Requirement of the tyrosines at residues 258 and 270 of MAIR-I in inhibitory effect on degranulation from basophilic leukemia RBL-2H3

Yasushi Okoshi1,2, Satoko Tahara-Hanaoka1, Chigusa Nakahashi1, Shin-ichiro Honda1, Akitomo Miyamoto1, Hiroshi Kojima2, Toshiro Nagasawa2, Kazuko Shibuya1 and Akira Shibuya1

1 Department of Immunology, Institute of Basic Medical Sciences and 2 Department of Clinical and Experimental Hematology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Japan

Correspondence to: A. Shibuya; E-mail: ashibuya{at}md.tsukuba.ac.jp


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mast cells and basophils express the high affinity receptor for IgE (Fc{varepsilon}RI) and play a central role for IgE-associated immediate hypersensitivity reactions and allergic disorders. Cross-linking of Fc{varepsilon}RI-bound IgE with multivalent antigen initiates the activation of mast cells and basophils, resulting in the degranulation from these cells. We have recently identified a novel inhibitory receptor, myeloid-associated immunoglobulin-like receptor (MAIR)-I, which is expressed on mast cells as well as other myeloid cell lineages. Co-ligation of Fc{varepsilon}RI and MAIR-I inhibits IgE-mediated degranulation from mast cells. However, MAIR-I-mediated signaling pathways involved in the inhibition remain undetermined. Here, we demonstrate that the transfectant of rat basophil leukemia RBL-2H3 expressing wild-type MAIR-I is tyrosine phosphorylated and recruits SHP-1 and SHIP upon cross-linking of MAIR-I. By using RBL-2H3 transfectants expressing variable mutant MAIR-I at Y233, Y258, Y270 and/or Y299, we further demonstrate that both Y258 and Y270, but not Y233 and Y299, were phosphorylated and were essentially required for inhibition of IgE-mediated degranulation from the RBL-2H3 transfectant.

Keywords: Fc{varepsilon}RI, inhibitory signal, ITIM, mast cell


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mast cells and basophils express the high affinity receptor for IgE (Fc{varepsilon}RI) on the cell surface and play a central role in IgE-associated immediate hypersensitivity reactions and allergic disorders (1,2). Crosslinking of Fc{varepsilon}RI-bound IgE with multivalent antigen initiates the activation of mast cells and basophils by promoting the aggregation of Fc{varepsilon}RI, resulting in degranulation, with the secretion of chemical mediators that are stored in the cytoplasmic granules of the cells, the de novo synthesis of pro-inflamatory lipid mediators and the synthesis and secretion of cytokines and chemokines [reviewed in (3)]. Fc{varepsilon}RI is a heterotetrameric receptor composed of an IgE-binding {alpha}-subunit, a four-transmembrane-spanning ß-subunit and two disulfide-bonded {gamma}-subunits ({alpha}ß{gamma}2). Aggregation of Fc{varepsilon}RI by crosslinking Fc{varepsilon}RI-bound IgE induces activation of the Src family protein tyrosine kinase (PTK) Lyn that binds to the ß subunit and Syk and then phosphorylation of the tyrosine residues in the immunoreceptor tyrosine-based activation motif (ITAM) of the ß- and {gamma}-subunits of Fc{varepsilon}RI (4). This early step of activation signaling leads to phosphorylation of adaptor proteins, including LAT, SLP76 and VAV and to the subsequent downstream signal pathways (3).

Mast cells and basophils also express the Fc{gamma}RIIB, which contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic region and blocks the early step of activation signals mediated by Fc{varepsilon}RI. When co-ligated with Fc{varepsilon}RI, the tyrosine residue of the Fc{gamma}RIIB is phosphorylated and recruits SH2 domain-containing inositol polyphosphate 5' phosphatase (SHIP) (5), resulting in inhibition of the Fc{varepsilon}RI-induced membrane recruitment and activation of BTK and PLC{gamma}. Other than the Fc{gamma}RIIB, mast cells and basophils express ITIM-containing inhibitory receptors, including gp49B (6,7), paired immunoglobulin-like receptor B (PIR-B) (810), mast-cell function-associated antigen (MAFA) (11,12) and signal regulatory protein {alpha} (SIRP{alpha}) (13) and recruit phosphatases such as SH2 domain-containing protein tyrosine phosphatase 1 (SHP-1), SHP-2 and/or SHIP.

