HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Adachi, T. Articles by Tsubata, T. Search for Related Content PubMed PubMed Citation Articles by Adachi, T. Articles by Tsubata, T.

CD72 Negatively Regulates Signaling Through the Antigen Receptor of B Cells¹

Takahiro Adachi^*, Chisato Wakabayashi^*, Toshinori Nakayama, Hidetaka Yakura and Takeshi Tsubata^2,*

^* Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan; Department of Molecular Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan; and Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The immunoreceptor tyrosine-based inhibition motif (ITIM) is found in various membrane molecules such as CD22 and the low-affinity Fc receptor for IgG in B cells and the killer cell-inhibitory receptor and Ly-49 in NK cells. Upon tyrosine phosphorylation at the ITIMs, these molecules recruit SH2 domain-containing phosphatases such as SH2-containing tyrosine phosphatase-1 and negatively regulate cell activity. The B cell surface molecule CD72 carries an ITIM and an ITIM-like sequence. We have previously shown that CD72 is phosphorylated and recruits SH2-containing tyrosine phosphatase-1 upon cross-linking of the Ag receptor of B cells (BCR). However, whether CD72 modulates BCR signaling has not yet been elucidated. In this paper we demonstrate that expression of CD72 down-modulates both extracellular signal-related kinase (ERK) activation and Ca²⁺ mobilization induced by BCR ligation in the mouse B lymphoma line K46µm, whereas BCR-mediated ERK activation was not reduced by the ITIM-mutated form of CD72. Moreover, coligation with CD72 with BCR reduces BCR-mediated ERK activation in spleen B cells of normal mice. These results indicate that CD72 negatively regulates BCR signaling. CD72 may play a regulatory role in B cell activation, probably by setting a threshold for BCR signaling.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ligation of the Ag receptor of B cells (BCR)³ activates the cytoplasmic kinase Syk and Src-family kinases such as Lyn (1). Those kinases then activate various signaling cascades including Ca²⁺ mobilization and mitogen-activated protein kinases (MAPK) such as extracellular signal-related kinase (ERK), resulting in cell proliferation, anergy, or apoptosis. BCR signaling is regulated either positively or negatively by various membrane molecules (2). For example, CD19, CD21, and CD45 are implicated to regulate BCR signaling positively. As for negative regulation, the low-affinity Fc receptor for IgG (FcRII), CD22, and paired Ig-like receptor B have been shown to down-modulate B cell activation when coligated with BCR (3, 4, 5, 6, 7, 8). Those inhibitory coreceptors carry the conserved immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic tail (9). ITIMs are also found in inhibitory receptors in other hemopoietic cell lineages such as NK cells (10). Upon tyrosine phosphorylation, ITIMs recruit and activate SH2-containing phosphatases such as SH2-containing tyrosine phosphatase-1 (SHP-1) and SH2-containing inositol 5-phosphatase (SHIP), which in turn down-regulate cell activity (9).

When B cells interact with Ags complexed with IgG, FcRII is coligated with BCR. The coligation induces phosphorylation of FcRII by the BCR-associated kinase Lyn (4, 11, 12). Phosphorylated FcRII then down-modulates Ca²⁺ mobilization and cell proliferation by recruiting and activating SHIP (4, 5). However, BCR cross-linking alone fails to induce phosphorylation of FcRII or its recruitment of SHIP. These findings indicate that FcRII inducibly down-modulates BCR signaling upon coligation with BCR by Ag-IgG immune complexes. This conclusion is also supported by a finding on mice deficient in FcRII (13). Those mice show enhanced B cell response to intact anti-Ig Abs interacting with both BCR and FcRII, whereas the response to F(ab')₂ fragments of anti-Ig Abs ligating BCR but not FcRII is comparable between FcRII-deficient and wild-type mice. Thus, FcRII may play a role in negative feedback regulation, in which activation of B cells is down-modulated once the specific IgG is produced and forms an immune complex with Ags. In contrast, a fraction of CD22 is associated with BCR, and BCR ligation induces phosphorylation of CD22 (14, 15, 16), indicating that CD22 is constitutively associated with BCR both physically and functionally. This notion is also supported by the finding that B cell activation induced by BCR ligation alone is enhanced in mice deficient in CD22 (17, 18, 19, 20). Upon phosphorylation, CD22 recruits SHP-1 and reduces both Ca²⁺ mobilization and MAPK activation (6, 17, 18, 19, 20, 21). Although CD22 constitutively associates with BCR, coligation of CD22 with BCR further reduces BCR signaling such as MAPK activation (21), probably because the coligation enhances association between CD22 and BCR. It is suggested that by down-modulating BCR signaling constitutively, CD22 sets a threshold for BCR ligation (22, 23, 24). Such a threshold may play a role in protecting B cells from BCR firing either spontaneously or by weak cross-reactive interaction with Ags.

