Receptor-Mediated Signaling Has

FCRL3, an Autoimmune Susceptibility Gene, Has Inhibitory Potential on B-Cell Receptor-Mediated Signaling

This information is current as Yuta Kochi, Keiko Myouzen, Ryo Yamada, Akari Suzuki, of October 2, 2021. Tomohiro Kurosaki, Yusuke Nakamura and Kazuhiko Yamamoto J Immunol 2009; 183:5502-5510; ; doi: 10.4049/jimmunol.0901982 http://www.jimmunol.org/content/183/9/5502 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

FCRL3, an Autoimmune Susceptibility Gene, Has Inhibitory Potential on B-Cell Receptor-Mediated Signaling1

Yuta Kochi,2* Keiko Myouzen,* Ryo Yamada,*† Akari Suzuki,* Tomohiro Kurosaki,‡ Yusuke Nakamura,§ and Kazuhiko Yamamoto*¶

A polymorphism that up-regulates the expression of Fc receptor-like 3 (FCRL3) gene has recently been described as predisposing for several human autoimmune diseases. FCRL3 is preferentially expressed on B cells and is unique in displaying both an ITAM and an ITIM in the cytosolic domain, suggesting signaling functions. Herein, we show that FCRL3 potentially inhibits BCR- mediated signaling, using murine Fc␥RIIB/human FCRL3 chimeric protein. Coligation of the chimeric protein with BCR leads to phosphorylation of tyrosine residues in the cytosolic domain. This coligation inhibits cell tyrosine phosphorylation and calcium mobilization in addition to activation-induced cell death mediated by BCR signaling. Mutational analysis showed the tyrosine residues in two potential ITIMs at 662 and 692 offer the main contributions to this inhibition, which is further supported by strong Downloaded from associations of SH-2 domain-containing phosphatases with the following phosphotyrosine motifs: SHIP with the ITIM-like motif at 662; and SHP-1 and -2 with the canonical ITIM at 692. These results, together with previous genetic data, suggest that augmented inhibition of BCR-mediated signaling by FCRL3 with the disease-risk genotype alter the activation threshold and promote tolerance breakdown in B cells. The Journal of Immunology, 2009, 183: 5502–5510.

utoimmune diseases such as rheumatoid arthritis (RA)3 variants are also reportedly associated with onset of autoimmune and systemic lupus erythematosus (SLE) are multifac- pancreatitis in Japanese populations (14). These findings together http://www.jimmunol.org/ A torial diseases where genetic and environmental factors indicate that FCRL3 associations with disease susceptibility are underlie disease pathogenesis (1). Although specific haplotypes of shared among different autoimmune diseases and ethnic groups. HLA genes are well known as the major determinants of suscep- Disease-associated polymorphism in the promoter region en- tibility for each disease, recent genome analyses of autoimmune hances binding of NF␬B and augments expression of FCRL3 in diseases have revealed several variants in non-HLA genes, such as the disease-risk allele (8). Considering the preferential expres- PTPN22 (2, 3) and CTLA4 (4) genes, that increase the risk of these sion of FCRL3 in germinal center B-cells (5), this gain-of-gene diseases. We have recently shown that a regulatory variant in function may influence B cell activity and promote autoimmune

