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The Journal of Immunology, 2007, 179, 7093 -7100
Copyright © 2007 by The American Association of Immunologists, Inc.
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Mast Cells and Basophils Are Selectively Activated In Vitro and In Vivo through CD200R3 in an IgE-Independent Manner1

Toshiyuki Kojima*, Kazushige Obata*, Kaori Mukai*, Shingo Sato*, Toshiyuki Takai{dagger}, Yoshiyuki Minegishi* and Hajime Karasuyama2,*

* Department of Immune Regulation, Tokyo Medical and Dental University Graduate School, Tokyo, Japan; and {dagger} Department of Experimental Immunology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mast cells and basophils have been implicated in the host defense system against pathogens and in the development of allergic disorders. Although IgE-dependent responses via Fc{epsilon}RI on these cells have been extensively studied, little is known about cell surface molecules that are selectively expressed by these cells and engaged in their activation via an IgE-independent mechanism. We have recently established two mAbs that reacted specifically with murine mast cells and basophils, and one of them selectively depleted basophils when administered in vivo. Biochemical and flow cytometric analyses revealed that both mAbs specifically recognized a CD200R-like protein, CD200R3, but not other CD200R family members. CD200R3 existed as a disulfide-linked dimer, unlike other CD200Rs, and was expressed on mast cells and basophils primarily in association with an ITAM-bearing adaptor DAP12. Cross-linking of CD200R3 with the mAbs induced degranulation in mast cells and production of the cytokine IL-4 in basophils in vitro. Administration of the nondepleting mAb in vivo elicited systemic and local anaphylaxis in a CD200R3-dependent manner. These results suggest that CD200R3 functions as an activating receptor on mast cells and basophils to regulate IgE-independent immune responses in cooperation with an inhibitory receptor CD200R, similar to the paired receptors expressed on NK cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mast cells and basophils have long been considered primary effector cells in inflammatory responses, particularly in IgE-mediated allergic reactions, such as systemic anaphylaxis, allergic rhinitis, and asthma (1, 2, 3, 4, 5). Accumulating evidence indicates that mast cells actively participate in the innate immune responses to many pathogens, including bacteria and viruses, via an IgE-dependent or IgE-independent mechanism (6). Recent studies demonstrated that mast cells could also be involved in the sensitization phase of some acquired immune responses, the pathogenesis of autoimmune disorders, and the induction of peripheral tolerance (7, 8, 9, 10, 11, 12), although the exact mechanisms by which mast cells exert such immunoregulatory effects remain largely unknown. The existence of basophils in the blood of many vertebrates has been known for a long time, since first described by Ehrlich in 1879 (49). However, our understanding of the in vivo roles of basophils is still very limited compared with that of mast cells (2, 13, 14). Basophils represent <1% of the peripheral blood leukocytes, making them one of the least common cell lineages found in the peripheral blood. Basophils are often recruited to the site of allergic inflammation (15, 16, 17, 18, 19), but under normal physiological conditions, they are usually confined to the circulation, unlike the tissue-resident mast cells. Moreover, basophils are candidate effector cells for the immune response against parasites and are a major source of the typical Th2 cytokine IL-4 during parasite infections (20, 21, 22, 23). However, until recently, no definitive evidence has been provided that basophils are critically involved in the pathogenesis of allergic disorders or in immunity to pathogens. The lack of proper animal models, including basophil-deficient mice, and the unavailability of mouse basophil-specific Abs have been practical obstacles to clarifying the critical roles of basophils in vivo.

We recently established a basophil/mast-cell-specific mAb, Ba103, by immunizing rats with mouse primary basophils (24). Ba103 selectively stains basophils from the peripheral blood, spleen, and bone marrow and mast cells from the peritoneal cavity and skin, but not from the intestine. The i.v. injection of Ba103 into mice results in a transient depletion of basophils but not mast cells (24). This made it possible to assess the roles of basophils in vivo. Treatment of mice with this mAb before allergen challenge completely abolished the development of IgE-mediated chronic allergic skin inflammation, characterized by massive eosinophil infiltration, even though basophils accounted for only ~2% of the infiltrating cells (24, 25). Treatment with the mAb during the progression of the dermatitis resulted in a drastic reduction in the numbers of infiltrating eosinophils and neutrophils, concomitant with the elimination of basophils from the skin lesions. These results along with our other previous observations (25) indicated that basophils play a nonredundant role in the development of IgE-mediated chronic allergic inflammation as an initiator rather than as an effector and function independent of mast cells and T cells (24). However, it remains to be determined which molecule was recognized by mAb Ba103.

