.05 was considered as statistically significant. Cal- IL-6 (clone MP5-32C11; BD Pharmingen), mouse-anti-human IFN-␥ culations were performed with a statistical package, StatView 5.0 for (clone 4S.B3; BD Pharmingen), mouse anti-human IL-5 (clone JES1- Macintosh (Acabus Concepts).

5A10; BD Pharmingen), mouse anti-human IL-8 (clone 18-S2; Endogen), Downloaded from mouse anti-human IL-13 (clone B69-2; BD Pharmingen), and polyclonal Results goat anti-human TNF (R&D Systems) were used for detection. The sen- sitivity limits for quantitative determinations were 2 pg/ml for human TNF, The CD200R regulates mouse peritoneal macrophage cytokine 3 pg/ml for mouse IL-6, and human IL-5, IL-8/CXCL8 and IL-13, 6 pg/ml production ␥ for human IFN- , and 10 pg/ml for mouse TNF. Resident murine peritoneal macrophages express the CD200R The CBA system (BD Pharmingen) was also used for simultaneous detection of cytokines and chemokines, according to the manufacturer’s (Fig. 1a) and agonistic mCD200Ig or anti-mCD200R Ab (DX109) instructions. The mouse inﬂammation (IL-6, IL-10, MCP-1/CCL2, IFN-␥, treatment inhibited IFN-␥-induced TNF secretion by these cells http://www.jimmunol.org/ TNF, and IL-12p70), human inﬂammation (IL-8/CXCL8, IL-1␤, IL-6, IL- (Fig. 1b). Similar effects were observed when macrophages were 10, TNF, and IL-12p70), human chemokine (IL-8/CXCL8, RANTES/ enriched by removing nonadherent cells. In contrast, comparable CCL5, MIG/CXCL9, MCP-1/CCL2, and IP-10/CXCL10) and human Th1/ Th2 (IL-2, IL-4, IL-5, IL-10, TNF, and IFN-␥) kits were used. The samples LPS-stimulated TNF secretion was not affected by either agonist were acquired on a FACSCalibur (BD Biosciences), and the data was an- (Fig. 1b). Elevated TNF responses induced by higher LPS doses alyzed using the BD Biosciences CBA software. were also not inhibited by CD200R agonists (data not shown). by guest on September 27, 2021

FIGURE 1. mCD200R agonists inhibit IFN-␥- and IL-17-induced peritoneal cell cytokine secretion. a, Expression of mCD200R on resident peritoneal F4/80ϩ macrophages. Resident peritoneal cells were stained with PE-conjugated anti-F4/80 and biotinylated anti-mCD200R (DX109, solid line) or biotinylated isotype control Ab (dotted line), followed by allophycocyanin-conjugated streptavidin. Similar results were seen when macrophages were phenotyped as CD11bϩ Gr-1low cells (data not shown). b, mCD200R agonists inhibit IFN-␥-, but not LPS-induced, peritoneal cell TNF secretion (p Ͻ 0.01 for both agonists, unpaired t test). After a 30-min incubation with 3 ␮g/ml control Ig (gray striped bars), mCD200Ig (black striped bars), rat IgG1 isotype control (gray bars), rat-anti-mouse CD200R (black bars), or no Ab (white bars), 1 ϫ 106 peritoneal cells were stimulated with 0.5 ng/ml recombinant murine IFN-␥ or 0.02 ng/ml Salmonella typhimurium LPS for 18 h. TNF secretion in supernatants was determined by ELISA. Results are presented as mean Ϯ SEM of triplicate cultures and are representative of six independent experiments. Ͻ, Below limit of detection (10 pg/ml). c, mCD200R agonists inhibit IFN-␥- and IL-17-induced peritoneal cell IL-6 secretion (p Ͻ 0.01 for both stimuli and agonists, unpaired t test). Cells were cultured as in b and IL-6 secretion determined by CBA. Results are presented as mean Ϯ SEM of triplicate cultures and are representative of two independent experiments. d, mCD200R agonists do not inhibit IFN-␥-induced peritoneal cell MCP-1 secretion. Cells were cultured as in b and MCP-1/CCL2 secretion determined by CBA. Results are presented as mean Ϯ SEM of triplicate cultures and are representative of two independent experiments. Ͻ, Below limit of detection (20 pg/ml). 194 CD200 RECEPTOR REGULATION OF MYELOID CELLS

