Characterization of the CD200 Receptor Family in Mice and Humans and Their Interactions with CD200

This information is current as Gavin J. Wright, Holly Cherwinski, Mildred Foster-Cuevas, of September 28, 2021. Gary Brooke, Michael J. Puklavec, Mike Bigler, Yaoli Song, Maria Jenmalm, Dan Gorman, Terri McClanahan, Man-Ru Liu, Marion H. Brown, Jonathon D. Sedgwick, Joseph H. Phillips and A. Neil Barclay

J Immunol 2003; 171:3034-3046; ; Downloaded from doi: 10.4049/jimmunol.171.6.3034 http://www.jimmunol.org/content/171/6/3034

References This article cites 39 articles, 20 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/171/6/3034.full#ref-list-1

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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 © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Characterization of the CD200 Receptor Family in Mice and Humans and Their Interactions with CD2001

Gavin J. Wright,2,3* Holly Cherwinski,3† Mildred Foster-Cuevas,* Gary Brooke,* Michael J. Puklavec,* Mike Bigler,† Yaoli Song,† Maria Jenmalm,† Dan Gorman,† Terri McClanahan,† Man-Ru Liu,† Marion H. Brown,* Jonathon D. Sedgwick,† Joseph H. Phillips,3,4† and A. Neil Barclay3,4*

CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a structurally related receptor (CD200R) expressed on rodent myeloid cells and is involved in regulation of macrophage function. We report the first characterization of human CD200R (hCD200R) and define its binding characteristics to hCD200. We also report the identification of a closely related

to hCD200R, designated hCD200RLa, and four mouse CD200R-related (termed mCD200RLa-d). CD200, CD200R, and Downloaded from CD200R-related genes were closely linked in humans and mice, suggesting that these genes arose by gene duplication. The distributions of the receptor genes were determined by quantitative RT-PCR, and expression was confirmed by a set of novel mAbs. The distribution of mouse and human CD200R was similar, with strongest labeling of macrophages and neutrophils, but also other leukocytes, including monocytes, mast cells, and T lymphocytes. Two mCD200 receptor-like family members, designated mCD200RLa and mCD200RLb, were shown to pair with the activatory adaptor protein, DAP12, suggesting that these

receptors would transmit strong activating signals in contrast to the apparent inhibitory signal delivered by triggering the http://www.jimmunol.org/ CD200R. Despite substantial with mCD200R, mCD200RLa and mCD200RLb did not bind mCD200, and presently have unknown ligands. The CD200 receptor gene family resembles the signal regulatory and killer Ig-related receptors in having receptor family members with potential activatory and inhibitory functions that may play important roles in immune regulation and balance. Because manipulation of the CD200-CD200R interaction affects the outcome of rodent disease models, targeting of this pathway may have therapeutic utility. The Journal of Immunology, 2003, 171: 3034–3046.

ell surface molecules containing Ig-like domains are one activation of macrophages in several tissues and a profound in- of the most abundant protein classes found in mamma- crease in susceptibility to autoimmune disease models affecting the C lian genomes (1), and the systematic characterization of brain and joints. These results indicated that CD200-CD200R in- by guest on September 28, 2021 these proteins expressed on leukocytes has provided many insights teractions are involved in the control of myeloid cellular function into their function. For example, the proteins that initiate intercel- (4). The broad tissue distribution of CD200 and changes in its level lular communication by forming receptor-ligand pairs and their of expression provide a mechanism for locally regulating myeloid ϭ ␮ interactions are typified by low affinities (KD 1Ð100 M) when cellular activity at appropriate sites, such as inflamed tissue (5, 6). measured in the monomeric state (2). Rat CD200 (OX2) is a The CD200-CD200R regulatory mechanism is an attractive target widely distributed cell surface protein that interacts with a receptor for immunomodulation because its manipulation can induce im- (CD200R) that is highly expressed on myeloid cells. Both proteins mune tolerance and autoimmune diseases. CD200-Fc fusion pro- contain two extracellular Ig-like domains, but the receptor differs teins have been shown to provide beneficial immunomodulatory in that it has a longer cytoplasmic tail containing known signaling effects in models of arthritis and allograft rejection (7, 8). motifs (3). The CD200-deficient mouse had observable alterations In this study, we have identified the homologue of the CD200 in the behavior of myeloid cells in tissues that normally expressed receptor in humans, and show that it is expressed at the surface of CD200. These included both an increase in number and state of myeloid cells and T cells and that it binds CD200. A second related gene was analyzed in humans and an additional four genes in mice that we have designated CD200 receptor-like (CD200RL)5 proteins. *Sir William Dunn School of Pathology, University of Oxford, Oxford, United King- Two of the mouse gene products were shown to pair with the immu- † dom; and DNAX Research Institute, Palo Alto, CA 94304 noreceptor tyrosine-based activation motif-containing adapter protein, Received for publication April 14, 2003. Accepted for publication July 9, 2003. DAP12, creating the potential for activating signal transduction. 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. Materials and Methods 1 This work was supported by the Medical Research Council. DNAX is supported by Cell lines and staining reagents Schering Plough. Human mast cells were derived from freshly isolated cord blood, as pre- 2 Current address: Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cam- viously described (9, 10). Mouse C57BL/6 mast cells were derived from bridge CB10 1SA, U.K. bone marrow of 2- to 3-wk-old mice. Bone marrow cells were cultured in 3 G.J.W., H.C., J.H.P., and A.N.B. contributed equally to this study. DMEM (BioWhittaker, Walkersville, MD) supplemented with 10% FCS 4 Address correspondence and reprint requests to Dr. A. Neil Barclay, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, U.K. E-mail address: [email protected], or Dr. Joseph H. Phillips, 5 Abbreviations used in this paper: CD200RL, CD200 receptor-like; EAE, experi- DNAX Research, 901 California Avenue, Palo Alto, CA 94304-1104. E-mail address: mental autoimmune encephalomyelitis; h, human; m, mouse; RU, response unit; EST, [email protected] expressed sequence tag.