We have recently identified a novel ITIM-containing inhibitory receptor, designated myeloid-associated immunoglobulin-like receptor (MAIR)-I, which is expressed on bone marrow-derived cultured mast cells and other primary myeloid cell lineages including macrophages, granulocytes and dendritic cells (14). MAIR-I gene is mapped to the proximal region of the E2 band of mouse chromosome 11 (14). Kitamura and colleagues also reported a gene cloned from a mast cell cDNA library encoding the same molecule as MAIR-I, which they called leukocyte mono-Ig-like receptor (LMIR)1 (15). MAIR-I comprises a family that includes another member MAIR-II [also called LMIR2 (15)], whose extracellular domain is highly conserved with that of MAIR-I (14,15). In contrast to MAIR-II whose cytoplasmic region is short without any signaling motif, MAIR-I has a long cytoplasmic region containing five tyrosine residues and is tyrosine phosphorylated upon stimulation with pervanadate (14,15). Moreover, co-ligation of Fc{varepsilon}RI and MAIR-I/LMIR1 inhibits IgE-mediated degranulation from bone marrow-derived cultured mast cells (14). However, MAIR-I-mediated signaling pathways involved in tyrosine phosphorylation of MAIR-I and inhibition of degranulation from mast cells have remained undetermined.

In the present report, we established the transfectants of rat basophil leukemia cell line (RBL-2H3) expressing variable mutant MAIR-I at tyrosine residues and examined MAIR-I-mediated signaling involved in inhibition of IgE-mediated degranulation.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cells, antibodies and reagents
The rat basophilic leukemia cell line, RBL-2H3, was obtained from Health Science Research Resources Bank (Osaka, Japan). Mouse anti-Flag mAb (M2 clone) and rabbit anti-Flag polyclonal antibody were purchased from Sigma-Aldrich (St Louis, MO). Anti-phosphotyrosine mAb (4G10) was purchased from Upstate Biotechnology (Lake Placid, NY). Mouse IgE anti-trinitrophenol (TNP) mAb was purchased from BD Bioscience (San Diego, CA). Anti-SHP-1, anti-SHP-2 and anti-SHIP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-MAIR mAb (TX10) that recognizes a common epitope of MAIR-I and MAIR-II was generated in our laboratory, as described (14). F(ab')2 fragments of anti-Flag mAb were prepared by digesting with immobilized pepsin (Pierce Chemical Co., Rockford, IL), according to the manufacturer's instruction. Purity of F(ab')2 fragments was determined by SDS–PAGE. For conjugation with TNP, OVA (Sigma-Aldrich) or antibodies were incubated with picrylsulfonic acid (TNBS) (pH 8.5) overnight at 4°C and then dialysed with PBS. Average numbers of TNP per molecule were 6.3 for TNP-OVA and 12.1 for TNP-F(ab')2 anti-Flag mAb, which were calculated as:

where x = absorbance of the protein after TNP conjugation at 348 nm (A348) / (0.1 x percent solution extinction coefficients of TNP x molecular weight (MW) of TNP) and y = (A280 – 0.2 x A348) / (0.1 x percent solution extinction coefficients of OVA or antibody x MW of OVA or antibody).

Plasmid constructs and transfectants
To generate site-specific MAIR-I mutants at residues Y233, Y258, Y270 or Y299, QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) was used, according to the manufacturer's instruction. In brief, sense PCR primers, which contained a codon for F (TTT or TTC) instead of Y233 (TAT), Y258 (TAT), Y270 (TAT) or Y299 (TAC) and their antisense oligonucleotide primers were designed. PCRs were performed with each pair of primers at 12 cycles of 30 s denaturation (95°C), 60 s annealing (55°C) and 14 min extension (68°C), using MAIR-I cDNA tagged with FLAG at the N-terminus in pMX retrovirus vector (14) as a template. To generate mutated MAIR-I at the multiple tyrosine residues, PCR was further performed using the mutant MAIR-I cDNA in pMX, generated as described above, as templates. MAIR-I cDNA lacking the cytoplasmic region just downstream from the S226 ({Delta}cyto) was generated by PCR and subcloned into pMX with cloning sites of BamH1 (5') and Sal1 (3'). All the mutant cDNAs were verified by sequencing. RBL-2H3 cells were transfected with the wild-type or mutant MAIR-I by retrovirus vector, as described previously (16) and Flag-expressing cells were purified by FACS sorting using anti-Flag mAb.

