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The Journal of Immunology, 2006, 176: 191-199.
Copyright © 2006 by The American Association of Immunologists

Regulation of Myeloid Cell Function through the CD200 Receptor1

Maria C. Jenmalm2, Holly Cherwinski, Edward P. Bowman3, Joseph H. Phillips and Jonathon D. Sedgwick3

SP Biopharma, Palo Alto CA 94022


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Myeloid cells play pivotal roles in chronic inflammatory diseases through their broad proinflammatory, destructive, and remodeling 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-{gamma}- 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 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Myeloid cells (i.e., macrophages, dendritic cells (DC),4 neutrophils, mast cells, and eosinophils) play important roles in maintaining chronic inflammation (1, 2, 3, 4, 5). They can be regulated through cell-cell interactions that trigger matched sets of activating and inhibitory receptors, in addition to being regulated by secreted factors (6). The regulation of myeloid cell activity by direct cell-cell contact allows a more localized control than that mediated by cytokines. The CD200-CD200R interaction also provides a cell-cell contact regulatory interaction for myeloid cells. The widely expressed glycoprotein CD200 is closely related structurally to the T cell costimulatory molecules CD80 and CD86 and is genetically linked to them on human chromosome 3 and mouse chromosome 16 (7, 8). Structurally, CD200 contains two Ig superfamily (IgSF) domains in a typical V/C2 arrangement (9).

Deletion of CD200 resulted in myeloid cell dysregulation and enhanced susceptibility to autoimmune inflammation such as experimental autoimmune encephalitis (EAE) and collagen-induced arthritis (CIA) (10), suggesting that CD200 normally induces immune suppression through CD200R. Consistent with this was the demonstration that a soluble form of CD200 (mouse (m) CD200Ig) administered to mice blocks autoimmune inflammation in vivo (11). In terms of mechanism of action, endogenous CD200 (and soluble CD200Ig) could send a signal that deviates the immune response away from a damaging effector pathway, as proposed (12), and/or it could directly inhibit effector cell function (10). As in vivo studies cannot discriminate between these possibilities, a mast cell model was established to test whether CD200R directly mediates cellular inhibition. IgE-mediated activation of mast cells was inhibited by mCD200Ig or an agonist rat-anti-mCD200R (DX109) when the cells were transfected with mCD200R, and by human (h) CD200Ig or agonist rat-anti-hCD200R (DX136 and DX153) when the cells were transfected with hCD200R (13). These agonists directly blocked mast cell degranulation as well as TNF and IL-13 secretion, with a typical IC50 of 0.2–1 nM. Thus, CD200 directly triggered cellular inhibition through CD200R engagement.

The CD200R is expressed at the surface of human and mouse myeloid cells, such as macrophages, DCs, neutrophils, and mast cells, and also on T cells (14, 15). CD200R is closely structurally related to CD200, located on the same chromosome, the genes probably evolved by gene duplication (15). However, the receptor is distinct from CD200 by virtue of a longer cytoplasmic tail containing three conserved tyrosine residues, one of which is contained within an NPXY motif (14, 15). Phosphorylated NPXY motifs bind phosphotyrosine-binding domains that are present in signaling adaptor molecules such as Shc (16), suggesting that the CD200R can signal after ligation by CD200. CD200R lacks an ITIM motif present in almost all immune inhibitory receptors (17). Using the mast cell system, we have shown that the NPXY motif is critical for receptor activity, and that signaling and cellular inhibition are linked to Dok1 and Dok2 phosphorylation and subsequent Ras/MAPK pathway inhibition (18).

The aim of the present study was to further characterize the regulation of myeloid cell function through the CD200R in vitro, focusing especially on the effects of the CD200R on macrophage and DC function in human and mouse.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
CD200R-binding reagents

Rat mAbs that were agonistic for mouse and human CD200R were generated in rats as previously described (15). Abs used were DX109 (ratIgG1-anti-mCD200R), DX136 (ratIgG2a-anti-hCD200R), and DX153 (ratIgG1-anti-hCD200R). DX109 does not bind the activating DNAX activating protein 12 (DAP-12)-paired mCD200RLa and mCD200RLb (15). hCD200RLa is not expressed and is probably nonfunctional (15).

Ligand (CD200)-Ig fusion proteins were generated as described (13) by fusion of the extracellular domain of human or mouse CD200 to the Fc domain of mIgG1 mutated in the CH2 domain (D265A) to inhibit binding to FcRs (19). mCD200Ig does not bind the activating DAP-12-paired mCD200RLa and mCD200RLb (15, 20).

All mAb and protein preparations used contained <0.1 ng of endotoxin/mg protein, as determined by the Limulus Amebocyte Lysate Pyrogen Testing kit, QCL-1000 (BioWhittaker).