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 3035

(HyClone, Logan, UT), 1.0 mM sodium pyruvate, 0.1 mM nonessential Analysis of the human and mouse genomes amino acids, 0.3 mg/ml of L-glutamine, 20 mM HEPES, 50 ␮M 2-ME, 50 ng/ml of recombinant stem cell factors (PeproTech, Rocky Hill, NJ), and The NCBI human and mouse genome databases were searched using 30 ng/ml of murine rIL-3. After 2 wk, the cells were cultured in RPMI BLAST (12), and corresponding genes and related genes were identified. 1640 medium containing the same supplements as above minus the stem Ability of CD200RL proteins to pair with DAP12 cell factor, but with the addition of 5 ng/ml of murine rIL-4. Ba/F3, a pro-B cell line, was kindly provided by T. Kitamura (University of Tokyo, To- cDNAs encoding full-length hCD200RLa, mCD200RLa, and kyo, Japan). A cDNA containing the CD8 leader segment followed by the mCD200RLb were subcloned into the pMX-pie retrovirus expression vec- FLAG peptide epitope and joined to the extracellular, transmembrane, and tor. Plasmid DNAs representing the various genes were transfected into the cytoplasmic domains of mouse DAP12 (mDAP12) was subcloned into the Phoenix ecotropic virus packaging cell line (G. Nolan), and the viruses retroviral vector pMX-neo (11). Plasmid DNA of this construct was trans- subsequently obtained were used to infect Ba/F3-mDAP12 cells, as previ- fected into the Pheonix ecotropic virus packaging cell line (G. Nolan, Stan- ously described (11). In the absence of a pairing partner, FLAG-tagged ford University, Stanford, CA), and viruses obtained were used to infect DAP12 remains within the cytoplasm of Ba/F3-mDAP12; however, if the Ba/F3 cells. Ba/F3-mDAP12 cells stably express cytoplasmic FLAG- CD200RL proteins can pair with DAP12, the FLAG epitope will appear on tagged mDAP12. Mouse and human peripheral blood leukocytes were pre- the cell surface of Ba/F3-mDAP12. After 1Ð2 wk in selection conditions, pared from peripheral blood by hypotonic lysis of RBC. Mouse splenic the Ba/F3-mDAP12 cells infected with the various CD200RL genes were leukocytes were prepared from single cell suspensions after hypotonic lysis analyzed by flow cytometry for the surface expression of the FLAG epitope of RBC. of DAP12 (anti-M2 Ab; Sigma-Aldrich, St. Louis, MO). These cells were The following anti-mouse leukocyte Abs were obtained from BD Bio- also stained with mAbs specific for the various CD200RL proteins. sciences (San Jose, CA): FITC-DX5, FITC-CD4, FITC-CD8, FITC-CD19, FITC-CD11b, and CyChrome-CD3. The following anti-human leukocyte CD200 binding to CD200R and CD200RL proteins Abs were obtained from BD Biosciences: FITC-CD3, FITC-CD4, FITC-

CD8, FITC-CD19, FITC-CD14, FITC-CD16, CyChrome-CD56, Cy- Fusion proteins, consisting of the extracellular domains of mCD200 and Downloaded from Chrome-CD3,andCyChrome-CD14.FITC-conjugatedanti-humanIgE(anti- hCD200 fused to the Fc region of hIg (mCD200-hIg, hCD200-hIg), were hIgE) was obtained from Kirkegaard & Perry Laboratories (Gaithersburg, used to investigate the ability of the mouse and human CD200R and MD). Anti-mouse CD200R (DX109, rat IgG1, OX110, rat IgG1), anti- CD200RL proteins to bind a soluble form of CD200. Stable transfectants mouse CD200RLa (DX87, rat IgG2c), and anti-mouse CD200RLb of Ba/F3-expressing mCD200R, hCD200R, and Ba/F3-mDAP12-express- (DX116, rat IgG1) were generated from rats using immunogenic fusion ing mCD200RLa or mCD200RLb were generated by retroviral infections, proteins consisting of the extracellular domains of the various CD200R as previously described. One microgram of mCD200-hIg, hCD200-hIg, or ϳ 6 genes fused to the Fc domain of hIg (mCD200R-hIg, mCD200RLa-hIg, control-hIg was used to stain 10 cells of the various receptor transfec- http://www.jimmunol.org/ mCD200RLb-hIg), except for OX110, in which mCD200RCD4d3ϩ4 was tants for 20 min at 4¡C. After washing, the cells were then stained with a used. Anti-human CD200R (DX136, rat IgG2a) was generated from a rat PE-conjugated goat anti-hIg Ab (20 min at 4¡C; Caltag), washed, and an- immunized with a fusion protein consisting of the extracellular domain of alyzed on a FACScan (BD Biosciences). For receptor Ab-blocking exper- hCD200R fused to the Fc domain of hIg (hCD200R-hIg). Anti-human iments, blocking mAbs against the CD200Rs were incubated with cells (2 ␮ 6 mCD200R (OX108, mouse IgG1) was generated from a BALB/c mouse g/10 cells) for 20 min at 4¡C before the addition of the mouse or human immunized with hCD200RCD4d3ϩ4 (see below). CD200-hIgs. Construction, expression, and purification of soluble Cloning of hCD200R recombinant proteins The National Center for Biotechnology Information (NCBI) expressed se- The CD200-hIg fusion proteins contain two CD200 extracellular regions, quence tag (EST) database was screened using the rat cDNA sequence of by guest on September 28, 2021 but for kinetic studies a monomeric hCD200CD4d3ϩ4 was expressed by CD200R (GenBank accession AF231392) utilizing the BLASTN program CHO.K1 cells using the expression vector pEE14 and subsequently puri- (12). A weak match was identified in the 3Ј nontranslated region with clone fied from spent tissue culture medium by immunoaffinity chromatography IMAGE:2054703. This clone was ordered from NCBI, and further se- using an OX68 mAb-Sepharose 4B column (3). Before BIAcore analysis, quencing revealed an open reading frame that contained an apparent in- the purified CD200 protein was fractionated by gel filtration on a Superdex sertion that affected the reading frame before the proposed transmembrane S-200 column (Pharmacia, Uppsala, Sweden) to exclude larger protein ag- domain. The full-length hCD200R sequence was then amplified from ϩ gregates that are known to influence binding measurements. The soluble cDNA generated from human lung poly(A) RNA (Clontech, Palo Alto, biotinylated forms of rat and mouse CD200R were produced, as described CA) by PCR using oligonucleotides cccactgttgatggggtaag (sense) and (3), and the human form was generated in an identical fashion after am- gactcgaggaaactgttcacacttgctcc (antisense with XhoI site underlined), and plifying the entire extracellular region (including the signal sequence) us- the products were reamplified in a nested PCR using the same antisense ing oligonucleotides gaaatctagaaaacagaaatgctctgcccttggag (sense with oligonucleotide, but gcagagcggccgcaaacagaaatgctc (sense) to introduce a XbaI site underlined) and tttggcagtcgacacaggaagtagctctatgtacagactc (anti- NotI site (underlined) for cloning. Products were cloned into appropriately sense with SalI site) in the pEF-BOS-CD4d3ϩ4bio-XB vector (14). The digested PCRScript (Stratagene, La Jolla, CA) vector, and sequencing of boundary of the hCD200R protein with CD4 domain 3 was ELLPVSTSIT three clones identified two alleles that differed at 3 aa in the extracellular (CD4 linker in bold). region (Fig. 1). Measurement of affinities using surface plasmon resonance Cloning of mCD200RL genes Affinity and kinetic data were collected using a BIAcore 2000 at 37¡C, as mCD200RLa was cloned from normal bone marrow-derived mast cells described (3). Briefly, ϳ5,000 response units (RU) of streptavidin were using a DAP12 trap technique, as previously described (13). In brief, a coupled to a CM5 research grade chip using amine coupling. In separate cDNA library was prepared from normal mouse mast cells in a pJEF14 experiments, biotinylated rat, mouse, or human CD200RCD4d3ϩ4 pro- vector. cDNA cloning by transient expression in 293T/FLAG-DAP12 cells teins were immobilized at high (ϳ1,600 RU), medium (ϳ850 RU), and was performed, as previously described (13). Transfected cells were low (ϳ400 RU) levels in three flow cells and control CD4d3ϩ4(ϳ2,200 screened for cDNA-encoded proteins that were capable of pairing with RU) in the fourth flow cell. Serially diluted monomeric hCD200CD4d3ϩ4 DAP12 and translocating FLAG-tagged DAP12 to the cell surface. Cell purified soluble chimeric proteins were then injected at the indicated active surface DAP12 was then visualized using the anti-FLAG M2 mAb (Kodak, concentrations over all four flow cells connected in series. The extinction Rochester, NY) and PE-conjugated goat anti-mouse second step (Caltag, coefficient, 40,534 MϪ1cmϪ1, was determined by amino acid analysis. The Burlingame, CA). Cells expressing surface FLAG-DAP12 were sorted on minimal fraction of purified protein able to bind the hCD200R was deter- a FACS cell sorter (BD Biosciences), and plasmid DNA was recovered. mined by depletion using avidin-Sepharose agarose beads (Sigma-Aldrich) After three rounds of cell sorter selection, single plasmid colonies were coated in biotinylated rat CD200RCD4d3ϩ4 as compared with biotinyl- used for transfection, and positive clones were selected and DNA inserts ated CD4d3ϩ4. Depleted and control fractions were resolved by SDS- sequenced. mCD200RLb was identified by homology search of the NCBI PAGE and densitometrically analyzed using ImageQuant software. At least EST database and a full-length cDNA isolated by standard PCR cloning 90% of hCD200 protein could be depleted by rat CD200R, and active techniques from mouse bone marrow-derived mast cells. mCD200RLc was protein concentration was calculated by taking this into account. KD values identified by homology search of the NCBI EST database. A full-length were obtained by both nonlinear curve fitting and Scatchard transforma- clone of mCD200RLc has not yet been isolated; however, mCD200RLc is tions to the binding data. The koff binding rate constant was obtained by ϳ90% identical at the amino acid level with mCD200RLa. fitting a 1:1 binding model to the kinetic data using the BIA evaluation 3.0 3036 HUMAN AND MOUSE CD200 RECEPTOR