FCRL3 gene (also known as FcRH3 (5), IRTA3 (6), and SPAP2 responses by B cells. Moreover, several clinical studies have by guest on October 2, 2021 (7)), which encodes the Fc receptor-like 3 (FCRL3) protein, is indicated that production of disease-associated autoantibodies associated with susceptibility for several autoimmune diseases in- was augmented in patients carrying the disease-susceptibility cluding RA, SLE, and autoimmune thyroiditis in the Japanese pop- genotype, supporting a signiﬁcant role of FCRL3 in B cell ulation (8). This association was followed by replication studies driven autoimmunity (8, 14). using RA cohorts in Japanese (9), Dutch Caucasian (10), and Ca- FCRL3 is a type I transmembrane protein in which the extra- nadian Caucasian (11) populations and multiple autoimmune thy- cellular domain, comprising six Ig-like domains, displays high ho- roiditis cohorts in U.K. Caucasian populations (12, 13). FCRL3 mology to the classical Fc␥Rs, although no ligands are known (5) (Fig. 1A). The intracellular domain, in contrast, contains four tyrosine residues at 650, 662, 692, and 722, which could generate *Laboratory for Autoimmune Diseases, Center for Genomic Medicine, RIKEN, To- kyo, Japan; †Laboratory of Functional Genomics, Human Genome Center, the Insti- both an ITAM and an ITIM. The sequence motif around tyrosines ‡ tute of Medical Science, the University of Tokyo, Tokyo, Japan; Laboratory of at 650 and 662 (Y650SNVNPGDSNPIY662SQI) closely resembles Lymphocyte Differentiation, Research Center for Allergy and Immunology, RIKEN, the canonical ITAM (YxxI/Lx YxxI/L) (15). A binding study Yokohama, Japan; §Laboratory of Molecular Medicine, Human Genome Center, In- (6–12) stitute of Medical Science, the University of Tokyo, Tokyo, Japan; and ¶Department using lysates of human T cell line Jurkat has shown that src-family of Allergy and Rheumatology, Graduate School of Medicine, the University of To- kinases Syk and ZAP70, which are involved in transmitting acti- kyo, Tokyo, Japan vation signals in lymphocytes, could be associated with this motif Received for publication June 22, 2009. Accepted for publication August 21, 2009. (7). Conversely, the tyrosine motif at 692 matches the canonical 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 consensus ITIM (I/L/VxYxxL/V) (16), and is predicted to function with 18 U.S.C. Section 1734 solely to indicate this fact. as an ITIM. The three other tyrosine motifs at 650, 662, and 722 1 This work was supported by a grant from the Japanese Millennium Project, a grant show high homology but not complete match, even if a more per- from CGM, RIKEN, and a grant from Ministry of Health, Labor and Welfare of Japan. missive consensus ITIM (I/L/V/S/TxYxxL/I/V) is adopted (16). 2 Address correspondence and reprint requests to Dr. Yuta Kochi, Laboratory for Src homology 2 domain-containing phosphatases (SHPs), SHP-1 Autoimmune Diseases, Center for Genomic Medicine, RIKEN, 7-3-1 Hongo, Bunkyo-Ku, Tokyo, Japan. E-mail address: [email protected] and SHP-2, have been described as associated with the ITIM and ITIM-like motif at 692 and 722 in the same study described above 3 Abbreviations used in this paper: RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; FCRL3, Fc receptor-like 3; SHP, Src homology 2 domain-containing (7). These associations of both kinases and phosphatases imply the phosphatase; HA, hemagglutinin; 7-AAD, 7-amino actinomycin D; CM-WT, chimera signaling potential of FCRL3 as a coreceptor on B cells that could wild type; CM-Mt, chimera mutant. either positively or negatively regulate B cells. Like other core- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 ceptors on B cells such as CD19, CD21, and CD22 (17), FCRL3 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901982 The Journal of Immunology 5503 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 1. FCRL3 and murine Fc␥RIIB chimeric proteins. A, Schematic structures of FCRL3 and murine Fc␥RIIB chimeric proteins. The black line ␥ represents FCRL3 sequence and the gray line represents the murine Fc RIIB sequence. HA (hemagglutinin)-tag was added at the NH2-end of chimeric protein. Each semicircular structure in the extracellular domain represents a single Ig-like domain. Tyrosine residues (Y) of the potential ITAM and ITIMs in FCRL3 and their positions are indicated. In the mutant construct (CM-Mt), tyrosines were displaced by phenylalanines (F). B, Expression of chimeric proteins was measured by ﬂow cytometry after staining with anti-HA. The black line represents anti-HA staining and the gray line represents the Ј isotype-control staining. C, Schematic representation of stimulation strategy. F(ab )2 of anti-IgM stimulates BCR only (left), while intact anti-IgM stimulates BCR in coligation with the chimeric protein (right).

could modulate BCR signaling and may consequently affect im- American Type Culture Collection (ATCC) and maintained in RPMI 1640 munological tolerance in B cells. Functional dissection of FCRL3 medium supplemented with 10% FCS, 2 mM L-glutamine, and 100 U/ml will thus facilitate an understanding of roles in the pathogenesis of penicillin/streptomycin. DT40 cells were obtained from the RIKEN Cell human autoimmune diseases. We report herein that FCRL3 has Bank and maintained using the medium described above with the addition of 1% chicken serum. Anti-hemagglutinin (HA) Ab (12CA5) was obtained inhibitory potential on BCR-mediated signaling, possibly through from Roche Diagnostics; anti-phospho-Syk (Tyr352) and anti-phospho- association of ITIMs with phosphatases SHIP, SHP-1, and SHP-2. PLC␥2 (Tyr1217) Abs from Cell Signaling Technologies; biotinylated anti- murine Fc␥RIIb (2.4G2) and HRP or PE-coupled anti-phosphotyrosine Materials and Methods Abs (PY20) from BD Biosciences; anti-PLC␥2, anti-SHP-1, anti-SHP-2, anti-SHIP and agarose-conjugated anti-HA Abs from Santa Cruz Biotech- Cells and Abs Ј nology; anti-ZAP70 Ab from eBioscience, and intact and F(ab )2 of rabbit ST486 cells, which represent human germinal center-derived B cell lym- anti-human IgM and goat anti-chicken IgY from Jackson ImmunoResearch phoma and lack expression of Fc␥Rs, and Jurkat cells were obtained from Laboratories. 5504 FCRL3 INHIBITS BCR SIGNALING

