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The Journal of Immunology, 2004, 173: 6786-6793.
Copyright © 2004 by The American Association of Immunologists

Molecular Mechanisms of CD200 Inhibition of Mast Cell Activation

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

DNAX Research Institute, Palo Alto, CA 94304


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD200 and its receptor CD200R are both type I membrane glycoproteins that contain two Ig-like domains. Engagement of CD200R by CD200 inhibits activation of myeloid cells. Unlike the majority of immune inhibitory receptors, CD200R lacks an ITIM in the cytoplasmic domain. The molecular mechanism of CD200R inhibition of myeloid cell activation is unknown. In this study, we examined the CD200R signaling pathways that control degranulation of mouse bone marrow-derived mast cells. We found that upon ligand binding, CD200R is phosphorylated on tyrosine and subsequently binds to adapter proteins Dok1 and Dok2. Upon phosphorylation, Dok1 binds to SHIP and both Dok1 and Dok2 recruit RasGAP, which mediates the inhibition of the Ras/MAPK pathways. Activation of ERK, JNK, and p38 MAPK are all inhibited by CD200R engagement. The reduced activation of these MAPKs is responsible for the observed inhibition of mast cell degranulation and cytokine production. Similar signaling events were also observed upon CD200R engagement in mouse peritoneal cells. These data define a novel inhibitory pathway used by CD200R in modulating mast cell function and help to explain how engagement of this receptor in vivo regulates myeloid cell function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The CD200 (originally OX2) is a membrane glycoprotein expressed by a broad range of cell types, including lymphoid cells, neurons, and endothelium (1). It is the ligand for a receptor (CD200R) whose expression is restricted to hemopoietic cells, particularly myeloid cells (1, 2, 3, 4). CD200 and its receptor both are type I membrane proteins and contain two extracellular Ig-like domains. Mouse CD200 has a short intracellular domain (19 aa) which lacks any known signaling motifs. Mouse CD200R, however, has a larger cytoplasmic domain (67 aa) containing three tyrosine residues (2).

Several in vivo studies have suggested that the interaction of CD200 with its receptor can deliver inhibitory signals to myeloid cells. For example, blocking CD200-CD200R interaction by either soluble CD200R protein or CD200R-blocking Ab augmented collagen-induced arthritis (CIA)2 in mice and exacerbated experimental autoimmune encephalomyelitis in rats (2, 5). These studies suggested that blocking the constitutive interactions of CD200R and CD200 decreased myeloid cell inhibitory thresholds, which in turn resulted in enhanced immune activation. Consistent with this hypothesis, mice receiving soluble CD200 protein were resistant to CIA induction (6, 7) and showed prolonged graft survival in both allo- and xenotransplantation models (8). The strongest supportive evidence for an inhibitory role of CD200 in immune modulation comes from studies of CD200-deficient mice (5). In these mice, there were increased numbers of activated macrophages in the spleen and the mesenteric lymph nodes and, more importantly, these mice showed a more rapid onset of experimental autoimmune encephalomyelitis and increased susceptibility to CIA. Recently, we provided direct evidence that CD200R is indeed an inhibitory receptor regulating mast cell activation in vitro and in vivo (48).

Most immune inhibitory receptors share a cytoplasmic amino acid sequence, termed the ITIM (9, 10, 11, 12). All ITIMs are composed of the consensus sequence (I/V/L/S)xYxx(L/V), which upon tyrosine phosphorylation recruits phosphatases (SRC homology region 2 domain-containing phosphatase (SHP) 1, SHP2, and/or SHIP). The binding of phosphatases to ITIM-containing receptors in turn suppresses cell activation by promoting dephosphorylation of activating receptors and downstream signaling molecules. Unlike the majority of immune inhibitor receptors, CD200R lacks an ITIM, but contains an NPXY sequence in its cytoplasmic domain. The NPXY motif has previously been shown to be a binding site for proteins with phosphotyrosine-binding (PTB) domain (13, 14). In the present study, we examined the intracellular signaling events associated with CD200R engagement in mouse bone marrow-derived mast cells (BMMCs) and primary peritoneal cells. Our results provide strong evidence for a novel inhibitory pathway used by CD200R to modulate myeloid cell function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Recombinant 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 from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-dual phosphorylated MAPK (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 were purchased from BD Pharmingen (San Diego, CA). SB203580 was obtained from Calbiochem (San Diego, CA).