Paired receptor families consisting of activating and inhibitory receptors are widely distributed in various cell types involved in immune responses (26). CD200R is a membrane glycoprotein possessing two Ig-like extracellular domains and functions as an inhibitory receptor for its ligand CD200 (27, 28). Although CD200 is broadly expressed on a variety of cell types, CD200R is primarily expressed on cells of the myeloid lineage, including mast cells and basophils. The engagement of CD200R by agonistic Abs or its ligand results in the potent inhibition of degranulation and cytokine production in mast cells that are stimulated by cross-linking the IgE receptor Fc{epsilon}RI (29). The activation of basophils via Fc{epsilon}RI is also inhibited by the binding of CD200 to CD200R (30). Thus, the recognition of CD200 by CD200R appears to play an important role in regulating immune responses, including allergic reactions (31). Additional CD200R-like proteins, designated CD200R2 (CD200RLc), CD200R3 (CD200RLb), CD200R4 (CD200RLa), and CD200R5, have recently been identified in mice (32, 33, 34). Unlike CD200R, these receptors contain in their transmembrane region the positively charged amino acid lysine, which has the potential to associate with the ITAM- or YxxM motif-bearing adaptor molecules such as DAP12, DAP10, FcR{gamma}, and CD3{zeta}, suggesting that they are capable of transducing stimulatory signals. Therefore, the CD200R-like receptors and CD200R may function as activating and inhibitory receptors, respectively, to balance immune responses, similar to the paired receptors expressed on NK cells (35, 36). However, the signaling capability and biological significance of each CD200R-like receptor remains to be determined.

In the present study, we performed the biochemical characterization, molecular identification, and functional analysis of a cell surface protein recognized by Ba103 and another basophil/mast cell-specific mAb. Our results clearly demonstrated that a CD200R-like receptor, CD200R3, functions as an activating receptor that is expressed exclusively on mast cells and basophils, suggesting that CD200R3 plays a role in IgE-independent immune responses mediated by these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

C57BL/6 and BALB/c mice (5–10 wk old) were purchased from CLEA Japan. DAP-12-deficient (37) and mast cell-deficient (Wsh/Wsh) C57BL/6 mice (38) were maintained in our animal facilities under specific pathogen-free conditions. All of the experiments in this study were performed according to the "Guidelines for Animal Use and Experimentation" as set out by our University.

Cell lines, cell preparation, and culture

Mouse mast cell lines, MC/9 (ATCC CRL 8306) and CFTL-15 (39), were cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 5 and 10% FCS (HyClone), respectively, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 x 10–5 M 2-ME, and rIL-3 (40). The fibroblast cell line NIH3T3 (ATCC CRL-1658) was grown in supplemented DMEM (Sigma-Aldrich), and the retroviral packaging cell line Plat-E (41) was maintained in the same medium with 1 µg/ml puromycin (Sigma-Aldrich) and 10 µg/ml blasticidin (Invitrogen Life Technologies). Mouse bone marrow cells were cultured in the supplemented RPMI 1640 with rIL-3 for 7–14 days to obtain cells that included bone marrow-derived basophils or for 3–6 wk to obtain bone marrow-derived mast cells (BMMC).3

Antibodies

FITC-conjugated mAbs specific for CD49b and c-kit, PE-conjugated anti-IL-4 mAb, and allophycocyanin-conjugated streptavidin were purchased from BD Pharmingen. PE-conjugated hamster anti-mouse Fc{epsilon}RI{alpha} was obtained from eBioscience. Purified and biotin-conjugated anti-FLAG M2 mAb and its agarose conjugates were purchased from Sigma-Aldrich. Anti-phospho-ERK Ab and HRP-conjugated goat anti-rabbit IgG were from Cell Signaling Technology. Anti-phospho-tyrosine mAb, anti-Erk1 mAb, and HRP-conjugated goat anti-rat and anti-mouse IgG were from Santa Cruz Biotechnology. The mAbs Ba91 and Ba103 were established as described previously (24).

Retroviral vectors and infection

cDNAs coding for mouse CD200R, CD200R2, CD200R3G, CD200R4, CD200R5, and DAP12 were generated by RT-PCR from MC/9, CFTL-15 cells, and C57BL/6 bone marrow cells and subcloned into the pBC(KS+) vector (Stratagene). The cDNAs and their FLAG-tagged versions were inserted into the retroviral vector pMX-IRES-GFP (41). The CD200-Fc fusion construct was prepared by combining the cDNA fragments coding for the extracellular region of mouse CD200 and the Fc region of mouse IgG1 and inserted into pMX-IRES-GFP. Plat-E cells were transfected with the retroviral vectors using FuGENE 6 (Roche) and their culture supernatants were collected 48 h later. NIH3T3 and CFTL-15 cells were infected by incubation with the Plat-E culture supernatant for 48 h in the presence of 5 µg/ml polybrene (Sigma-Aldrich).

Semiquantitative RT-PCR analysis

Total RNA was prepared from cells and subjected to first-strand cDNA synthesis with reverse transcription using oligo(dT) primers. PCR was performed with 3- or 5-fold serially diluted cDNA templates using following primers: for CD200R3, forward 5'-gtgcttaacctgactccactcc-3' for all splice variants and reverse 5'-gttcttcactacgtaggagccaa-3' (A/D variants), 5'-tctccgttcacccaagagcttct-3' (B/E variants), 5'-tctcctcaggagccttctggca-3' (C/F variants) and 5'-ctctccacagtcatgacactgtt-3' (G/H variants), and for IL-4 and HPRT as described elsewhere (25).