Biochemistry
To analyze tyrosine phosphorylation of MAIR-I and association of MAIR-I with phosphatases, RBL-2H3 transfectant expressing wild-type or mutant MAIR-I was stimulated or not with 100 mM sodium pervanadate for 10 min at 37°C and lysed with a lysis buffer containing 1% digitonin (Calbiochem, San Diego, CA), 0.12% Triton-X (Sigma-Aldrich), 150 mM NaCl, 20 mM triethanolamine and protease and phosphatase inhibitors. Lysates were immunoprecipitated with anti-Flag M2 affinity gel (Sigma-Aldrich), anti-SHIP, anti-SHP-1 or anti-SHP-2.

To examine whether co-ligation of Fc{varepsilon}RI with MAIR-I is required for inhibition, 4 x 106 RBL-2H3 transfectant cells expressing wild-type MAIR-I were incubated with 400 ng of IgE anti-TNP mAb for 30 min on ice and washed twice with cold PBS. Cells were then suspended in 50 µl of cold HEPES–Tyrode's BSA buffer (25 mM HEPES buffer, pH 7.4, 140 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 5.6 mM D-glucose, 12 mM NaHCO3, 0.37 mM NaH2PO4, 0.49 mM MgCl2, 0.1% BSA) containing 40 ng of TNP-conjugated OVA plus either unlabeled (coligation, –) or TNP-conjugated (coligation, +) F(ab')2 fragments of anti-Flag mAb (80 ng/106 cells) for 20 min on ice. Afterwards, cells were incubated at 37°C and lysed with digitonin lysis buffer. Lysates were immunoprecipitated with anti-MAIR mAb (TX10)-binding Protein L agarose beads (ImmunoPure Immobilized Protein L, Pierce Chemical Co.). To verify co-ligation of Fc{varepsilon}RI and MAIR-I, RBL-2H3 transfectant cells expressing wild-type MAIR-I were incubated with biotin-labeled IgE anti-TNP, followed by TNP-conjugated OVA plus either unlabeled (co-ligation, –) or TNP-conjugated (co-ligation, +) F(ab')2 fragments of anti-Flag mAb. Cells were lysed with 1% digitonin lysis buffer and immunoprecipitated with control Ig or anti-MAIR-I.

Immunoprecipitates were separated by SDS–PAGE under reducing conditions, transferred (80 V, 1 h in 25 mM Tris, 195 mM glycine and 20% methanol) to PVDF membranes (Immobilon-P; Millipore, Billerica, MA). Membranes were incubated overnight in TBST (10 mM Tris-buffered saline containing 0.5% Tween 20, 0.5 g/l MgCl2, pH 8.0) containing 3% bovine serum albumin (BSA) for blocking and incubated with primary antibodies. Proteins were detected by using HRP-conjugated anti-rabbit Igs or streptavidin (Amersham Biosciences, Piscataway, NJ) or mouse IgG TrueBlot (e-Bioscience, San Diego, CA) and were developed with SuperSignal CL-HRP substrate (Pierce Chemical Co.). Chemiluminescence was detected by HAMA II (Hamamatsu Photonics, Shizuoka, Japan) and analyzed by Image Quant (Molecular Dynamics, Sunnyvale, CA). For stripping and re-blotting, Restore Western Blot Stripping Buffer (Pierce Chemical Co.) was used according to the manufacturer's instruction.