A fusion protein consisting of the extracellular domain of mouse CD200 fused 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 mIg and 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 as previously described (4). Anti-mouse CD200RLa Ab (DX89, rat IgM) was generated from a rat immunized with a fusion protein consisting of the extracellular domain of mouse CD200RLa fused to the Fc region of human IgG1 as previously described (4). Cross-linking CD200RLa with DX89 induces a strong dose-dependent degranulation response in mouse mast cells (data not shown). In the present study, mAb DX89 was used to activate mouse mast cells because the IgM isotype (Iso) did not require additional cross-linking reagents to activate the receptor and did not bind FcRs for IgG.

Cell culture, gene transduction, and flow cytometry

Mouse BMMCs were generated from bone marrow of 2- to 3-wk-old C57BL/6 mice as previously described (4). Mast cells overexpressing CD200R and CD200RLa (DT733) were generated by retroviral transduction of BMMCs. A cDNA containing the CD8 leader segment followed by the Flag epitope tag (DYKDDDDK) and joined to the extracellular, transmembrane and cytoplasmic domains of mouse CD200RLa was subcloned into the pMXneo retroviral vector (15). Plasmid DNA was transfected into Phoenix ecotropic retrovirus packaging cells (a gift from G. Nolan, Stanford University, Palo Alto, CA) using Lipofectamine (Invitrogen Life Technologies, Carlsbad, CA). Two days later, BMMCs were infected by coculture with the transfected packaging cell line. After 30 h, the nonadherent BMMCs were removed and put into fresh medium and after 72 h were switched to selection medium containing 1 mg/ml G418 (Roche Molecular Biochemicals, Indianapolis, IN). Cells were sorted for CD200RLa expression using the anti-Flag Ab M2 (Sigma-Aldrich, St. Louis, MO). A cDNA containing the CD8 leader segment followed by the c-Myc epitope tag (EQKLISEEDL) and joined to the extracellular, transmembrane and cytoplasmic regions of mouse CD200R were subcloned into the retroviral vector pMXneo. The resultant construct was then introduced into the mast cell transfectant expressing the Flag-tagged CD200RLa by retroviral infection as described above. Cells were sorted for cell surface CD200R expression using the anti-Myc Ab 9E10 and subsequently verified for gene expression using anti-CD200R mAb. Cells ≥95% positive and having similar levels of expression for both CD200R and CD200RLa were used for the biochemical analyses and degranulation assays as described below.

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

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

Surface expression of CD200R and CD200RLa was analyzed by standard flow cytometry techniques. In brief, mast cells were washed once with PBS and stained with either FITC-conjugated anti-CD200R (DX109, rat IgG1) or PE-conjugated anti-CD200RLa (DX87, rat IgG2c) Ab. After incubation 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, San Jose, CA).

Degranulation assays and cytokine ELISA

Mast cell degranulation was determined using a {beta}-hexosaminidase release assay as previously described.3 The degranulation was triggered by incubating mast cells with indicated amount of DX89 Ab, which binds to the activating receptor CD200RLa and induces a strong degranulation response. Briefly, 1 x 106 cells/ml mast cells were treated with indicated amounts of DX89 Ab for 1 h in RPMI 1640 medium in 96-well plates. Supernatants were assayed for {beta}-hexosaminidase activity. For CD200-mediated inhibition, the cells were pretreated with indicated amounts of control mIg or CD200-mIg for 30 min before activation. For FcR blockage experiments, cells were pretreated with the FcR-blocking Ab 2.4G2 (20 µg/ml) for 30 min before subsequent incubations with control mIg or CD200-mIg. U0126 and SB203580 were used at 10 µM. TNF and IL-13 were measured by ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

