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The Journal of Immunology, 2005, 175: 2469-2474.
Copyright © 2005 by The American Association of Immunologists

Recombinant CD200 Protein Does Not Bind Activating Proteins Closely Related to CD200 Receptor1

Deborah Hatherley*, Holly M. Cherwinski{dagger}, Mehrdad Moshref{dagger} and A. Neil Barclay2,*

* Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom; and {dagger} DNAX Research Institute, Palo Alto, CA 94304


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD200 (OX2) is a cell surface glycoprotein that interacts with a structurally related receptor (CD200R) expressed mainly on myeloid cells and is involved in regulation of macrophage and mast cell function. In mouse there are up to five genes related to CD200R with conflicting data as to whether they bind CD200. We show that mouse CD200 binds the inhibitory receptor CD200R with a comparable affinity (Kd = 4 µM) to those found for the rat and human CD200 CD200R interactions. CD200 gave negligible binding to the activating receptors, CD200RLa, CD200RLb, and CD200RLc, by direct analysis at the protein level using recombinant monomeric and dimeric fusion proteins or to CD200RLa and CD200RLb when expressed at the cell surface. An additional potential activating gene, CD200RLe, found in only some mouse strains also did not bind CD200. Thus, the CD200 receptor family consists of both activatory and inhibitory members like several other paired ligand receptors, such as signal regulatory protein, killer cell Ig-like receptor/KAR, LY49, dendritic cell immunoreceptor/dendritic cell immunoactivating receptor, and paired Ig-like type 2 receptor. Although the ligand for the inhibitory product is a widely distributed host protein, the ligands of the activating forms remain to be identified, and one possibility is that they are pathogen components.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cell surface molecules containing Ig-like domains are particularly common in mammalian genomes (1). They are often involved in the intercellular communication of leukocytes, and their interactions are typified by low affinities (Kd = 1–100 µM) when measured in the monomeric state (2). CD200 (OX2) is a widely distributed cell surface protein that interacts with a receptor (CD200R) that is highly expressed on myeloid cells. Both proteins contain two extracellular Ig-like domains, but the receptor differs in that it has a longer cytoplasmic tail containing known signaling motifs (3). Studies with the CD200 knockout (4, 5) and in vitro analysis (6, 7, 8, 9) indicated that CD200-CD200R interactions are involved in the control of myeloid cellular function. The broad tissue distribution of CD200 and changes in its level of expression provide a mechanism for locally regulating myeloid cellular activity at appropriate sites, such as inflamed tissue (10, 11). The CD200-CD200R regulatory mechanism is an attractive target for immunomodulation, because its manipulation can induce either immune tolerance or autoimmune diseases. CD200-Fc fusion proteins have been shown to provide beneficial immunomodulatory effects in models of arthritis and allograft rejection (12, 13).

In addition to the inhibitor CD200R, several related genes have been identified in the mouse. These were termed CD200RL (for receptor like) (14), CD200R1, CD200R2, etc. (15, 16). These are predicted to be associated with DNAX activating protein (DAP)12, and this has been shown for two of the mouse genes, CD200RLa (CD200R4) and CD200RLb (CD200R3) (14), and also independently for CD200R3 (16). Thus, these proteins are expected to give activating signals and, hence, are similar to the many examples of paired receptors where closely related genes contain members that can give inhibitory signals and those that give activating signals through adaptors such as DAP12. In some cases, the ligands for the inhibitory forms do not bind the activating forms, such as with the signal regulatory proteins (SIRPs)3 (17, 18). The NKAT2 inhibitory p58 binds the HLA-C peptide complex with much higher affinity than the activating p50 (19); however, both activating Ly49H and inhibitory Ly49I can bind the same m157 mouse CMV protein, although affinities have not been measured (20). Both paired Ig-like type 2 receptor (PILR){alpha} (inhibitory) and PILR{beta} (activating) bind a CD99-related protein (21). With regard to CD200, it was reported that CD200-Fc fusion proteins did not bind to CD200RLa and CD200RLb (14), but a contradictory report suggested that similar reagents bound all the activating proteins as well as the inhibitory form (15). Clearly this has important functional implications and possibilities for therapies using CD200-Fc fusion proteins.