IFN-␥- and IL-17-induced IL-6 secretion was also inhibited by observed when hCD200R was cross-linked on monocytes, but not mCD200Ig and anti-mCD200R Ab (Fig. 1c). CD200R inhibition when the hCD200R was cross-linked on CD4ϩ T cells (Fig. 4). As of IFN-␥-induced activation was not universal, as MCP-1/CCL2 controls, IL-5 and IL-13 secretion was also inhibited when the production was not affected (Fig. 1d). IL-17 did not induce detect- CD200R was cross-linked on both monocytes and T cells, and in able TNF and MCP-1/CCL2 production. PBMC cultures performed in parallel (Fig. 4). In this experiment, IFN-␥ secretion was not inhibited after CD200R ligation. CD200R expression on human peripheral blood leukocytes We then explored the role of CD200R in the human system. Effects of hCD200R agonists on DC activation CD200R expression was ﬁrst investigated on human blood cells. ϩ Ϫ CD200R was expected to be functionally important on monocyte- Fresh ex vivo CD11c lin DCs expressed the highest CD200R ϩ ϩ ϩ derived DCs, since they express high CD200R levels. Thus, we levels, followed by CD14 monocytes, CD4 T cells, and CD8 investigated the effects of CD200R agonists on these cells with and T cells (Fig. 2). B cells did not express CD200R (15). Monocyte- without further cross-linking. DCs were activated with LPS, derived DCs also expressed high CD200R levels (Fig. 2), and IFN-␥, CD40L and IFN-␥, or TNF and IL-1␤. We analyzed ex- slightly down-regulated CD200R expression after further matura- pression of the cell surface markers CD1a, CD32, CD80, CD83, tion with CD40L or TNF and IL-1␤ (data not shown). CD86, and HLA-DR, as well as secretion of IL-1␣, IL-1␤, IL-6, Inhibition of TT-induced cytokine secretion from PBMC by IL-8/CXCL8, IL-10, IL-12p70, IP-10/CXCL10, MCP-1/CCL2, CD200R triggering MIG/CXCL9, MIP-1␣/CCL3, RANTES/CCL5, and TNF. We did

not observe any effect of CD200R agonists on these parameters Downloaded from We next investigated whether Ag-induced cytokine responses, re- (data not shown). CD200R agonists also had no effect on alloge- quiring cooperation between Ag-presenting monocytes and T cells, ϩ ϩ neic DC-induced CD4 T cell proliferation or CD4 T cell cyto- were modulated by CD200R agonists. Thus, PBMC from recently kine production using TT-loaded DC (data not shown). It is likely TT-vaccinated donors were pretreated with CD200R agonists be- that human DC will also be modulated through CD200R given the fore stimulation with TT. TT dose-dependently induced IL-5, IL- recent data showing effects of CD200Ig on mouse DC function 13, IFN-␥ , and TNF (data not shown), but not IL-2, IL-4, IL-10, (24). However, to date, we have not identiﬁed the relevant in vitro http://www.jimmunol.org/ and IL-12p70 secretion (Fig. 3). CD200R agonists inhibited pro- conditions needed to observe CD200R-mediated modulation of duction of IL-5 and IL-13, but only when the agonists were further human DC function. Ј cross-linked by F(ab )2 goat anti-mouse/rat IgG (Fig. 3 and Table I). The effects on IFN-␥ (Fig. 3c) and TNF (data not shown) were lesser and more variable. No effect of CD200R stimulation was Inhibition of hCD200R-transfected U937 cell cytokine observed at suboptimal TT doses. Data from four independent do- production by CD200R agonists nors are summarized in Table I. Human monocytic U937 cells were transfected with hCD200R to The inhibitory activity of CD200R agonists could be due to a further explore CD200R regulation of human myeloid cell activa- direct or indirect effect on T cell activation, since both PBMC tion. U937 cells did not express the receptor endogenously, but CD4ϩ T cells and monocytes express CD200R. Each cell type was when infected with a retrovirus encoding hCD200R, they express by guest on September 27, 2021 isolated and separately treated with CD200R agonists to investi- hCD200R at high levels (Fig. 5a). IFN-␥ stimulation resulted in gate whether the inhibition of TT-induced cytokine secretion re- increased IL-8/CXCL8, IP-10/CXCL10, and MIG/CXCL9 secre- quired triggering of the hCD200R on monocytes, on CD4ϩ T cells, tion, whereas TNF, IL-1␤, IL-6, IL-10, and IL-12p70 production or on both cell types. Each cell type was incubated with an anti- was not detectable (data not shown). CD200R agonists dose-de- hCD200R Ab and a cross-linking Ab and then mixed before stim- pendently inhibited IFN-␥-induced IL-8/CXCL8 (Fig. 5b), MIG/ ulation with TT. Inhibition of IL-5 and IL-13 secretion was only CXCL9, and IP-10/CXCL10 (data not shown) secretion from the