Immunoprecipitation and immunoblotting

Mast cells (1–2 x 107 cells/ml) were stimulated at 37°C with control mIg or CD200-mIg (3 µg/ml) for various times as indicated. For CD200-mediated inhibition, the cells were pretreated with control mIg or CD200-mIg (3 µg/ml) for 30 min at 37°C and then stimulated with DX89 Ab (20 ng/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 (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 5 mM EGTA, 50 mM NaF, 1 mM Na3VO4, plus protease inhibitor mixtures) for 20 min on ice. Lysates were clarified at 14,000 rpm for 10 min. The protein concentration 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 Western blotting, primary Abs were detected with HRP-conjugated secondary Abs and chemiluminescence (Pierce, Rockford, IL). For immunoprecipitations, Abs were incubated with 0.5–1 mg of cell lysate for 2 h at 4°C. The immune complexes were recovered by incubation with protein A-agarose beads or protein G Plus-agarose beads (Santa Cruz Biotechnology) for 1 h at 4°C. After washing three times in lysis buffer and once in PBS containing 1 mM Na3VO4, the immune complexes were dissociated in SDS sample buffer. The samples were analyzed by Nu-PAGE and Western blotting as described above.

Peptide synthesis and in vitro-binding assay

N-terminal biotinylated peptides (DEMQPYASYTEKSNPLYDTVT) encompassing the three tyrosine residues (Y286, Y289, and Y297) in the cytoplasmic domain of mouse CD200R were synthesized and phosphorylated at tyrosine in all possible combinations at the Biomolecular Resource Center (University of California, San Francisco, CA). Mast cells (1 x 107 cells/ml) were stimulated at 37°C with control mIg or CD200-mIg (3 µg/ml) for 5 min and then lysed as described above. Cell lysates were precleared with 50 µl of avidin-agarose beads at room temperature for 30 min (Vector Laboratories, Burlingame, CA) and then incubated with 5 µg of biotinylated peptides at 4°C for 1 h, followed by incubation with 50 µl of avidin-agarose beads at 4°C for 30 min. The protein complexes were washed extensively and subjected to PAGE and Western blotting analysis as described above.

Ras activation assay

Ras activation was measured using a Ras activation assay kit (Upstate Biotechnology) according to the manufacturer’s instructions. Briefly, after stimulation, cells were lysed and GTP-bound Ras was pulled down by GST fusion protein containing the Ras-binding domain of Raf-1 bound to glutathione-agarose. The precipitated Ras-GTP was detected by Western blot.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD200R engagement inhibits mast cell activation

CD200R is highly expressed on myeloid cells such as mast cells, macrophage, and neutrophils (4). As reported elsewhere,3 engagement of the mouse CD200R by agonist Abs or ligand results in a potent inhibition of mast cell degranulation and cytokine secretion in mouse mast cells overexpressing CD200R. To study the molecular mechanisms of CD200 inhibition, mouse mast cells were generated from C57BL/6 bone marrow. As shown in Fig. 1A, these BMMCs expressed relatively high level of CD200R and CD200RLa. Engagement of the CD200R by soluble CD200-mIg, however, did not inhibit mast cell degranulation induced by the activating Ab DX89 (Fig. 1B). To increase the sensitivity of the assay, mast cells were induced to overexpress CD200R and CD200RLa by retroviral gene transfer. As shown in Fig. 1, DT733 cells expressed higher levels of both CD200R and CD200RLa and demonstrated an enhanced degranulation response, which was strongly inhibited by CD200R engagement. Pretreatment of DT733 cells with CD200-mIg also inhibited mast cell degranulation induced by Ag cross-linking of Fc{epsilon}RI (data not shown). But, unlike other inhibitory immune receptors, CD200R-mediated inhibition of mast cell degranulation did not require coligation of CD200R with the activating receptor. Because mouse mast cells express FcRs for IgG, it was important to verify that the Fc-mutated CD200-mIg did not functionally trigger FcR to manifest inhibitory signals. Pretreatment of mast cells overexpressing CD200R and CD200RLa with the FcR-blocking Ab 2.4G2 before triggering the CD200R with CD200-mIg had no effects on the CD200 induced inhibition of mast cell degranulation (data not shown). Mast cells overexpressing CD200R and CD200RLa (DT733) were used subsequently in all studies to dissect the molecular mechanisms of CD200R signaling.



