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of Mast Cell ActivationMolecular Mechanisms of CD200 Inhibition

Joseph H. PhillipsShuli Zhang, Holly Cherwinski, Jonathon D. Sedgwick and

http://www.jimmunol.org/content/173/11/6786doi: 10.4049/jimmunol.173.11.6786

2004; 173:6786-6793; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2004 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Molecular Mechanisms of CD200 Inhibition of MastCell Activation

Shuli Zhang, Holly Cherwinski, Jonathon D. Sedgwick, and Joseph H. Phillips1

CD200 and its receptor CD200R are both type I membrane glycoproteins that contain two Ig-like domains. Engagement ofCD200R by CD200 inhibits activation of myeloid cells. Unlike the majority of immune inhibitory receptors, CD200R lacks anITIM in the cytoplasmic domain. The molecular mechanism of CD200R inhibition of myeloid cell activation is unknown. In thisstudy, we examined the CD200R signaling pathways that control degranulation of mouse bone marrow-derived mast cells. Wefound that upon ligand binding, CD200R is phosphorylated on tyrosine and subsequently binds to adapter proteins Dok1 andDok2. Upon phosphorylation, Dok1 binds to SHIP and both Dok1 and Dok2 recruit RasGAP, which mediates the inhibition of theRas/MAPK pathways. Activation of ERK, JNK, and p38 MAPK are all inhibited by CD200R engagement. The reduced activationof these MAPKs is responsible for the observed inhibition of mast cell degranulation and cytokine production. Similar signalingevents were also observed upon CD200R engagement in mouse peritoneal cells. These data define a novel inhibitory pathway usedby CD200R in modulating mast cell function and help to explain how engagement of this receptor in vivo regulates myeloid cellfunction. The Journal of Immunology, 2004, 173: 6786–6793.

T he CD200 (originally OX2) is a membrane glycoproteinexpressed by a broad range of cell types, including lym-phoid cells, neurons, and endothelium (1). It is the ligand

for a receptor (CD200R) whose expression is restricted to hemo-poietic cells, particularly myeloid cells (1–4). CD200 and its re-ceptor both are type I membrane proteins and contain two extra-cellular Ig-like domains. Mouse CD200 has a short intracellulardomain (19 aa) which lacks any known signaling motifs. MouseCD200R, however, has a larger cytoplasmic domain (67 aa) con-taining three tyrosine residues (2).

Several in vivo studies have suggested that the interaction ofCD200 with its receptor can deliver inhibitory signals to myeloidcells. For example, blocking CD200-CD200R interaction by eithersoluble CD200R protein or CD200R-blocking Ab augmented col-lagen-induced arthritis (CIA)2 in mice and exacerbated experimen-tal autoimmune encephalomyelitis in rats (2, 5). These studies sug-gested that blocking the constitutive interactions of CD200R andCD200 decreased myeloid cell inhibitory thresholds, which in turnresulted in enhanced immune activation. Consistent with this hy-pothesis, mice receiving soluble CD200 protein were resistant toCIA induction (6, 7) and showed prolonged graft survival in bothallo- and xenotransplantation models (8). The strongest supportiveevidence for an inhibitory role of CD200 in immune modulationcomes from studies of CD200-deficient mice (5). In these mice,there were increased numbers of activated macrophages in thespleen and the mesenteric lymph nodes and, more importantly,these mice showed a more rapid onset of experimental autoim-mune encephalomyelitis and increased susceptibility to CIA. Re-

cently, we provided direct evidence that CD200R is indeed aninhibitory receptor regulating mast cell activation in vitro and invivo (48).

Most immune inhibitory receptors share a cytoplasmic aminoacid sequence, termed the ITIM (9–12). All ITIMs are composedof the consensus sequence (I/V/L/S)xYxx(L/V), which upon ty-rosine phosphorylation recruits phosphatases (SRC homology re-gion 2 domain-containing phosphatase (SHP) 1, SHP2, and/orSHIP). The binding of phosphatases to ITIM-containing receptorsin turn suppresses cell activation by promoting dephosphorylationof activating receptors and downstream signaling molecules. Un-like the majority of immune inhibitor receptors, CD200R lacks anITIM, but contains an NPXY sequence in its cytoplasmic domain.The NPXY motif has previously been shown to be a binding sitefor proteins with phosphotyrosine-binding (PTB) domain (13, 14).In the present study, we examined the intracellular signaling eventsassociated with CD200R engagement in mouse bone marrow-de-rived mast cells (BMMCs) and primary peritoneal cells. Our re-sults provide strong evidence for a novel inhibitory pathway usedby CD200R to modulate myeloid cell function.