Cells and cell culture

Mouse peritoneal cells. Resident peritoneal cells were obtained by peritoneal lavage of 8- to 12-wk-old C57BL/6 mice (The Jackson Laboratory) with 5 ml of DMEM (Mediatech). A total of 1 x 106 cells were cultured in 1 ml of DMEM with 10% heat-inactivated FCS (HyClone) and 1% penicillin and streptomycin (Sigma-Aldrich) in Falcon 24-well plates (BD Biosciences Discovery Labware) at 37°C in a humidified incubator containing 5% CO2. In some experiments, nonadherent cells were removed after 2 h to obtain a more pure macrophage population. After a 30-min incubation with mCD200Ig, control Ig, rat-anti-mCD200R (DX109), or rat IgG1 isotype control (BD Pharmingen), cells were stimulated with recombinant murine IFN-{gamma} (R&D Systems), recombinant murine IL-17 (DNAX), or Salmonella typhimurium LPS (Sigma-Aldrich) for 18 h. Cell supernatants were collected and analyzed for IL-6, IL-10, IL-12p70, MCP-1/CCL2, IFN-{gamma}, and TNF secretion by ELISA or cytometric bead array (CBA) as described below.

Human peripheral blood cells. PBMC were isolated from heparinized blood of recently tetanus toxoid (TT)-vaccinated healthy donors, using Ficoll-Paque density gradient (Amersham Biosciences). A total of 1 x 106 PBMC were cultured in 1 ml of RPMI 1640 with 10% heat-inactivated FCS and 1% penicillin and streptomycin in Falcon 24-well 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 inactivated Clostridium tetani TT (Calbiochem) for 6 days. In some experiments, cells were washed after the 30-min incubation, and cell-bound hCD200IgG or rat-anti-hCD200R were cross-linked with 5 µg/ml endotoxin-depleted F(ab')2 goat anti-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-{gamma}, and TNF secretion was analyzed by ELISA, CBA (BD Pharmingen, or the LINCOplex Simultaneous Multianalyte Detection Systems (Linco Research) as described below.

Both human blood monocytes and CD4+ T cells express the CD200R, and Ag-induced cytokine responses require cooperation between Ag-presenting monocytes and T cells (15). To study the relative contribution of cross-linking the CD200R on monocytes or CD4+ T cells before TT stimulation, CD14+ monocytes were positively selected from PBMC on an autoMACS (Miltenyi Biotec) using CD14 microbeads (Miltenyi Biotec), according to the manufacturer’s protocol. CD4+ T cells were negatively selected on an autoMACS from the CD14– fraction using the CD4+ T cell Isolation kit II (Miltenyi Biotec), according to the manufacturer’s protocol. Cells were >95% pure after isolation, as analyzed by flow cytometry as described below. A total of 250,000 monocytes/well and 750,000 CD4+ T cells/well were incubated separately, in the presence of rat-anti-hCD200R (DX153) and cross-linking Ab as above. After washing, the monocytes and T cells were then mixed and stimulated with TT.

Human DCs. Monocytes were negatively selected from PBMC on an autoMACS using the Monocyte Isolation kit II (Miltenyi Biotec), according to the manufacturer’s protocol. To generate immature DCs, 2 x 106 monocytes were cultured in 100 ng/ml GM-CSF (Schering-Plough) and 5 ng/ml IL-4 (Schering-Plough) for 5 days in 4 ml of RPMI 1640 with 10% heat-inactivated FCS and 1% penicillin and streptomycin in Falcon 6-well plates at 37°C in a humidified incubator containing 5% CO2. After 5 days in culture, cells were CD14–, CD11c+, and HLA-DR+.

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 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 (R&D Systems) and rIFN-{gamma}, rIFN-{gamma} alone or rTNF (R&D Systems), and rIL-1{beta} (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-anti-hCD200R were cross-linked with 5 µg/ml endotoxin-depleted F(ab')2 goat anti-mouse/rat IgG, and incubated for another 30 min before addition of stimuli. Supernatants were collected and IL-1{alpha}, IL-1{beta}, IL-6, IL-8/CXCL 8 (CXCL8), IL-10, IL-12p70, IFN-inducible protein 10 (IP-10)/CXCL10, MCP-1/CCL2, monokine induced by IFN-{gamma} (MIG)/CXCL9, MIP-1{alpha}/CCL3, RANTES/CCL5, and TNF secretion was analyzed by ELISA, CBA, or the LINCOplex Simultaneous Multianalyte Detection System as described below. Expression of the cell surface activation markers CD1a, CD32, CD80, CD83, CD86, and HLA-DR was analyzed by flow cytometry as described below.