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  		FIGURE 1. Expression of CD200R and CD200RLa and degranulation in mouse mast cells. A, Expression of CD200R and CD200RLa on mouse mast cell BMMCs and DT733 as analyzed by flow cytometry. B,. Inhibition of mast cell degranulation by CD200-mIg (CD200), as compared with control mIg fusion protein (C). The degranulation was measured as described in Materials and Methods. Results are expressed as the mean of triplicates from one of three experiments.

 
CD200R is tyrosine phosphorylated upon CD200 binding

Unlike the majority of inhibitory receptors, CD200R lacks an ITIM, but contains three tyrosine residues Y286, Y289, and Y297 in the cytoplasmic domain of mouse receptor. Sodium pervanadate pretreatment of cells expressing CD200R has been shown to result in CD200R tyrosine phosphorylation (2). To determine whether engagement of CD200R by its ligand induced CD200R phosphorylation, we treated DT733 cells with CD200-mIg and examined the phosphorylation of CD200R. As shown in Fig. 2A, CD200 stimulation 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 same treatment in BMMCs did not result in significant phosphorylation of CD200R. However, cross-linking the CD200-mIg with a secondary goat anti-mouse Ab induced the receptor phosphorylation (data not shown).



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  		FIGURE 2. CD200 induced its receptor tyrosine phosphorylation. A, DT733 cells were stimulated with control mIg (C) at 3 µg/ml for 1 min or CD200-mIg (CD200) at 3 µg/ml for indicated time periods. CD200R was immunoprecipitated from cell lysates using a rat anti-CD200R Ab and immunoblotted with anti-phosphotyrosine Ab (pY). The same membrane was 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 analyzed by flow cytometry. The cells were stained for anti-CD200 Ab (bold line) or an Iso control Ab (dotted line). C, NIH3T3 (3T3), CD200-negative NIH3T3 (CD200–), or CD200-overexpressing NIH3T3 (CD200+) cells were incubated with ether BMMCs or DT733 cells at 37°C for 10 min. The phosphorylation of CD200R was determined as described above.

 
Since CD200 is a transmembrane protein, we then examined whether the membrane form of CD200 could induce the receptor phosphorylation in both BMMCs and mast cells overexpressing CD200R. NIH3T3 cells expressing the membrane form of mouse CD200 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 endogenous CD200, and CD200 was highly expressed on the transductants. Incubation of mast cells overexpressing CD200R with CD200-expressing NIH3T3 cells resulted in strong tyrosine phosphorylation of CD200R (Fig. 2C). The weak phosphorylation of CD200R induced by NIH3T3 cells was due to the low level expression of CD200 in these cells. Although soluble CD200-mIg fusion protein did not induce tyrosine phosphorylation of CD200R in low-expressing BMMCs, the membrane form of CD200 expressed on NIH3T3 cells was able to trigger the CD200R phosphorylation in BMMCs. These results suggest that membrane CD200 would achieve natural cross-linking of the CD200R.

Dok1 and Dok2 are tyrosine phosphorylated upon CD200R engagement 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 made synthetic biotinylated peptides encompassing the three tyrosine residues: Y286, Y289, and Y297, which were phosphorylated in all possible combinations (Fig. 3A). After stimulation with control mIg or CD200-mIg, the cells were lysed and cell lysates were precleared with avidin-agarose beads, then incubated with each peptide. The protein complexes were pulled down by avidin-agarose beads and subjected to PAGE and Western blotting analysis. As shown in Fig. 3B, peptides 4 and 6 pulled down two clearly phosphorylated proteins with a molecular weight of 60 and 50 kDa. Abs specific for PTB domain proteins were then used to identify the proteins binding to CD200R peptides 4 and 6. Among the known PTB domain proteins, Dok1, Dok2, and Shc were found to bind to both peptides. Other PTB domain proteins such as IRS-1 were not detected, suggesting that the binding to these peptides is specific for Dok and Shc proteins. Interestingly, unlike Dok1, Dok2 weakly bound to peptide 7, whereas Shc bound equally to peptides 4, 6, and 7. The binding to peptides 4 and 6 did not require phosphorylation of Doks and Shc since peptide 4 pulled down equal levels of Dok1, Dok2, and Shc from control mIg and CD200-mIg stimulated cells. However, phosphorylation of Doks and Shc only occurred after stimulation with CD200-mIg. These results clearly indicated that phosphorylation of Y297 on CD200R was required for the binding of Dok1 and Dok2 to the receptor.