Materials and MethodsRecombinant protein, cytokines, and Abs

Recombinant mouse stem cell factor was purchased from PeproTech(Rocky Hill, NJ). Rabbit polyclonal anti-Shc, Dok2, SHIP Ab, and anti-phosphotyrosine mAb (4G10) were obtained from Upstate Biotechnology(Lake Placid, NY). Rabbit polyclonal anti-Dok1 Ab was purchased fromSanta Cruz Biotechnology (Santa Cruz, CA). Anti-dual phosphorylatedMAPK (ERK, JNK, and p38 MAPK), control anti-MAPK, anti-Myc Ab,and MEK inhibitor U0126 were obtained from Cell Signaling Technology(Beverly, MA). Monoclonal anti-Ras, Dok1, Dok2, and RasGAP Ab werepurchased from BD Pharmingen (San Diego, CA). SB203580 was obtainedfrom Calbiochem (San Diego, CA).

A fusion protein consisting of the extracellular domain of mouse CD200fused to the Fc domain of mouse IgG1 mutated in the CH2 domain(D265A) to inhibit binding to FcRs (CD200-mIg) and control mutant Ig(control mIg) were generated as previously described.3 Both control mIgand CD200-mIg do not bind to FcRs for IgG as analyzed by flow cytometry(data not shown). Anti-mouse CD200R Ab (DX109, rat IgG1) and anti-mouse CD200RLa Ab (DX87, rat IgG2c), which recognizes a Dap12-linked activating receptor homologous to CD200R (4), were generated aspreviously described (4). Anti-mouse CD200RLa Ab (DX89, rat IgM) was

DNAX Research Institute, Palo Alto, CA 94304

Received for publication April 7, 2004. Accepted for publication September 29, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 Address correspondence and reprint requests to Dr. Joseph H. Phillips, DNAX Re-search Institute, 901 California Avenue, Palo Alto, CA 94304-1104. E-mail address:joe.phillips@dnax.org2 Abbreviations used in this paper: CIA, collagen-induced arthritis; SHP, Src homol-ogy region 2 domain-containing phosphatase; PTB, phosphotyrosine; mIg, mutant Ig;BMMC, bone marrow-derived mast cell.

The Journal of Immunology

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00

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generated from a rat immunized with a fusion protein consisting of theextracellular domain of mouse CD200RLa fused to the Fc region of humanIgG1 as previously described (4). Cross-linking CD200RLa with DX89induces a strong dose-dependent degranulation response in mouse mastcells (data not shown). In the present study, mAb DX89 was used to ac-tivate mouse mast cells because the IgM isotype (Iso) did not require ad-ditional cross-linking reagents to activate the receptor and did not bindFcRs for IgG.

Cell culture, gene transduction, and flow cytometry

Mouse BMMCs were generated from bone marrow of 2- to 3-wk-oldC57BL/6 mice as previously described (4). Mast cells overexpressingCD200R and CD200RLa (DT733) were generated by retroviral transduc-tion of BMMCs. A cDNA containing the CD8 leader segment followed bythe Flag epitope tag (DYKDDDDK) and joined to the extracellular, trans-membrane and cytoplasmic domains of mouse CD200RLa was subclonedinto the pMXneo retroviral vector (15). Plasmid DNA was transfected intoPhoenix ecotropic retrovirus packaging cells (a gift from G. Nolan, Stan-ford University, Palo Alto, CA) using Lipofectamine (Invitrogen LifeTechnologies, Carlsbad, CA). Two days later, BMMCs were infected bycoculture with the transfected packaging cell line. After 30 h, the nonad-herent BMMCs were removed and put into fresh medium and after 72 hwere switched to selection medium containing 1 mg/ml G418 (Roche Mo-lecular Biochemicals, Indianapolis, IN). Cells were sorted for CD200RLaexpression using the anti-Flag Ab M2 (Sigma-Aldrich, St. Louis, MO). AcDNA containing the CD8 leader segment followed by the c-Myc epitopetag (EQKLISEEDL) and joined to the extracellular, transmembrane andcytoplasmic regions of mouse CD200R were subcloned into the retroviralvector pMXneo. The resultant construct was then introduced into the mastcell transfectant expressing the Flag-tagged CD200RLa by retroviral in-fection as described above. Cells were sorted for cell surface CD200Rexpression using the anti-Myc Ab 9E10 and subsequently verified for geneexpression using anti-CD200R mAb. Cells �95% positive and having sim-ilar levels of expression for both CD200R and CD200RLa were used forthe biochemical analyses and degranulation assays as described below.

NIH3T3 mouse fibroblast cells expressing the membrane form of mouseCD200 (16) were generated by retroviral transduction. The surface expres-sion of CD200 was determined by flow cytometry using PE-conjugated ratanti-mouse CD200 (clone MRC OX90; Serotec, Oxford, U.K.) Ab. Thenegative expressing cells were sorted and used as negative control. Theparental NIH3T3 and transductants were cultured in six-well plates andincubated with either BMMCs or DT733 cells upon confluence. The acti-vation of CD200R was determined as described below.

Mouse peritoneal cells were isolated from 8- to 10-wk-old C57BL/6mice by standard protocol (17). Cells (4 � 107 cells/ml) were stimulatedwith control mIg or CD200-mIg (10 �g/ml) for 10 min, and then immu-noprecipitation and Western blot were done as described below.