Flow cytometric analysis and cell sorting

Cells stained with the indicated mAbs were analyzed with a FACSCalibur (BD Biosciences). To assay for CD200-Fc binding, cells were incubated with the culture supernatant of the NIH3T3 transfectant of CD200-Fc and then stained with biotinylated anti-mouse IgG1 mAb, which was revealed by allophycocyanin-streptavidin. For intracellular cytokine staining, cells were stimulated with the indicated Abs in the presence of 2 µM monensin for 6 h, followed by fixation and cytoplasmic staining with PE-conjugated anti-IL4 mAb or isotype-matched control. For RT-PCR analyses, CD49b+Fc{epsilon}RI+ bone marrow basophils and c-kit+Fc{epsilon}RI+ peritoneal mast cells were sorted with a FACSVantage (BD Biosciences).

Cell surface biotinylation, immunoprecipitation, and immunoblotting

Mast cells were incubated with EZ-Link Sulfo-NHS-LC-Biotin (Pierce 21335) in PBS (pH 8.0) for 30 min at room temperature, followed by blocking with RPMI 1640 medium for 5 min. Cells were lysed with 1% Nonidet P-40-containing lysis buffer with proteinase inhibitor mixture. The cell lysates were incubated with the indicated mAbs and immunoprecipitates were subjected to SDS-PAGE. The blotted biotinylated proteins were visualized by streptavidin-HRP conjugate (Amersham Pharmacia Biotech). Cell lysates were prepared from NIH3T3 and CFTL-15 transfectants using lysis buffer containing 1% n-dodecyl β-D-maltoside (Sigma-Aldrich), and subjected to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated Abs and HRP-conjugated secondary Abs. Proteins were visualized by an ECL system (Immobilon Western; Millipore).

Mast cell degranulation assay

MC/9 cells were incubated with the indicated Abs or A23187 (Sigma-Aldrich) in Tyrode’s buffer (42) at 37°C for 20 min. After harvesting culture supernatants, cell pellets were lysed with Tyrode’s buffer containing 0.1% TritonX-100. β-Hexosaminidase activity in cell lysates and supernatants was measured as described previously (42). The extent of degranulation was calculated by dividing the activity in the supernatant by the sum of the activity in the supernatant and cell lysate.

Induction of systemic and local anaphylaxis

To induce systemic anaphylaxis, mice were treated with an i.v. injection of 100 µg of Ba91, Ba103, or control rat IgG in 0.2 ml of PBS, and their body temperature was monitored with a rectal probe (Shibaura Electronics). To induce local anaphylaxis, mice were first treated with an intradermal injection of 20 µg of Ba91 or control rat IgG in 0.02 ml of PBS in the ear and 3 min later with an i.v. injection of 0.5% Evans blue dye (Sigma-Aldrich) in 0.2 ml of PBS. The mice were sacrificed 10 min later, and the dye was extracted from each dissected ear in 0.7 ml of formamide at 63°C overnight. The absorbance of the dye in the extracts was measured with a spectrophotometer at 620 nm.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Characterization of molecules recognized by mAbs Ba91 and Ba103, which specifically react with basophils and mast cells

We previously established mAb Ba103 (rat IgG2b), which specifically stains mouse basophils and mast cells, by immunizing rats with basophil-enriched mouse bone marrow cells (24). Ba91 (rat IgG2a), another mAb established using the same protocol, showed a cellular reactivity similar to that of Ba103 (representative data are shown in Fig. 1A). Both Ba91 and Ba103 stained bone marrow basophils, peritoneal mast cells, bone marrow-derived basophils, and BMMC. However, these two mAbs did not compete with each other in the staining of basophils or mast cells (data not shown), suggesting they recognized different molecules or different epitopes on the same molecule, which was expressed exclusively by these cells. The mAbs stained one mast cell line, MC/9, but not another, CFTL-15 (Fig. 1A), even though both cell lines expressed the high-affinity IgE receptor Fc{epsilon}RI on their cell surface (Fig. 1B). This indicated that the molecule(s) recognized by the mAbs was distinct from Fc{epsilon}RI.


Figure 1
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  		FIGURE 1. Flow cytometric and biochemical characterization of molecules recognized by the mAbs Ba91 and Ba103. A, Basophils were identified as Fc{epsilon}RI+CD49b+ cells in bone marrow cells freshly prepared from BALB/c mice or those cultured in vitro with IL-3 for 10 days. Mast cells were identified as Fc{epsilon}RI+c-kit+ cells in freshly prepared peritoneal cells or bone marrow cells cultured in vitro for 4 wk (BMMC). These basophils and mast cells as well as mast cell lines MC/9 and CFTL-15 were stained with Ba91 or Ba103. Histograms with black and gray lines show the staining with Ba91/103 and an isotype-matched control, respectively. B, MC/9 and CFTL-15 cells were surface biotinylated and their lysates were subjected to immunoprecipitation with Ba91, Ba103, anti-Fc{epsilon}RI{alpha}, or control Ab, followed by SDS-PAGE under reducing (R) or nonreducing (NR) conditions. Biotinylated proteins were detected by immunoblotting with streptavidin-conjugated HRP. C, Total cell lysates were prepared from CFTL-15, MC/9, and BMMC and subjected to SDS-PAGE under reducing or nonreducing conditions, followed by immunoblotting with Ba103.