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

 
To verify the peptide binding results in intact cells, mast cells were stimulated with control mIg or CD200-mIg at 37°C for indicated time periods. Dok1, Dok2, Shc, and SHIP were then immunoprecipitated from the cell lysates and separated by 8% Nu-PAGE, transferred to polyvinylidene difluoride membranes, and blotted with anti-phosphotyrosine Ab. As shown in Fig. 4A, triggering CD200R resulted in strong tyrosine phosphorylation of both Dok1 and Dok2. The membrane was then stripped and reblotted for associated CD200R, RasGAP, and SHIP. As seen in Fig. 4A, after CD200R engagement, phosphorylated Dok1 and Dok2 were associated with CD200R and RasGAP. Interestingly, Dok1, but not 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 Shc and SHIP. As shown in Fig. 4B, CD200R stimulation slightly increased the tyrosine phosphorylation of SHIP and induced the association of SHIP with phosphorylated Dok1. Unlike Dok proteins, the phosphorylation state of Shc was not changed after CD200R engagement, and we could not detect association of CD200R with Shc or SHIP. These results showed that upon CD200R engagement, phosphorylated CD200R binds to adapter proteins Dok1 and Dok2, which in turn recruit RasGAP and SHIP.



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

 
Engagement of CD200R inhibited activation of Ras/MAPK pathways

Dok proteins have been shown to mediate inhibitory signaling by recruiting inhibitory effectors such as RasGAP, SHIP, and Csk (18, 19, 20, 21, 22, 23). Association of Dok-1 with RasGAP has been shown to attenuate Ras activity, leading to the inhibition of downstream MAPK pathways in B cells and mast cells (18, 23). The association of Dok proteins with CD200R and RasGAP suggested a potential inhibitory pathway for CD200 signaling. To investigate this potential inhibitory pathway, we first examined whether CD200R triggering inhibited MAPK activation. Normally growing IL-3-dependent DT733 cells were incubated with either control mIg or CD200-mIg for various periods of time. Equal amounts of cell lysates were immunoblotted with anti-phospho ERK Ab that recognizes the dual-phosphorylated ERK1 and ERK2. As shown in Fig. 5A, CD200-mIg strongly inhibited constitutively activated ERKs which were induced by IL-3 in the culture medium and the inhibition occurred within 1 min after CD200R triggering. Control mIg had no effect even after a 30-min incubation (data not shown).



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  		FIGURE 5. Inhibition of Ras/MAPK activation by CD200. A, Normal growing DT733 cells were stimulated with control mIg (C) or CD200-mIg (CD200) at 3 µg/ml for indicated time periods. The activation of ERK was detected 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 µg/ml at 37°C for 30 min or nontreated (thick line) and then stimulated with either Iso control Ab or DX89 Ab (20 ng/ml) for 5 min. The activation of ERK, JNK, and p38 MAPK was detected by immunoblotting with the indicated Abs. C, Growth factor-starved DT733 cells were pretreated with control mIg (C) or CD200-mIg (CD200) and then stimulated with either Iso control Ab or DX89 Ab as shown above. Ras activation was measured as described in Materials and Methods. The numbers below the blot indicate the relative intensity as measured by densitometry analysis.

 
Experiments were then performed to determine whether triggering CD200R would inhibit activation of Ras/MAPK pathways induced by DX89 Ab. Mast cells overexpressing CD200R were factor starved for 16 h in medium without IL-3 or serum. After pretreatment with control mIg or CD200-mIg, the cells were stimulated with DX89 and the activation of ERK, p38, JNK, and Ras was examined. Activation of mouse mast cells via the DAP12 pairing receptor CD200RLa caused a pronounced phosphorylation of ERK, p38, and JNK that was directly associated with a potent degranulation response. Preincubation of mast cells with CD200-mIg markedly reduced the activation-dependent phosphorylation of ERK, p38, and JNK (Fig. 5B). Consistent with the dephosphorylation of ERK, CD200R triggering also significantly reduced Ras activation (Fig. 5C). This was not unique to the CD200RLa activation process, because activation of ERK and p38 MAPK induced by aggregation of Fc{epsilon}RI was also inhibited by CD200R engagement (data not shown).