Human monocyte cell line. Human U937 monocytic cells (American Type Culture Collection) were transfected with hCD200R. cDNA encoding full-length hCD200R was subcloned into the pMX-pie retrovirus expression vector (21) containing a puromycin resistance gene, an internal ribosomal entry site (IRES) element, and an enhanced GFP gene. Plasmid DNA was then transfected into the Phoenix ecotropic virus packaging cell line (a gift from G. Nolan, Stanford University, Palo Alto, CA). The U937 cells were infected by coculture with the transfected packaging cell line. After drug selection, the resulting hCD200R or control vector transfectants were isolated by FACS on a FACSVantage System (BD Immunocytometry Systems) based on GFP expression. The hCD200R transfectant was further sorted into very low, low, medium, and high hCD200R-expressing cells after staining with PE-labeled anti-hCD200R (DX136). Stable expression of the hCD200R was seen over several months of culture. The cell lines were grown in RPMI 1640 (Mediatech) with 10% heat-inactivated FCS, 1% penicillin and streptomycin, 20 mM HEPES buffer (Fisher Chemical), 1 mM sodium pyruvate (BioWhittaker), and 0.1 mM nonessential amino acids (Invitrogen Life Technologies) at 37°C in a humidified incubator containing 5% CO2, and split every 3–4 days.

A total of 1 x 106 hCD200R or vector control-transfected U937 cells 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% CO2. After a 30-min incubation with hCD200Ig, control Ig, rat-anti-hCD200R (DX153), or rat IgG1 isotype control, hCD200R or vector control-transfected U937 cells were stimulated with 100 U/ml recombinant human IFN-{gamma} (R&D Systems) for 18 h. In some experiments, cells were washed after the 30-min incubation and the cell-bound hCD200Ig or rat-anti-hCD200R were cross-linked with 5 µg/ml endotoxin-depleted F(ab')2 goat anti-mouse/rat IgG (Jackson ImmunoResearch Laboratories), and incubated for another 30 min before addition of IFN-{gamma}. Cell supernatants were collected and analyzed for IL-1{beta}, IL-6, IL-8/CXCL8, IL-10, TNF, IL-12p70, IP-10/CXCL10, MCP-1/CCL2, MIG/CXCL9, and RANTES/CCL5 secretion by ELISA or CBA as described below.

Flow cytometry

Cells were stained and washed in PBS containing 0.1% NaN3 (Sigma-Aldrich) and either 1% heat-inactivated FCS for mouse cells or 1% heat-inactivated normal human serum (Jackson ImmunoResearch Laboratories) for human cells. To block Fc{gamma}Rs, 1 x 106 cells/tube were incubated with 0.1 µg of Fc block (anti-CD16/CD32 (clone 2.4G2); BD Pharmingen) for mouse cells or 10% heat-inactivated normal human serum for human cells. The cells were then stained with FITC-, PE-, PerCP-, allophycocyanin-, or biotin-conjugated Abs (BD Pharmingen, except as indicated) for 30 min 4°C in the dark. Biotinylated Abs were secondarily labeled with streptavidin-allophycocyanin (BD Pharmingen). Cells were stained with Abs to mouse CD11b (clone M1/70), mouse CD200R (DX109; DNAX), mouse F4/80 (clone CI:A3-1; Caltag Laboratories), mouse Gr-1 (Ly-6G/Ly-6C, clone RB6-8C5), human CD1a (clone HI149), human CD3 (clone HIT3a), human CD4 (clone RPA-T4), human CD8 (clone HIT8a), human CD11c (clone B-ly6), human CD14 (clone M5E2), human CD19 (clone HIB19), human CD20 (clone L27), human CD32 (clone FLI8.26), human CD80 (clone L307.4), human CD83 (clone HB15e), human CD86 (clone FUN-1), human CD200R (DX136; DNAX), human HLA-DR (clone L243), lin 1 (mixture of CD3 (clone SK7), CD14 (clone M{phi}P9), CD16 (clone 3G8), CD19 (clone SJ25C1), CD20 (clone L27), and CD56 (clone NCAM16.2)) and corresponding mouse IgG1, mouse IgG2a, mouse IgG2b, rat IgG1, and rat IgG2a isotype controls. Flow cytometry was performed on a FACSCalibur flow cytometer (BD Biosciences) or an LSR II flow cytometer (BD Biosciences). Data was analyzed using CellQuest Pro Software (BD Biosciences).