 
To identify the molecule(s) recognized by the mAbs, MC/9 cells were surface biotinylated and their cell lysates were reacted with each mAb (Fig. 1B, left panel). Both Ba91 and Ba103 but not control rat IgG immunoprecipitated a major band with an apparent molecular mass of 38 kDa under reducing conditions and a major band of 74 kDa under nonreducing conditions. This indicated that the molecule(s) recognized by the mAbs mainly existed as a disulfide-linked dimer on the cell surface. Neither band, under reducing or nonreducing conditions, was immunoprecipitated by these mAbs from CFTL-15 cells that were analyzed the same way (Fig. 1B, right panel), consistent with the staining profiles shown in Fig. 1A. In addition, these bands were detected when the whole-cell lysates of unlabeled MC/9 cells and BMMC but not those of CFTL-15 cells were subjected to immunoblotting with Ba103 (Fig. 1C). Ba91 did not work for immunoblotting under our experimental conditions.

CD200R3 is a target molecule of Ba91 and Ba103

To clarify the nature of the molecule recognized by Ba103, the 38-kDa protein that was immunoprecipitated with Ba103 from MC/9 cell lysates was subjected to time-of-flight mass spectrometric analysis. The amino acid sequences of five polypeptides were obtained: AQLFCHPSPSK, AQLFCHPSPSKEATLR, VPAHH QSSDLPIK, IETTDGIFQER, and NYFR. All of the sequences corresponded to parts of the mouse CD200R3 protein, indicating that Ba103 recognizes CD200R3. CD200R3 is a CD200R-like transmembrane protein, for which eight different splice variants (hereafter referred to as CD200R3A-H, see Discussion for details) have been reported (Fig. 2, A and B) (32, 33, 34). RT-PCR analysis indicated that bone marrow basophils and peritoneal mast cells expressed CD200R3G at higher levels than others (Fig. 2C).


Figure 2
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  		FIGURE 2. Primary basophils and mast cells express CD200R3, particularly G-type splice variants. A, A schematic view of the genomic organization of the mouse CD200R3 gene. B, A schematic view of eight splice variants of CD200R3. C, Semiquantitative RT-PCR analysis for CD200R3 splice variants was performed by using RNA prepared from sorted bone marrow basophils and peritoneal mast cells. Data shown are representative of three repeated experiments.

 
To confirm that Ba103 recognizes CD200R3, NIH3T3 fibroblasts were infected with a retroviral vector carrying the cDNA for FLAG-tagged CD200R3G, as a representative CD200R3 variant, alone or coexpressed with an adaptor protein, DAP12. Consistent with a previous report using 293T transfectants (33), the cell surface expression of FLAG-tagged CD200R3 in NIH3T3 cells was detected with an anti-FLAG mAb only when DAP12 was coexpressed (Fig. 3A, first and second rows). Both Ba91 and Ba103 stained the CD200R3/DAP12 transfectants while neither showed any significant reactivity with the mock (vector alone) transfectants, regardless of DAP12 coexpression (Fig. 3A, fourth and fifth rows), demonstrating that the mAbs recognized native forms of CD200R3. In transfected NIH3T3 cells, CD200R, CD200R2, and CD200R5 were all detected on the cell surface regardless of DAP12 coexpression (Fig. 3A, first and second rows). In contrast, the surface expression of CD200R4, like that of CD200R3, was detected only when DAP12 was coexpressed (Fig. 3A, second row), in agreement with previous reports (33, 34). The chimeric molecule CD200-Fc, composed of mouse CD200 (a ligand of CD200R) and mouse IgG1-Fc, bound CD200R as expected, but did not bind any other members of the CD200R family expressed on the transfectants (Fig. 3A, third row). Neither Ba91 nor Ba103 stained transfectants expressing CD200R, CD200R2, CD200R4, or CD200R5 (Fig. 3A, fourth and fifth rows). Thus, both mAbs specifically recognized CD200R3 and no other CD200R family members.


Figure 3
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  		FIGURE 3. Ba91 and Ba103 recognize CD200R3 but not the other members of the CD200R family. A, NIH3T3 cells were infected with a panel of retroviral vectors carrying the GFP gene plus a cDNA encoding individual FLAG-tagged CD200R family members or vector alone (mock), without or in combination with the DAP12 gene. Two days after the infection, the cells were stained with anti-FLAG mAb, CD200-Fc, Ba91, or Ba103. Histograms are shown for the GFP+-infected cells and histograms with gray lines show the control staining with isotype-matched mAbs. B, Cell lysates were prepared from NIH3T3 cells infected with cDNA encoding individual FLAG-tagged CD200R family members or vector alone (mock) in combination with the DAP12 gene and reacted with anti-FLAG-conjugated agarose. The immunoprecipitated proteins were subjected to SDS-PAGE under reducing (R) or nonreducing (NR) conditions, followed by immunoblotting (IB) with Ba103 (upper panel) or anti-FLAG mAb (lower panel). The band at 42 kDa detected in all of the lanes of immunoblots with the anti-FLAG mAb (lower panels) is a nonspecific background signal.