Generation of chimeric mutants of FCRL3 and stable sciences) at 37°C for 10 min, permeabilized with BD Phosflow Perm transfectants Buffer II (BD Biosciences) for 30 min at on ice, and then stained with PE-conjugated anti-phosphotyrosine Abs (PY20) before FACS analysis. The cDNAs encoding transmembrane and intracellular domains of human FCRL3 were fused to cDNA of the extracellular domain of murine Results Fc␥RIIB and cloned into pDisplay vector (Invitrogen). A NotI site was introduced between the extracellular domain of Fc␥RIIB and the trans- Construction of chimeric proteins membrane domain of FCRL3, translating into a 3-alanine spacer. Site- To investigate the function of FCRL3 in B cells, we constructed a directed mutagenesis was performed by PCR according to standard proto- cols. For lentiviral transduction, cDNAs encoding the chimeric proteins chimeric protein which comprised the extracellular domain of mu- were excised from pDisplay and cloned into pLenti6 vector (Invitrogen). rine Fc␥RIIB and the transmembrane and intracellular domains of All constructs were verified by DNA sequencing. Transfection of ST486 FCRL3 (designated as chimera wild type (CM-WT)) (Fig. 1A). In cells and DT40 cells was performed according to the instructions of the addition, the intracellular domain of the chimeric protein was mu- manufacturer. Virus-containing supernatant was mixed with polybrene to a tated by replacing all 4 tyrosine residues with phenylalanine (des- final concentration of 3 ␮g/ml and added to 6 ϫ 105 cells. Lentivirally transduced cells were incubated for 10 days in a medium containing blas- ignated as chimera mutant (CM-Mt)) to analyze the potential func- ticidine S (10 ␮g/ml) and stably transfected cells were purified using anti- tion of motifs around the residues. These constructs were stably HA Ab and Pan Mouse IgG Dynabeads (Dynal). Expression of chimeric transfected to ST486, which is a germinal-center derived B cell proteins was determined using a FACSCalibur flow cytometer (BD line and lacks expression of Fc␥Rs. Surface expression of the chi- Biosciences). meric proteins was confirmed by flow cytometer (Fig. 1B). Trans- Calcium measurements fectants were stimulated with intact anti-IgM Abs, resulting in colli- Ј ϫ 6 ␮ gation of BCR and the chimeric proteins. As a control, the F(ab )2 of

For ST486 cells, 5 10 cells were loaded with 5 g of fura-2/AM Downloaded from (Dojin) in RPMI 1640 medium. After incubation for 30 min at 37°C, anti-IgM Abs was used to stimulate BCR alone (Fig. 1C). cells were washed twice and resuspended in 0.5 ml HBSS with 1 mM