software, and these data are shown in Table I. In addition, koff rate con- the extracellular region (Fig. 1). In addition, an alternative variant stants were also determined by fitting a first order exponential decay curve (hCD200Ri) was identified that contained an extra segment of se- to normalized data over the dissociation phase (see Fig. 4c), and these were quence contiguous to the signal peptide that corresponded to an in good agreement. exon and thus presumably represents an alternatively spliced vari- Tissue expression of CD200R and related genes by mRNA ant cDNA (Fig. 1). The hCD200R is a type I cell surface glyco- analysis protein containing two Ig-like domains in the common V/C2 set For the determination of , previously described cDNA li- arrangement (17) with a hydrophobic transmembrane sequence braries generated from a variety of mouse and human cell types and tissues and a substantial cytoplasmic region. A comparison of this se- were used (15). Gene expression was determined by real-time quantitative quence with that of the rodent CD200 receptors showed that all the PCR using an ABI GeneAmp 5700 sequence detection system and the extracellular cysteine residues were conserved and the protein was reporter fluorescent dye, SYBR (PerkinElmer Applied Biosystems, Foster also highly glycosylated, containing eight potential N-linked gly- City, CA). In brief, 20 ng of cDNA in PCR buffer contained 200 ␮M ␮ cosylation sites (Fig. 1). All three tyrosine residues that are pre- dATP, dCTP, and dGTP; 400 M dUTP; 4 mM MgCl2;1.25UofAm- pliTaq DNA polymerase; 0.5 U Amp-Erase uracil-N-glycocylase; 900 nM dicted to lie in the cytoplasmic portion of the molecule were also of each primer; and 250 nM probe. The thermal cycling conditions were: conserved, and the rat protein has been shown to be phosphory- 50¡C for 2 min, 95¡C for 10 min, followed by 40 cycles of amplification lated upon pervanadate treatment (3). Phylogenetic analysis of at 95¡C for 15 s and 60¡C for 1 min for denaturing and anneal extension, respectively. PCR amplification of the housekeeping gene ubiquitin was CD200R and proteins shown to have the highest levels of simi- performed for each sample to control for sample loading and to allow for larity from database searches revealed some sequence similarity to normalization between samples, according to the manufacturer’s instruc- the ligand, CD200, and other Ig superfamily members (Fig. 2), as tions (PerkinElmer). Each data point was evaluated for integrity by analysis reported previously (3). hCD200R showed closest similarity to Downloaded from of amplification plots and dissociation curves and the ubiquitin normalized human herpesvirus entry protein HveC and related proteins HveB data were expressed as the fold gene expression. Primers used in this study were designed with Primer Express software (PerkinElmer). The fol- (nectin2) and HveD (poliovirus receptor or CD155). HveC, previ- lowing primers were used (sense primer is given first): mCD200R, AGGAG ously known as poliovirus-related protein PRR1, permits entry of GATGAAATGCAGCCTTA, TGCCTCCACCTTAGTCACAGTATC; herpes simplex viruses through interaction with the envelope gly- mCD200RLa, GGAGGACTCTGGCTTTGATGTTA, CCCGTCCTTTCAC coprotein D (18, 19). TGAACAAC; mCD200RLb, CCATAGAACTGAGTCAAGGTACAATGA, TCCTACGTTAAGAAGAATAATCACCAAA; hCD200R, TGGGAGGTC http://www.jimmunol.org/ CACAATGTGTCTA, TGTACAGACTCTTGTTGCCAGTCA. Analysis of the interaction between CD200 and CD200R in Flow cytometry human, mouse, and rat Flow cytometric analysis was performed on freshly isolated peripheral To analyze the ability of mouse and human CD200R to bind blood leukocytes from normal 5- to 7-wk C57BL/6 mice, normal human CD200, we used soluble fusion proteins consisting of the extra- peripheral blood leukocytes, and human monocyte-derived dendritic cells, cellular domains of mouse and human CD200 fused to the Fc as previously described (16). C57BL/6 splenocytes were also analyzed for the expression of mCD200R and mCD200RL proteins. Cells were incu- binding domain of hIg and Ba/F3 cell transfectants expressing ei- bated with biotinylated rat mAbs specific for mouse or human CD200 ther mCD200R or hCD200R. As shown in Fig. 3, mCD200 and receptor family members (or appropriate isotype controls) for 20 min at hCD200 bound to their respective receptors, and ligand binding by guest on September 28, 2021 4oC, washed, and then stained with a PE-conjugated streptavidin (CalTag, could be completely blocked by specific mAb to these receptors Burlingame, CA). After blocking with a cocktail of mouse and rat serum, (see below). the cells were incubated for 20 min with FITC-conjugated and CyChrome- conjugated mAbs against leukocyte differentiation Ags, after which the The interaction between human CD200R and CD200 was charac- cells were washed, fixed, and analyzed using a FACScan (BD terized biochemically by making recombinant proteins corresponding Biosciences). to their extracellular regions and quantified in real time using surface plasmon resonance in a BIAcore (14). hCD200R-CD4d3ϩ4-biotin Results protein and a negative control (CD4d3ϩ4-biotin) were immobilized Cloning and sequence analysis of the hCD200R in separate flow cells on a streptavidin-coated sensor chip before in- EST databases were screened using rat CD200R cDNA, and a jecting purified soluble hCD200-CD4d3ϩ4 protein. hCD200- candidate sequence for the hCD200R was identified and named CD4d3ϩ4 bound to the hCD200R-CD4d3ϩ4 flow cell in compari- hCD200R. cDNA clones were isolated from human lung cDNA by son with the control reference cell (Fig. 4A). This binding was PCR, and sequencing revealed two alleles that differed at 3 aa in quantified by calculating the difference in RU observed in the hCD200R and control flow cells once equilibrium had been reached and is plotted as a binding curve (Fig. 4B). The equilibrium-binding affinities (K ) were calculated both by nonlinear curve fitting (Fig. Table I. Kinetic data for hCD200-binding human, rat, and mouse D CD200Ra 4B) and Scatchard transformations (Fig. 4B, inset) of the binding data giving similar values. The CD200 interacted with the hCD200R with ϳ ␮⌴ Kinetics an equilibrium-binding affinity of 0.5 at 37¡C. Kinetic analysis ϳ Ϫ1 Ϫ of the interaction yielded an off rate 0.1 s (equivalent to a t1/2 ␮ Ϫ1 hCD200 Binding to: KD ( M) Off Rate (s ) t1/2 (s) of 7 s) at different levels of hCD200R immobilization, indicating that Human CD200R 0.49 Ϯ 0.08 0.09 Ϯ 0.004 7.7 kinetic measurements were not grossly affected by rebinding or mass Rat CD200R 0.59 Ϯ 0.07 0.24 Ϯ 0.03 2.9 transport effects (Fig. 4C). These measurements were repeated using Mouse CD200R 7.0 Ϯ 0.33 2.10 Ϯ 0.2 0.3 soluble biotinylated forms of both the rat and mouse CD200R pro- a duced in an identical manner, and the data are presented in Table I. The KD values were determined by averaging those calculated from each of the three different CD200R immobilization levels obtained by nonlinear curve fitting This revealed that the hCD200 interacted with CD200R with similar (note that the curves shown in Fig. 4B are from high levels of human CD200R kinetics to the equivalent interaction in rats and mice. hCD200 immobilization alone). The koff values were determined by averaging the values ob- tained by fitting the dissociation phases of the binding curves to a simple 1:1 binding showed some cross species binding to both mouse and rat CD200R, model (see Materials and Methods) using two different concentrations of ligand over but binding to mCD200R was weaker, with about a 10-fold faster ϭ three levels of receptor immobilization (i.e., n 6). In all cases, the level of receptor dissociation rate that explained why hCD200-hIg did not bind immobilization had negligible effects on koff, indicating that mass transport and re- binding effects were minimal. mCD200R (data not shown). The Journal of Immunology 3037 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. The human and mouse CD200R and related sequences. The hCD200R sequences have been aligned to the rodent orthologs (accession