 
CD200R3 is unique among the CD200R family in its glycosylation and formation of a disulfide-linked dimer

Immunoblotting with Ba103 detected a 38- to 40-kDa protein under reducing conditions in NIH3T3 cells infected with CD200R3G/DAP12, as observed in MC/9 cells and BMMC, but not in those infected with empty vector (Fig. 3B, upper left panel). In accordance with the flow cytometric analysis shown in Fig. 3A, Ba103 showed no detectable cross-reactivity with the other members of the CD200R family in immunoblots (Fig. 3B, upper left panel). Even though the expected molecular mass of the core protein of each CD200R family member is within the range between 25 and 33 kDa, the apparent molecular mass of each member varied greatly, ranging from 38 to 90 kDa under reducing conditions (Fig. 3B, lower panel). The apparent size of CD200R3 was much smaller than that of the other members, suggesting that CD200R3 is the least glycosylated.

As observed in MC/9 cells and BMMC, the apparent molecular mass of CD200R3G detected by Ba103 or anti-FLAG mAb in the NIH3T3 transfectants shifted from 38 to 40 kDa to ~80 kDa under nonreducing conditions (Fig. 3B, right panels), demonstrating that CD200R3 exists as disulfide-linked dimer. In contrast, no disulfide-linked dimer formation was detected for CD200R, CD200R2, CD200R4, or CD200R5 expressed in NIH3T3 cells (Fig. 3B, lower panels).

CD200R3 expression on primary mast cells and basophils is primarily dependent on its association with DAP12

When CD200R3G was coexpressed with DAP12 in NIH3T3 cells, DAP12 coprecipitated with CD200R3G and vice versa (Fig. 4A), indicating that CD200R3 and DAP12 physically associated with each other. Primary basophils express DAP10 and the FcR{gamma} chain in addition to DAP12, and a previous study using 293T transfectants showed that CD200R3 could also pair with DAP10, albeit less efficiently than with DAP12 (33). Therefore, we examined the DAP12 dependency of CD200R3 expression on the surface of primary basophils and mast cells by comparing its expression in DAP12–/– and wild-type mice (Fig. 4B). Fc{epsilon}RI+CD49b+ basophils were detected in the bone marrow of DAP12–/– mice at similar levels as in wild-type mice. However, little or no CD200R3 was detected on basophils from DAP12–/– mice, unlike those from wild-type mice, when analyzed with Ba91 or Ba103 (Fig. 4B, left panels). This was also true for CD200R3 expression on Fc{epsilon}RI+c-kit+ mast cells in the peritoneal cavity (Fig. 4B, right panels). Thus, the surface expression of CD200R3 was primarily dependent on its association with DAP12. These results also implied that CD200R3/DAP12 was dispensable for the development and survival of basophils and mast cells in vivo.


Figure 4
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  		FIGURE 4. CD200R3 is expressed on the cell surface of basophils and mast cells primarily in association with DAP12. A, NIH3T3 cells were infected with empty vector (mock), CD200R3G vector, FLAG-tagged DAP12 (F-DAP12) vector, or the combination of CD200R3G and F-DAP12. The cell lysates were reacted with Ba91, Ba103, or anti-FLAG mAb, and the immunoprecipitates were subjected to SDS-PAGE under reducing conditions, followed by immunoblotting with Ba103 or anti-FLAG mAb. B, Basophils and mast cells were identified, respectively, as Fc{epsilon}RI+CD49b+ cells in the bone marrow of wild-type and DAP12–/– mice and as Fc{epsilon}RI+c-kit+ cells in the peritoneal cavity, and their staining profiles with Ba91 and Ba103 are shown as histograms. Black lines indicate staining with Ba91 or Ba103 and gray lines indicate staining with isotype-matched controls.

 
CD200R3 functions as an activating receptor in mast cells and basophils in vitro and in vivo

The association of CD200R3 with the ITAM-bearing DAP12 strongly suggested that CD200R3 could transduce activating signals. We first examined the possible activation of the CD200R3/DAP12-infected NIH3T3 and CFTL-15 cells by cross-linking CD200R3 with Ba91 or Ba103. In both types of cells, the cross-linking of CD200R3 with Ba91 induced the tyrosine phosphorylation of DAP12 (Fig. 5, A and B). Treatment with Ba103 also induced DAP12 phosphorylation, albeit to a lesser extent. The phosphorylation of ERK was induced when the CFTL transfectants were treated with Ba91, just as observed when the cells were stimulated with an anti-Fc{epsilon}RI{alpha} mAb. Ba103 elicited the same effect, but to a lesser extent than Ba103 (Fig. 5B, left panels). The ERK phosphorylation was dependent on the surface expression of CD200R3 and not due to the nonspecific stimulation of cells with the mAbs, because it was not detected when untransfected CFTL-15 cells were treated the same way (Fig. 5B, right panels). MC/9 cells expressing endogenous CD200R3 also showed a transient ERK phosphorylation that peaked 2 or 3 min after they were reacted with Ba91 or Ba103 (Fig. 5C). These results indicated that CD200R3 could transduce activating signals.