Cytokine and chemokine assays

The levels of mouse TNF and IL-6, and human IFN-{gamma}, IL-5, IL-8/CXCL8, IL-13, and TNF were determined by ELISA, as described in (22), except that Ultra-3,3',5,5'-tetramethylbenzidine substrate (Pierce) was used as substrate. The following coating Abs were used: polyclonal goat anti-mouse TNF (R&D Systems), rat anti-mouse IL-6 (clone MP5-20F3; BD Pharmingen), mouse-anti-human IFN-{gamma} (clone NIB42; BD Pharmingen), mouse anti-human IL-5 (clone TRFK 5; BD Pharmingen), mouse anti-human IL-8 (clone 3IL8-H10; Endogen), mouse anti-human IL-13 (clone 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 Pharmingen), recombinant human IFN-{gamma}, IL-13, and TNF (R&D Systems), and recombinant human IL-8 (Endogen) were used for standards. Biotinylated polyclonal goat anti-mouse TNF (R&D Systems), rat anti-mouse IL-6 (clone MP5-32C11; BD Pharmingen), mouse-anti-human IFN-{gamma} (clone 4S.B3; BD Pharmingen), mouse anti-human IL-5 (clone JES1-5A10; BD Pharmingen), mouse anti-human IL-8 (clone 18-S2; Endogen), mouse anti-human IL-13 (clone B69-2; BD Pharmingen), and polyclonal goat anti-human TNF (R&D Systems) were used for detection. The sensitivity limits for quantitative determinations were 2 pg/ml for human TNF, 3 pg/ml for mouse IL-6, and human IL-5, IL-8/CXCL8 and IL-13, 6 pg/ml for human IFN-{gamma}, and 10 pg/ml for mouse TNF.

The CBA system (BD Pharmingen) was also used for simultaneous detection of cytokines and chemokines, according to the manufacturer’s instructions. The mouse inflammation (IL-6, IL-10, MCP-1/CCL2, IFN-{gamma}, TNF, and IL-12p70), human inflammation (IL-8/CXCL8, IL-1{beta}, IL-6, IL-10, TNF, and IL-12p70), human chemokine (IL-8/CXCL8, RANTES/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-{gamma}) kits were used. The samples were acquired on a FACSCalibur (BD Biosciences), and the data was analyzed using the BD Biosciences CBA software.

Human cytokines and chemokines (IL-1{alpha}, IL-1{beta}, IL-2, IL-4, IL-5, IL-6, IL-8/CXCL8, IL-10, IL-12p70, IL-13, IP-10/CXCL10, MCP-1/CCL2, MIP-1{alpha}/CCL3, RANTES/CCL5, TNF, and IFN-{gamma}) were also analyzed using the LINCOplex Simultaneous Multianalyte Detection System (Linco Research), according to the manufacturer’s instructions. Cytokine levels were quantitated using the Luminex 100 system. Values were expressed as picograms per milliliter deduced from the mean fluorescence intensity of the standard curves after subtracting the blanks, using a 5-parameter logistic algorithm with MasterPlex QT Quantitation Software version 2.0 (MiraiBio).

Gene expression analysis using TaqMan real-time quantitative PCR

Methods were essentially as described in Ref.23 .

Statistical methods

Comparisons between groups were assessed with the two-tailed unpaired t test. A p value of <0.05 was considered as statistically significant. Calculations were performed with a statistical package, StatView 5.0 for Macintosh (Acabus Concepts).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The CD200R regulates mouse peritoneal macrophage cytokine production

Resident murine peritoneal macrophages express the CD200R (Fig. 1a) and agonistic mCD200Ig or anti-mCD200R Ab (DX109) treatment inhibited IFN-{gamma}-induced TNF secretion by these cells (Fig. 1b). Similar effects were observed when macrophages were enriched by removing nonadherent cells. In contrast, comparable LPS-stimulated TNF secretion was not affected by either agonist (Fig. 1b). Elevated TNF responses induced by higher LPS doses were also not inhibited by CD200R agonists (data not shown). IFN-{gamma}- and IL-17-induced IL-6 secretion was also inhibited by mCD200Ig and anti-mCD200R Ab (Fig. 1c). CD200R inhibition of IFN-{gamma}-induced activation was not universal, as MCP-1/CCL2 production was not affected (Fig. 1d). IL-17 did not induce detectable TNF and MCP-1/CCL2 production.