ß-Hexosaminidase release assay
1~2 x 105 RBL-2H3 cells in log-phased proliferation were cultured overnight in 0.1% gelatin (Sigma-Aldrich)-coated 24-well plates and incubated with 500 ng/ml (2.6 nM) of mouse IgE anti-TNP in medium without supplement for an hour. After washing with PBS twice, cells were stimulated with 50 ng/ml (1.1 nM) TNP-conjugated OVA and various concentrations of TNP-conjugated or unconjugated F(ab')2 fragments of anti-Flag antibody (100, 200, 400 ng/ml, i.e. 0.9, 1.8, 3.6 nM, respectively) in HEPES–Tyrode's BSA buffer for 45 min at 37°C. The ß-hexosaminidase in the supernatants was quantified by hydrolysis of p-nitrophenyl-N-acetyl-ß-D-glucosaminide (Sigma-Aldrich) in 0.4 M citric acid–0.2 M sodium phosphate buffer (pH 4.5) for 3 h at 37°C. The reaction was stopped by adding 0.2 M glycine–NaOH (pH 10.7) and absorbance at 415 nm was determined. Spontaneous release (supernatant from unstimulated cells) was subtracted from each test well and the percentage of ß-hexosaminidase release was calculated as: OD415 of test well/OD415 of the well where MAIR-I were not stimulated with F(ab')2-anti-Flag mAb. For statistical analysis, paired t-test was performed.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
MAIR-I is tyrosine phosphorylated and recruits SHP-1, SHP-2 and SHIP in RBL-2H3
We have previously reported that MAIR-I was tyrosine phosphorylated and recruits SHIP in bone marrow (BM)-derived cultured mast cells upon stimulation with pervanadate. Moreover, cross-linkinig MAIR-I inhibited IgE-mediated degranulation from mast cells, suggesting that MAIR-I mediates inhibitory signals. To explore MAIR-I-mediated signaling pathways in mast cells or basophils, we generated RBL-2H3 transfectant stably expressing wild-type (WT) MAIR-I tagged with Flag peptide at the N-terminus (Fig. 1A). Stimulation of the RBL-2H3 transfectant with pervanadate induced tyrosine phosphorylation of MAIR-I (Fig. 2A), consistent with our previous report (14). MAIR-I was co-immunoprecipitated with SHP-1, SHP-2 and SHIP after stimulation with pervanadate (Fig. 2A), indicating that MAIR-1 can potentially recruit all these phosphatases in the RBL-2H3 transfectant upon stimulation with pervanadate.



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Fig. 1. Establishment of RBL-2H3 transfectants expressing Flag-tagged wild-type and mutant MAIR-I. (A) Wild-type (WT) MAIR-I contains the tyrosines (Y) at residues 233, 258, 270, 299 and 313 (not shown) in the cytoplasmic region. {Delta}cyto MAIR-I is a deletion mutant in which the cytoplasic region containing all the tyrosines was deleted. The site-specific mutant MAIR-I, in which one or more of the tyrosines were replaced with phenylalanine(s) (F), is also shown. (B) RBL-2H3 transfectants expressing Flag-tagged WT and mutant MAIR-I were stained with anti-Flag mAb, followed by FITC-conjugated secondary antibody and analyzed by flow cytometry. (C) RBL-2H3 transfectants were incubated with biotin-conjugated IgE, followed by streptavidin–APC and analyzed by flow cytometry.

 


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Fig. 2. Tyrosine phosphorylation of MAIR-I and recruitment of the phosphatases. RBL-2H3 transfectants expressing WT and mutant MAIR-I were stimulated or not with pervanadate (VO4) and lysed with 1% digitonin buffer. MAIR-I was immunoprecipitated with anti-Flag mAb and immunoblotted with the antibodies indicated. Data are representative of three or more independent experiments.