Figure 5
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  		FIGURE 5. Cross-linking of surface CD200R3 induces the tyrosine phosphorylation of signaling molecules. A, After a 16-h culture without FCS, NIH3T3 cells infected with both CD200R3G and FLAG-tagged DAP12 were harvested, and single-cell suspensions were reacted with biotinylated Ba91, Ba103, or isotype-matched control IgG on ice for 30 min, followed by incubation with streptavidin at 37°C for 2 min. Cell lysates were prepared and subjected to immunoprecipitation with anti-FLAG-conjugated agarose, followed by immunoblotting with anti-phosphotyrosine (upper panel) or anti-FLAG mAb (lower panel). B, After a 5-h culture without IL-3 or FCS, uninfected CFTL-15 cells or CFTL-15 cells infected with both CD200R3G and FLAG-tagged DAP12 were reacted with biotinylated anti-Fc{epsilon}RI mAb, Ba91, Ba103, or isotype-matched control (hamster IgG, rat IgG2a, or rat IgG2b) on ice for 30 min, followed by incubation with streptavidin at 37°C for 2 min. Cell lysates were prepared and subjected to immunoblotting with anti-phospho-ERK (first row), anti-ERK (second row), or first immunoprecipitated with anti-FLAG-conjugated agarose followed by immunoblotting with anti-phosphotyrosine (third row) or anti-FLAG mAb (fourth row). C, After a 5-h culture without IL-3 or FCS, MC/9 cells were reacted with biotinylated Ba91, Ba103, or control IgG on ice for 30 min, followed by incubation with streptavidin at 37°C for the indicated time. Cell lysates were prepared and subjected to immunoblotting with anti-phospho-ERK or anti-ERK.

 
We next examined functional consequences of cross-linking CD200R3 on mast cells and basophils in vitro, ex vivo, and in vivo. Stimulation of MC/9 cells with Ba91 or Ba103 induced their degranulation (Fig. 6A). The extent of degranulation induced by the mAbs was comparable to or even higher than that induced by IgE plus Ags (data not shown). RT-PCR analysis revealed that the level of IL-4 mRNA was greatly up-regulated in bone marrow basophils stimulated ex vivo with Ba91 or Ba103, as observed when they were stimulated with an anti-Fc{epsilon}RI{alpha} mAb (Fig. 6B). The production of IL-4 protein was indeed detected in a fraction of CD49b+ bone marrow-derived basophils stimulated with Ba91 or Ba103 (Fig. 6C). All of these results suggested a stimulatory role of CD200R3 in mast cells and basophils and prompted us to perform in vivo experiments using the mAbs.


Figure 6
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  		FIGURE 6. Ba91 and Ba103 activate mast cells and basophils to degranulate and produce cytokines. A, MC/9 cells were incubated with 2.5 µg/ml Ba91, Ba103, or control rat IgG, 1 µM calcium ionophore (A23187) or its control (0.1% ethanol vehicle) at 37°C for 20 min, and the release of β-hexosaminidase was measured. Percent release of β-hexosaminidase is shown as the mean ± SEM (n = 3). B, CD49b+ cell-enriched bone marrow cells including basophils (~10%) were reacted ex vivo with 10 µg/ml Ba91, Ba103, anti-Fc{epsilon}RI{alpha} mAb, or control rat or hamster IgG at 37°C for 1 h. Their RNAs were prepared and subjected to semiquantitative RT-PCR analysis for gene expression of IL-4 and HPRT. C, Cultured bone marrow cells containing basophils (bone marrow-derived basophils) were incubated with 10 µg/ml Ba91, Ba103, anti-Fc{epsilon}RI{alpha} mAb, or control rat IgG in the presence of monensin at 37°C for 6 h and then subjected to staining for cell surface CD49b and cytoplasmic IL-4.

 
The i.v. injection of 100 µg of Ba91 into C57BL/6 mice induced systemic anaphylaxis with a rapid drop in rectal temperature by nearly 4°C within 30 min (Fig. 7A). The same dose of Ba103 also elicited systemic anaphylaxis, albeit with a much less drastic drop of rectal temperature and quicker return to the basal level. Systemic anaphylaxis was not observed when DAP12-deficient mice were treated with Ba91 (Fig. 7A), indicating that the mAb induced systemic anaphylaxis in a CD200R3-dependent manner. The Ba91-induced systemic anaphylaxis was not elicited in mast cell-deficient Wsh/Wsh mice (Fig. 7A), suggesting that mast cells played a major role in the systemic anaphylaxis. Ba91 also induced local anaphylaxis when injected intradermally into the ears, as judged by increased vascular permeability and dye extravasation (Fig. 7B). Thus, the treatment of mice with Ba91 induced systemic and local anaphylactic reactions in an IgE/Fc{epsilon}RI-independent manner.