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  		FIGURE 1. mCD200R agonists inhibit IFN-{gamma}- and IL-17-induced peritoneal cell cytokine secretion. a, Expression of mCD200R on resident peritoneal F4/80+ 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 CD11b+ Gr-1low cells (data not shown). b, mCD200R agonists inhibit IFN-{gamma}-, but not LPS-induced, peritoneal cell TNF secretion (p < 0.01 for both agonists, unpaired t test). After a 30-min incubation with 3 µg/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 x 106 peritoneal cells were stimulated with 0.5 ng/ml recombinant murine IFN-{gamma} or 0.02 ng/ml Salmonella typhimurium LPS for 18 h. TNF secretion in supernatants was determined by ELISA. Results are presented as mean ± SEM of triplicate cultures and are representative of six independent experiments. <, Below limit of detection (10 pg/ml). c, mCD200R agonists inhibit IFN-{gamma}- and IL-17-induced peritoneal cell IL-6 secretion (p < 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 ± SEM of triplicate cultures and are representative of two independent experiments. d, mCD200R agonists do not inhibit IFN-{gamma}-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 ± SEM of triplicate cultures and are representative of two independent experiments. <, Below limit of detection (20 pg/ml).

 
CD200R expression on human peripheral blood leukocytes

We then explored the role of CD200R in the human system. CD200R expression was first investigated on human blood cells. Fresh ex vivo CD11c+lin– DCs expressed the highest CD200R levels, followed by CD14+ monocytes, CD4+ T cells, and CD8+ T cells (Fig. 2). B cells did not express CD200R (15). Monocyte-derived DCs also expressed high CD200R levels (Fig. 2), and slightly down-regulated CD200R expression after further maturation with CD40L or TNF and IL-1{beta} (data not shown).



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  		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.

 
Inhibition of TT-induced cytokine secretion from PBMC by CD200R triggering

We next investigated whether Ag-induced cytokine responses, requiring cooperation between Ag-presenting monocytes and T cells, were modulated by CD200R agonists. Thus, PBMC from recently TT-vaccinated donors were pretreated with CD200R agonists before stimulation with TT. TT dose-dependently induced IL-5, IL-13, IFN-{gamma}, and TNF (data not shown), but not IL-2, IL-4, IL-10, and IL-12p70 secretion (Fig. 3). CD200R agonists inhibited production of IL-5 and IL-13, but only when the agonists were further cross-linked by F(ab')2 goat anti-mouse/rat IgG (Fig. 3 and Table I). The effects on IFN-{gamma} (Fig. 3c) and TNF (data not shown) were lesser and more variable. No effect of CD200R stimulation was observed at suboptimal TT doses. Data from four independent donors are summarized in Table I.



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  		FIGURE 3. hCD200R agonists inhibit TT-induced PBMC cell IL-5 and IL-13 secretion. After a 30-min incubation with 10 µg/ml control Ig (gray striped bars), hCD200Ig (black striped bars), rat IgG1 isotype control (gray bars), or rat-anti-human CD200R (black bars), 1 x 106 PBMC were washed and the hCD200Ig or rat-anti-human CD200R were cross-linked with 5 µg/ml endotoxin-depleted F(ab')2 goat anti-mouse/rat IgG, and incubated for another 30 min before addition of 1, 10, or 100 ng/ml TT. Supernatants were harvested after 6 days. IL-5 (a), IL-13 (b), and IFN-{gamma} (c) secretion was determined by ELISA. Results are presented as mean ± SEM of triplicate cultures and are representative of three to four independent experiments. Statistically significant inhibition of IL-5 and IL-13 secretion by hCD200R agonists was observed (p < 0.01 for both agonists and cytokines, unpaired t test).

 

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

 
The inhibitory activity of CD200R agonists could be due to a direct or indirect effect on T cell activation, since both PBMC CD4+ T cells and monocytes express CD200R. Each cell type was isolated and separately treated with CD200R agonists to investigate whether the inhibition of TT-induced cytokine secretion required triggering of the hCD200R on monocytes, on CD4+ T cells, or on both cell types. Each cell type was incubated with an anti-hCD200R Ab and a cross-linking Ab and then mixed before stimulation with TT. Inhibition of IL-5 and IL-13 secretion was only observed when hCD200R was cross-linked on monocytes, but not when the hCD200R was cross-linked on CD4+ T cells (Fig. 4). As controls, IL-5 and IL-13 secretion was also inhibited when the CD200R was cross-linked on both monocytes and T cells, and in PBMC cultures performed in parallel (Fig. 4). In this experiment, IFN-{gamma} secretion was not inhibited after CD200R ligation.



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  		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 CD4+ T cells. Monocytes and CD4+ T cells were isolated by MACS to >95% purity. After separate incubation of 250,000 monocytes/well and 750,000 CD4+ T cells/well with 10 µg/ml rat IgG1 isotype control (gray bars) or rat-anti-human CD200R (black bars), cells were washed and the rat-anti-human CD200R was cross-linked with 5 µg/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 ± SEM of triplicate cultures, was determined by the LINCOplex Simultaneous Multianalyte Detection System using a Luminex. <, Below limit of detection (3 pg/ml). Inhibition was only observed when hCD200R was cross-linked on monocytes, or on both monocytes and T cells or in PBMC (p < 0.01 for both agonists and cytokines in all three cultures, unpaired t test).