Surface expression of CD200R and CD200RLa was analyzed by stan-dard flow cytometry techniques. In brief, mast cells were washed once withPBS and stained with either FITC-conjugated anti-CD200R (DX109, ratIgG1) or PE-conjugated anti-CD200RLa (DX87, rat IgG2c) Ab. After in-cubation at 4°C for 20 min, cells were washed twice in PBS with 0.5%BSA and analyzed on a FACScan flow cytometer (BD Biosciences, SanJose, CA).

Degranulation assays and cytokine ELISA

Mast cell degranulation was determined using a �-hexosaminidase releaseassay as previously described.3 The degranulation was triggered by incu-bating mast cells with indicated amount of DX89 Ab, which binds to theactivating receptor CD200RLa and induces a strong degranulation re-sponse. Briefly, 1 � 106 cells/ml mast cells were treated with indicatedamounts of DX89 Ab for 1 h in RPMI 1640 medium in 96-well plates.Supernatants were assayed for �-hexosaminidase activity. For CD200-me-diated inhibition, the cells were pretreated with indicated amounts of con-trol mIg or CD200-mIg for 30 min before activation. For FcR blockageexperiments, cells were pretreated with the FcR-blocking Ab 2.4G2 (20�g/ml) for 30 min before subsequent incubations with control mIg orCD200-mIg. U0126 and SB203580 were used at 10 �M. TNF and IL-13were measured by ELISA kits (R&D Systems, Minneapolis, MN) accord-ing to the manufacturer’s instructions.

Immunoprecipitation and immunoblotting

Mast cells (1–2 � 107 cells/ml) were stimulated at 37°C with control mIgor CD200-mIg (3 �g/ml) for various times as indicated. For CD200-me-diated inhibition, the cells were pretreated with control mIg or CD200-mIg(3 �g/ml) for 30 min at 37°C and then stimulated with DX89 Ab (20ng/ml) for indicated times. Cells were then rinsed once with ice-cold PBS

containing 1 mM Na3VO4 and lysed in lysis buffer (50 mM Tris-HCl (pH8.0), 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 5 mM EGTA, 50mM NaF, 1 mM Na3VO4, plus protease inhibitor mixtures) for 20 min onice. Lysates were clarified at 14,000 rpm for 10 min. The protein concen-tration of the supernatant was determined by a Bio-Rad protein assay kit(Bio-Rad, Hercules, CA). Equal amounts of protein were analyzed by Nu-PAGE (Invitrogen Life Technologies) and Western blotting. For Westernblotting, primary Abs were detected with HRP-conjugated secondary Absand chemiluminescence (Pierce, Rockford, IL). For immunoprecipitations,Abs were incubated with 0.5–1 mg of cell lysate for 2 h at 4°C. Theimmune complexes were recovered by incubation with protein A-agarosebeads or protein G Plus-agarose beads (Santa Cruz Biotechnology) for 1 hat 4°C. After washing three times in lysis buffer and once in PBS contain-ing 1 mM Na3VO4, the immune complexes were dissociated in SDS sam-ple buffer. The samples were analyzed by Nu-PAGE and Western blottingas described above.

Peptide synthesis and in vitro-binding assay

N-terminal biotinylated peptides (DEMQPYASYTEKSNPLYDTVT) en-compassing the three tyrosine residues (Y286, Y289, and Y297) in the cyto-plasmic domain of mouse CD200R were synthesized and phosphorylatedat tyrosine in all possible combinations at the Biomolecular Resource Cen-ter (University of California, San Francisco, CA). Mast cells (1 � 107

cells/ml) were stimulated at 37°C with control mIg or CD200-mIg (3 �g/ml) for 5 min and then lysed as described above. Cell lysates were pre-cleared with 50 �l of avidin-agarose beads at room temperature for 30 min(Vector Laboratories, Burlingame, CA) and then incubated with 5 �g ofbiotinylated peptides at 4°C for 1 h, followed by incubation with 50 �l ofavidin-agarose beads at 4°C for 30 min. The protein complexes werewashed extensively and subjected to PAGE and Western blotting analysisas described above.

Ras activation assay

Ras activation was measured using a Ras activation assay kit (UpstateBiotechnology) according to the manufacturer’s instructions. Briefly, afterstimulation, cells were lysed and GTP-bound Ras was pulled down by GSTfusion protein containing the Ras-binding domain of Raf-1 bound to glu-tathione-agarose. The precipitated Ras-GTP was detected by Western blot.