Since mast cell degranulation and cytokine secretion are dependent on both ERK and p38 MAPK activation (24, 25, 26), we then tested whether inhibition of these two pathways by pharmacological inhibitors would recapitulate the inhibition seen with CD200R engagement. The cells were pretreated with U0126 (a MEK inhibitor) and SB203580 (a p38 MAPK inhibitor) and then stimulated with DX89. Activation of MAPKs, degranulation response, and cytokine production were then measured. As shown in Fig. 6A, U0126 strongly inhibited the activation of ERK, JNK, and p38 MAPK while SB203580 only inhibited the activation of p38 MAPK and JNK. Pretreatment with either U0126 or SB203580 inhibited mast cell degranulation and the secretion of TNF and IL-13 (Fig. 6, B and C). Interestingly, U0126 was more potent than SB20358 to inhibit degranulation and cytokine production. This correlated with its broad and potent inhibition of MAPKs. These results are consistent with the hypothesis that the activation-dependent phosphorylation of CD200R recruits phosphorylated Dok proteins which subsequently bind RasGAP and SHIP. The incorporation of RasGAP and SHIP into the CD200R complex leads to downstream inhibition of the Ras/MAPK pathways and a functional reduction in mast cell degranulation and cytokine secretion.



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  		FIGURE 6. Inhibition of MAPK activation, mast cell degranulation, and cytokine secretion by ERK and p38 MAPK inhibitors. A, Growth factor-starved DT733 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 min and 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 by immunoblotting with the indicated Abs. B and C, Inhibition of mast cell degranulation (B) and cytokine secretion (C). DT733 cells were pretreated with DMSO, 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 in Materials and Methods.

 
Dok1, and Dok2 are tyrosine phosphorylated upon CD200R engagement in mouse peritoneal cells

To confirm our findings using mouse mast cells in cells expressing normal CD200R ex vivo, we examined whether CD200 stimulation induced Doks phosphorylation in primary mouse peritoneal cells. These cells mainly contain macrophages and mast cells, both of which express CD200R. Resting mouse peritoneal cells were isolated from C57BL/6 mice. After stimulation with control mIg or CD200-mIg, Dok1 and Dok2 were immunoprecipitated from the cell lysates and separated by 8% Nu-PAGE, transferred to polyvinylidene difluoride membranes, and blotted with anti-phosphotyrosine Ab. As shown in Fig. 7, triggering CD200R resulted in tyrosine phosphorylation of Dok1 and Dok2. These results provide direct evidence that CD200R engagement leads to the phosphorylation of Dok1 and Dok2 in primary peritoneal myeloid cells.



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  		FIGURE 7. CD200R stimulation induced tyrosine phosphorylation of Dok1 and Dok2 in mouse peritoneal cells. Mouse peritoneal cells were stimulated with control mIg (C) or CD200-mIg (CD200) at 10 µg/ml for 10 min. Dok1 and Dok2 were immunoprecipitated from cell lysates and immunoblotted with the indicated Abs.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CD200R is an inhibitory receptor expressed highly on myeloid cells and some T cells (4). Most inhibitory receptors on immune cells contain a consensus amino acid sequence, termed the ITIM, in the cytoplasmic domain (9, 10, 11, 27). The prototype ITIM consists of the sequence (I/V/L/S)-x-Y-x-x-(L/V), where x denotes any amino acid. Ligand-induced clustering of these ITIM-containing receptors results in tyrosine phosphorylation, often by a Src family kinase, which provides a docking site for the recruitment of tyrosine phosphatases SHP1 (and occasionally SHP2) and the inositol phosphatase SHIP. As defined by sequences in their extracellular domain, ITIM-bearing receptors belong to either the Ig superfamily or C-type lectin superfamily. By recruiting phosphatase SHP1 and/or SHIP, ITIM-bearing inhibitory receptors suppress cell activation by promoting dephosphorylation reactions. Unlike other myeloid inhibitor receptors, such as Fc{epsilon}RIIB (28, 29), gp49B1 (30, 31), paired Ig-like receptor {beta} (32, 33), and mast cell function-associated Ag (34, 35), CD200R lacks an ITIM, but contains three tyrosine residues in the cytoplasmic domain. One tyrosine (Y297) is located in a NPxY motif, which may represent a potential PTB domain protein-binding motif. Engagement of CD200R by soluble CD200-mIg fusion protein resulted in rapid CD200R tyrosine phosphorylation in mouse mast cells overexpressing CD200R, but not in wild-type BMMCs. This result combined with the degranulation response as shown in Fig. 1B clearly show that the CD200R density on mast cells determines the threshold of inhibition. In BMMCs, CD200R have to be cross-linked with a secondary Ab to trigger the receptor and inhibit cell activation. Since CD200 is naturally expressed as a membrane protein, the cell surface density of the ligand and its presentation to the CD200R may be different from the soluble form in that the membrane-bound CD200 will achieve a high degree of natural cross-linking of the CD200R. Our results support this hypothesis because the membrane form of CD200 expressed on NIH3T3 cells was able to trigger the CD200R phosphorylation in both BMMCs and mast cells overexpressing the CD200R (Fig. 2C).