AF231392 and AF231393), and the sequence has been split according to predicted domain organization. The NH2 terminus is based on protein data for rat CD200R. The superscript bars predict the extent of the ␤-strands characteristic of the Ig fold by comparison with solved structures. The sequence of the allele used in all binding experiments is shown with the three positions of allelic polymorphism shown above the sequence (accession AF283760). The form with an additional spliced exon is termed CD200Ri (accession NM 138806), and only the extra sequence is shown as other residues are identical with hCD200R. A second human gene termed CD200RLa was identified by EST and cDNA analysis. Additional mouse genes include two forms isolated through their association with DAP12 (mCD200RLa and mCD200RLb; accession XM156177 and XM148097). Residues identical in four or more sequences are boxed (three for cytoplasmic regions) apart from CD200Ri form, as this is known to be a splice variant and CD200RLc and CD200RLb (incomplete sequences). One sequence, CD200RLc, was only isolated as a partial cDNA clone, and additional residues were deduced from genomic sequence and are shown in italics. The partial sequence CD200RLd was only identified as genomic sequence and is shown in italics. Potential N-linked glycosylation sites (N) and the lysine residue (K) in the transmembrane are shown in bold.

Identification and structural characterization of genes related to short cytoplasmic regions devoid of known signaling motifs. Un- CD200R in mice and humans like mCD200R, the transmembrane regions of these two genes In addition to the mCD200R cloned previously (3), two cDNAs for contained a positively charged amino acid, lysine (Fig. 1). Because genes closely related to CD200R were isolated from normal bone mCD200RLa was also isolated by DAP12 trapping (see Materials marrow-derived mouse mast cells. These two proteins, named and Methods), the lysine in the transmembrane was expected to mCD200RLa and mCD200RLb, showed extensive sequence sim- form a salt bridge with DAP12, allowing these proteins to pair. ilarity in the extracellular regions to the mCD200R, but displayed Transduction of mCD200RLa and mCD200RLb into Ba/F3 cells 3038 HUMAN AND MOUSE CD200 RECEPTOR

amino acid in the transmembrane domain, again suggesting pairing with DAP12. A full-length cDNA of mCD200RLc, however, has not yet been isolated. A fifth partial gene sequence, mCD200RLd, was also identified in the mouse genome (Fig. 1), and the arrange- ment of the genes on 16 is shown in Fig. 6. All five members of the mCD200 receptor gene family are located on a short section of chromosome 16 (ϳ200 kbp) and within ϳ400 kbp of the CD200 gene itself. In contrast to the mouse genome, analysis of the showed only two CD200R-like genes: one corresponding to the CD200R form originally described in mouse and rat (3), and a second, termed hCD200RLa, which showed the most sequence homology to the mCD200RLa. hCD200RLa contains a short cy- toplasmic region, and the charged residue in the transmembrane suggests that it can also interact with DAP12. The human CD200, CD200R, and CD200RL genes are located on , agreeing with previous cytogenetic analysis for CD200 (20). In- terestingly, although both CD200 and its receptor are transcribed in the same direction in humans, this is not the case in mice (Fig. Downloaded from 6). Given the similarities in sequence, it seems likely that the CD200RL genes evolved from recent gene duplications and that these and CD200 evolved from a common ancestral precursor.

Distribution of CD200R and CD200RL gene products by mRNA http://www.jimmunol.org/ FIGURE 2. Phylogenetic analysis of CD200R. The extracellular two analysis Ig-like domains of hCD200R; hCD200; human herpesvirus entry proteins The distribution of CD200R was determined by a highly sensitive HveB, C, and D; and other hIg-like proteins (CD56, CD80, and CD86) quantitative RT-PCR method in a panel of cDNA libraries from a were aligned using ClustalW (42), manually refined, and then a Neighbor- variety of purified cell types and normal tissues from both mice Joining tree constructed with 1000 bootstrap trials. In Hve proteins and and humans (Figs. 7 and 8). Using PCR primers that specifically CD56, only the membrane distal two domains were compared. identify CD200R mRNA, it was clear that CD200R shared a sim- ilar cellular and tissue distribution in both species. The highest

stably expressing a FLAG-tagged murine DAP12 resulted in the levels of expression were observed in bone marrow-derived mac- by guest on September 28, 2021 cell surface expression of DAP12 and the associated CD200RL rophages from the mouse, and monocyte-derived dendritic cells in proteins (Fig. 5). humans. Significant gene expression, however, was also seen in Immunoprecipitation experiments using mAbs against polarized Th2 T cells, mast cells, and dendritic cells of both mice mCD200RLa and mCD200RLb further verified that these two and humans. Interestingly, although mouse bone marrow-derived genes are naturally pairing with DAP12 in normal mouse mast macrophages expressed very high levels of CD200R mRNA, cells (data not shown). A fourth form (mCD200RLc) was also freshly isolated human peripheral blood monocytes expressed only identified from EST database analysis, which has a high degree of low levels of CD200R mRNA. The differentiation status of these identity to mCD200RLa and also contains a positively charged particular cell types is, however, not comparable, and it is thus