ResultsCD200R engagement inhibits mast cell activation

CD200R is highly expressed on myeloid cells such as mast cells,macrophage, and neutrophils (4). As reported elsewhere,3 engage-ment of the mouse CD200R by agonist Abs or ligand results in apotent inhibition of mast cell degranulation and cytokine secretionin mouse mast cells overexpressing CD200R. To study the mo-lecular mechanisms of CD200 inhibition, mouse mast cells weregenerated from C57BL/6 bone marrow. As shown in Fig. 1A, theseBMMCs expressed relatively high level of CD200R andCD200RLa. Engagement of the CD200R by soluble CD200-mIg,however, did not inhibit mast cell degranulation induced by theactivating Ab DX89 (Fig. 1B). To increase the sensitivity of theassay, mast cells were induced to overexpress CD200R andCD200RLa by retroviral gene transfer. As shown in Fig. 1, DT733cells expressed higher levels of both CD200R and CD200RLa anddemonstrated an enhanced degranulation response, which wasstrongly inhibited by CD200R engagement. Pretreatment ofDT733 cells with CD200-mIg also inhibited mast cell degranula-tion induced by Ag cross-linking of Fc�RI (data not shown). But,unlike other inhibitory immune receptors, CD200R-mediated in-hibition of mast cell degranulation did not require coligation ofCD200R with the activating receptor. Because mouse mast cellsexpress FcRs for IgG, it was important to verify that the Fc-mu-tated CD200-mIg did not functionally trigger FcR to manifest in-hibitory signals. Pretreatment of mast cells overexpressingCD200R and CD200RLa with the FcR-blocking Ab 2.4G2 beforetriggering the CD200R with CD200-mIg had no effects on theCD200 induced inhibition of mast cell degranulation (data notshown). Mast cells overexpressing CD200R and CD200RLa

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(DT733) were used subsequently in all studies to dissect the mo-lecular mechanisms of CD200R signaling.

CD200R is tyrosine phosphorylated upon CD200 binding

Unlike the majority of inhibitory receptors, CD200R lacks anITIM, but contains three tyrosine residues Y286, Y289, and Y297 inthe cytoplasmic domain of mouse receptor. Sodium pervanadatepretreatment of cells expressing CD200R has been shown to resultin CD200R tyrosine phosphorylation (2). To determine whetherengagement of CD200R by its ligand induced CD200R phosphor-

ylation, we treated DT733 cells with CD200-mIg and examinedthe phosphorylation of CD200R. As shown in Fig. 2A, CD200stimulation induced strong tyrosine phosphorylation of CD200R,which was detected as early as 1 min after receptor engagement,and CD200R remained phosphorylated for �30 min. The sametreatment in BMMCs did not result in significant phosphorylationof CD200R. However, cross-linking the CD200-mIg with a sec-ondary goat anti-mouse Ab induced the receptor phosphorylation(data not shown).

Since CD200 is a transmembrane protein, we then examinedwhether the membrane form of CD200 could induce the receptorphosphorylation in both BMMCs and mast cells overexpressingCD200R. NIH3T3 cells expressing the membrane form of mouseCD200 were generated by retroviral transduction. The CD200-negative expressing cells were sorted and used as negative control.As shown in Fig. 2B, NIH3T3 cells expressed low levels of en-dogenous CD200, and CD200 was highly expressed on the trans-ductants. Incubation of mast cells overexpressing CD200R withCD200-expressing NIH3T3 cells resulted in strong tyrosine phos-phorylation of CD200R (Fig. 2C). The weak phosphorylation ofCD200R induced by NIH3T3 cells was due to the low level ex-pression of CD200 in these cells. Although soluble CD200-mIgfusion protein did not induce tyrosine phosphorylation of CD200Rin low-expressing BMMCs, the membrane form of CD200 ex-pressed on NIH3T3 cells was able to trigger the CD200R phos-phorylation in BMMCs. These results suggest that membraneCD200 would achieve natural cross-linking of the CD200R.

Dok1 and Dok2 are tyrosine phosphorylated upon CD200Rengagement and associate with Ras-GAP, SHIP, and CD200R

CD200R cytoplasmic domain has a potential PTB motif,NPXY297. To test which proteins may bind to CD200R, we madesynthetic biotinylated peptides encompassing the three tyrosineresidues: Y286, Y289, and Y297, which were phosphorylated in allpossible combinations (Fig. 3A). After stimulation with controlmIg or CD200-mIg, the cells were lysed and cell lysates wereprecleared with avidin-agarose beads, then incubated with eachpeptide. The protein complexes were pulled down by avidin-aga-rose beads and subjected to PAGE and Western blotting analysis.As shown in Fig. 3B, peptides 4 and 6 pulled down two clearlyphosphorylated proteins with a molecular weight of 60 and 50

FIGURE 1. Expression of CD200R and CD200RLa and degranulationin mouse mast cells. A, Expression of CD200R and CD200RLa on mousemast cell BMMCs and DT733 as analyzed by flow cytometry. B,. Inhibi-tion of mast cell degranulation by CD200-mIg (CD200), as compared withcontrol mIg fusion protein (C). The degranulation was measured as de-scribed in Materials and Methods. Results are expressed as the mean oftriplicates from one of three experiments.