CaCl2, 1 mM MgCl2, and 0.1% BSA. Cytosolic calcium concentrations FCRL3 engagement inhibits BCR-stimulated tyrosine of 5 ϫ 105 cells were measured using a CAF 110 spectrophotometer phosphorylation and Ca2ϩ influx ␮ ␮ Ј (Jasco), after addition of 15.0 g/ml intact anti-IgM, 10.0 g/ml F(ab )2 of anti-IgM, or biotinylated 5.0 ␮g/ml anti-murine Fc␥RIIB (2.4G2) To elucidate the function of FCRL3 in B cells, and associations followed by 10.0 ␮g/ml streptavidin. For DT40 cells, cells were instead with BCR signaling in particular, we first examined BCR-stimu- loaded with 5 ␮g of fluo-3/AM (Dojin). Cytosolic calcium concentra- lated tyrosine phosphorylation in ST486 transfectants of chimeric http://www.jimmunol.org/ tions in 1 ϫ 106 cells were measured using the FACSCalibur flow ␮ proteins. Although few proteins were phosphorylated at the ty- cytometer, after addition of 50.0 g/ml intact anti-chicken IgY or 50.0 Ј ␮ Ј rosine residues under resting status, BCR ligation with F(ab )2 g/ml F(ab )2 of anti-anti-chicken IgY. anti-IgM strongly induced tyrosine phosphorylation in all trans- Apoptosis assay fectants (Fig. 2A). BCR-induced phosphorylation was modestly Stably transfected ST486 cells were incubated in the presence of anti-IgM inhibited in CM-WT transfectants, but not in mock transfectants, ␮ Ј ␮ Abs (10.0 g/ml F(ab )2 and 15.0 g/ml for intact IgG). Dead cells were when intact anti-IgM was used. Phosphorylation of adaptor pro- determined using 7-amino actinomycin D (7-AAD) staining. Cells were teins in BCR complex, such us Syk and PLC␥2, was also inhibited also stained with PE-labeled annexin V to detect those in the apoptotic when BCR was stimulated with intact anti-IgM in CM-WT trans- phase, according to the specifications of the manufacturer for the Guava by guest on October 2, 2021 nexin assay kit (GE Healthcare). Stained cells were counted using a Guava fectants (Fig. 2B). The same inhibition was not observed in PCA96 analytical system (GE Healthcare). CM-Mt transfectants, in which all tyrosine residues had been replaced, suggesting that tyrosine motifs in CM-WT have inhibitory Cellular activation, Western blotting, affinity precipitation, and effects on tyrosine phosphorylation in BCR signaling. immunoprecipitation We then examined whether the chimeric proteins themselves To monitor the effects of transduced receptors on BCR-induced signaling, were phosphorylated through BCR stimulation. Chimeric proteins ϫ 6 5 10 cell aliquots were washed twice and incubated for2hinmedium were immunoprecipitated after BCR stimulation, then immuno- lacking FCS and supplemented with 20 mM HEPES (pH 7.2), before stim- ␮ Ј ␮ blotted to detect phosphorylation status at the tyrosine residues ulation with intact anti-IgM (15 g/ml) or anti-IgM F(ab )2 (10 g/ml). Western blotting, affinity precipitation and immunoprecipitation were per- (Fig. 2C). Whereas CM-WT proteins were not phosphorylated in Ј formed as described (18) with some modifications. In brief, cells were spun resting status, BCR stimulation alone by F(ab )2 anti-IgM induced down before incubation in RIPA buffer containing 1% Nonidet P-40, 50 slight phosphorylation in tyrosine residues in chimeric proteins. When mM Tris-HCl (pH 7.4), 1 mM EDTA, 150 mM NaCl, and the addition of CM-WT transfectants were stimulated with intact anti-IgM, tyrosine complete protease inhibitor mixture (Roche Diagnostics) and phosphatase phosphorylation of CM-WT protein was greatly enhanced. This sug- inhibitors Na3VO4 (1 mM) and NaF (1 mM). Whole-cell lysate proteins were quantitated using BCA reagent (Pierce) and resolved by SDS/PAGE gests that chimeric proteins were colligated to BCR, and that co- before transfer to PDVF membranes (Millipore), which were then probed localization with BCR allowed tyrosine kinases in the BCR com- with the indicated Abs, visualized using ECL reagent (GE Healthcare), and plex to phosphorylate tyrosine residues of CM-WT chimeric detected by LAS-3000 (Fuji Film). For affinity precipitation, cells were stimulated with 100 ␮M pervanadate (Sigma-Aldrich) prepared as de- proteins. This phosphorylation of tyrosine residues may have led to scribed (19). Whole-cell lysates of ST486 and Jurkat cells in 1 ml RIPA recruitment of adapter proteins to the tyrosine motifs of chimeric buffer were incubated at 4°C for 6 h with 20 ␮l of NeutrAvidin-agarose proteins, in turn reducing phosphorylation of proteins in the cells. beads (Pierce) saturated by biotinylated phosphor-peptides (Operon Bio- Ligation of chimeric proteins alone did not induce cell tyrosine ϫ 7 technology). For immunoprecipitation of chimeric proteins, 4 10 stable phosphorylation (data not shown), although tyrosine residues at transfectants were lysed with RIPA buffer after stimulation with intact anti-IgM (50 ␮g/ml) for 3 min. The lysate was then treated with 10 ␮gof 650 and 662 display strong similarity to ITAM and may have agarose-conjugated anti-HA Ab and incubated at 4°C for 4 h. Beads were activation potential. Together, these results suggest that the cyto- then washed three times with 1 ml of lysis buffer and boiled, and Western plasmic domain of FCRL3, in total, displays inhibitory potential blotting was performed as described above. for tyrosine phosphorylation when colligated with BCR. 2ϩ Flow cytometric analysis of cellular phosphorylation Next, we examined Ca mobilization as a downstream event of the phosphorylation of PLC␥2 in the BCR complex (Fig. 2D). DT40 stable transfectants were incubated at 37°C for 2 h before stimula- Ј tion. The cells (5 ϫ 106) were then stimulated with 50.0 ␮g/ml intact Although BCR ligation with the F(ab )2 of anti-IgM resulted in a ␮ Ј characteristic calcium influx in all transfectants, this was partially anti-chicken IgY or 50.0 g/ml F(ab )2 of anti-anti-chicken IgY. Finally, treated cells were fixed with 200 ␮l of Phosflow Fix Buffer (BD Bio- inhibited by intact anti-IgM in CM-WT transfectants but not in The Journal of Immunology 5505 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. Coligation of Fc␥RIIB/FCRL3 chimeric protein inhibits BCR-induced tyrosine phosphorylation and Ca2ϩ influx in ST486 cells. A, ST486 Ј ␮ ␮ transfectants of chimeric proteins were stimulated with F(ab )2 of anti-IgM (10.0 g/ml) or intact anti-IgM (15 g/ml) for 3 min before analysis of whole-cell phosphorylation of tyrosine residues detected by antiphosphotyrosine Ab (PY20). Blots were striped and reprobed using anti-␤-actin Ab. The densities of phoshotyrosine proteins were quantified and shown on the top of figure, where each transfectant stimulated with F(ab)2 anti-IgM for 3 min is set to be 1.00. B, Whole lysates from A were immunoblotted against phosphor-Syk (Tyr352) and phosoho-PLC␥2 (Tyr1217). Blots were striped and reprobed with anti-Syk and PLC␥2 Ab, respectively. The densities of phosphor-Syk and phosoho-PLC␥2 were quantified and shown on the top of figure, where each transfectant stimulated with F(ab)2 anti-IgM is set to be 1.00. C, Whole lysates from A were immunoprecipitated with anti-HA Ab and immunoblotted with anti-phosphotyrosine Ab. Blots were striped and reprobed with anti-HA Ab. Data are representative of four separate experiments. D, ␮ Ј ␮ ST486 transfectants of indicated chimeric proteins were loaded with fura-2 and stimulated with 10.0 g/ml F(ab )2 of anti-IgM (solid thick line) or 15 g/ml intact anti-IgM (dotted line). Chimeric proteins were also ligated alone with biotinylated 5.0 ␮g/ml anti-murine Fc␥RIIB (2.4G2) followed by 10.0 ␮g/ml streptavidin (solid thin line). The results are representative of three independent experiments. mock transfectants. When BCR was colligated with CM-Mt, no Abs. Inhibition of tyrosine phosphorylation in CM-WT transfec- inhibition of Ca2ϩ influx was observed, suggesting the inhibitory tants was again observed when colligated with BCR, as in ST486 effects of tyrosine motifs in CM-WT on Ca2ϩ mobilization. We transfectants (Fig. 3A). We also examined BCR-induced Ca2ϩ in- also examined whether the chimeric proteins could induce Ca2ϩ flux in DT40 cells (Fig. 3B). Inhibition of Ca2ϩ influx in CM-WT mobilization by ligating the chimeric proteins alone (Fig. 2D). No transfectants was even more obvious in DT40 cells when colli- Ca2ϩ influx was observed for any of the transfectants, indicating gated with chimeric proteins, while moderate inhibition was also the absence of ability to mobilize Ca2ϩ in FCRL3. observed in CM-Mt transfectants, unlike ST486 cells. To evaluate To further confirm the inhibition of BCR-induced signaling by each individual effect of tyrosine motifs on Ca2ϩ influx, additional the chimeric proteins, another set of stable transfectants was ex- mutational constructs were made by replacing three tyrosine res- amined using DT40 cells (Fig. 3 and supplemental Fig. 1B). Ty- idues with phenylalanine and leaving a single tyrosine residue in- rosine phosphorylation induced by BCR cross-linking in DT40 tact (designated as CM-Y650, CM-Y662, CM-Y692, and CM- cells was evaluated by flow cytometry using anti-phosphotyrosine Y722). We also evaluated the effect of a potential ITAM motif 5506 FCRL3 INHIBITS BCR SIGNALING