CD72 is a 45-kDa type II membrane protein containing a C-type lectin-like domain and is expressed on most B-lineage cells as a homodimer (25, 26, 27). CD72 carries an ITIM and an ITIM-like sequence in the cytoplasmic tail. We (28) and Wu et al. (29) have previously demonstrated that CD72 is tyrosine-phosphorylated and recruits SHP-1 upon BCR ligation, as is the case for CD22. However, it has not yet been known whether CD72 negatively regulates BCR signaling. In this paper we demonstrate that expression of CD72 inhibits ERK activation and Ca²⁺ mobilization by BCR cross-linking even in the absence of coligation of CD72 with BCR. This result strongly suggests that CD72 constitutively down-modulates BCR signaling and sets a signaling threshold for B cell activation, as is the case for CD22.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice and cells

DBA/2 and BALB/c mice were purchased from Sankyo (Tokyo, Japan). Spleen B cells were purified as described previously (30). K46µm, a transfectant of the mouse B lymphoma line K46 expressing both the µ H and L chains of IgM specific for hapten (4-hydroxy-3-nitrophenyl) acetyl (NP), was kindly provided by Drs. M. Reth and J. Wienands (31). The mouse B lymphoma line WEHI-231.5 was described previously (28). Cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 50 µM 2-ME, and 1 mM glutamine. cDNA encoding a mutated CD72, in which tyrosine residues at 7 and 39 were replaced by phenylalanine (SHP-1Y/F), was generated by site-directed mutagenesis using mouse pBSCD72 carrying the CD72 cDNA (28) as a template and was inserted into the expression vector pMKITneo (a gift from Dr. K. Maruyama, Tokyo Medical and Dental University) (pMKITCD72Y/F). The expression plasmids pMikCD72 (28) and pMKITCD72Y/F were transfected by electroporation. K46µm and its transfectants were stimulated with NP₁₅-coupled BSA (NP-BSA). For coligation of BCR with CD72 on normal B cells, purified spleen B cells from DBA/2 mice were pretreated with 10 µg/ml of mAb 2.4G2 (a gift from Dr. N. Sorimachi) reacting to mouse FcRII on ice for 5 min. Cells were then added with either 10 µg/ml of anti-mouse CD72^a mAb 9-6.1 (mouse IgG2b, ) (32) or the same amount of an isotype-matched control mAb (Zymed, San Francisco, CA) before incubation on ice for 5 min. After washing with PBS, cells were suspended in medium at 37°C and added with rat anti-mouse -chain mAb 187.1 (final concentration, 20 µg/ml). Alternatively, spleen B cells from BALB/c mice were pretreated with 10 µg/ml of anti-mouse CD72^b mAb CT72.2 (mouse IgM; Cedarlane Laboratories, Hornby, Ontario, Canada) or with 10 µg/ml anti-NP mAb B1-8 (mouse IgM) (33) before incubation on ice for 5 min. After washing with PBS, cells were suspended in medium at 37°C and added with F(ab')₂ fragments of goat anti-mouse IgM Ab (ICN Pharmaceuticals, Aurora, OH) (final concentration, 20 µg/ml).

Flow cytometry

Cells were stained using the following reagents: FITC-labeled goat anti-mouse µ-chain Ab (Southern Biotechnology Associates, Birmingham, AL), FITC-labeled goat anti-mouse -chain Ab (Southern Biotechnology Associates), biotin-labeled anti-mouse CD72^a mAb 9-6.1 (32), biotin-labeled anti-mouse CD72^b mAb CT72.2 (Cedarlane), and FITC-labeled streptavidin (Dako, Glostrup, Denmark). Cells were analyzed by flow cytometry using a FACSCalibur (Becton Dickinson, San Jose, CA).