FIGURE 2. CD200 induced its receptor tyrosinephosphorylation. A, DT733 cells were stimulated withcontrol mIg (C) at 3 �g/ml for 1 min or CD200-mIg(CD200) at 3 �g/ml for indicated time periods.CD200R was immunoprecipitated from cell lysatesusing a rat anti-CD200R Ab and immunoblotted withanti-phosphotyrosine Ab (pY). The same membranewas stripped and reblotted with anti-CD200R Ab. B,Surface expression of CD200 on NIH3T3 (3T3),CD200-negative NIH3T3 (CD200�), and CD200-overexpressing NIH3T3 (CD200�) cells was ana-lyzed by flow cytometry. The cells were stained foranti-CD200 Ab (bold line) or an Iso control Ab (dot-ted line). C, NIH3T3 (3T3), CD200-negative NIH3T3(CD200�), or CD200-overexpressing NIH3T3(CD200�) cells were incubated with ether BMMCs orDT733 cells at 37°C for 10 min. The phosphorylationof CD200R was determined as described above.

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kDa. Abs specific for PTB domain proteins were then used toidentify the proteins binding to CD200R peptides 4 and 6. Amongthe known PTB domain proteins, Dok1, Dok2, and Shc were foundto bind to both peptides. Other PTB domain proteins such as IRS-1

were not detected, suggesting that the binding to these peptides isspecific for Dok and Shc proteins. Interestingly, unlike Dok1,Dok2 weakly bound to peptide 7, whereas Shc bound equally topeptides 4, 6, and 7. The binding to peptides 4 and 6 did not requirephosphorylation of Doks and Shc since peptide 4 pulled downequal levels of Dok1, Dok2, and Shc from control mIg andCD200-mIg stimulated cells. However, phosphorylation of Doksand Shc only occurred after stimulation with CD200-mIg. Theseresults clearly indicated that phosphorylation of Y297 on CD200Rwas required for the binding of Dok1 and Dok2 to the receptor.

To verify the peptide binding results in intact cells, mast cellswere stimulated with control mIg or CD200-mIg at 37°C for in-dicated time periods. Dok1, Dok2, Shc, and SHIP were then im-munoprecipitated from the cell lysates and separated by 8% Nu-PAGE, transferred to polyvinylidene difluoride membranes, andblotted with anti-phosphotyrosine Ab. As shown in Fig. 4A, trig-gering CD200R resulted in strong tyrosine phosphorylation of bothDok1 and Dok2. The membrane was then stripped and reblottedfor associated CD200R, RasGAP, and SHIP. As seen in Fig. 4A,after CD200R engagement, phosphorylated Dok1 and Dok2 wereassociated with CD200R and RasGAP. Interestingly, Dok1, butnot Dok2, was also found to bind tyrosine-phosphorylated SHIP.The binding was transient and only induced by CD200 stimulation.CD200-triggered mast cells were also immunoprecipitated for Shcand SHIP. As shown in Fig. 4B, CD200R stimulation slightly in-creased the tyrosine phosphorylation of SHIP and induced the as-sociation of SHIP with phosphorylated Dok1. Unlike Dok pro-teins, the phosphorylation state of Shc was not changed afterCD200R engagement, and we could not detect association ofCD200R with Shc or SHIP. These results showed that upon CD200Rengagement, phosphorylated CD200R binds to adapter proteinsDok1 and Dok2, which in turn recruit RasGAP and SHIP.

Engagement of CD200R inhibited activation of Ras/MAPKpathways

Dok proteins have been shown to mediate inhibitory signaling byrecruiting inhibitory effectors such as RasGAP, SHIP, and Csk

FIGURE 3. Pull-down assay by phosphopeptides. A, The sequences ofbiotinylated peptides. �, Denotes phosphorylation and the underlined is theNPxY motif. B, DT733 cells were stimulated with control mIg (C) orCD200-mIg (CD200) for 5 min. Cell lysates were precleared with avidin-agarose beads and then used in the pull-down assay with beads alone ordifferent peptides. TCL, Total cell lysate. The protein complexes wereseparated in Nu-PAGE and blotted with different Abs.

FIGURE 4. Tyrosine phosphorylation of Doks, SHIP, and Shc upon CD200R engagement. A, Growth factor-starved DT733 cells were stimulated withcontrol mIg (C) for 2 min or CD200-mIg (CD200) for indicated time periods. Dok1 and Dok2 were immunoprecipitated from cell lysates and immuno-blotted with the indicated Abs. CD200R was detected by using anti-Myc Ab. Results are one representative of three experiments. B, Growth factor-starvedDT733 cells were stimulated with control mIg (C) or CD200-mIg (CD200) for 5 min. SHIP and Shc were immunoprecipitated from cell lysates andimmunoblotted with the indicated Abs. Results are one representative of two experiments.

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(18–23). Association of Dok-1 with RasGAP has been shown toattenuate Ras activity, leading to the inhibition of downstreamMAPK pathways in B cells and mast cells (18, 23). The associa-tion of Dok proteins with CD200R and RasGAP suggested a po-tential inhibitory pathway for CD200 signaling. To investigate thispotential inhibitory pathway, we first examined whether CD200Rtriggering inhibited MAPK activation. Normally growing IL-3-de-pendent DT733 cells were incubated with either control mIg orCD200-mIg for various periods of time. Equal amounts of celllysates were immunoblotted with anti-phospho ERK Ab that rec-ognizes the dual-phosphorylated ERK1 and ERK2. As shown inFig. 5A, CD200-mIg strongly inhibited constitutively activatedERKs which were induced by IL-3 in the culture medium and theinhibition occurred within 1 min after CD200R triggering. ControlmIg had no effect even after a 30-min incubation (data not shown).