FIGURE 3. Coligation of Fc␥RIIB/ FCRL3 chimeric protein inhibits BCR- induced tyrosine phosphorylation and Ca2ϩ influx in DT40 cells. A, DT40 transfectants of chimeric proteins were unstimulated (dotted line) or stimulated ␮ Ј with 50.0 g/ml F(ab )2 of anti-IgY Downloaded from (solid thick line) or 50.0 ␮g/ml intact anti-IgY (gray line) for 10 min before analysis of cellular phosphorylation of tyrosine residues detected using anti- phosphotyrosine Ab (PY20) by flow cytometry. B, DT40 transfectants of indicated chimeric proteins and mutant http://www.jimmunol.org/ proteins were loaded with Iono-3 and ␮ Ј stimulated with 50.0 g/ml F(ab )2 of anti-IgY (black line) or 50.0 ␮g/ml intact anti-IgY (gray line). The Ca2ϩ influx was detected by flow cytometry. The results given are representative of three independent experiments. by guest on October 2, 2021

comprising tyrosines at 650 and 662, by mutating the remaining death in CM-WT transfectants (Fig. 4A, Annexin Vϩ 7-AADϩ tyrosines (named CM-Y650/Y662). Among these constructs, in- cells). Interestingly, colligation to CM-Mt chimeric proteins en- hibition of BCR-induced Ca2ϩ influx was observed in CM-Y662, hanced BCR-induced cell death. To evaluate individual effects of CM-Y692, and CM-Y650/Y662. Intriguingly, and contrary to our the four tyrosine residues (Y650, Y662, Y692, and Y722), addi- expectations, Ca2ϩ influx was enhanced when colligated with chi- tional mutated constructs of CM-WT were created (supplemental meric proteins in transfectants of CM-Y650 and CM-Y722. Fig. S1A; each tyrosine residue was replaced with phenylalanine and constructs were designated CM-F650, CM-F662, CM-F692, FCRL3 engagement inhibits BCR-induced cell death and CM-F722, respectively). In comparison with CM-WT, inhibi- Activation-induced cell death occurs in germinal center B cells, in tion of cell death was reduced in CM-F662 and partially reduced which FCRL3 is highly expressed, following prolonged ligation of in CM692, while mutations in CM-F650 and CM-F722 had little BCR by Abs. This mimics the negative selection process of self- effect (Fig. 4B). This suggests that tyrosine motifs at 662 and 692 reactive B-cells induced by strong BCR ligation with self-Ags play critical roles in the inhibitory function of FCRL3 on activa- (20). This also occurs in germinal center-derived ST486 cells (21). tion-induced cell death. We therefore examined BCR-induced cell death in ST486 transfectants of chimeric proteins. BCR was stimulated alone or with Association of tyrosine kinases and phosphatases with FCRL3 coligation to chimeric proteins. Compared with mock transfec- Given that the intracellular domain of FCRL3 can inhibit tyrosine tants, colligation to chimeric proteins significantly reduced cell phosphorylation of cell proteins, calcium mobilization, and cell The Journal of Immunology 5507

FIGURE 4. Coligation of Fc␥RIIB/ FCRL3 chimeric protein inhibits acti-

vation-induced cell death by BCR stim- Downloaded from ulation. A, ST486 transfectants were incubated in medium alone (Ⅺ), with ␮ Ј f 10.0 g/ml F(ab )2 of anti-IgM ( ), or with 15 ␮g/ml intact anti-IgM (u) for 24 h. Cells were then stained with PE- labeled annexin V and 7-AAD to detect ϩ early apoptotic cells (annexin V , http://www.jimmunol.org/ 7-AAD Ϫ, and dead cells (annexin Vϩ 7AADϩ). B, ST486 transfectants with the chimeric protein and mutants (each tyrosine residue was replaced with phenylalanine and designated as CM-F650, CM-F662, CM-F692, and CM-F722, respectively) were incubated with 10.0 ␮ Ј g/ml F(ab )2 of anti-IgM or with 15 ␮g/ml intact anti-IgM for 48 h. Reduc- by guest on October 2, 2021 tion in the proportion of dead cells (7- AAD positive cells) in the latter (intact anti-IgM) compared with the former Ј (F(ab )2 of anti-IgM) is shown with a solid bar. Data represent mean Ϯ SD of p Ͻ 0.01), and ,ء) quadricate cultures are representative of three independent experiments.