 
The tyrosines at residues 258 and 270 are responsible for phosphorylation of MAIR-I and recruitment of the phosphatases in RBL-2H3
MAIR-I contains the five tyrosines at residues 233, 258, 270, 299 and 313 (Y233, Y258, Y270, Y299 and Y313) in the cytoplasmic domain (Fig. 1A). While Y258 and Y270 correspond to the sequences (VEY258STL and LHY270SSV, respectively) that fit the consensus ITIM [(I/V/L/S)xYxx(L/V)], CQY233VNL, AEY299SEI and DLY313L do not fit it (14). To study the role of the tyrosines in MAIR-I-mediated signaling, we also generated RBL-2H3 transfectants stably expressing variable mutant MAIR-I, in which the cytoplasmic region was deleted ({Delta}cyto) or Y233, Y258, Y270 and/or Y299 were replaced with phenylalanines (F) (Fig. 1A). These transfected MAIR-I were tagged with the Flag peptide epitope at the N-terminus. Expressions of MAIR-I and Fc{varepsilon}RI on the transfectants were comparable with each other, as determined by flow cytometry using anti-Flag and IgE mAbs, respectively (Fig. 1B and C).

In contrast to the transfectant expressing WT MAIR-I, stimulation of the transfectants expressing {Delta}cyto MAIR-I or MAIR-I mutated at all Y233, Y258, Y270 and Y299 (FFFF) with pervanadate did not induce tyrosine phosphorylation (Fig. 2A). Because Y258 and Y270 are most likely to be phosphorylated, the transfectants expressing MAIR-I mutated at residues 258 and 270 (YFFY) or at residues 233 and 299 (FYYF) were stimulated with pervanadate and examined for tyrosine phosphorylation. As expected, although the FYYF MAIR-I was significantly phosphorylated to a similar extent as WT MAIR-I, we could not detect tyrosine phosphorylation of YFFY MAIR-I (Fig. 2A). Moreover, co-immunoprecipitation with the phosphatases SHP-1, SHP-2 and SHIP was observed in the FYYF MAIR-I, but not in the YFFY MAIR-I. These results suggested that either one of Y258 and Y270, or both, are responsible for tyrosine phosphorylation of MAIR-I and recruitment of the phosphatases in RBL-2H3 upon stimulation with pervanadate.

To further address this issue, we generated the RBL-2H3 transfectants expressing mutant MAIR-I at Y233, Y258 and Y299 (FFYF); Y233, Y270 and Y299 (FYFF); Y258 (YFYY); and Y270 (YYFY) (Fig. 1A). As shown in Fig. 2(B), all these mutant MAIR-I were tyrosine phosphorylated (Fig. 2B), indicating that both Y258 and Y270 were phosphorylated upon stimulation with pervanadate. Furthermore, we observed co-immunoprecipitation of SHP-1, SHP-2 and SHIP with the FFYF, YFYY and YYFY mutant MAIR-I, suggesting that both Y258 and Y270 associated with all these phosphatases. However, FYFF mutant MAIR-I was co-immunoprecipitated with SHP-1 alone. Although the reason for this discrepancy is not clear at present, one possibility is that it might be caused by a conformational change of FYFF mutant MAIR-I that affects binding with SHIP and SHP-2. Nonetheless, these results suggested that both Y258 and Y270 are tyrosine phosphorylated and involved in phosphatase recruitment.

Co-ligation of MAIR-I with Fc{varepsilon}RI is essential to the MAIR-I-mediated inhibitory signal for degranulation from RBL-2H3
We previously demonstrated that cross-linking MAIR-I and Fc{varepsilon}RI with anti-MAIR-I mAb and IgE antibody, followed by co-ligation with a common secondary antibody, induced inhibition of IgE-mediated degranulation from BM-derived cultured mast cells (14). To examine whether co-ligation of Fc{varepsilon}RI and MAIR-I with a common secondary antibody is essential to the MAIR-I-mediated inhibitory signal, we established a system that co-ligated Fc{varepsilon}RI and MAIR-I using TNP-conjugated OVA and F(ab')2 fragments of anti-MAIR-I (Fig. 3A). In this system, co-ligation was verified by the co-immunoprecipitation of MAIR-I with Fc{varepsilon}RI-bound biotin-labeled IgE (Fig. 3B). By using this system, we first examined tyrosine phosphorylation of WT MAIR-I in RBL-2H3 transfectant after cross-linking with antibodies with or without co-ligation. As demonstrated in Fig. 3(C), tyrosine phosphorylation of MAIR-I was detected 1~3 min after cross-linking of Fc{varepsilon}RI and MAIR-I, regardless of whether with or without the co-ligation. Moreover, cross-linking of MAIR-I recruited SHP-1 and SHIP even without co-ligation (Fig. 3D). However, although IgE-mediated ß-hexosaminidase release from RBL-2H3 transfectant was inhibited by the co-ligation of the receptors, it did not change when these receptors were independently cross-linked without the co-ligation (Fig. 3E), indicating that tyrosine phosphorylation of MAIR-I and recruitment of phosphatases does not always mediate inhibitory signal against IgE-mediated degranulation. These results demonstrate that co-ligation of MAIR-I with Fc{varepsilon}RI is essential to the MAIR-I-mediated inhibitory signal for degranulation from RBL-2H3.