FIGURE 2. Expression of hCD200R on human peripheral blood leukocytes. Human peripheral blood leukocytes were stained with biotinylated anti-hCD200R (DX136, solid line) or biotinylated isotype control Ab (dotted line), followed by allophycocyanin-conjugated streptavidin. For fresh ex vivo DCs, cells were also stained with anti-lin-FITC (mixture of Abs to CD3, CD14, CD16, CD19, CD20, and CD56) and anti- CD11c-PE, for monocytes with anti-CD14-FITC, for monocyte-derived DCs with anti-CD14-FITC and anti- CD11c-PE and for T cells with anti-CD3-FITC and anti- CD4-PE or anti-CD8-PE. The Journal of Immunology 195

hCD200R-transfected U937 cells. Transcriptional regulation probably contributed to these effects, as rat-anti-human CD200R de- creased IFN-␥-induced IL-8/CXCL8, IP-10/CXCL10, and MIG/ CXCL9 mRNA expression (data not shown). No effect of CD200R agonists was seen on vector control-transfected cells (data not shown). LPS and IL-17 did not stimulate any cytokine responses (data not shown). The bulk hCD200R-expressing U937 cell line was sorted into four sublines expressing very low, low, medium, and high hCD200R levels to study the effects of varying hCD200R levels on inhibition of responses. The sorted cells stably expressed the receptor over several months of culture (Fig. 6a). CD200R agonists (hCD200Ig shown in Fig. 6b) inhibited spontaneous and IFN-␥- induced IL-8/CXCL8 secretion proportionally to the hCD200R expression on the cells. Thus, high CD200R-expressing cells were more readily inhibited by hCD200R agonists than cells with medium expression levels. Low expressing cells were barely inhibited and no inhibition was seen with very low expressing cells. In a second set of studies, minimally responsive low hCD200R- Downloaded from expressing cells were incubated with CD200R agonists but with cross-linking before IFN-␥ stimulation to determine whether further cross-linking of CD200R could convert a subtherapeutic inhibitory signal into a fully inhibitory signal. IFN-␥-stimulated IL- 8/CXCL8 production was inhibited by CD200R agonists when actively cross-linked (Fig. 6c). Furthermore, suboptimal levels of http://www.jimmunol.org/ CD200R agonists induced stronger inhibition in medium and high expressing U937 cells in the presence of cross-linking Ab (data not shown).

Discussion The present study shows that triggering of CD200R suppresses

myeloid cell function in vitro. Thus, after stimulation with the by guest on September 27, 2021 myeloid cell-activating cytokine IFN-␥, cytokine secretion from mouse peritoneal macrophages and human myeloid U937 cells transfected with the hCD200R was inhibited by triggering of the CD200R. IL-17-induced IL-6 production from mouse peritoneal macrophages was also inhibited by CD200R triggering. By con- FIGURE 3. hCD200R agonists inhibit TT-induced PBMC cell IL-5 and trast, responses after LPS stimulation were not affected. CD200R- IL-13 secretion. After a 30-min incubation with 10 ␮g/ml control Ig (gray striped bars), hCD200Ig (black striped bars), rat IgG1 isotype control (gray mediated inhibition correlated with levels of CD200R on U937 bars), or rat-anti-human CD200R (black bars), 1 ϫ 106 PBMC were washed cells. In cells expressing low CD200R levels, inhibition was only and the hCD200Ig or rat-anti-human CD200R were cross-linked with 5 ␮g/ml observed if the CD200R agonists were further cross-linked. TT- Ј endotoxin-depleted F(ab )2 goat anti-mouse/rat IgG, and incubated for another induced PBMC IL-5 and IL-13 secretion was also inhibited by 30 min before addition of 1, 10, or 100 ng/ml TT. Supernatants were harvested cross-linked CD200R agonists. This inhibition required cross-link- after 6 days. IL-5 (a), IL-13 (b), and IFN-␥ (c) secretion was determined by ing the CD200R on monocytes, but not cross-linking the CD200R Ϯ ϩ ELISA. Results are presented as mean SEM of triplicate cultures and are on CD4 T cells. In all, our in vitro data agree well with the in representative of three to four independent experiments. Statistically signiﬁ- vivo observations showing that targeted deletion of CD200 re- cant inhibition of IL-5 and IL-13 secretion by hCD200R agonists was observed (p Ͻ 0.01 for both agonists and cytokines, unpaired t test). sulted in dysregulation of myeloid cells and enhanced susceptibility to autoimmune inﬂammation such as EAE and CIA (10).