FIGURE 3. Flow cytometry showing binding of CD200-Ig to mCD200R and hCD200R. Ba/F3 cells ex- pressing either the human (A) or the mouse (D) CD200R were stained with anti-CD200R mAbs or iso- type controls, followed by a goat anti-rat PE-conjugated secondary Ab. CD200-Ig or control Igs were allowed to bind to CD200R-expressing Ba/F3 cells in the presence of a control Ab (B, E) or specific anti-CD200R- blocking Abs (C, DX136; F, DX109). The CD200 Igs were detected with a monoclonal rat anti-human PE- conjugated secondary reagent that did not cross-react with mouse or rat IgG, and analyzed by flow cytometry. The Journal of Immunology 3039 Downloaded from

FIGURE 5. mCD200RLa and mCD200RLb can associate with DAP12. Ba/F3 cells stably expressing FLAG-tagged murine DAP12 were infected with either mCD200RLa or mCD200RLb containing retroviruses. Trans- http://www.jimmunol.org/ duction of mCD200RLa or mCD200RLb into these cells resulted in the pairing of these receptors with FLAG-tagged DAP12 and the cell surface expression of the complexes. This was confirmed by flow cytometry using either an anti-FLAG Ab or mAbs specific for mCD200RLa or mCD200RLb. A, BaF3 cells stably expressing murine DAP12 were stained with control Ab (----)oranti-FLAG Ab (⅐⅐⅐⅐⅐). B, Ba/F3-DAP12 cells infected with murine CD200RLa were stained with control Ab (- - - -), anti- FLAG Ab (⅐⅐⅐⅐⅐), or anti-mCD200RLa Ab (——). C, Ba/F3-DAP12 cells

infected with murine CD200RLb were stained with control Ab (- - - -), anti- by guest on September 28, 2021 FLAG Ab (⅐⅐⅐⅐⅐), or anti-mCD200RLb Ab (——).

possible that CD200R expression is up-regulated on tissue mac- rophages. B cells, fibroblasts, and endothelial cells from both mice and humans expressed little, if any, detectable CD200R mRNA. In whole tissues, the highest expression of CD200R mRNA was ob- served in bone marrow, lymph nodes, spleen, and lung, while the lowest expression was seen in liver, spinal cord, and kidney (Figs. 7 and 8). Tissue macrophages of the CNS (microglia) are known to express low levels of CD200R in the rat (3); however, in whole spinal cord tissues, the level of mRNA contributed by these cells was negligible. The thymus was the only tissue that showed dif- ferential expression between mice and humans. Mouse thymus showed significant expression of CD200R mRNA, whereas human thymus had essentially undetectable levels. The cellular distribution of mCD200RLa and mCD200RLb was also quantified by PCR on the same panel of mouse cDNA librar- ies (Fig. 7). The highest level of mCD200RLa was observed in FIGURE 4. The equilibrium affinity and kinetics of hCD200R interact- unstimulated mast cells. This expression was, however, signifi- ing with hCD200 protein. A, The indicated active concentrations (␮M) of cantly decreased in mast cells triggered via Fc⑀RI. Strong hCD200CD4d3ϩ4 were injected at 15 ␮l/min and 37¡C through flow cells with 1815 RU of hCD200RCD4d3ϩ4-biotin (solid line) or 2185 RU of mCD200RLa expression was also seen in bone marrow-derived CD4d3ϩ4-biotin (control dotted line) immobilized. The amount of hCD200CD4d3ϩ4 that bound at each concentration was calculated as the difference between the response at equilibrium in the hCD200RCD4d3ϩ4 (2.0 ␮M) at 100 ␮l/min over immobilized hCD200RCD4d3ϩ4-biotin at and control flow cells, and these are plotted as binding curves (B). B, The high (1815 RU) and medium (895 RU) levels and also a negative control curved lines in the main plot are nonlinear curve fits to the data and cor- CD4d3ϩ4-biotin (2185 RU), and data were collected at 10 Hz. The data respond to an affinity of 0.5 ␮M. A Scatchard transformation of the same were then normalized (100% at the start of the dissociation phase), and first binding data is shown inset in B, and the linear fit shown corresponds to a order exponential decay curves were fitted to the dissociation data ␮⌴ 1 KD of 0.5 . C, The dissociation rate constant of the hCD200-hCD200R (hCD200R medium) and yielded a koff value of 0.09 s . For clarity, only interaction was measured by injecting 10 ␮l of soluble hCD200CD4d3ϩ4 every tenth data point is shown. 3040 HUMAN AND MOUSE CD200 RECEPTOR

FIGURE 6. Organization of CD200 and receptor genes in the human and mouse ge- nomes. The approximate positions of the CD200 and CD200R and related genes are shown as oblongs in their approximate po- sitions on the genome. The numbering of the bases is from the current NCBI human and mouse databases. Arrows indicate direction of transcription. Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. Expression of mCD200R, mCD200RLa, and mCD200RLb by mRNA analysis in various cell lines (A) and tissues from C57BL/6 mice (B). Results are presented as fold expression relative to normalized ubiquitin levels. The Journal of Immunology 3041

FIGURE 8. Expression of hCD200R by mRNA Downloaded from analysis in various cell lines (A) and normal human autopsy tissues (B). Results are presented as fold ex- pression relative to normalized ubiquitin levels. http://www.jimmunol.org/ by guest on September 28, 2021

macrophages and dendritic cells, and to a lesser extent in polarized Gr-1Ϫ) with weaker labeling of most T cells (CD3ϩ) (Fig. 9). T Th2 T cells. mCD200RLb, however, was primarily expressed cell staining for CD200R was primarily restricted to CD4ϩ T cells in mast cells and polarized Th2 T cells and to a lesser degree in (data not shown). Weak mCD200R expression was observed on a bone marrow-derived dendritic cells. Unlike mCD200R and subset of NK cells (DX5ϩ, CD3Ϫ), NKT cells (CD3ϩ, DX5ϩ), mCD200RLa, mCD200RLb was undetectable in bone marrow-de- and B cells (CD19ϩ). In the spleen, mCD200R showed an iden- rived macrophages and was expressed at relatively low levels in tical cellular expression pattern to that observed in the peripheral bone marrow-derived dendritic cells. cDNA for hCD200RLa was blood, although with somewhat higher levels of expression of isolated from peripheral blood, but was not found in significant CD200R on splenic T cells (data not shown). Consistent results levels in cDNA libraries. Analysis of the amino acid sequence were obtained with another rat anti-mouse CD200R mAb OX110 revealed that it lacked two cysteines when compared with the (data not shown). mouse homologue and other genes in this group (Fig. 1). This may In human peripheral blood, hCD200R (DX136) displayed a sim- explain why hCD200RLa was not expressed even in the presence ilar cellular distribution to that observed in mouse peripheral blood of DAP12, DAP10, Fc⑀R1␥,orCD3␥. Indeed, reintroducing the (Fig. 10). The majority of human peripheral blood T cells (both cysteine residues restored expression in the presence of DAP12 (G. CD8ϩ and CD4ϩ), all neutrophils (CD14ϩϩ, CD16ϩϩ), and all Brooke, unpublished data). It is likely therefore that hCD200RLa (CD9ϩϩ,Fc⑀RIϩϩ; data not shown) expressed high lev- is nonfunctional in humans. els of hCD200R. Similar to mouse peripheral blood, the majority of human monocytes expressed moderate levels of hCD200R; this Distribution of mouse and human CD200R by mAb staining expression was significantly up-regulated when monocytes were mAb specific for mCD200R and hCD200R were generated to in- induced to differentiate into dendritic cells in vitro (CD14ϩ/Ϫ, vestigate the cellular distribution of the CD200R protein. In mouse HLA-DRϩϩ) in the presence of GM-CSF and IL-4 (data not peripheral blood, mCD200R (DX109) was strongly expressed on shown). The majority of NK cells (CD56ϩ, CD3Ϫ) did not express all granulocytes (CD11bϩ, Gr-1ϩϩ) and monocytes (CD11bϩϩ, hCD200R. Low levels of hCD200R expression, however, were 3042 HUMAN AND MOUSE CD200 RECEPTOR