Western blot analysis

Cells were lysed in SDS-PAGE sample buffer and Western blot analysis was done as described previously (28) using anti-phospho-ERK Ab (New England Biolabs, Beverly, MA), rabbit anti-ERK2 Ab (Santa Cruz Biotechnology, Santa Cruz, CA), or rabbit anti-mouse CD72 Ab generated against the GST-CD72 fusion protein carrying the cytoplasmic tail of CD72 conserved among different CD72 allotypes (28).

Measurement of intracellular Ca²⁺ concentration

Cells (1 x 10⁵) of K46µm and its transfectants were incubated in culture medium containing 5 µM Fluo-3/AM (Molecular Probes, Eugene, OR) and 0.02% (w/v) pluronic F-127 (Sigma, St. Louis, MO) at 37°C for 30 min. After washing, cells were suspended in HEPES buffer (10 mM HEPES (pH 7.4), 140 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 0.6 mM MgCl₂, 1 mg/ml BSA, and 1 mg/ml D-glucose). Fluo-3 fluorescence of cells was measured continuously by flow cytometry using a FACSCalibur (Becton Dickinson). Cells were added with NP-BSA or a calcium ionophore A23187, and data was collected for a total of 300 s.

Purified spleen B cells (1 x 10⁶) were incubated in culture medium containing 1 µM Indo-1/AM (Molecular Probes) and 0.02% (w/v) pluronic F-127 at 37°C for 30 min. After washing three times, cells were incubated in Hanks’ solution containing either 10 µg/ml of anti-mouse CD72^b mAb CT72.2 (mouse IgM; Cedarlane) or the same amount of anti-NP mAb B1-8 (mouse IgM) for 15 min on ice. After washing once, cells were suspended in Hanks’ solution and incubated at 37°C for 10 min, and the ratio of Indo-1 violet/blue of cells was measured continuously by flow cytometry using a FACSVantage (Becton Dickinson) as described (34). Cells were added with F(ab')₂ fragments of goat anti-mouse IgM Abs (ICN Pharmaceuticals) (final concentration, 20 µg/ml), and data was collected for a total of 200 s.

In vitro kinase assay

Cells (2 x 10⁶) were lysed in 400 µl of lysis buffer (25 mM Tris-HCl (pH 7.4), 137 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 10% glycerol, 1 mM DTT, 1 mM Na₃VO₄, 10 mM NaF, and 1 mM PMSF). Cleared cell lysates were then incubated with 1 µg of anti-ERK2 Ab (Santa Cruz Biotechnology) and 30 µl of protein G-Sepharose beads (Pharmacia Biotech, Uppsala, Sweden). After washing twice with lysis buffer, once with washing buffer (25 mM Tris-HCl (pH 8.0), and 500 mM LiCl), and once with kinase buffer (25 mM HEPES (pH 7.4), 10 mM MgCl₂, 10 mM MnCl₂, and 2 mM DTT), the beads were incubated in 20 µl of kinase buffer containing 5 µg of bovine myelin basic protein (MBP) (Sigma) and 5 µCi of [-³²P]ATP (Amersham, Amersham, U.K.) at room temperature for 20 min. The reaction was terminated by adding SDS-PAGE sample buffer, and proteins were separated by SDS-PAGE before autoradiography. Phosphorylation of MBP was quantitated by a BAS-2500 (Fuji Photo Film, Tokyo, Japan).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD72 negatively regulates both ERK activation and Ca²⁺ mobilization induced by BCR ligation in the K46µm B lymphoma cells

To investigate the signaling function of CD72, we assessed CD72 expression on the surface of B cell lines by flow cytometry. The B lymphoma line K46µm was not stained by Ab to CD72^b (Fig. 1), although this line is derived from a BALB/c mouse carrying the CD72^b allotype. This indicated that K46µm does not express CD72 on the surface. Moreover, Western blot analysis using rabbit Ab reacting to the cytoplasmic region conserved among different CD72 allotypes showed that CD72 was undetectable in the total cell lysates of K46 (data not shown). Taken together, K46µm is likely to lack CD72 production. We then transfected K46µm with pMikCD72 containing the CD72^a cDNA. For further analysis, we chose two transfectants, K46µmCD72-4 and K46µmCD72-6 because they expressed a significant amount of CD72^a on the surface and expressed similar amounts of surface IgM (µ and ) to the parent K46µm cells (Fig. 1).