Experiments were then performed to determine whether trigger-ing CD200R would inhibit activation of Ras/MAPK pathways in-duced by DX89 Ab. Mast cells overexpressing CD200R were fac-tor starved for 16 h in medium without IL-3 or serum. Afterpretreatment with control mIg or CD200-mIg, the cells were stim-ulated with DX89 and the activation of ERK, p38, JNK, and Ras

was examined. Activation of mouse mast cells via the DAP12pairing receptor CD200RLa caused a pronounced phosphorylationof ERK, p38, and JNK that was directly associated with a potentdegranulation response. Preincubation of mast cells with CD200-mIg markedly reduced the activation-dependent phosphorylationof ERK, p38, and JNK (Fig. 5B). Consistent with the dephosphor-ylation of ERK, CD200R triggering also significantly reduced Rasactivation (Fig. 5C). This was not unique to the CD200RLa acti-vation process, because activation of ERK and p38 MAPK inducedby aggregation of Fc�RI was also inhibited by CD200R engage-ment (data not shown).

Since mast cell degranulation and cytokine secretion are depen-dent on both ERK and p38 MAPK activation (24–26), we thentested whether inhibition of these two pathways by pharmacolog-ical inhibitors would recapitulate the inhibition seen with CD200Rengagement. The cells were pretreated with U0126 (a MEK inhib-itor) and SB203580 (a p38 MAPK inhibitor) and then stimulatedwith DX89. Activation of MAPKs, degranulation response, andcytokine production were then measured. As shown in Fig. 6A,U0126 strongly inhibited the activation of ERK, JNK, and p38MAPK while SB203580 only inhibited the activation of p38MAPK and JNK. Pretreatment with either U0126 or SB203580inhibited mast cell degranulation and the secretion of TNF andIL-13 (Fig. 6, B and C). Interestingly, U0126 was more potent thanSB20358 to inhibit degranulation and cytokine production. Thiscorrelated with its broad and potent inhibition of MAPKs. Theseresults are consistent with the hypothesis that the activation-de-pendent phosphorylation of CD200R recruits phosphorylated Dokproteins which subsequently bind RasGAP and SHIP. The incor-poration of RasGAP and SHIP into the CD200R complex leads todownstream inhibition of the Ras/MAPK pathways and a func-tional reduction in mast cell degranulation and cytokine secretion.

Dok1, and Dok2 are tyrosine phosphorylated upon CD200Rengagement in mouse peritoneal cells

To confirm our findings using mouse mast cells in cells expressingnormal CD200R ex vivo, we examined whether CD200 stimula-tion induced Doks phosphorylation in primary mouse peritonealcells. These cells mainly contain macrophages and mast cells, bothof which express CD200R. Resting mouse peritoneal cells wereisolated from C57BL/6 mice. After stimulation with control mIg orCD200-mIg, Dok1 and Dok2 were immunoprecipitated from thecell lysates and separated by 8% Nu-PAGE, transferred to poly-vinylidene difluoride membranes, and blotted with anti-phosphotyrosine Ab. As shown in Fig. 7, triggering CD200R re-sulted in tyrosine phosphorylation of Dok1 and Dok2. Theseresults provide direct evidence that CD200R engagement leads tothe phosphorylation of Dok1 and Dok2 in primary peritoneal my-eloid cells.

DiscussionCD200R is an inhibitory receptor expressed highly on myeloidcells and some T cells (4). Most inhibitory receptors on immunecells contain a consensus amino acid sequence, termed the ITIM,in the cytoplasmic domain (9–11, 27). The prototype ITIM con-sists of the sequence (I/V/L/S)-x-Y-x-x-(L/V), where x denotesany amino acid. Ligand-induced clustering of these ITIM-contain-ing receptors results in tyrosine phosphorylation, often by a Srcfamily kinase, which provides a docking site for the recruitment oftyrosine phosphatases SHP1 (and occasionally SHP2) and the ino-sitol phosphatase SHIP. As defined by sequences in their extra-cellular domain, ITIM-bearing receptors belong to either the Ig

FIGURE 5. Inhibition of Ras/MAPK activation by CD200. A, Normalgrowing DT733 cells were stimulated with control mIg (C) or CD200-mIg(CD200) at 3 �g/ml for indicated time periods. The activation of ERK wasdetected by immunoblotting with anti-phospho ERK Ab. B, Growth factor-starved DT733 cells were either pretreated with control mIg (C) or CD200-mIg (CD200) at 3 �g/ml at 37°C for 30 min or nontreated (thick line) andthen stimulated with either Iso control Ab or DX89 Ab (20 ng/ml) for 5min. The activation of ERK, JNK, and p38 MAPK was detected by im-munoblotting with the indicated Abs. C, Growth factor-starved DT733cells were pretreated with control mIg (C) or CD200-mIg (CD200) andthen stimulated with either Iso control Ab or DX89 Ab as shown above.Ras activation was measured as described in Materials and Methods. Thenumbers below the blot indicate the relative intensity as measured by den-sitometry analysis.