FIGURE 9. Expression of mCD200R by flow cytometric analysis of peripheral blood leukocytes. A, Mouse peripheral blood leukocytes were stained with FITC-conjugated anti-DX5, CyChrome-con- jugated anti-CD3, and biotinylated isotype control Abs or biotinylated anti-mCD200R (followed by PE-conjugated streptavidin), and then electroni- cally gated by forward and 90¡ scatter on lym- phocytes (A). Various leukocyte populations were isolated by specific phenotype gating and analyzed for the expression of isotype control (----)and anti-mCD200R (——). The following leukocyte populations were studied: NK cells were DX5ϩ, Downloaded from CD3Ϫ; NKT cells were DX5ϩ, CD3ϩ; T cells were CD3ϩ; and B cells were DX5Ϫ, CD3Ϫ (Ͼ93% CD19ϩ; data not shown). B, Mouse peripheral blood leukocytes were stained with FITC-conju- gated anti-CD11b, CyChrome-conjugated Gr-1, and biotin-conjugated isotype control Ab or biotin-

conjugated anti-mCD200R (followed by PE-conju- http://www.jimmunol.org/ gated streptavidin). Monocytes were identified by forward and 90¡ light scatter and were phenotype gated as CD11bϩ, Gr-1Ϫ (data not shown). Gran- ulocytes were also identified by distinctive forward and 90¡ light scatter and were phenotype gated, CD11bϩ Gr-1ϩϩ (data not shown). Monocytes and granulocytes were analyzed for the expression of isotype control Ab (----)andmCD200R (——). by guest on September 28, 2021

observed on a subset of the CD56 bright NK cells (CD56ϩϩ, Binding of mCD200 to mCD200RLa and mCD200RLb CD3Ϫ) as well as NKT cells (CD56ϩ, CD3ϩ). A small subset of ϩ To analyze the ability of mCD200 to bind mCD200RLa and B cells (CD19 ) demonstrated hCD200R expression in most do- mCD200RLb, we used soluble fusion proteins consisting of the nors analyzed. hCD200R was not expressed on freshly isolated extracellular domains of mCD200 fused to the Fc binding domain peripheral blood platelets or RBC (data not shown). Similar results of hIg and Ba/F3 cell transfectants expressing either mCD200RLa were obtained with an anti-human mCD200R mAb OX108 (data or mCD200RLb. Although mCD200-hIg readily bound to trans- not shown). fectants expressing the mCD200R protein (Fig. 3), we were unable to demonstrate any significant binding of mCD200 to either the Distribution of mouse CD200RLa and CD200RLb by mAb mCD200RLa or mCD200RLb proteins (data not shown). Attempts staining to demonstrate mCD200 binding to these receptors by varying Rat mAb specific for mCD200RLa (DX87) and mCD200RLb mCD200 concentrations, binding times, and binding temperatures (DX116) were generated to investigate the expression of these pro- have also failed to show specific receptor binding. Presumably, teins on mouse leukocytes. Unlike mCD200R, mCD200RLa was mCD200RLa and mCD200RLb bind alternative ligands despite not expressed on T cells (Fig. 11). Strong expression of the relatively high sequence similarity to the mCD200R. mCD200RLa was observed on NK cells, monocytes, and a subset of NKT cells, while low expression was seen on B cells and gran- Discussion ulocytes. Although data for peripheral blood leukocytes are shown, We report on the characterization of the CD200 receptor family in similar expression patterns were also observed on splenic popula- both humans and mice. Analysis of the human and mouse genomes tions of leukocytes (data not shown). mCD200RLb, although together with cDNA sequence analysis indicates that there has strongly expressed on bone marrow-derived mast cells (data not been extensive gene duplication in the CD200R genes with up to shown), was not significantly expressed on peripheral blood leu- five related genes in mice on chromosome 16 and two genes in kocytes (Fig. 11). humans on chromosome 3. The human homologue of mCD200R, The Journal of Immunology 3043

FIGURE 10. Expression of hCD200R by flow cy- tometry of peripheral blood leukocytes. A, Human peripheral blood leukoctyes were stained with FITC- conjugated anti-CD3, CyChrome-conjugated anti- CD56, and biotinylated isotype control Abs or biotinylated anti-hCD200R (followed by PE-conju- gated streptavidin) and then electronically gated by forward and 90¡ light scatter on lymphocytes (A). Downloaded from Various leukocyte populations were identified by spe- cific phenotype gating and analyzed for the expres- sion of isotype control (----)and anti-hCD200R (——). The following leukocyte populations were studied: NK cells, CD56ϩ, CD3Ϫ; CD56 bright NK cells, CD56ϩϩ, CD3Ϫ; T cells, CD3ϩ; NKT cells, CD56ϩ, CD3ϩ; and B cells, CD56Ϫ, CD3Ϫ, Ͼ90% http://www.jimmunol.org/ CD19ϩ (not shown). B, Human peripheral blood leukocytes were stained with FITC-conjugated anti- CD14, CyChrome-conjugated CD16, and biotin- conjugated isotype control Ab or biotin-conjugated anti-hCD200R (followed by PE-conjugated streptavi- din). Monocytes were identified by forward and 90¡ light scatter and were phenotype gated as CD14ϩ, CD16Ϫ (data not shown). Granulocytes were also

identified by distinctive high forward and 90¡ light by guest on September 28, 2021 scatter and were phenotype gated as CD14Ϫ CD16ϩϩ (not shown). Monocytes and granulocytes were then analyzed for the expression of isotype control Ab (----)andhCD200R (——).