View larger version (49K): [in this window] [in a new window]   FIGURE 1. Expression of IgM and CD72 on cell surface. Cells of K46µm (A–D) and its CD72a transfectants (K46µmCD72-4 (E–H) and K46µmCD72-6 (I–L)) were stained with FITC-labeled goat anti-mouse µ-chain Ab (A, E, and I) or FITC-labeled goat anti-mouse -chain Ab (B, F, and J). Alternatively, cells were reacted with biotinylated anti-mouse CD72a mAb 9.6.1 (C, G, and K) or biotinylated anti-mouse CD72b mAb CT72.2 (D, H, and L) before staining with FITC-labeled streptavidin. As a positive control for CD72b expression, WEHI-231.5 cells were stained with the combination of biotinylated CT72.2 and FITC-labeled streptavidin (M). Cells reacted without Abs and those reacted with biotinylated anti-CD72 mAb alone were used as negative controls for expression of IgM (µ and ) and CD72, respectively (gray histograms). Cells were analyzed by flow cytometry using a FACSCalibur (Becton Dickinson).

View larger version (40K): [in this window] [in a new window]   FIGURE 2. CD72 expression reduces BCR-mediated phosphorylation of ERK in K46µm cells. Cells (5 x 105) of K46µm and its CD72a transfectants (K46µmCD72-4 and K46µmCD72-6) were treated with the indicated amounts of NP-BSA for 3 min (A) or with 10 µg/ml of NP-BSA for the indicated times (B) at 37°C. As negative controls, cells were treated with medium alone. Cells were subsequently lysed and subjected to Western blot analysis using anti-phospho-ERK Ab. Please note that the data on ERK1 and ERK2 were taken from the same membrane with different exposure times because the intensity of the phospho-ERK1 band in each lane was much less than that of the phospho-ERK2 band. Numbers under each lane indicate relative intensities of phospho-ERK2 band. The same blots were reprobed with anti-ERK2 Ab to ensure equal loading. Representative data of three experiments are shown.

View larger version (40K): [in this window] [in a new window]   FIGURE 3. In vitro kinase assay for ERK2. Cells (2 x 106) of K46µm and its CD72a transfectants (K46µmCD72-4 and K46µmCD72-6) were treated with medium alone or with 10 µg/ml of NP-BSA for the indicated times at 37°C. Cells were then lysed in lysis buffer, and cleared cell lysates were incubated with 1 µg of anti-ERK2 Ab together with 30 µl of protein G-Sepharose beads. After washing, half of the beads were incubated in kinase buffer containing 5 µg of MBP and 5 µCi of [-32P]ATP at room temperature for 20 min. The reaction was terminated by adding SDS-PAGE sample buffer, and proteins were separated by SDS-PAGE before autoradiography. Numbers under each lane indicate the cumulative fold increase in ERK2 activity over that found in unstimulated K46µm. The other half of each anti-ERK2 immunoprecipitates were analyzed by Western blotting using anti-ERK2 Ab to ensure the existence of ERK2 in the immunoprecipitates. The immunoprecipitate from the parent K46µm contained a smaller amount of ERK2 than the CD72 transfectants did, probably because the number of K46µm cells used for this experiment was less than that initially estimated. However, please note that the variation of the amount of ERK did not weaken the difference of ERK activity between K46µm and its CD72 transfectant as shown by the fact that the immunoprecipitate of K46µm contained a smaller amount of ERK2 but showed higher ERK2 kinase activity compared with the same indicators for CD72 transfectants. Representative data of three experiments are shown.