Table I. hCD200R agonists inhibit tetanus toxoid-induced PBMC IL-5 and IL-13 secretiona

IL-5 IL-13 IFN-␥ TNF

Donor cIg CD200Ig rIgG1 DX153 cIg CD200Ig rIgG1 DX153 cIg CD200Ig rIgG1 DX153 cIg CD200Ig rIgG1 DX153

1 115.5 25.4 160.3 63.0 58.5 61.2 63.7 46.4 2 144.2 71.0 103.2 24.4 133.6 68.2 112.9 49.5 169.2 122.8 322.6 245.4 300.3 160.7 220.7 73.0 3 214.2 118.4 215.9 131.2 191.8 96.7 299.7 92.8 151.4 106.0 255.3 151.1 200.5 121.1 215.7 136.7 4 269.9 117.6 188.9 105.2 128.0 112.1 458.4 408.3

a A total of 1 ϫ 106 PBMCs were cultured in 1 ml of RPMI 1640 with 10% heat-inactivated FCS, 1% penicillin and streptomycin at 37°C in a humidiﬁed incubator containing 5% CO2. After a 30-min incubation with control-Ig, hCD200Ig, rat IgG1 isotype control, or rat-anti-human CD200R, cells were washed and the hCD200Ig or rat-anti-human ␮ Ј CD200R were cross-linked with 5 g/ml endotoxin-cleaned F(ab )2 goat anti-mouse/rat IgG, and incubated for another 30 min before addition of tetanus toxoid. Supernatants were harvested after 6 days. IL-5, IL-13, IFN-␥, or TNF secretion was determined by ELISA, CBA, or Luminex. Data from the optimal tetanus toxoid dose (10 ng/ml for donor 1 and 100 ng/ml for donors 2, 3, and 4) are presented as mean values picograms per milliliter of triplicate cultures. 196 CD200 RECEPTOR REGULATION OF MYELOID CELLS

FIGURE 4. TT-induced IL-5 and IL-13 secretion is inhibited when the hCD200R is cross-linked on monocytes, but not when the hCD200R is cross-linked on CD4ϩ T cells. Monocytes and CD4ϩ T cells were isolated by MACS to Ͼ95% purity. After separate incubation of 250,000 monocytes/well and 750,000 CD4ϩ T cells/well with 10 ␮g/ml rat IgG1 isotype control (gray bars) or rat-anti-human CD200R (black bars), cells were washed and the rat-anti-human CD200R was ␮ Ј cross-linked with 5 g/ml endotoxin-cleaned F(ab )2 goat anti-mouse/rat IgG, and incubated for another 30 min before addition of 100 ng/ml TT. IL-5 (a) and IL-13 (b), mean Ϯ SEM of triplicate cultures, was determined by the LINCOplex Simultaneous Multianalyte Detection System using a Luminex. Ͻ, Below limit of detection (3 pg/ml). Inhibition was only observed when

hCD200R was cross-linked on monocytes, or on both Downloaded from monocytes and T cells or in PBMC (p Ͻ 0.01 for both agonists and cytokines in all three cultures, unpaired t test). http://www.jimmunol.org/

As macrophages play pivotal roles in chronic inflammatory dis- by macrophages, such as via anti-TNF therapy for rheumatoid ar- eases through their broad proinflammatory, destructive, and re- thritis, Crohn’s disease, and psoriasis (25). Triggering inhibitory modeling capacities (1–3, 5), regulation of their function is critical receptors represents an alternative way of regulating myeloid cells for treatment of these disorders. One way of controlling inflam- (6), potentially superior to focused anti-cytokine therapies given mation is through blocking proinflammatory cytokines produced the ability to affect a number of effector pathways via cellular by guest on September 27, 2021