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Fig. 3. Tyrosine phosphorylation of MAIR-I and degranulation from RBL-2H3 after stimulation with or without co-ligation of Fc{varepsilon}R and MAIR-I. RBL-2H3 transfectants expressing Flag-tagged wild-type MAIR-I were incubated with a biotin unlabeled (A) or labeled (B) mouse IgE anti-TNP antibody and then stimulated with TNP-conjugated OVA and various concentrations of TNP-conjugated (co-ligation, +) or unconjugated (co-ligation, –) F(ab')2 fragments of anti-Flag antibody (A), as described in the Methods. After stimulation, RBL-2H3 transfectants were lysed and MAIR-I was immunoprecipitated with anti-MAIR-I mAb and immunoblotted with streptavidin (B) anti-phosphotyrosine (C and D), anti-phosphatases indicated (C and D) or anti-Flag antibodies (B–D). ß-Hexosaminidase release in the supernatants was also measured after stimulation of RBL-2H3 transfectant and releases were calculated, as described in the Methods (E). Error bar indicates 1 SD. Data are representative of three or more independent experiments.

 
The tyrosines at residues 258 and 270 are required for inhibition of degranulation from RBL-2H3
To confirm that the tyrosine(s) in the MAIR-I cytoplasmic region is responsible for inhibitory signal for IgE-mediated degranulation, we examined the RBL-2H3 transfectant expressing {Delta}cyto MAIR-I. In contrast to the transfectant expressing WT MAIR-I (Fig. 4A), ß-hexosaminidase release did not change from {Delta}cyto MAIR-I transfectant after the co-ligation of Fc{varepsilon}RI with MAIR-I (Fig. 4B). However, it was inhibited in the transfectant expressing FYYF, but not YFFY, MAIR-I after the co-ligation (Fig. 4C and D), indicating that either Y258 or Y270, or both, are involved in the inhibition. These results are consistent with the observation that Y258 and Y270 of MAIR-I were phosphorylated and recruited the phosphatases upon stimulation with pervanadate or cross-linking of MAIR-I (Figs 2A and 3C and D). Further studies demonstrated that ß-hexosaminidase release was inhibited in all the transfectant expressing FFYF, FYFF, YFYY and YYFY MAIR-I after the co-ligation (Fig. 4E–G). These results indicate that both Y258 and Y270 are essentially required for inhibition of degranulation from RBL-2H3.



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Fig. 4. Degranulation from RBL-2H3 transfectants expressing mutant MAIR-I after co-ligation of Fc{varepsilon}RI and MAIR-I. RBL-2H3 transfectant expressing Flag-tagged wild-type or mutant MAIR-I were incubated with a mouse IgE anti-TNP antibody and then stimulated with TNP-conjugated OVA and various concentrations of TNP-conjugated F(ab')2 fragments of anti-Flag antibody. After stimulation, ß-hexosaminidase release in the supernatants was measured and percentage releases were calculated, as described in the Methods. Percentage ß-hexosaminidase releases in the absence or presence (400 ng/ml) of TNP-conjugated F(ab')2 fragments of anti-Flag antibody were compared with each other. Error bar indicates 1 SD. Data are representative of three or more independent experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this report, we established RBL-2H3 transfectants expressing WT and variable mutant MAIR-I at Y233, Y258, Y270 and/or Y299 to explore MAIR-I-mediated signaling pathways involved in inhibition of IgE-mediated degranulation from mast cells. By using these transfectants, we have formally demonstrated that Y258 and Y270 that consist of the consensus ITIM sequence, but not Y233 and Y299, were phosphorylated and essentially required for inhibitory signal for IgE-mediated degranulation from RBL-2H3. Although MAIR-I recruited SHP-1, SHP-2 and SHIP in RBL-2H3 transfectants upon stimulation with pervanadate, cross-linking of MAIR-I (a more physiological condition than pervanadate stimulation) recruited SHP-1 and SHIP, but not SHP-2. The recuitment of SHIP and SHP-1 was reported in the case of PIR-B (9), an inhibitory receptor on myeloid cells and B cells recognizing MHC class-I (17). However, we cannot formally exclude the possibility at present that any of the phosphatases SHP-1, SHP-2 and SHIP that bind to a docking site at Y258 and Y270 show phosphatase activity for Fc{varepsilon}RI-mediated signaling in RBL-2H3. Further studies using mice deficient in these phosphatases should address a phosphatase required for inhibition of IgE-mediated degranulation from mast cells and basophils.