View larger version (24K): [in this window] [in a new window]   FIGURE 4. CD72 reduces Ca2+ mobilization induced by BCR ligation. Cells (1 x 105) of K46µm, K46µmCD72-4, and K46µmCD72-6 were loaded with 5 µM Fluo-3 in the presence of 0.02% pluronic F-127 at 37°C for 30 min. After washing, cells were suspended in HEPES buffer. Intracellular free Ca2+ concentration was measured by a FACSCalibur. Cells were added with the indicated concentrations of NP-BSA (A–C) or with 5 mM A23187 (D) at 30 sec (indicated by arrows), and measurement of free Ca2+ concentration was continued for 360 s. Representative data of five experiments are shown.

View larger version (26K): [in this window] [in a new window]   FIGURE 5. Expression of an ITIM-mutated form of CD72 (CD72Y/F) fails to reduce BCR-mediated phosphorylation of ERK in K46µm cells. A and B, CD72 expression on the cell surface. Cells of a K46µm CD72Y/F transfectant (B) were reacted with biotinylated anti-mouse CD72a mAb 9.6.1 before staining with FITC-labeled streptavidin. As a control, K46µmCD72-4 cells (A) were stained as parallel. Cells reacted without Abs were used as negative controls (gray histograms). Cells were analyzed by flow cytometry using a FACSCalibur (Becton Dickinson). C and D, Phosphorylation of ERK. Cells (5 x 105) of K46µm and its CD72Y/F transfectant were treated with the indicated amounts of NP-BSA for 3 min (C) or with 10 µg/ml of NP-BSA for the indicated times (D) at 37°C. As negative controls, cells were treated with medium alone. Cells were subsequently lysed and subjected to Western blot analysis using anti-phospho-ERK Ab. Please note that the data on ERK1 and ERK2 were taken from the same membrane with different exposure times because the intensity of the phospho-ERK1 band in each lane was much less than that of the phospho-ERK2 band. Numbers under each lane indicate the relative intensities of the phospho-ERK2 bands. The same blots were reprobed with anti-ERK2 Ab to ensure equal loading. Representative data of three experiments are shown.

To investigate whether CD72 negatively regulates BCR signaling in normal B cells, we isolated spleen B cells from 10-wk-old BALB/c mice carrying the CD72^b allotype and cross-linked BCR together with CD72 to enhance the regulatory effect of CD72 on BCR signaling. We treated B cells with either anti-CD72^b mAb CT72.2 (mouse IgM) or an isotype-matched control mAb B1-8 on ice before addition of F(ab')₂ fragments of anti-mouse IgM Ab. The treatment with the combination of CT72.2 and anti-IgM Ab coligated CD72 with BCR as anti-IgM Ab reacted to both CT72.2 and BCR (surface IgM) of B cells. In contrast, treatment with the combination of the control Ab and anti-IgM Ab ligated BCR alone. Because CT72.2, B1-8, and F(ab')₂ fragments of anti-IgM Ab do not contain Fc, these treatments did not coligate FcR with BCR. After treatment with anti-IgM Ab at 37°C for 5 min, we collected B cells as BCR ligation induced the maximal ERK2 phosphorylation at this time point (Fig. 6A). Western blotting of total cell lysates using anti-phospho-ERK Ab showed that both ERK1 and ERK2 were phosphorylated by either BCR ligation alone or coligation of BCR and CD72 (Fig. 6B). However, BCR ligation induced stronger ERK phosphorylation than coligation of CD72 with BCR did, indicating that BCR ligation-induced phosphorylation of ERK is down-modulated when CD72 is coligated with BCR. To confirm this observation, we coligated CD72 with BCR on spleen B cells from DBA/2 mice carrying CD72^a using anti-CD72^a and anti-mouse mAb. Because both anti-CD72^a mAb and anti- mAb contain Fc, we blocked FcR by pretreating B cells with anti-FcR mAb 2.4G2. Phosphorylation of both ERK1 and ERK2 induced by coligation of BCR and CD72 was weaker than that induced by BCR ligation alone (Fig. 6C), indicating that coligation with CD72 reduced BCR ligation-mediated phosphorylation of ERK in DBA/2 spleen cells.