Because CD200R could be tyrosine phosphorylated upon ligand engagement, this suggests that it may bind to downstream target proteins, in particular, the PTB domain proteins. To determine whether the NPxY motif was functional and which PTB domain proteins may bind to CD200R, we made biotinylated peptides encompassing the three tyrosine residues (Y286, Y289, and Y297), which were phosphorylated in all possible combinations, and performed the in vitro-binding assay. Among the known PTB domain proteins, Dok1, Dok2, and Shc were found to bind to the peptide in which Y297 was phosphorylated. IRS-1, another PTB domain protein, does not bind to any peptide in the experiment, suggesting that the binding is specific for Dok and Shc proteins. We were also unable to detect the binding of SHIP and SHP1 to any of the phosphorylated peptides. Interestingly, peptide 8, in which all three tyrosines are phosphorylated, did not bind Dok1, Dok2, or Shc. Phosphorylation of all three tyrosines may create steric hindrance, thus preventing the binding of Dok and Shc. The binding of Dok1 and Dok2 to CD200R was confirmed in mouse mast cells. CD200 stimulation of mouse mast cells induced tyrosine phosphorylation of Dok1 and Dok2 and their subsequent association with CD200R. Not only do Doks bind to CD200R after receptor stimulation, they also recruit RasGAP. Interesting, Dok1 but not Dok2 also binds to phosphorylated SHIP after CD200R stimulation. Unlike Dok, the phosphorylation of Shc did not change following CD200 stimulation, and we could not detect the association of Shc with CD200R in intact cells, even though Shc was found to bind phosphorylated peptides in the in vitro-binding assay. These results show that upon ligand binding CD200R specifically recruits Dok1 and Dok2 to the receptor complex which in turn binds RasGAP and SHIP. We are currently examining which kinase mediates phosphorylation of CD200R and Doks. Considering the role of Src family kinases Lyn and Fyn in mast cell activation (36, 37, 38), they may be involved in the phosphorylation of CD200R and Doks.

Among the PTB domain proteins, Dok proteins have been shown to mediate inhibitory signaling (18, 19, 20, 21, 22, 23). The Dok family comprises five known members; Dok-1, Dok-2 (also termed Dok-R and FRIP), Dok-3 (also named Dok-L), Dok-4, and Dok-5 (22, 39, 40, 41, 42). These molecules contain an amino-terminal pleckstrin homology domain, a central PTB domain, and a carboxyl-terminal region with multiple potential tyrosine phosphorylation sites and proline-rich regions, which may serve as docking sites for Src homology 3 domains. Dok proteins undergo tyrosine phosphorylation in response to a variety of stimuli, such as immunoreceptor ligation, growth factors, and cytokines. This phosphorylation triggers Src homology 2 domain-mediated interactions with inhibitory effectors including RasGAP, SHIP, and Csk. In B cells, both Dok-1 and Dok-3 undergo rapid tyrosine phosphorylation in response to B cell receptor engagement and negatively regulate cell activation (18, 22). Dok1 has been shown to inhibit MAPK activation and cell proliferation upon coaggregation of B cell receptor and Fc{gamma}RIIB (18, 21). Dok2 negatively regulates T cell development by recruiting RasGAP and Nck (43). In mast cells, co-cross-linking of Fc{gamma}RIIB with Fc{epsilon}RI stimulates Dok1 tyrosine phosphorylation and association with SHIP and RasGAP (23). Overexpression of Dok1 in the mast cell line RBL-2H3 inhibited Fc{epsilon}RI-mediated Ras/Raf1/ERK signaling and the de novo synthesis of TNF-{alpha} (44). These studies have established that Dok family proteins are inhibitory adaptor molecules, presumably due to their ability to recruit inhibitory effectors RasGAP, SHIP, and Csk. Our results show that CD200R also binds to Dok1 and Dok2, which then recruit RasGAP and SHIP.