FIGURE 5. hCD200R agonists dose-dependently inhibit IFN-␥-induced hCD200R-transfected U937 cell IL-8/CXCL8 secretion. a, Expression of hCD200R on hCD200R- and vector control-transfected U937 cells. The transfected U937 cells were stained with biotinylated anti-hCD200R (DX136, solid line) or biotinylated isotype control Ab (dotted line), followed by allophycocyanin-conjugated streptavidin. b, hCD200R agonists inhibit IFN-␥-induced hCD200R-transfected U937 cell IL-8/CXCL8 secretion. After a 30-min incubation with control Ig (circles with no connecting line), hCD200Ig (circles with connecting lines), rat IgG1 isotype control (squares with no connecting line), rat-anti-human CD200R (squares with connecting lines), 1 ϫ 106 hCD200R- transfected U937 cells were stimulated with 100 U/ml recombinant human IFN-␥ (filled symbols with solid connecting lines) or cultured in medium alone (open symbols with dashed connecting lines) for 18 h. IL-8/CXCL8 secretion in supernatants was determined by ELISA and is presented as mean Ϯ SEM of triplicate cultures. Statistically significant inhibition (unpaired t test) was observed from 0.3 ␮g/ml hCD200Ig and 0.1 ␮g/ml rat-anti-hCD200R. The Journal of Immunology 197 Downloaded from http://www.jimmunol.org/ FIGURE 6. Inhibition of IFN-␥-induced IL-8/CXCL8 secretion by hCD200Ig correlates with hCD200R expression levels in hCD200R-transfected U937 cells, and is dependent on hCD200R agonist cross-linking in low expressing cells. a, Expression of hCD200R on sorted hCD200R-transfected U937 cells. hCD200R-transfected U937 cells were sorted into very low, low, medium, and high hCD200R-expressing cells, and then stained with biotinylated anti-hCD200R (DX136, solid line) or biotinylated isotype control Ab (dotted line), followed by allophycocyanin-conjugated streptavidin. b, Inhibition of IFN-␥-induced IL-8/CXCL8 secretion by hCD200Ig correlates with hCD200R expression levels in hCD200R-transfected U937 cells. After a 30-min incubation with hCD200Ig, 1 ϫ 106 hCD200R-transfected cells with very low (diamonds), low (triangles), medium (circles), or high (squares) hCD200R expression were stimulated with 100 U/ml recombinant human IFN-␥ (filled symbols with solid connecting lines) or cultured in medium alone (open symbols with dashed connecting lines) for 18 h. IL-8/CXCL8 secretion in supernatants was determined by ELISA and is presented as mean Ϯ SEM of triplicate cultures. Statistically significant inhibition (unpaired t test) of spontaneous secretion was observed from 0.1 ␮g/ml hCD200Ig for high expressing cells and 1 ␮g/ml hCD200Ig for medium and low expressing cells. For IFN-␥-induced IL-8 secretion, statistically significant inhibition (unpaired t test) by guest on September 27, 2021 was observed from 0.3 ␮g/ml hCD200Ig for high and medium expressing cells and 1 ␮g/ml hCD200Ig for low expressing cells. c, Inhibition of IFN-␥-induced IL-8/CXCL8 secretion by hCD200R agonists is dependent on hCD200R agonist cross-linking in low expressing cells (p Ͻ 0.001 for hCD200Ig and p ϭ 0.02 for rat-anti-hCD200R, unpaired t test, in the presence of cross-linking). After a 30-min incubation with 1 ␮g/ml hCD200R agonists or isotype controls, 1 ϫ 106 hCD200R-transfected cells with low hCD200R expression were washed and the hCD200Ig or rat-anti-human CD200R were ␮ Ј cross-linked with 5 g/ml endotoxin-cleaned F(ab )2 goat anti-mouse/rat IgG (marked as x-l in figure), and incubated for another 30 min before addition of 100 U/ml recombinant human IFN-␥. Supernatants were harvested after 18 h. IL-8/CXCL8 secretion in supernatants was determined by ELISA and is presented as mean Ϯ SEM of triplicate cultures. targets. Many of these inhibitory receptors are members of the following stimulation of mouse macrophages with IFN-␥, a very IgSF of integral membrane proteins. Data is now emerging that important activator of macrophage function (28–30). TNF plays a signaling through these receptors is essential for normal myeloid central role in autoimmune disease (25). TNF has several proin- cell regulation in the peripheral immune system. Thus, disruption flammatory activities such as up-regulation of adhesion molecule of signaling through the myeloid inhibitory receptors SIRP␣ and expression, induction of cytokines and chemokines, angiogenesis Fc␥RIIB leads to potentiation of cellular function with consequent and bone resorption (1, 