View larger version (19K): [in this window] [in a new window]   FIGURE 6. BCR-mediated phosphorylation of ERK and Ca2+ mobilization in spleen B cells of normal mice. A, Time course analysis of BCR-mediated ERK2 activation. Purified spleen B cells (3 x 106) from 10-wk-old BALB/c mice were incubated with 20 µg/ml of rabbit anti-mouse -chain Ab at 37°C for the indicated times. Cells were then lysed and subjected to Western blot analysis using anti-phospho-ERK Ab. The blots were then reprobed with anti-ERK2 Ab to ensure equal loading. B, Coligation of CD72 with BCR reduces BCR-mediated ERK activation in BALB/c B cells. Purified spleen B cells from 10-wk-old BALB/c mice were incubated with 10 µg/ml of anti-mouse CD72b mAb CT72.2 or with an isotype-matched control mAb B1-8 on ice for 5 min. After washing, cells were incubated with or without 20 µg/ml of goat anti-mouse IgM Ab at 37°C for 5 min. Cells were then lysed and subjected to Western blot analysis using anti-phospho-ERK Ab. Numbers under each lane indicate relative intensities of the phospho-ERK2 bands. The blots were then reprobed with anti-ERK2 Ab to ensure equal loading. Representative data of three experiments are shown. C, Coligation of CD72 with BCR reduces BCR-mediated ERK activation in DBA/2 spleen B cells. Purified spleen B cells from 10-wk-old DBA/2 mice were preincubated with 10 µg/ml of anti-mouse FcRII mAb 2.4G2 on ice for 5 min. Cells were then added with 10 µg/ml of anti-mouse CD72a mAb 9-6.1 or with an isotype-matched control mAb and incubated on ice for an additional 5 min. After washing, cells were incubated with or without 20 µg/ml of anti-mouse -chain mAb 187.1 at 37°C for 5 min. Cells were then lysed and subjected to Western blot analysis using anti-phospho-ERK Ab. Numbers under each lane indicate the relative intensities of phospho-ERK2 bands. The blots were then reprobed with anti-ERK2 Ab to ensure equal loading. Representative data of three experiments are shown. D, Coligation of CD72 with BCR reduces BCR-mediated Ca2+ mobilization. Purified spleen B cells (3 x 106) from 10-wk-old BALB/c mice were loaded with 1 µM Indo-1/AM in the presence of 0.02% pluronic F-127 at 37°C for 30 min. After washing, cells were incubated with either anti-CD72b mAb CT72.2 (mouse IgM) or with isotype-matched control mAb B1-8 for 15 min on ice. Intracellular free Ca2+ concentration was measured by a FACSVantage (Becton Dickinson). Cells were added with 20 µg/ml of anti-IgM Ab at the time point indicated by an arrow, and measurement of free Ca2+ concentration was continued for a total of 200 s.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
By taking advantage of the finding that the B lymphoma line K46µm expresses no detectable CD72, we have established K46µm transfectants expressing either wild-type CD72 or ITIM-mutated CD72 and demonstrated that expression of CD72 diminishes both ERK activation and Ca²⁺ mobilization induced by BCR ligation, whereas the ITIM-mutated form of CD72 does not reduce BCR-mediated ERK activation. Moreover, coligation of CD72 with BCR down-modulates both BCR-mediated ERK activation and Ca²⁺ mobilization in normal spleen B cells. These results strongly suggest that CD72 negatively regulates BCR signaling in B cell lines and normal mature B cells and that the ITIM and/or the ITIM-like sequence in CD72 are crucial for its negative regulatory effect on BCR signaling. Because induced coligation of CD72 with BCR is not required for negative regulation of BCR signaling by CD72 in K46µm cells, CD72 may interact with BCR and down-modulate its signaling constitutively. This observation is in agreement with the previous finding that BCR ligation alone induces both phosphorylation of CD72 and recruitment of SHP-1 to CD72 (28, 29), indicating that CD72 functionally interacts with BCR even in the absence of coligation of CD72 with BCR. However, induced coligation of CD72 with BCR by using Abs to those molecules diminishes BCR signaling in spleen B cells (Fig. 6). Coligation of CD72 with BCR may enhance interaction of CD72 with BCR, resulting in further down-modulation of BCR signaling. This observation is in agreement with the finding on CD22 that coligation of CD22 with BCR further reduces BCR signaling (21), although several lines of evidence indicate that CD22 negatively regulates BCR signaling constitutively (22, 23, 24). Taken together, CD72 appears to constitutively down-modulate BCR signaling, but its negative regulatory effect is further enhanced by coligation of CD72 with BCR.