 
Effects of hCD200R agonists on DC activation

CD200R was expected to be functionally important on monocyte-derived DCs, since they express high CD200R levels. Thus, we investigated the effects of CD200R agonists on these cells with and without further cross-linking. DCs were activated with LPS, IFN-{gamma}, CD40L and IFN-{gamma}, or TNF and IL-1{beta}. We analyzed expression of the cell surface markers CD1a, CD32, CD80, CD83, CD86, and HLA-DR, as well as secretion of IL-1{alpha}, IL-1{beta}, IL-6, IL-8/CXCL8, IL-10, IL-12p70, IP-10/CXCL10, MCP-1/CCL2, MIG/CXCL9, MIP-1{alpha}/CCL3, RANTES/CCL5, and TNF. We did not observe any effect of CD200R agonists on these parameters (data not shown). CD200R agonists also had no effect on allogeneic DC-induced CD4+ T cell proliferation or CD4+ T cell cytokine production using TT-loaded DC (data not shown). It is likely that human DC will also be modulated through CD200R given the recent data showing effects of CD200Ig on mouse DC function (24). However, to date, we have not identified the relevant in vitro conditions needed to observe CD200R-mediated modulation of human DC function.

Inhibition of hCD200R-transfected U937 cell cytokine production by CD200R agonists

Human monocytic U937 cells were transfected with hCD200R to further explore CD200R regulation of human myeloid cell activation. U937 cells did not express the receptor endogenously, but when infected with a retrovirus encoding hCD200R, they express hCD200R at high levels (Fig. 5a). IFN-{gamma} stimulation resulted in increased IL-8/CXCL8, IP-10/CXCL10, and MIG/CXCL9 secretion, whereas TNF, IL-1{beta}, IL-6, IL-10, and IL-12p70 production was not detectable (data not shown). CD200R agonists dose-dependently inhibited IFN-{gamma}-induced IL-8/CXCL8 (Fig. 5b), MIG/CXCL9, and IP-10/CXCL10 (data not shown) secretion from the hCD200R-transfected U937 cells. Transcriptional regulation probably contributed to these effects, as rat-anti-human CD200R decreased IFN-{gamma}-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).



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  		FIGURE 5. hCD200R agonists dose-dependently inhibit IFN-{gamma}-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-{gamma}-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 x 106 hCD200R-transfected U937 cells were stimulated with 100 U/ml recombinant human IFN-{gamma} (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 ± SEM of triplicate cultures. Statistically significant inhibition (unpaired t test) was observed from 0.3 µg/ml hCD200Ig and 0.1 µg/ml rat-anti-hCD200R.

 
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-{gamma}-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.



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  		FIGURE 6. Inhibition of IFN-{gamma}-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-{gamma}-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 x 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-{gamma} (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 ± SEM of triplicate cultures. Statistically significant inhibition (unpaired t test) of spontaneous secretion was observed from 0.1 µg/ml hCD200Ig for high expressing cells and 1 µg/ml hCD200Ig for medium and low expressing cells. For IFN-{gamma}-induced IL-8 secretion, statistically significant inhibition (unpaired t test) was observed from 0.3 µg/ml hCD200Ig for high and medium expressing cells and 1 µg/ml hCD200Ig for low expressing cells. c, Inhibition of IFN-{gamma}-induced IL-8/CXCL8 secretion by hCD200R agonists is dependent on hCD200R agonist cross-linking in low expressing cells (p < 0.001 for hCD200Ig and p = 0.02 for rat-anti-hCD200R, unpaired t test, in the presence of cross-linking). After a 30-min incubation with 1 µg/ml hCD200R agonists or isotype controls, 1 x 106 hCD200R-transfected cells with low hCD200R expression were washed and the hCD200Ig or rat-anti-human CD200R were cross-linked with 5 µg/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-{gamma}. Supernatants were harvested after 18 h. IL-8/CXCL8 secretion in supernatants was determined by ELISA and is presented as mean ± SEM of triplicate cultures.