We have shown that co-ligation of Fc{varepsilon}RI and MAIR-I was required for the inhibition of degranulation from RBL-2H3. This result was consistent with the situations of other inhibitory receptors expressed on mast cells, such as Fc{gamma}RIIB (5), gp49B1 (6,7), PIR-B (9,18), MAFA (11,12,19) and SIRP{alpha} (13). Aggregation of Fc{varepsilon}RI induces its association with lipid rafts and subsequent activation signaling via Fc{varepsilon}RI (20). A recent report demonstrated that co-localization of Fc{varepsilon}RI, a protein tyrosine kinase Lyn and Csk-binding protein (Cbp), a negative regulator for Fc{varepsilon}RI signaling, in lipid rafts is important for the inhibitory signal (21), suggesting a hypothesis that co-ligation of Fc{varepsilon}RI and MAIR-I also aggregates signaling molecules in lipid rafts that efficiently interact with each other and mediate an inhibitory signal. At present, however, the physiological significance of the requirement of co-ligation of Fc{varepsilon}RI and MAIR-I is unclear because the ligand for MAIR-I has not yet been identified.

It was of note that MAIR-I was tyrosine phosphorylated and recruited SHIP and SHP-1 after cross-linking Fc{varepsilon}RI and MAIR-I with antibodies without co-ligating each other. At present, a protein tyrosine kinase responsible for tyrosine phosphorylation of MAIR-I has remained undetermined. However, while tyrosine phosphorylation of MAIR-I occurred as early as 1 min after co-ligation of both receptors, it was detected at 3 min after cross-linking of MAIR-I without co-ligation. Because degranulation from mast cells is complete within 2 min after Fc{varepsilon}RI cross-linking in bone marrow mast cells (22), tyrosine phosphorytaion of MAIR-I at 3 min may not be able to effectively induce negative signals.

In conclusion, we have shown that co-ligation of Fc{varepsilon}RI and MAIR-I with antibodies mediates an inhibitory signal for IgE-mediated degranulation, for which Y258 and Y270 of the cytoplasmic region of MAIR-I are responsible. Recent reports have demonstrated ligands for inhibitory receptors expressed on mast cells that are endogenous cell surface proteins, including MHC class I for PIR-B (17) and non-MHC proteins for gp49B1 (23) and SIRP{alpha} (24,25). Thus, mast cell activation by cross-linking of Fc{varepsilon}RI-bound IgE with multivalent antigen may be regulated by interaction between these inhibitory receptors and their ligands. To evaluate the physiological role of MAIR-I in regulation of mast cell activation, identification of a MAIR-I ligand is required.


    Acknowledgements
 
We thank Yurika Soeda and Satoshi Yamazaki for secretarial assistance and preparing F(ab')2 fragments of anti-Flag mAb, respectively. Y.O. is a Research Fellow of the Japan Society for the Promotion of Science. This research was supported in part by a Grant-in-Aid for Scientific Research provided by the Ministry of Education, Culture, Sports and Technology of Japan and Special Coordination Funds of the Science and Technology Agency of the Japanese Government.


    Notes
 
Transmitting editor: T. Takai

Received 10 August 2004, accepted 19 October 2004.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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