Like other inhibitory receptors such as Fc{gamma}RIIB, gp49B1, paired Ig-like receptor {beta}, and mast cell function-associated Ag in mast cells, CD200R engagement inhibits mast cell degranulation and cytokine production. This inhibition is likely mediated by the reduced activation of MAPKs: ERK, p38 MAPK, and JNK, since CD200R engagement inhibited activation of all three MAPKs. Unlike the majority of myeloid inhibitory receptors, the inhibition does not require co-cross-linking of CD200R with an activating receptor because CD200 ligand alone could inhibit the activation of ERK1/2 induced by IL-3. This suggests that CD200R is a novel inhibitory receptor that employs recruitment of RasGAP to directly inhibit Ras activation. However, other molecules may also be involved in the inhibition because CD200 also inhibits p38 MAPK and JNK activation which are not dependent on a Ras pathway. It is likely that SHIP also plays a role in CD200-mediated inhibition because SHIP has been shown to act as a negative regulator of mast cell and B cell activation (45, 46, 47). The role of SHIP in CD200 inhibition is currently under investigation.

Besides mast cells, CD200R is also highly expressed on macrophages (4). CD200-CD200R interaction has been shown to be important for regulation of the macrophage lineage. In CD200-deficient mice, there were increased numbers of macrophages in the spleen and the mesenteric lymph nodes, and these macrophages show increased activation (5). Our data now show that CD200R engagement induced tyrosine phosphorylation of Dok1 and Dok2 in primary mouse peritoneal cells. This confirms the findings in mast cells and underlines the importance of CD200 in modulating myeloid cell activation broadly. Our separate studies show that engagement of CD200R by agonist Ab and soluble CD200-Ig fusion protein inhibited mast cell degranulation in the passive cutaneous anaphylaxis model, suggesting the inhibitory role of CD200R in vivo.3

In conclusion, we have shown that Dok1 and Dok2 are mediators 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 the inhibition because CD200 also inhibits p38 MAPK and JNK activation which do not depend on Ras. Our results suggest a novel inhibitory pathway used by CD200R in modulating myeloid cell function (Fig. 8). That this pathway is triggered and active without need for co-cross-linking to an activating receptor provides a unique opportunity for the use of CD200R as an anti-inflammatory target in vivo.



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  		FIGURE 8. Proposed model for CD200R-mediated inhibition of mast cell activation. Engagement of ITAM-containing activating receptors such as Fc{epsilon}RI and CD200RLa leads to the activation of downstream Ras/MAPK pathways. Triggering CD200R by CD200 induces its phosphorylation and recruitment of Dok1 and Dok2. Dok1 and Dok2 are phosphorylated and bind to RasGAP and SHIP, which leads to inhibition of Ras and ERK activation. Other unknown molecules may mediate the inhibition of p38 MAPK and JNK activation.

 


   Acknowledgments
 
We thank Mike Bigler and Yaoli Song for assistance and Janet Wagner and Sandra Zurawski for making the fusion proteins and mAbs. We also thank Maria Jenmalm for help and discussion.


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

1 Address correspondence and reprint requests to Dr. Joseph H. Phillips, DNAX Research Institute, 901 California Avenue, Palo Alto, CA 94304-1104. E-mail address: joe.phillips{at}dnax.org Back

2 Abbreviations used in this paper: CIA, collagen-induced arthritis; SHP, Src homology region 2 domain-containing phosphatase; PTB, phosphotyrosine; mIg, mutant Ig; BMMC, bone marrow-derived mast cell. Back

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


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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