death induced by BCR ligation, we sought to identify the adaptor (Fig. 5A). As tyrosines at 650 and 662 could potentially be com- proteins that can mediate these inhibitory effects. To precipitate ponents of a single ITAM or two separate ITIMs, a peptide ex- adaptor proteins from cell lysates, phosphopeptides corresponding tending from 644 to 678 was synthesized (designated Y650Y662). to the ITAM and ITIMs in the FCRL3 cytosolic domain were used Two other peptides corresponding to potential ITIMs at 692 and 5508 FCRL3 INHIBITS BCR SIGNALING

FIGURE 5. Tyrosine motifs of FCRL3 associate with kinases and phosphatases. A, Phosphopeptides were synthesized according to the sequences around the tyrosine residues of the

FCRL3 cytosolic domain. Amino acid Downloaded from “pY” represents phosphorylated tyrosine. Consensus ITAM (italic) and ITIM (underlined) motifs are indicated. B, ST486 cells and Jurkat cells were ac- tivated with pervanadate, then lysed. Using whole cell lysates, proteins were afﬁnity precipitated with phoshopep- http://www.jimmunol.org/ tide-coated beads, and separated by SDS/PAGE, and blots were probed with speciﬁc Abs. C,Fc␥RIIB/FCRL3 chimeric proteins in ST486 transfectants stimulated with intact anti-IgM (50 ␮g/ml) for 3 min were immunoprecipitated with ant-HA Abs and separated by SDS/PAGE. The resulting

blots were probed with speciﬁc Abs. D, by guest on October 2, 2021 Fc␥RIIB/FCRL3 mutational chimeric proteins in ST486 transfectants were immunoprecipitated under stimulation of BCR for 3 min, as described in C. Results are representative of four independent experiments.