25). IL-6 is the most strikingly elevated effects on inflammatory disease processes (17, 26). cytokine in human rheumatoid arthritis (1, 31), and it is required The CD200-CD200R interaction provides a new regulatory for experimentally induced autoimmune diseases, including CIA mechanism for myeloid cells. The IgSF glycoprotein CD200, for- (32) and EAE (31, 33). Clinical trials with a humanized anti-IL- merly known as OX-2, is expressed widely, including on thymo- 6R-antibody for treatment of rheumatoid arthritis and Crohn’s dis- cytes, B cells, endothelial cells, smooth muscle cells, and neurons ease are ongoing (34). Proinflammatory effects of IL-6 include (6, 27). CD200R is more restricted and is predominantly expressed enhancing autoantibody production, bone absorption and trigger- on myeloid cells and T cells (14, 15). CD200 deletion resulted in ing of acute phase responses (31), and blocking regulatory T cell myeloid cell dysregulation and enhanced susceptibility to autoim- suppression (35). mune inflammation such as EAE and CIA (10), suggesting that The observation that IL-17-induced IL-6 production was sup- CD200 normally induces immune suppression through CD200R. pressed by CD200R agonists is also relevant to the in vivo mech- Soluble CD200R protein, binding CD200 and preventing CD200- anism of CD200R. This is the first report that IL-17 activates CD200R interactions, increased the incidence of CIA in normally mouse macrophages, although enhancement of IL-6 production resistant mice (10). Mice receiving soluble CD200 were con- has been observed in human rheumatoid arthritis synovial cultures versely resistant to CIA induction (11). and human macrophages after IL-17 stimulation (36, 37). IL-17 is One potential mechanism through which CD200 may control a T cell-derived cytokine that exhibits pleiotropic biological ac- inflammation in vivo is via inhibition of TNF and IL-6 production tivities on various types of cells, such as fibroblasts, endothelial 198 CD200 RECEPTOR REGULATION OF MYELOID CELLS cells, and epithelial cells, mediating a wide range of responses, cross-linking of the CD200R. Inhibition of secretion of the che- mostly proinflammatory and hemopoietic (38). Increased levels of motactic factors IL-8, IP-10, and MIG would reduce recruitment of IL-17 have been found in several human inflammatory disorders, neutrophils and Th1 cells to sites of inflammation (46). including multiple sclerosis (39), rheumatoid arthritis (40), psori- In conclusion, we provide direct evidence in vitro that the asis (41), and asthma (42). Neutralizing IL-17 reduces the severity CD200-CD200R interaction controls monocyte/macrophage func- of CIA (43) and EAE (44), and its production is triggered by IL- tion in both mice and humans and that the efficiency of CD200R- 23, an essential cytokine in the development of autoimmune in- mediated inhibition of cellular functions is proportional to the re- flammation (44). ceptor density at the cell surface. Our data solidify the concept that CD200R triggering did not result in universal macrophage in- CD200R agonism may provide a novel approach to the treatment hibition, since LPS-induced TNF responses were not affected. This of chronic inflammatory diseases. could be a safety advantage in a therapeutic setting, especially if this holds true for other pathogen-derived TLR ligands. The dif- Acknowledgments ferential effects of CD200R-mediated inhibition on IFN-␥-, IL-17-, We thank Janet Wagner and Sandra Zurawski for production of fusion and LPS-induced cytokine responses is not surprising, as different proteins and mAbs. signaling pathways are induced by these factors (30, 38, 45). We also explored the role of CD200R in human systems in vitro Disclosures after showing that mouse macrophages were functionally inhibited The authors have no financial conflict of interest. by CD200R triggering. As blood APCs and CD4ϩ T cells express the CD200R, we first investigated whether Ag-induced cytokine References Downloaded from responses, requiring cooperation between Ag-presenting mono- 1. Kinne, R. W., R. Brauer, B. Stuhlmuller, E. Palombo-Kinne, and cytes and T cells, could be modulated by CD200R agonists. We G. R. Burmester. 2000. Macrophages in rheumatoid arthritis. Arthritis Res. 2: 189–202. found that TT-induced PBMC IL-5 and IL-13 secretion was in- 2. 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