 
In a second set of studies, minimally responsive low hCD200R-expressing cells were incubated with CD200R agonists but with cross-linking before IFN-{gamma} stimulation to determine whether further cross-linking of CD200R could convert a subtherapeutic inhibitory signal into a fully inhibitory signal. IFN-{gamma}-stimulated IL-8/CXCL8 production was inhibited by CD200R agonists when actively cross-linked (Fig. 6c). Furthermore, suboptimal levels of CD200R agonists induced stronger inhibition in medium and high expressing U937 cells in the presence of cross-linking Ab (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The present study shows that triggering of CD200R suppresses myeloid cell function in vitro. Thus, after stimulation with the myeloid cell-activating cytokine IFN-{gamma}, 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 contrast, responses after LPS stimulation were not affected. CD200R-mediated inhibition correlated with levels of CD200R on U937 cells. In cells expressing low CD200R levels, inhibition was only observed if the CD200R agonists were further cross-linked. TT-induced PBMC IL-5 and IL-13 secretion was also inhibited by cross-linked CD200R agonists. This inhibition required cross-linking the CD200R on monocytes, but not cross-linking the CD200R on CD4+ T cells. In all, our in vitro data agree well with the in vivo observations showing that targeted deletion of CD200 resulted in dysregulation of myeloid cells and enhanced susceptibility to autoimmune inflammation such as EAE and CIA (10).

As macrophages play pivotal roles in chronic inflammatory diseases through their broad proinflammatory, destructive, and remodeling capacities (1, 2, 3, 5), regulation of their function is critical for treatment of these disorders. One way of controlling inflammation is through blocking proinflammatory cytokines produced by macrophages, such as via anti-TNF therapy for rheumatoid arthritis, Crohn’s disease, and psoriasis (25). Triggering inhibitory receptors represents an alternative way of regulating myeloid cells (6), potentially superior to focused anti-cytokine therapies given the ability to affect a number of effector pathways via cellular targets. Many of these inhibitory receptors are members of the IgSF of integral membrane proteins. Data is now emerging that signaling through these receptors is essential for normal myeloid cell regulation in the peripheral immune system. Thus, disruption of signaling through the myeloid inhibitory receptors SIRP{alpha} and Fc{gamma}RIIB leads to potentiation of cellular function with consequent effects on inflammatory disease processes (17, 26).

The CD200-CD200R interaction provides a new regulatory mechanism for myeloid cells. The IgSF glycoprotein CD200, formerly known as OX-2, is expressed widely, including on thymocytes, B cells, endothelial cells, smooth muscle cells, and neurons (6, 27). CD200R is more restricted and is predominantly expressed on myeloid cells and T cells (14, 15). CD200 deletion resulted in myeloid cell dysregulation and enhanced susceptibility to autoimmune inflammation such as EAE and CIA (10), suggesting that CD200 normally induces immune suppression through CD200R. Soluble CD200R protein, binding CD200 and preventing CD200-CD200R interactions, increased the incidence of CIA in normally resistant mice (10). Mice receiving soluble CD200 were conversely resistant to CIA induction (11).

One potential mechanism through which CD200 may control inflammation in vivo is via inhibition of TNF and IL-6 production following stimulation of mouse macrophages with IFN-{gamma}, a very important activator of macrophage function (28, 29, 30). TNF plays a central role in autoimmune disease (25). TNF has several proinflammatory activities such as up-regulation of adhesion molecule expression, induction of cytokines and chemokines, angiogenesis and bone resorption (1, 25). IL-6 is the most strikingly elevated cytokine in human rheumatoid arthritis (1, 31), and it is required for experimentally induced autoimmune diseases, including CIA (32) and EAE (31, 33). Clinical trials with a humanized anti-IL-6R-antibody for treatment of rheumatoid arthritis and Crohn’s disease are ongoing (34). Proinflammatory effects of IL-6 include enhancing autoantibody production, bone absorption and triggering of acute phase responses (31), and blocking regulatory T cell suppression (35).

The observation that IL-17-induced IL-6 production was suppressed by CD200R agonists is also relevant to the in vivo mechanism of CD200R. This is the first report that IL-17 activates mouse macrophages, although enhancement of IL-6 production has been observed in human rheumatoid arthritis synovial cultures and human macrophages after IL-17 stimulation (36, 37). IL-17 is a T cell-derived cytokine that exhibits pleiotropic biological activities on various types of cells, such as fibroblasts, endothelial cells, and epithelial cells, mediating a wide range of responses, mostly proinflammatory and hemopoietic (38). Increased levels of IL-17 have been found in several human inflammatory disorders, including multiple sclerosis (39), rheumatoid arthritis (40), psoriasis (41), and asthma (42). Neutralizing IL-17 reduces the severity of CIA (43) and EAE (44), and its production is triggered by IL-23, an essential cytokine in the development of autoimmune inflammation (44).

CD200R triggering did not result in universal macrophage inhibition, since LPS-induced TNF responses were not affected. This could be a safety advantage in a therapeutic setting, especially if this holds true for other pathogen-derived TLR ligands. The differential effects of CD200R-mediated inhibition on IFN-{gamma}-, IL-17-, and LPS-induced cytokine responses is not surprising, as different signaling pathways are induced by these factors (30, 38, 45).