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superfamily or C-type lectin superfamily. By recruiting phospha-tase SHP1 and/or SHIP, ITIM-bearing inhibitory receptors sup-press cell activation by promoting dephosphorylation reactions.Unlike other myeloid inhibitor receptors, such as Fc�RIIB (28, 29),gp49B1 (30, 31), paired Ig-like receptor � (32, 33), and mast cellfunction-associated Ag (34, 35), CD200R lacks an ITIM, but con-tains three tyrosine residues in the cytoplasmic domain. One ty-rosine (Y297) is located in a NPxY motif, which may represent apotential PTB domain protein-binding motif. Engagement ofCD200R by soluble CD200-mIg fusion protein resulted in rapidCD200R tyrosine phosphorylation in mouse mast cells overex-pressing CD200R, but not in wild-type BMMCs. This result com-bined with the degranulation response as shown in Fig. 1B clearlyshow that the CD200R density on mast cells determines the thresh-old of inhibition. In BMMCs, CD200R have to be cross-linked witha secondary Ab to trigger the receptor and inhibit cell activation.Since CD200 is naturally expressed as a membrane protein, thecell surface density of the ligand and its presentation to theCD200R may be different from the soluble form in that the mem-brane-bound CD200 will achieve a high degree of natural cross-linking of the CD200R. Our results support this hypothesis be-cause the membrane form of CD200 expressed on NIH3T3 cellswas able to trigger the CD200R phosphorylation in both BMMCsand mast cells overexpressing the CD200R (Fig. 2C).

Because CD200R could be tyrosine phosphorylated upon ligandengagement, this suggests that it may bind to downstream targetproteins, in particular, the PTB domain proteins. To determinewhether the NPxY motif was functional and which PTB domain

proteins may bind to CD200R, we made biotinylated peptides en-compassing the three tyrosine residues (Y286, Y289, and Y297),which were phosphorylated in all possible combinations, and per-formed the in vitro-binding assay. Among the known PTB domainproteins, Dok1, Dok2, and Shc were found to bind to the peptidein which Y297 was phosphorylated. IRS-1, another PTB domainprotein, does not bind to any peptide in the experiment, suggestingthat the binding is specific for Dok and Shc proteins. We were alsounable to detect the binding of SHIP and SHP1 to any of thephosphorylated peptides. Interestingly, peptide 8, in which allthree tyrosines are phosphorylated, did not bind Dok1, Dok2, orShc. Phosphorylation of all three tyrosines may create steric hin-drance, thus preventing the binding of Dok and Shc. The bindingof Dok1 and Dok2 to CD200R was confirmed in mouse mast cells.CD200 stimulation of mouse mast cells induced tyrosine phos-phorylation of Dok1 and Dok2 and their subsequent associationwith CD200R. Not only do Doks bind to CD200R after receptorstimulation, they also recruit RasGAP. Interesting, Dok1 but notDok2 also binds to phosphorylated SHIP after CD200R stimula-tion. Unlike Dok, the phosphorylation of Shc did not change fol-lowing CD200 stimulation, and we could not detect the associationof Shc with CD200R in intact cells, even though Shc was found tobind phosphorylated peptides in the in vitro-binding assay. Theseresults show that upon ligand binding CD200R specifically recruitsDok1 and Dok2 to the receptor complex which in turn binds Ras-GAP and SHIP. We are currently examining which kinase medi-ates phosphorylation of CD200R and Doks. Considering the roleof Src family kinases Lyn and Fyn in mast cell activation (36–38),

FIGURE 6. Inhibition of MAPK activation, mast cell degranulation, and cytokine secretion by ERK and p38 MAPK inhibitors. A, Growth factor-starvedDT733 cells were pretreated with either DMSO (2), control mIg (C, 3), CD200-mIg (CD200, 4), U0126 (U, 5), or SB203580 (SB, 6) at 37°C for 30 minand then stimulated with Iso control Ab (1) or DX89 Ab (2–6; 20 ng/ml) for 5 min. The activation of ERK, JNK, and p38 MAPK was detected byimmunoblotting with the indicated Abs. B and C, Inhibition of mast cell degranulation (B) and cytokine secretion (C). DT733 cells were pretreated withDMSO, control mIg (C), CD200-mIg (CD200), U0126, or SB203580 (SB) for 30 min at 37°C and then stimulated with either Iso control Ab or DX89 Ab(20 ng/ml) for 1 h in degranulation and 24 h in cytokine production, respectively. Degranulation and cytokine secretion were measured as described inMaterials and Methods.

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they may be involved in the phosphorylation of CD200Rand Doks.