In this study we critically examine binding of CD200-Fc and other CD200 fusion proteins to CD200RL, proteins expressed at the cell surface and directly at the protein level, to determine the affinities of the interactions at 37°C. This establishes that CD200 is not a significant ligand for four CD200R-related proteins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Construction, expression, and purification of soluble recombinant proteins

A fusion protein consisting of the extracellular domain of mouse (m) CD200 fused to the Fc domain of mouse IgG1 mutated in the CH2 domain (D265A) to inhibit binding to FcRs (CD200-Fc) and control mutant IgG (cFc) were generated as previously described (22). The inability of the fusion proteins to bind FcRs was confirmed by staining the FcR-positive cell line J774 (data not shown). The clonings of CD200RLa and CD200RLb were described previously (14). Full-length CD200RLc was not obtained in that study. The complete extracellular region of CD200RLc was isolated by PCR from cDNA purified from mouse peritoneal exudate cells, and the extracellular region had the same sequence as that previously published (accession no. NM_206535). CD200RLe (accession no. BAC40774) was isolated by PCR from cDNA prepared from bone marrow mast cells derived from CD-1 mice (Charles River Laboratories) as previously described (14). The mCD200R 5' primer (5'-gtgcttaaccagattccactcc-3') and 3' primer (5'-tgttttcttgtattgtgtcatcagac-3') were used. Fusion proteins for mouse CD200R, CD200RLa, CD200RLb, and CD200RLc and CD200RLe with domains 3 and 4 of rat CD4 plus a biotinylation signal were prepared as previously described (14, 23). The boundary between the CD200RLa part and CD4 was MTTPRSTSIT, for CD200RLb TSILPSTSIT, for CD200RLc TTPSTSIT and for CD200RLe QGSTSIT (CD4 linker is underlined). Mouse CD200CD4d3+4 (4) was produced in CHO.K1 cells using the expression vector pEE14 and was subsequently purified from spent tissue culture medium by immunoaffinity chromatography using an OX68 mAb-Sepharose 4B column (3, 24). Before BIAcore analysis, the purified CD200 protein was fractionated by gel filtration on a Superdex S-200 column (Pharmacia Biotech) to exclude larger protein aggregates that are known to influence binding measurements (25).

Flow cytometry

Ba/F3 cells expressing mouse CD200R, CD200RLa, and CD200RLb and mAb recognizing mCD200R (DX109), mCD200RLa (DX87), and mCD200RLb (DX116) were generated as previously described (14). Surface expression of the CD200 receptors was analyzed by flow cytometry using the specific CD200 receptor mAb described above, followed by goat anti-rat PE-conjugated secondary Ab (Caltag Laboratories). CD200-Fc or cFc (2 µg/5 x 105 cells) was incubated on ice for 30 min at 4°C in PBS containing 1% BSA and 0.05% sodium azide. Cells were washed, and Fc was detected using a PE-conjugated rat anti-mouse IgG1 secondary Ab (clone A85-1; BD Biosciences). Analyses were performed on a FACScan flow cytometer (BD Biosciences).

Measurement of affinities of interactions of CD200 using surface plasmon resonance

Affinity and kinetic data were collected using a Biacore 2000 (Biacore) at 37°C as previously described (3). Briefly, ~2500 response units (RU) of streptavidin was coupled to a CM5 research grade chip using amine coupling. Biotinylated mCD200RCD4d3+4-related proteins and control CD4d3+4 were each immobilized at ~1000 RU. For kinetic analysis, serially diluted monomeric CD200CD4d3+4 purified proteins were injected at the indicated active concentrations over all four flow cells connected in series. The extinction coefficient, 53,590 M–1cm–1, was calculated. Kd values were obtained by both nonlinear curve fitting and Scatchard transformations to the binding data. The following were then passed over all four flow cells sequentially: cIg, CD200-Fc, specific CD200R mAb, and OX68 mAb recognizing CD4d3+4 common to all proteins (all at 20 µg/ml).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Analysis of the interaction of CD200 with CD200R and related proteins expressed at the cell surface

Fig. 1 shows the similarity in the amino acid sequences of the extracellular regions of mCD200R and related proteins. Ba/F3 cells were transfected with constructs for CD200R, CD200RLa, or CD200RLb, and the three receptors were expressed at high levels, as shown by binding of three specific mAb (Fig. 2, A–C). A DAP12 construct was cotransfected with CD200RLa and CD200RLb, because previous studies had shown that this was necessary to obtain expression of CD200RLa and CD200RLb at the cell surface (14). Soluble fusion proteins consisting of the extracellular domains of mCD200 fused to the Fc binding domain of mouse IgG gave very strong binding to cells expressing CD200R and not to cells expressing the activating receptors CD200RLa and CD200RLb (Fig. 2, D–F). No binding could be obtained for these proteins using a variety of CD200-Fc fusion protein concentrations, labeling times, and temperatures, indicating that CD200 is a ligand for CD200R, but not for the related activating gene products, CD200RLa and CD200RLb.