Figure 7
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  		FIGURE 7. Treatment of mice with Ba91 induces systemic and local anaphylaxis. A, Wild-type, DAP12-deficient, or Wsh/Wsh mutant C57BL/6 mice were treated with an i.v. injection of 100 µg of Ba91 or Ba103, and their rectal temperature was measured at the indicated times after the injection. Data are expressed as the mean ± SEM of three mice in each group. The injection of the same amount of control rat IgG did not induce any significant change of the temperature in any of the mouse types (data not shown). B, BALB/c mice were given an intradermal injection of 20 µg of Ba91 or control rat IgG in 0.02 ml of PBS in the ear and then 3 min later with an i.v. injection of Evans blue. Ten minutes later, photographs of the ears were taken, and the extravasated dye was extracted from the ear skin and measured by spectrophotometry. Data are expressed as the mean ± SEM of four mice in each group. *, p = 0.013.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The high-affinity IgE receptor Fc{epsilon}RI, composed of {alpha}-, β-, and {gamma}2-chains, is expressed exclusively on mast cells and basophils and plays an essential role in the activation of these cells in IgE-mediated immune responses (3, 4, 43). In contrast, little has been known about the cell surface molecules engaged in the IgE-independent activation of mast cells and basophils. The CD200R3 gene was first identified as a CD200R-related gene and reportedly expressed at the mRNA level primarily in mast cells and Th2 cells and, to a lesser extent, in bone marrow-derived dendritic cells (34). It was then reported that polyclonal and mAbs raised against CD200R3 peptides stained as many as ~35% of mouse bone marrow cells (32). Independently, a comparative microarray analysis of basophils and eosinophils identified CD200R3 as being preferentially expressed in basophils (33). Thus, the pattern of CD200R3 expression on primary cells remained to be defined at the protein level using reliable reagents. The present study, along with our previous report (24), clearly demonstrated that the CD200R3 protein is expressed exclusively on the surface of primary mast cells and basophils. No significant expression of CD200R3 was detected on other cell lineages, including T cells and dendritic cells by flow cytometric analysis with the two mAbs that recognized different epitopes on CD200R3. Only ~0.5% of the nucleated cells in mouse bone marrow, spleen, and peripheral blood expressed CD200R3 on their surface, and virtually all of them were basophils. Interestingly, mast cells display heterogeneity in CD200R3 expression, depending on their anatomical localization (24). Those from the peritoneal cavity express relatively high levels of CD200R3, while those from the intestine hardly express it and those from the ear skin show intermediate expression.

The present study revealed that CD200R3 exists as a disulfide-linked dimer, most likely a homodimer, in sharp contrast with the other members of the CD200R family. Furthermore, the extent of glycosylation on CD200R3 is much lower than on the other CD200R family members, reflecting the relative paucity of potential N-glycosylation sites on CD200R3, which has only two, compared with the 6–11 sites of the other CD200Rs. A comparison of amino acid sequences demonstrated that CD200R3 is more distantly related to the other members of the CD200R family than they are to one another (33). Interestingly, the N terminus of the putative C-set Ig domain of CD200R3 lacks nine amino acids (LVPPEVTYF) that are shared by all of the other members. Instead, CD200R3 has 15–16 extra amino acids upstream of the putative V-set Ig domain, unlike the others. Taken together, these data indicate that CD200R3 is unique among the CD200R family, not only in its basophil/mast-cell-restricted expression pattern, but also in its protein structure.

The cloning of cDNAs encoding CD200R3 performed independently by three groups revealed heterogeneity in the sequences (32, 33, 34). Voehringer et al. (33) reported six different splice variants of CD200R3 (CD200R3A–F), which included one reported by Wright et al. (34). Gorczynski et al. (32) reported another CD200R3 clone (here designated CD200R3G) that is similar to the RIKEN clone 4733401I18Rik (here designated CD200R3H) but has additional sequences. We compared the nucleotide sequences of these eight different clones with the available genomic sequences and found a previously unrecognized exon (the ninth exon) encoding the cytoplasmic tail of CD200R3G and CD200R3H. Thus, the CD200R3 gene is composed of nine exons instead of eight exons (33), and at least eight different splice variants are generated through different combinations of the exons (Fig. 2, A and B). Four among them (A, B, C, and G) share the extracellular and transmembrane portions and are all expressed on the cell surface, even though they differ in their cytoplasmic tail. The other four variants (D, E, F, and H) do not possess the second Ig-like domain due to the skipping of the fourth exon and hence are not expressed on the cell surface (33). In the present study, we mainly examined CD200R3G as a representative splice variant, because it was the predominant form expressed in primary basophils and mast cells at the mRNA level. Because the mAbs Ba91 and Ba103 stained not only CD200R3G transfectants but also CD200R3A transfectants (data not shown), it is likely that the mAbs also react with CD200R3B and CD200R3C. It remains to be determined whether there is any functional difference among CD200R3A, B, C, and G.