Among the PTB domain proteins, Dok proteins have beenshown to mediate inhibitory signaling (18–23). The Dok familycomprises five known members; Dok-1, Dok-2 (also termedDok-R and FRIP), Dok-3 (also named Dok-L), Dok-4, and Dok-5(22, 39–42). These molecules contain an amino-terminal pleck-strin homology domain, a central PTB domain, and a carboxyl-terminal region with multiple potential tyrosine phosphorylationsites and proline-rich regions, which may serve as docking sites forSrc homology 3 domains. Dok proteins undergo tyrosine phos-phorylation in response to a variety of stimuli, such as immuno-receptor ligation, growth factors, and cytokines. This phosphory-lation triggers Src homology 2 domain-mediated interactions withinhibitory effectors including RasGAP, SHIP, and Csk. In B cells,both Dok-1 and Dok-3 undergo rapid tyrosine phosphorylation inresponse to B cell receptor engagement and negatively regulatecell activation (18, 22). Dok1 has been shown to inhibit MAPKactivation and cell proliferation upon coaggregation of B cell re-ceptor and Fc�RIIB (18, 21). Dok2 negatively regulates T celldevelopment by recruiting RasGAP and Nck (43). In mast cells,co-cross-linking of Fc�RIIB with Fc�RI stimulates Dok1 tyrosinephosphorylation and association with SHIP and RasGAP (23).Overexpression of Dok1 in the mast cell line RBL-2H3 inhibitedFc�RI-mediated Ras/Raf1/ERK signaling and the de novo synthe-sis of TNF-� (44). These studies have established that Dok familyproteins are inhibitory adaptor molecules, presumably due to theirability to recruit inhibitory effectors RasGAP, SHIP, and Csk. Ourresults show that CD200R also binds to Dok1 and Dok2, whichthen recruit RasGAP and SHIP.

Like other inhibitory receptors such as Fc�RIIB, gp49B1, pairedIg-like receptor �, and mast cell function-associated Ag in mastcells, CD200R engagement inhibits mast cell degranulation andcytokine production. This inhibition is likely mediated by the re-duced activation of MAPKs: ERK, p38 MAPK, and JNK, sinceCD200R engagement inhibited activation of all three MAPKs. Un-like the majority of myeloid inhibitory receptors, the inhibitiondoes not require co-cross-linking of CD200R with an activatingreceptor because CD200 ligand alone could inhibit the activationof ERK1/2 induced by IL-3. This suggests that CD200R is a novelinhibitory receptor that employs recruitment of RasGAP to directlyinhibit Ras activation. However, other molecules may also be in-volved in the inhibition because CD200 also inhibits p38 MAPKand JNK activation which are not dependent on a Ras pathway. Itis likely that SHIP also plays a role in CD200-mediated inhibitionbecause SHIP has been shown to act as a negative regulator ofmast cell and B cell activation (45–47). The role of SHIP inCD200 inhibition is currently under investigation.

Besides mast cells, CD200R is also highly expressed on mac-rophages (4). CD200-CD200R interaction has been shown to beimportant for regulation of the macrophage lineage. In CD200-deficient mice, there were increased numbers of macrophages inthe spleen and the mesenteric lymph nodes, and these macrophagesshow increased activation (5). Our data now show that CD200Rengagement induced tyrosine phosphorylation of Dok1 and Dok2in primary mouse peritoneal cells. This confirms the findings inmast cells and underlines the importance of CD200 in modulatingmyeloid cell activation broadly. Our separate studies show thatengagement of CD200R by agonist Ab and soluble CD200-Ig fu-sion protein inhibited mast cell degranulation in the passive cuta-neous anaphylaxis model, suggesting the inhibitory role ofCD200R in vivo.3

In conclusion, we have shown that Dok1 and Dok2 are media-tors of CD200R inhibitory signaling. They both bind to RasGAP,leading to the inhibition of Ras and downstream ERK activation.It is possible that other molecules may also be involved in theinhibition because CD200 also inhibits p38 MAPK and JNK ac-tivation which do not depend on Ras. Our results suggest a novelinhibitory pathway used by CD200R in modulating myeloid cellfunction (Fig. 8). That this pathway is triggered and active withoutneed for co-cross-linking to an activating receptor provides aunique opportunity for the use of CD200R as an anti-inflammatorytarget in vivo.

AcknowledgmentsWe thank Mike Bigler and Yaoli Song for assistance and Janet Wagner andSandra Zurawski for making the fusion proteins and mAbs. We also thankMaria Jenmalm for help and discussion.

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FIGURE 8. Proposed model for CD200R-mediated inhibition of mastcell activation. Engagement of ITAM-containing activating receptors suchas Fc�RI and CD200RLa leads to the activation of downstream Ras/MAPKpathways. Triggering CD200R by CD200 induces its phosphorylation andrecruitment of Dok1 and Dok2. Dok1 and Dok2 are phosphorylated andbind to RasGAP and SHIP, which leads to inhibition of Ras and ERKactivation. Other unknown molecules may mediate the inhibition of p38MAPK and JNK activation.

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