722 were also used (designated Y692 and Y722, respectively). In Jurkat cells, while only Syk but not ZAP-70 was associated in addition, peptides phosphorylated in tyrosine residues were also ST486 cells (Fig. 5B). When either of two tyrosines in the peptides prepared (designated, for example, as pY650Y662: pY stands for was not phosphorylated, this association was diminished. Tyrosine phosphorylated tyrosine). Lysates of Jurkat cells were used in ad- phosphatases, in contrast, were also associated with the phos- dition to ST486 cells for comparison with data previously reported phopeptides of FCRL3 cytosolic domain. SHP-1 and -2 were as- by Xu et al. (7), in which GST fusion proteins of FCRL3 and sociated with both Y650pY662 and pY692, although stronger as- Jurkat cell lysates were used for pull-down assays. sociation was observed between SHP-1 and pY692, particularly in Affinity precipitation with these peptides showed that Src-family ST486 cells, and between SHP-2 and Y650pY662 in both ST486 kinases Syk and ZAP-70 were associated with pY650pY662 in cells and Jurkat cells. This further supported the finding by Xu The Journal of Immunology 5509 et al. that the tyrosine motif at 692 had recruited SHP-1 and might totic effects on B-cells when coligated with BCR (22). As CM-Mt function as an ITIM. Another SH2 domain-containing phospha- protein did not show to enhance tyrosine-phosphorylation or cal- tase, SHIP, was preferentially bound to Y650pY662 in ST486 cium influx in ST486 and DT40 cells, this proapototic effect may cells, but not in Jurkat cells, in which SHIP expression was absent. be independent of these signaling events. Although these observa- Because Xu et al. used lysates of Jurkat cells and did not detect tions indicate additional biological functions of the intracellular binding of SHIP to FCRL3, this is the first report to identify that domain of FCRL3 other than the inhibitory potential of tyrosine SHIP binds to the phosphotyrosine motif at 662 of FCRL3. motifs at 662 and 692, overall potential seems inhibitory. The To confirm the associations of these adaptor proteins with function of FCRL3, however, may be altered depending on the cell FCRL3, we also examined binding of these adaptor proteins to environment, particularly expression levels of adaptor proteins, as chimeric protein in ST486 transfectants under the condition of suggested by the specific binding of ZAP-70 and SHIP in Jukat BCR signaling. Chimeric proteins were immunoprecipitated alone and ST486 cells, respectively. or after colligation with BCR using intact anti-IgM Abs. Although Genes for the six Fc receptor-like molecules (FCRL1–6) lie in SHIP and SHP-1 and -2 were detected binding to chimeric proteins the 1q21–23 chromosomal region and share some characteristics in which tyrosine residues were phosphorylated, Syk association (23). The proteins are preferentially expressed in B cell lineages, was below detectable levels (Fig. 5C). We also immunoprecipi- showing homologous structures consisting of Ig-like extracellular tated mutational proteins (CM-F650, CM-F662, CM-F692, and domains and potential ITIM and/or ITAM motifs in the cytoplas- CM-F722) to examine the role of individual tyrosine motifs. Bind- mic tails. FCRL1, which has two potential ITAM motifs, is the ing of SHIP and SHP-1 was highly reduced in CM-F662 and CM- only member that has been shown to have an activating coreceptor F692, respectively, matching the results of peptide experiments. In function on B-cells. FCRL4 and FCRL5, in contrast, have been contrast, binding of SHP-2 was substantially reduced in CM-F692, shown to exhibit inhibitory potential on BCR-signaling by recruit- Downloaded from despite the greatest binding capacity of peptide pY662, indicating ing tyrosine phosphatases including SHP-1, SHP-2, and SHIP to that SHP-2 mainly binds to tyrosine motif at 692 under physio- the ITIM motifs. Interestingly, FCRL5 has both ITIMs and an logical conditions. ITAM, just like FCRL3, and has been shown to inhibit B cell activation via SHP-1 recruitment of tyrosine phosphatase to the Discussion ITIM (24). The dominant effect of ITIM over ITAM in FCRL5 In the present study using B cell transfectants of FCRL3 chimeric could be explained by the lower binding capacity of Syk binding http://www.jimmunol.org/ proteins, which display the unique property of having both ITAM to ITAM or the dominant antagonistic effect of phosphatases on and ITIM, we demonstrated the inhibitory potential of FCRL3 on kinases, as was the case in FCRL3. Together with differential ex- BCR-mediated signaling. When colligated with BCR, FCRL3 chi- pression of FCRLs in the B cell lineage and the potential for both meric protein inhibited BCR-mediated cell tyrosine phosphoryla- activatory and inhibitory potential on B cells, these mechanisms tion and calcium mobilization. FCRL3 also inhibited activation- provide sophisticated control of the differentiation of B-cells and induced cell death followed by prolonged BCR signaling. Analysis may influence the fate of individual B cell clones. with mutational chimeric proteins showed tyrosine residues in the Tolerance susceptibility of B cells and thresholds of BCR sig- cytosolic domain were essential for these inhibitory effects of naling can be modulated by coreceptors, as demonstrated by sev- by guest on October 2, 2021 FCRL3. Binding assays using phosphopeptides of the tyrosine mo- eral transgenic and knockout mice models (17). For instance, tifs displayed that SHIP associated with the phosphotyrosine motif transgenic mice that overexpress human CD19, an activating co- at 662, while SHP-1 and SHP-2 bound to phosphotyrosine motifs receptor on B cells, exhibit increased levels of autoantibodies (25), at 662 and 692 (stronger association at 692 under physiological while gene targeting of Fcgr2b that has an inhibitory potential on conditions). Both of the tyrosine motifs at 662 and 692 displayed BCR signaling renders several murine strains susceptible to auto- inhibitory potential on BCR-induced cell death according to the immune disorders (26, 27). In humans, polymorphism in the trans- study of the mutational constructs. Taken together, the two ty- membrane region of FCGR2B gene is reportedly associated with rosine motifs at 662 and 692 participate in the inhibitory effects of susceptibility to SLE (28). As the mutant allele of FCGR2B cannot FCRL3 on BCR-mediated signaling through binding of phospha- associate with the lipid rafts where the BCR complex resides, loss tases SHIP, SHP-1, and SHP2. Although the potential ITAM in- of inhibition in BCR signaling may promote autoimmune response cluded tyrosines at 650 and 662 bound with Syk in ST486 cells, (29). These findings indicate enhancement or loss of inhibition in which play important roles in initiating BCR-signaling, no activa- BCR signaling by coreceptors augments autoimmune response in tion signaling was observed in ST486 transfectant when the both mice and humans. FCRL3 chimeric protein were ligated alone. This may be ex- Intriguingly, and contrary to this, the results presented in this plained by the functional predominance of phosphatases SHIP, study show an opposing role for FCRL3, with inhibitory potential SHP-1, and SHP-2, which could antagonize BCR-signaling initi- on BCR signaling, where gain of gene function due to elevated ated by Syk and other phosohoproteins in the signalosome. The expression leads to autoimmunity. Supporting evidence for our lack of detectable levels of Syk association in coprecipitation with observation that reduced BCR-signaling leads to autoimmunity has FCRL3 chimeric protein further implicates relatively lower acti- recently been reported by Kumar et al. using the NZM240 murine vation potential of the ITAM in FCRL3. lupus model (30). By crossing a congenic strain bearing the Sle1 The enhanced calcium influx observed in CM-Y650 and CM- locus of lupus susceptibility with the HEL/Ig-HEL transgenic Y722 transfectants in DT40 cells implies additional biological ef- strain, they demonstrated that B cell tolerance was critically reg- fect of FCRL3 cytosolic domain in the absence of tyrosine motifs ulated by Ly108 gene, a member of the Slam gene family. Lupus- at 662 and 692, which may be mediated by unknown adaptor mol- associated allele Ly108.1 reduced the strength of BCR signaling ecules binding to FCRL3. As this enhancement was not observed compared with the normal allele Ly108.2. This reduced BCR sig- in CM-Mt transfectant, at least one intact tyrosine-motif at either naling resulted in incomplete induction of anergy and deletion in 662 or 692 is needed. In contrast, enhanced apoptosis was ob- autoreactive B cells, leading to breakdown of tolerance. This observed in the CM-Mt tranfectant in ST486 cells. Similar observa- servation led us to consider that augmented expression of FCRL3 tions were reported for FCGR2B, where a mutant FCGR2B protein in individuals with the disease-susceptibility genotype may inter- lacking SHIP recruitment to the mutated ITIM showed proapop- fere with the induction of B cell tolerance by up-regulating the 5510 FCRL3 INHIBITS BCR SIGNALING activation threshold of BCR signaling. More interestingly, parallel 10. Thabet, M. M., J. Wesoly, P. E. Slagboom, R. E. Toes, and T. W. Huizinga. 2007. situations have been described in T cell-mediated autoimmunities FCRL3 promoter 169 CC homozygosity is associated with susceptibility to rheumatoid arthritis in Dutch Caucasians. Ann. Rheum. Dis. 66: 803–806. in both mouse and human autoimmune diseases. In SKG mice, a 11. Newman, W. G., Q. Zhang, X. Liu, E. Walker, H. Ternan, J. Owen, B. Johnson, new model for rheumatoid arthritis, inadequate TCR signaling due W. Greer, D. P. Mosher, W. P. Maksymowych, et al. 2006. Rheumatoid arthritis association with the FCRL3–169C polymorphism is restricted to PTPN22 1858T- to a mutation in Zap70 caused tolerance breakdown of T cells (31). homozygous individuals in a Canadian population. Arthritis Rheum. 54: In several human autoimmune diseases, a mutation in PTPN22, 3820–3827. which encodes tyrosine phosphatases in the TCR complex, report- 12. Simmonds, M. J., J. M. Heward, J. Carr-Smith, H. Foxall, J. A. Franklyn, and S. C. Gough. 2006. Contribution of single nucleotide polymorphisms within edly reduces TCR response in T cells and increases the risk of FCRL3 and MAP3K7IP2 to the pathogenesis of Graves’ disease. J. Clin. Endo- diseases (2, 3, 32). Given that T and B cells show strong similar- crinol. Metab. 91: 1056–1061. ities in Ag-receptor signaling and mechanisms of tolerance induc- 13. Burton, P. R., D. G. Clayton, L. R. Cardon, N. Craddock, P. Deloukas, A. Duncanson, D. P. Kwiatkowski, M. I. McCarthy, W. H. Ouwehand, tion, the notion that reduction of Ag receptor signaling may be a N. J. Samani, et al. 2007. Association scan of 14,500 nonsynonymous SNPs in common mechanism of tolerance breakdown in both T and B cells four diseases identifies autoimmunity variants. Nat. Genet. 39: 1329–1337. is intriguing; autoreactive lymphocytes would be rescued from tol- 14. Umemura, T., M. Ota, H. Hamano, Y. Katsuyama, K. Kiyosawa, and S. Kawa. 2006. Genetic association of Fc receptor-like 3 polymorphisms with autoimmune erance mechanisms due to incomplete induction of anergy or de- pancreatitis in Japanese patients. Gut 55: 1367–1368. letion that should have been induced by self Ags. 15. Fodor, S., Z. Jakus, and A. Mocsai. 2006. ITAM-based signaling beyond the adaptive immune response. Immunol. Lett. 104: 29–37. In conclusion, FCRL3 displays inhibitory potential on BCR- 16. Daeron, M., S. Jaeger, L. Du Pasquier, and E. Vivier. 2008. Immunoreceptor mediated signaling, and greater inhibition in cells with the disease- tyrosine-based inhibition motifs: a quest in the past and future. Immunol. Rev. risk genotype may lead to breakdown of tolerance. Blockade of 224: 11–43. FCRL3 function might thus restore tolerance in autoreactive B 17. Poe, J. C., M. Hasegawa, and T. F. Tedder. 2001. CD19, CD21, and CD22:

multifaceted response regulators of B lymphocyte signal transduction. Int. Rev. Downloaded from cells and could represent a potential therapy for autoimmune dis- Immunol. 20: 739–762. eases. To further clarify the involvement of FCRL3 in autoimmu- 18. Yamashita, Y., M. Ono, and T. Takai. 1998. Inhibitory and stimulatory functions of paired Ig-like receptor (PIR) family in RBL-2H3 cells. J. Immunol. 161: nity and the potency of FCRL3-tageted therapy, identification of 4042–4047. the unknown ligands and analysis in animal disease models are 19. Wienands, J., O. Larbolette, and M. Reth. 1996. Evidence for a preformed trans- needed. ducer complex organized by the B cell antigen receptor. Proc. Natl. Acad. Sci. USA 93: 7865–7870. 20. Galibert, L., N. Burdin, C. Barthelemy, G. Meffre, I. Durand, E. Garcia, Acknowledgments P. Garrone, F. Rousset, J. Banchereau, and Y. J. Liu. 1996. Negative selection of http://www.jimmunol.org/ We are grateful to Y. Hayashi, E. Kanno, and all other members of the human germinal center B cells by prolonged BCR cross-linking. J. Exp. Med. Laboratory for Autoimmune Diseases, Center for Genomic Medicine 183: 2075–2085. 21. Kaptein, J. S., C. K. Lin, C. L. Wang, T. T. Nguyen, C. I. Kalunta, E. Park, (CGM, former SNP Research Center), RIKEN for their advice and tech- F. S. Chen, and P. M. Lad. 1996. Anti-IgM-mediated regulation of c-myc and its nical assistance. possible relationship to apoptosis. J. Biol. Chem. 271: 18875–18884. 22. Ono, M., H. Okada, S. Bolland, S. Yanagi, T. Kurosaki, and J. V. Ravetch. 1997. Disclosures Deletion of SHIP or SHP-1 reveals two distinct pathways for inhibitory signaling. Cell 90: 293–301. The authors have no financial conflict of interest. 23. Davis, R. S. 2007. Fc receptor-like molecules. Annu. Rev. Immunol. 25: 525–560. 24. Haga, C. L., G. R. Ehrhardt, R. J. Boohaker, R. S. Davis, and M. D. Cooper. 2007. 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