We also explored the role of CD200R in human systems in vitro after showing that mouse macrophages were functionally inhibited by CD200R triggering. As blood APCs and CD4+ T cells express the CD200R, we first investigated whether Ag-induced cytokine responses, requiring cooperation between Ag-presenting monocytes and T cells, could be modulated by CD200R agonists. We found that TT-induced PBMC IL-5 and IL-13 secretion was inhibited by CD200R agonists after further cross-linking. Effects on IFN-{gamma} and TNF secretion were more minor and variable. Cytokine responses were not affected by CD200R agonists in the absence of further cross-linking. The requirement for cross-linking is not surprising, as CD200R is normally present as a cell surface protein. Engagement of CD200 and CD200R as a cell-cell interaction is likely to lead to multimerization or aggregation of receptor, with enhanced receptor signaling potential. The inhibition of IL-5 and IL-13 secretion was dependent on cross-linking the CD200R on monocytes, but not on CD4+ T cells. It is presently unclear how triggering the CD200R on monocytes leads to a stronger inhibition of IL-5 and IL-13 responses than of IFN-{gamma} and TNF responses, although effects may be mediated through cell surface molecules involved in monocyte-T cell interaction. Preliminary results indicate that CD40, CD80, CD86, or HLA-DR expression levels are not changed by CD200R triggering, however. Interestingly, we have recently shown that in vivo administration of rat-anti-mCD200R Ab (DX109) selectively decreases IgE Ab responses to OVA in OVA-sensitized mice, suggesting inhibition of Th2-like responses in vivo (our unpublished data).

To further explore the role of CD200R in regulating human APC function, we investigated the effects of CD200R triggering on monocyte-derived DCs, as these cells express high CD200R levels. We have not yet been able to observe any effects of CD200R agonists on a broad range of cell surface markers or cytokine/chemokine secretion, however, after stimulation of the cells with LPS, IFN-{gamma}, CD40L and IFN-{gamma}, or TNF and IL-1{beta}. CD200R agonists also had no effects on allogeneic DC-induced CD4+ T cell proliferation or CD4+ T cell cytokine production when DCs were used to present TT. It is possible that other aspects of DC activation may be affected (e.g., via indoleamine 2,3-dioxygenase-based effects (24)), or that the in vitro conditions do not permit effects of CD200R triggering (e.g., contact with other cells and extracellular matrix proteins present in the in vivo environment are absent).

Human hCD200R-expressing monocytic U937 cells were used to further explore the CD200R regulation of human myeloid cell activation. CD200R engagement inhibited IFN-{gamma}-stimulated secretion of IL-8/CXCL8, IP-10/CXCL10, and MIG/CXCL9. Inhibition of cytokine production positively correlated with CD200R expression levels. Inhibition of low CD200R-expressing cells was only observed if the CD200R agonists were further cross-linked. This may explain why inhibition of TT-induced secretion required cross-linking of the CD200R. Inhibition of secretion of the chemotactic factors IL-8, IP-10, and MIG would reduce recruitment of neutrophils and Th1 cells to sites of inflammation (46).

In conclusion, we provide direct evidence in vitro that the CD200-CD200R interaction controls monocyte/macrophage function in both mice and humans and that the efficiency of CD200R-mediated inhibition of cellular functions is proportional to the receptor density at the cell surface. Our data solidify the concept that CD200R agonism may provide a novel approach to the treatment of chronic inflammatory diseases.


   Acknowledgments
 
We thank Janet Wagner and Sandra Zurawski for production of fusion proteins and mAbs.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 SP Biopharma (previously DNAX Research) is supported by Schering Plough, New Jersey. Back

2 Current address: Department of Molecular and Clinical Medicine, Division of Pediatrics, and Clinical Research Centre, Faculty of Health Sciences, Linköping University, Sweden. Back

3 Address correspondence and reprint requests to Dr. Edward P. Bowman, SP Biopharma, 901 California Avenue, Palo Alto, CA 94022; E-mail address: eddie.bowman{at}dnax.org or Dr. Jonathon D. Sedgwick at the current address: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285; E-mail address: sedgwickjd{at}lilly.com Back

4 Abbreviations used in this paper: DC, dendritic cell; IgSF, Ig superfamily; EAE, experimental autoimmune encephalitis; CIA, collagen-induced arthritis; h, human; m, mouse; CBA, cytometric bead array; IP-10, interferon-inducible protein 10; MIG, monokine induced by IFN-{gamma}; TT, tetanus toxoid. Back

Received for publication April 28, 2005. Accepted for publication October 7, 2005.


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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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