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  		FIGURE 1. The mCD200R and related sequences. The NH2 terminus is based on protein data for rat CD200R. The superscript bars predict the extent of the {beta} strands characteristic of the Ig fold by comparison with solved structures. Additional mouse genes include two forms isolated through their association with DAP12 (mCD200RLa and mCD200RLb; accession no. XM156177 and XM148097). A sequence found in some mouse strains, but not the C57BL genome (BAC40774), is named CD200RLe. This and mCD200RLc (NM_206535) are predicted to be associated with DAP12. Residues identical in four or more sequences are boxed (three for cytoplasmic regions). An additional sequence, termed CD200RLd, is not shown, because it is incomplete and may not give an expressed protein (14 ).

 


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  		FIGURE 2. Flow cytometry showing binding of CD200-Fc to CD200R, but not to CD200RLa and CD200RLb. Ba/F3 cells expressing mCD200R (A and D), Ba/F3 cells expressing DAP12 and CD200RLa (B and E), and Ba/F3 cells expressing DAP12 and CD200RLb (C and F) were stained with specific mAb (A–C) or CD200-Fc (D–F). All three constructs were expressed at high levels (A–C), and CD200-Fc fusion protein gave strong specific binding to CD200R (D), but not to CD200RLa (E) or CD200RLb (F). Controls for mAb labeling (cIgG; A–C) and Fc fusion protein (cFc; D–F) gave no labeling.

 
Analysis of interaction of CD200 with CD200R and related proteins at the protein level

The failure of CD200-Fc protein to bind to CD200RLa and CD200RLb is unlikely to be due to a lack of sensitivity in the assay, because very high levels of expression of all three receptors were obtained (Fig. 2, A–C), and there was strong binding of CD200-Fc to the control CD200R (Fig. 2D). However, analysis using purified proteins allowed a quantitative comparison of interacting proteins to be determined. CD200R, CD200RLa, CD200RLb, and two gene products, CD200RLc and CD200RLe (see below), for which specific mAb were not available, were expressed as chimeric proteins consisting of the extracellular regions of these proteins together with two domains of rat CD4 as an antigenic tag and a sequence that allows biotinylation. The proteins were expressed transiently, biotinylated, concentrated, and bound to streptavidin-coated BIAcore chips along with a control protein, CD4d3+4, consisting of the two domains of CD4 present in each construct. Mouse CD200CD4d3+4 was expressed in Chinese hamster ovary cell lines and purified by affinity chromatography, followed by gel filtration to remove any aggregates that might produce anomalous binding. The resultant preparation was passed over the four proteins. Clear binding to CD200R was obtained, but not to CD200RLa, CD200RLb, or the control CD4d3+4 (Fig. 3A), confirming the data on binding to cells (Fig. 2). The interactions were weak as expected with rapid dissociation over a few seconds. Thus, high concentrations of mCD200 were used, which gave rise to a bulk effect as the protein passed over the chip. For the control protein, CD4d3+4, this was seen as signal that quickly reached equilibrium and was washed out rapidly. The specific binding of CD200 to CD200R showed an increased signal compared with the control, which also quickly reached equilibrium and then dissociated rapidly. CD200RLb gave the same signal as the control protein, indicating that there was no specific binding to CD200; trace binding was observed with CD200RLa, and this was quantitated further (see below and Fig. 4A).



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  		FIGURE 3. Analysis by surface plasmon resonance shows mCD200 interacts with mCD200R, but not with related proteins. A, CD200CD4d3+4 (40 µM) was injected at 20 µl/min at 37°C over four flow cells in sequence that had been coupled with CD200RCD4d3+4-biotin (1167 RU) or CD200RLaCD4d3+4-biotin (1064 RU), CD200RLbCD4d3+4-biotin (1145 RU), and CD4d3+4-biotin (1278 RU) immobilized on BIAcore chips to which streptavidin (2500 RU) had been previously coupled. The monomeric CD200CD4d3+4 binds well to CD200R, trace amounts bind to CD200RLa, and does not bind to CD200RLb or the control, where the signal is due to the bulk effect of the high protein concentration. B, CD200-Fc fusion protein binds to CD200CD4d3+4, binds well to CD200R, but does not bind to CD200RLa, CD200RLb, or the control, giving a similar bulk effect to the control Fc. C, DX109 mAb binds only to CD200R. D, DX87 mAb gives minimal binding to CD200RLa (see text). E, DX116 mAb binds only CD200RLb. F, CD200R, CD200RLa, CD200RLb, and control CD4d3+4 protein bind OX68 mAb recognizing the CD4 tag present in all proteins, giving additional evidence for the presence of well-folded chimeric proteins on the chip. The bars above the trace indicate the duration of the injection of the protein named.