designated hCD200R, shows a high degree of sequence and struc- Analysis of mRNA expression in mouse and human cDNA li- tural similarity to its mouse counterpart, and indeed interacts with braries reveals that CD200R is expressed in a variety of cell types hCD200 protein in an analogous manner to that reported in rodents of the myeloid/monocytic lineage, in particular, macrophages, den- (3). Of particular interest are the highly conserved tyrosines and dritic cells, and mast cells. Interestingly, CD200R mRNA is also associated amino acids in the cytoplasmic domain of both mouse highly expressed in polarized Th2 T cells in both mice and hu- and human CD200R. The high degree of identity in the tyrosine- mans, indicating that this receptor may have a more extensive im- rich regions of the cytoplasmic tails of mCD200R and hCD200R munological role than previously anticipated because it has a suggests that these proteins recruit similar signal transduction ma- broader distribution than observed with the original anti-rat chinery. The molecular identity of the cytoplasmic receptor sig- CD200R mAb (3). mAbs specific for mCD200R and hCD200R naling proteins is presently unknown; however, both mouse and were generated to investigate the expression of CD200R protein on human CD200R have a conserved phosphotyrosine binding site for freshly isolated leukocyte populations. In both mice and humans, Shc (NPXY) in the cytoplasmic domains (21, 22). Studies are pres- CD200R is prominently expressed on peripheral blood neutrophils. ently underway to delineate the mechanisms and the structural re- Because neutrophils play a major role in most inflammatory re- quirements for signal transduction via the CD200R. sponses (23), the high expression levels of CD200R suggest that 3044 HUMAN AND MOUSE CD200 RECEPTOR

FIGURE 11. Expression of mCD200RLa and mCD200RLb by flow cytometric analysis of mouse peripheral blood leukocytes. Mouse peripheral blood leukocyte subsets were identified and pheno- type gated, as described in Fig. 9. Specific leuko- cyte populations were then analyzed for the expres- sion of isotype control Abs (- - - -), anti-mCD200RLa (——), and anti-mCD200RLb (- ⅐ - ⅐ -). The follow-

ing leukocyte populations were analyzed for the ex- Downloaded from pression of mCD200RLa and mCD200RLb: T cells, CD3ϩ; NKT cells, CD3ϩ, DX5ϩ; NK cells, CD3Ϫ, DX5ϩ; B cells, CD3Ϫ, DX5Ϫ; monocytes, CD11bϩ, Gr-1Ϫ; and granulocytes, CD11bϩ, Gr-1ϩϩ. http://www.jimmunol.org/ by guest on September 28, 2021