Analysis with DAP12-deficient mice revealed that the expression of CD200R3 on the surface of primary mast cells and basophils was primarily dependent on the coexpression of DAP12, even though a previous study suggested DAP10 had the potential to associate with CD200R3 (33). Biochemical analyses using NIH3T3 transfectants expressing both CD200R3G and DAP12 clearly demonstrated that the two molecules were associated with each other, in agreement with a previous observation that a CD200R3C-GFP fusion protein assembles with DAP12 (33). The cytoplasmic domain of DAP12 contains a consensus ITAM sequence, suggesting that the CD200R3/DAP12 complex can transduce activating signals (44). Voehringer et al. (33) reported that the CD200R3-DAP12 complex could transduce signals, by demonstrating that the surface cross-linking of FLAG-tagged CD200R3C with the anti-FLAG Ab induced GFP expression in a CD200R3C-transfected proB cell line that was engineered to express GFP under the control of NFAT promoter elements. However, it remained uncertain whether CD200R3 could transduce signals in primary cells. We demonstrated in the present study that the surface cross-linking of CD200R3 with specific mAbs induced degranulation and cytokine production in vitro in mast cells and basophils, respectively, and elicited systemic and local anaphylaxis in vivo, indicating that CD200R3 indeed functions as an activating receptor.

We previously demonstrated that the treatment of mice with an i.v. injection of 30 µg of Ba103 resulted in a transient depletion of basophils (24). In contrast, the treatment with Ba91 even at higher doses, up to 300 µg, did not show any significant effect on the numbers of basophils or mast cells (data not shown). The basophil depletion by Ba103 was complement independent but FcR{gamma} chain dependent, suggesting the involvement of the Ab-dependent cellular cytotoxicity or FcR-mediated phagocytosis (24). The difference of Ba91 and Ba103 in their ability to deplete basophils in vivo may be attributed to the different IgG subclasses (IgG2a vs IgG2b) or possible difference in their affinity to CD200R3. Treatment of mice with Ba103 elicited systemic anaphylaxis, albeit to a much lesser extent than did the Ba91 treatment as shown in Fig. 7A. Nevertheless, the Ba103 pretreatment did not show any apparent effect on IgE-mediated anaphylaxis when analyzed 1 day later, even though basophils were efficiently depleted (24).

The identity of the CD200R3 ligand has been controversial. One research group demonstrated that none of the activating-type CD200R family members, including CD200R3, could bind CD200, a ligand of the inhibitory-type member CD200R (34, 45), whereas another group reported that CD200 was a ligand for all of the CD200R members (32). To address this issue, we expressed each CD200R family member in the NIH3T3 cells and examined its ability to bind CD200-Fc, a soluble form of CD200. CD200-Fc reacted with CD200R, but not with the other members. Therefore, CD200 does not seem to be a ligand for CD200R3. Our preliminary experiments using the soluble CD200R3 have so far failed to identify its possible ligand on a panel of mouse cell lines and freshly isolated primary mouse cells (data not shown). Given that mast cells and basophils are important players in the first defense against pathogens, including parasites, it is intriguing to speculate that CD200R3 functions as an activating receptor that recognizes certain pathogen-associated molecules, independent of or synergistically with IgE/Fc{epsilon}RI. In this regard, it is notable that the m157 protein of mouse CMV (MCMV), which displays a structural similarity to MHC class I, is recognized by Ly49I, an inhibitory NK receptor, in certain MCMV-susceptible mouse strains, whereas the same protein is recognized by Ly49H, an activating NK receptor, in an MCMV-resistant mouse strain (46, 47). This finding led to the hypothesis that MCMV acquired m157 as a ligand for the inhibitory receptor Ly49I to escape attack by NK cells and subsequently the immune system in mice evolved the activating Ly49H to protect against viral infection (48). Importantly, viral CD200 homologs such as human herpesvirus-8 CD200 have been shown to counteract Fc{epsilon}RI-mediated basophil activation (30). Therefore, one might propose that CD200R3 evolved to function as an activating type of receptor expressed exclusively on mast cells and basophils to protect the host against virulent pathogens that had acquired CD200-related molecules. Recent work has demonstrated that mast cells actively participate in the innate immune responses to many pathogens including bacteria and viruses (6), even though little is known about the roles of basophils in these processes. Our results strongly suggest that CD200R3 participates in IgE-independent immune responses mediated by mast cells and basophils, whereas Fc{epsilon}RI is involved in IgE-dependent ones. Activation of these cells through CD200R3 could also contribute to the pathogenesis of allergic or autoimmune disorders.


   Acknowledgments
 
We thank H. Hayashi and A. Yoshida for assistance in flow cytometric analysis; S. J. Galli, S. Nakae, and H. Suto for providing Wsh/Wsh-C57BL/6 mice; T. Kitamura for providing pMX-IRES-GFP and Plat-E; and S. Ikemizu for valuable discussion with regard to the possible structure of CD200R3.


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


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

1 This work was supported by Grants-in-Aid 17047013, 173709, 18659109, and 19390110 from the Japanese Ministry of Education, Culture, Sports, Science and Technology. Back

2 Address correspondence and reprint requests to Dr. Hajime Karasuyama, Department of Immune Regulation, Tokyo Medical and Dental University Graduate School, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail address: karasuyama.mbch{at}tmd.ac.jp Back

3 Abbreviations used in this paper: BMMC, bone marrow-derived mast cells; MCMV, mouse CMV. Back

Received for publication July 23, 2007. Accepted for publication September 10, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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