 


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  		FIGURE 4. Equilibrium binding data for CD200 binding to CD200R, CD200RLa, CD200RLb, CD200RLc, and CD200RLe. A, A series of concentrations of CD200CD4d3+4 was passed over the proteins as described in Fig. 3A. The amount of CD200CD4d3+4 that bound at each concentration was calculated as the difference between the responses at equilibrium in the CD200RCD4d3+4 and control flow cells. The curved lines in the main plot are nonlinear curve fits to the data and correspond to Kd = 4.8 µM for CD200 binding CD200R. No binding was obtained to CD200RLb, and trace amounts of binding to CD200RLa indicate a Kd of >500 µM. C, Comparable experiment showing that CD200 did not bind CD200RLe, and there was only trace binding to CD200RLc. A similar affinity (Kd = 3.8 µM) was found for CD200 binding CD200R as in A. B and D, Scatchard analysis of data in A and C, showing good linearity and comparable Kd values to curve fitting for CD200 binding to CD200R.

 
The dimeric CD200-Fc fusion protein used in cell binding assays was passed over the flow cells with similar results, namely good binding to CD200R, but not to CD200RLa and CD200RLb (Fig. 3B). The fusion protein was at a much lower concentration (~0.2 µM) than the monomeric CD200 protein in Fig. 3A and, as expected, gave more avid binding, with a slower on rate; equilibrium was not reached, and dissociation occurred over ~5 min.

The conditions of protein production and immobilization permit the minimum of handling and are unlikely to cause denaturation. Three mAb specific for the three proteins were passed over the flow cells, showing specific binding of DX109 to CD200R and of DX116 to CD200RLb (Fig. 3, C and E). The DX87 mAb did not label CD200R and CD200RLb as expected, but only gave marginal labeling of CD200RLa (20 RU; Fig. 3D). However, this mAb did give very good labeling of CD200RLa expressed at the cell surface (Fig. 2). It is possible that this mAb reacts better with cell surface-expressed protein that might have different glycosylation compared with the soluble extracellular region of the fusion protein. (This phenomenon has been observed for another highly glycosylated cell surface protein, SIRP{gamma} (18).) This protein and indeed all the CD200Rs were reactive with the OX68 mAb that binds the CD4 tag present in the chimeric proteins (Fig. 3F), suggesting that all the proteins are correctly folded. In the unlikely event that the CD200RLa was completely inactive, the data from Fig. 2, B and E, clearly showed no CD200 binding to antigenically active CD200RLa expressed at the cell surface.

The experiment was repeated with a range of concentrations of CD200-CD4d3+4 protein, and the equilibrium binding levels are plotted in Fig. 4A. The affinity for CD200 binding to CD200R was calculated (Kd = ~4 µM at 37°C) from curve fitting (Fig. 4A) and Scatchard analysis (Fig. 4B) and was comparable to that determined for the equivalent interaction in rats (Kd = 1 µM at 37°C) (3) and humans (Kd = 0.5 µM at 37°C) (14). The binding to CD200RLa was too weak to get accurate values, but extrapolation of the data would indicate a Kd >500 µM at 37°C, which is unlikely to be significant physiologically. There was no detectable binding of CD200-CD4d3+4 to CD200RLb.

Comparable experiments were conducted for CD200RLc, although there are no specific mAb recognizing this protein, and it is not known whether this protein is expressed on normal cells (14). This gene product also gave no significant binding to CD200 (Fig. 4C), whereas CD200R gave values similar to those shown in Fig. 4A. One CD200R mAb, OX110, cross-reacted weakly on this protein (data not shown).