this receptor may play an important regulatory role in neutrophil Our previous studies in the mouse have implicated CD200R in biology; this concept has been reviewed (5). Experiments are pres- the regulation of normal macrophage/myeloid function (3, 4). Con- ently underway to investigate the functional role of CD200R in sistent with these earlier studies, mCD200R was strongly ex- neutrophil phagocytosis, superoxide production, and chemokine pressed on peripheral blood monocytes and splenic macrophages. responses. Indeed, preliminary immunocytochemistry analysis demonstrates As expected from the mRNA distribution analysis, CD200R is the expression of CD200R on subsets of macrophages from normal ϩ also expressed on freshly isolated CD4 T cells from mouse and mouse skin, lung, and intestine. mAb staining in the mouse clearly human peripheral blood. The mRNA analysis showed a restricted shows that, although CD200R is associated with the macrophage/ expression of CD200R to polarized Th2 cells; however, cellular expression of the protein indicates that the majority of CD4ϩ T cells in the mouse and human peripheral blood expressed CD200R. It is possible that CD200R is down-regulated by the cytokines Table II. Summary of tissue distribution of human and mouse CD200R required to polarize Th1 cells, while remaining stably expressed on a committed Th2 cells. The expression of CD200R on Th2 cells has and related proteins important implications for immune regulation. Th2 cells have been strongly implicated in variety of pathological conditions, including Tissue hCD200R mCD200R mCD200RLa mCD200RLb allergy, asthma, and hypersensitivity (24Ð27). Proinflammatory Monocytes ϩϩ ϩϩϩ ϩϩϩ ϩ/Ϫ cytokines, such as IL-3, IL-4, IL-5, IL-13, and GM-CSF, are pro- Granulocytes ϩϩϩ ϩϩϩ ϩ/ϪϪ ϩϩϩ ϩ ϩ ϩ duced by Th2 cells and are believed to be the major factors in Dendritic cells Macrophage ϩϩ ϩϩϩ ϩϩ Ϫ allergic pathologies, such as inflammation, mucous hypersecre- Mast cells ϩϩϩϩϩϩϩ tion, and airway constriction. The strong expression of CD200R, T cells ϩϩ ϩϩ Ϫ Ϫ an inhibitory receptor, on Th2 cells may indicate that this receptor T cell clones Th1 ϩϪ Ϫ Ϫ plays an important regulatory role in Th2-mediated responses. The T cell clones Th2 ϩϩ ϩ ϩ/Ϫϩ ϩ Ϫϩ ϩ ϩ expression of CD200R on Th2 cells also suggests that therapeutic B cells / NK cells Ϫ Ϫ ϩϩ Ϫ strategies directed toward triggering CD200R on Th2 cells may NKT ϩϩ ϩ Ϫ function to inhibit allergic inflammation and associated patholo- a Compilation of data from expression studies by mRNA analysis and mAb stain- gies. The distribution of CD200R and related proteins is summa- ing on normal tissues and cell lines (see Figs. 7Ð11). Human macrophage data are rized in Table II. from skin (data not shown). The Journal of Immunology 3045 myeloid lineage, it is not ubiquitously expressed on all tissue- there is clearly a form that can give an activating signal through bound myeloid cells. In the human, however, CD200R is ex- DAP12, and in this regard it resembles other gene families such as pressed on the majority of monocyte/myeloid lineage cells in the signal regulatory proteins and killer Ig-related receptors that have peripheral blood. In addition to neutrophils and monocytes, both activating and inhibitory forms (38Ð41). Manipulating the CD200R was strongly expressed on basophils and dendritic cells CD200R has been shown to affect immune responses, but these derived from monocytes. Likewise, preliminary immunocyto- data indicate that the cells being affected may include both myeloid chemical analysis demonstrates a strong expression of CD200R on cells and T cells. Importantly, the definition of a family of closely myeloid cells of normal human skin and lung (data not shown). related CD200R-like proteins (at least in the mouse) suggests that Mast cells, which are derived from hemopoietic progenitors, different effects in vivo and in vitro may be expected using play a critical effector role in allergic diseases and IgE-dependent CD200-Fc fusion proteins, shown in this study to be specific for immune responses. Engagement of the high affinity FcR for IgE on CD200R, and Abs against the various related members that may mast cells and basophils triggers a series of biochemical events show different cross-reactivities. resulting in the secretion of a variety of inflammatory mediators, including histamine, sulfated proteoglycans, proteinases, and cy- Acknowledgments tokines (28). The prominent expression of CD200R on both mouse We are grateful to Tony Willis for amino acid analysis, and Sandra Zuraw- and human mast cells suggests that this receptor may function as ski, Janet Wagner, and Lisa Oldham for the construction and purification of a constitutive regulator of mast cell biological responses. Although CD200-Ig fusion proteins. we have not yet investigated the expression of CD200R on freshly isolated mast cells, mast cells derived from hemopoietic progeni- References Downloaded from tors clearly express both high levels of specific mRNA for 1. Barclay, A. N. 2003. Membrane proteins with immunoglobulin-like domains: a CD200R as well as cell surface protein (data not shown). Previous master superfamily of interaction molecules. Semin. Immunol. 15:215. in vivo studies have implicated the CD200R in the regulation of 2. Van der Merwe, P. A., and A. N. Barclay. 1996. Analysis of cell-adhesion mol- ecule interactions using surface plasmon resonance. Curr. Opin. Immunol. 8:257. experimental autoimmune encephalomyelitis (EAE) and collagen- 3. Wright, G. J., M. J. Puklavec, A. C. Willis, R. M. Hoek, J. D. Sedgwick, induced arthritis (3, 4, 7). The conclusion from these studies was M. H. Brown, and A. N. Barclay. 2000. Lymphoid/Neuronal cell surface OX2 glycoprotein recognizes a novel receptor on macrophages implicated in the con- that CD200R played a major role in the regulation of these auto- http://www.jimmunol.org/ trol of their function. Immunity 13:233. immune models by modulating macrophage function. Macro- 4. Hoek, R. M., S. R. Ruuls, C. A. Murphy, G. J. Wright, R. Goddard, phages are known to contribute to the onset and severity of brain S. M. Zurawski, B. Blom, M. E. Homola, W. J. Streit, M. H. Brown, et al. 2000. inflammation in the EAE model (29). Several recent studies using Down-regulation of the macrophage lineage through interaction with OX2 (CD200). Science 290:1768. mast cell-deficient mice, however, have shown that EAE and collag- 5. Nathan, C., and W. A. Muller. 2001. Putting the brakes on innate immunity: a en-induced arthritis are dependent upon functional mast cells (30, 31). regulatory role for CD200? Nat. Immun. 2:17. It is possible, therefore, that some of the effects of CD200R in these 6. Barclay, A. N., G. J. Wright, G. Brooke, and M. H. Brown. 2002. CD200 and interactions in the control of myeloid cells. Trends Immunol. models are manifested through the regulation of mast cell functions. 23:285. The molecular and biological functions of CD200R expression on 7. Gorczynski, R. M., Z. Chen, K. Yu, and J. Hu. 2001. CD200 immunoadhesin mast cells are presently a major focus of ongoing studies. suppresses collagen-induced arthritis in mice. Clin. Immunol. 101:328. by guest on September 28, 2021 8. Gorczynski, R. M., M. S. Cattral, Z. Chen, J. Hu, J. Lei, W. P. Min, G. Yu, and Two of the mouse genes of the CD200 receptor family J. Ni. 1999. An immunoadhesin incorporating the molecule OX-2 is a potent (mCD200RLa and mCD200RLb) associate with DAP12 and re- immunosuppressant that prolongs allo- and xenograft survival. J. Immunol. quire DAP12 for stable cell surface expression. DAP12 is a small 163:1654. 9. Valent, P., E. Spanblochl, W. R. Sperr, C. Sillaber, K. M. Zsebo, H. Agis, disulfide-bonded homodimer that is structurally similar to Fc⑀RI␥ H. Strobl, K. Geissler, P. Bettelheim, and K. Lechner. 1992. Induction of differ- chain and the TCR␨ chain (32, 33). The transmembrane domain of entiation of human mast cells from bone marrow and peripheral blood mononu- clear cells by recombinant human stem cell factor/-ligand in long term culture. DAP12 contains a negatively charged aspartic acid residue that Blood 80:2237. allows pairing with a variety of cell surface receptors with posi- 10. Mitsui, H., T. Furitsu, A. M. Dvorak, A. M. A. Irani, L. B. Schwartz, N. Inagaki, tively charged amino acid residues in their transmembrane regions. M. Takei, K. Ishizaka, K. M. Zsebo, S. Gills, and T. Ishizaka. 1993. Development of human mast cells from umbilical cord blood cells by recombinant human and The cytoplasmic domain of DAP12 contains a consensus immu- murine c-kit ligand. Proc. Natl. Acad. Sci. USA 90:735. noreceptor tyrosine-based activation motif, which when phosphor- 11. Onihsi, M., S. Kinoshi, Y. Morikawa, A. Shibuya, J. H. Phillips, L. L. Lanier, ylated recruits protein tyrosine kinases (32Ð35). Pairing of D. Gorman, G. P. Nolan, A. Miyajima, and T. Kitamura. 1996. Applications of retrovirus-mediated expression cloning. Exp. Hematol. 24:324. mCD200RLa and mCD200RLb with DAP12 clearly defines these 12. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic proteins as potentially activating receptors. mCD200RLa and local alignment search tool. J. Mol. Biol. 215:403. mCD200RLb do not bind mCD200, despite extensive sequence 13. Bakker, A. B., E. Baker, G. R. Sutherland, J. H. Phillips, and L. L. Lanier. 1999. Myeloid DAP12-associating lectin (MDL)-1 is a cell surface receptor involved in similarity in the extracellular regions at least for CD200RLa (84 the activation of myeloid cells. Proc. Natl. Acad. Sci. USA 96:9792. and 39% identity for CD200RLa and CD200RLb with CD200R, 14. Brown, M. H., K. Boles, P. A. van der Merwe, V. Kumar, P. A. Mathew, and respectively). The ligands of CD200RLa and CD200RLb and the A. N. Barclay. 1998. 2B4, the NK and T cell immunoglobulin superfamily sur- face protein is a ligand for CD48. J. Exp. Med. 188:2083. functional and biological significance of these receptors are un- 15. Halfon, S., J. Ford, J. Foster, L. Dowling, L. Lucian, M. Sterling, Y. Xu, known; however, it is possible that these receptors have evolved to M. Weiss, M. Ikeda, D. Liggett, et al. 1998. Leukocystatin, a new class II cystatin interact with viral proteins in a manner similar to that recently expressed selectively by hematopoietic cells. J. Biol. Chem. 273:16400. 16. Vassili, S., P. A. Reche, H. Kanzler, W. Yuan, G. Edward, B. Homey, M. Gilliet, described for a DAP12 pairing member of the Ly-49 family and S. Ho, S. Antonenko, A. Lauerma, et al. 2002. Human epithelial cells trigger mouse CMV (36, 37). The human homologue of mCD200RLa, dendritic cell-mediated allergic inflammation by producing TSLP. Nat. Immun. hCD200RLa, is structurally very similar to the mouse gene, in- 3:673. 17. Barclay, A. N., M. H. Brown, S. K. A. Law, A. J. McKnight, M. G. Tomlinson, cluding the positively charged residue in the transmembrane do- and P. A. van der Merwe. 1997. Leucocyte Antigens Factsbook, 2nd Ed. Aca- main. It seems likely that hCD200RLa is a nonfunctional gene, as demic Press, London. 18. Geraghty, R. J., C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and no expression could be detected despite many attempts with a va- P. G. Spear. 1998. Entry of alphaherpesviruses mediated by poliovirus receptor- riety of adapter molecules. related protein 1 and poliovirus receptor. Science 280:1618. CD200R is a member of a group of proteins expressed on my- 19. Lopez, M., F. Cocchi, L. Menotti, E. Avitabile, P. Dubreuil, and G. Campadelli-Fiume. 2000. Nectin2␣ (PRR2␣ or HveB) and nectin2␦ are low- eloid cells, but we now report expression on many T cells in mouse efficiency mediators for entry of herpes simplex virus mutants carrying the and human in contrast to earlier data in the rat (3). In the mouse, Leu25Pro substitution in glycoprotein D. J. Virol. 74:1267. 3046 HUMAN AND MOUSE CD200 RECEPTOR

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