A new CD200R-like gene (CD200RLe or CD200R5) present in some strains of mice

This gene (accession no. BAC40774) was characterized as an expressed sequence tag from the NOD mouse, noted in database searches (16), and termed CD200R5 (16). No mRNA expression was found in C57BL/6 mice (16), and we found no evidence of this gene in searches of the mouse genome (C57BL/6J; <www.ensembl.org/Mus_musculus/>), through established sequence tag searches, or through functional analysis. However, it was expressed in the CD1 mouse strain. The extracellular region of this protein was expressed as described above, and binding to CD200 was analyzed. This protein also did not bind CD200 (Fig. 4C). We term this gene product CD200RLe (RL stands for receptor like), rather than CD200R5, because it is not a receptor of CD200. It should be noted that it is probably not present in all mouse strains, and likewise, the expression of all the other gene products has not been tested in this mouse.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We present clear evidence that CD200 does not bind CD200RLa, CD200RLb, CD200RLc, or CD200RLe, but gives good binding to CD200R. CD200RLa and CD200RLb are known to be expressed on normal leukocytes (14). These data are in agreement with and extend those reported previously (14), but disagree with a recent paper by Gorczynski et al. (15), who found that CD200RLa, CD200RLb, and CD200RLc bound CD200 in addition to CD200R. In the latter, it is argued that the CD200RLb used (14) was not full length, because they identified a Met further upstream. In fact, the construct used previously (14) and in this paper also included this Met, so there is no difference in the protein, just the interpretation of which Met might be used as an initiator. By analogy with the other genes, the earlier Met is more likely to be used, as suggested by Gorczynski et al. (15). Similar Fc fusion proteins are used to recognize CD200R and related genes transfected into cell lines. The data we present show that there is no reaction at the cell surface using similar reagents, and virtually no binding at the protein level using carefully controlled reagents. The mostly likely explanation for the discrepancy centers on the fact that we (14) have shown that the activating CD200RLa and CD200RLb were only expressed in the presence of DAP12, and others (16) have shown the same for CD200RLb (CD200R3). In the study by Gorczynski et al. (15) this does not seem to have been done, and it seems unlikely that specific binding of CD200 to these products is being observed. DAP12 is a small disulfide-bonded homodimer that is structurally similar to the Fc{epsilon}RI {gamma}-chain and the TCR {zeta}-chain (26, 27) that contains a negatively charged residue in its transmembrane that allows pairing with a variety of cell surface receptors with positively charged amino acid residues in their transmembrane regions. The cytoplasmic domain of DAP12 contains a consensus ITAM motif, which, when phosphorylated, recruits protein tyrosine kinases (26, 27, 28, 29). There is a charged residue in all the CD200RL gene products, and they probably all interact with DAP12 and have the potential to produce activating signals. The current analysis uses well-characterized mAb that are highly specific for the gene products and recognize the proteins on normal cells, whereas in the study by Gorczynski et al. (15) the Abs are less well characterized antipeptide reagents.

Analysis of the sequence similarities of the extracellular regions of the receptors shows that CD200R is most similar to the strain-specific CD200RLe (91% amino acid sequence identity), but is also similar to CD200RLa (84%) and CD200RLc (83%, although this lacks part of the N-terminal region), and is less similar to CD200RLb (39%). Mutagenesis analysis of the human CD200R has identified residues in predicted {beta} strands C and F important for binding human CD200 (30). Analysis of this region in the mouse shows that it is highly conserved between the binding CD200R and the nonbinding CD200RLe. However, a single residue change can eliminate human CD200R-CD200 binding (30), and this sensitivity of the binding site to small changes in sequence may be important in the evolution of activating receptors from inhibitory receptors.

CD200R resembles other gene families, such as SIRP, killer cell Ig-like receptor, NKG2A/C, LY49, dendritic cell immunoreceptor/dendritic cell immunoactivating receptor, and PILR, that have both activating and inhibitory forms (31, 32, 33, 34, 35). The numbers of genes can vary considerably between species and strains, and this is also observed with CD200R, for which there are several related genes in the mouse, but only one in the human, and the number in mice may vary. Like some other paired receptors, the inhibitory CD200R binds a host protein. In addition, there is an example where a CD200 homologue (K14) in the HHV8 virus binds the inhibitory receptor (9). The ligands of CD200RLa, CD200RLb, CD200RLc, and CD200RLe and the functional and biological significance of these receptors remain unknown; however, it is possible that these receptors have evolved to interact with bacterial or viral components in a manner similar to that recently described for a DAP12 pairing member of the Ly49 family and mouse CMV m157 protein (20, 36).


   Acknowledgments
 
We are grateful to Marion H. Brown and Dan Gorman for helpful discussions.


   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 This work was supported by the Medical Research Council and the Cellular Immunology Unit hybridoma fund. DNAX is supported by Schering Plough Corp. Back

2 Address correspondence and reprint requests to Dr. A. Neil Barclay, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, U.K. OX1 3RE. E-mail address: neil.barclay{at}path.ox.ac.uk Back

3 Abbreviations used in this paper: SIRP, signal-regulatory protein; cFc, control mutant IgG; RU, response unit; DAP, DNAX activating protein; PILR, paired Ig-like type 2 receptor. Back

Received for publication March 28, 2005. Accepted for publication June 6, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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