Treatment of B cells with anti-CD72 Abs has been shown to enhance activation and proliferation of normal mature B cells induced by BCR ligation (32, 35). However, this observation may not contradict the idea of a negative regulatory role of CD72 on BCR signaling. Indeed, BCR-mediated B cell activation is enhanced by treatment with Abs to CD22 (36, 37), whose inhibitory role on BCR signaling has already been established by lines of evidence including that on CD22-deficient mice (17, 18, 19, 20). Anti-CD72 Abs may disrupt interaction between CD72 and BCR, resulting in enhancement of BCR signaling in the absence of the negative regulatory effect of CD72 on BCR signaling. Alternatively, CD72 transmits a stimulatory signal independent of BCR when CD72 is ligated by anti-CD72. This is consistent with the recent finding that CD72 ligation activates Src-family kinases Lyn and Blk in the absence of activation of Syk, which is essential for BCR signaling (38).

Both motheaten mice deficient in SHP-1 and Lyn-deficient mice show a marked increase in the number of plasma cells and development of autoimmune disease associated with autoantibody production (39, 40, 41, 42, 43). Thus, SHP-1 and Lyn may prevent development of autoimmune disease, probably by inhibiting B cell hyperactivity. This inhibitory role of SHP-1 and Lyn appears to involve CD22. Indeed, CD22 is a substrate of Lyn and induces activation of SHP-1 (12, 44), suggesting that CD22 is a component of a signaling pathway including Lyn and SHP-1. This notion is also supported by the genetic evidence obtained using mice with heterozygous deficiency in SHP-1, Lyn, or CD22 (45). Although CD22-deficient mice show B cell hyperresponsiveness, the severity of the defects in CD22-deficient mice is much milder than that of SHP-1-deficient motheaten mice. Thus, other ITIM-containing molecules in B cells may play a role in maintaining the normal immune response together with CD22 by activating SHP-1. FcRII may not be involved in this pathway, as shown by the fact that the inhibitory function of FcRII is mostly ascribed to SHIP and not SHP-1 (5, 46). In contrast, CD72 negatively regulates BCR signaling in a manner similar to that of CD22. Indeed, both CD22 and CD72 constitutively associate with BCR (15, 16, 47), are substrates of Lyn (12, 28, 44), recruit SHP-1 upon BCR ligation, and negatively regulate BCR signaling such as Ca²⁺ mobilization even in the absence of coligation with BCR ( Figs. 2–4 and 6 and Refs. 17, 18, 19, 20, 21). Moreover, the cytoplasmic tails of both CD22 and CD72 carry ITIMs essential for recruitment of SHP-1 and negative regulation of BCR signaling (Fig. 5 and Refs. 6, 28). Thus, CD72 may carry a function redundant with CD22 and, together with CD22, may maintain normal humoral immunity by activating an inhibitory signaling pathway involving Lyn and SHP-1. As defects in this pathway cause autoimmune disease with autoantibody production, defects in CD72 may be involved in development of autoimmune diseases.

CD72 may interact with its natural ligands through the extracellular region containing a C-type lectin-like domain. Interaction with the ligands probably modulates B cell activation induced by BCR ligation and may be involved in activation of B cells in certain humoral immune responses. CD5 expressed on T cells and B-1 cells has been shown to be a ligand for CD72 (48). However, this is controversial because Biancone et al. (49) and Bikah et al. (50) have recently demonstrated that CD5 fails to bind to CD72. Further elucidation of the role of CD72-mediated regulation of BCR signaling and its modification by CD72 ligands may be crucial for understanding the molecular mechanisms for normal and abnormal humoral immune responses.

	Acknowledgments

 
We thank Drs. M. Reth and J. Wienands (Freiburg University) for K46µm, Drs. N. Sorimachi (Tokyo Metropolitan Institute of Medical Science), K. Maruyama (Tokyo Medical and Dental University), and Y. Aiba for reagents, and Ms. Y. Shimokawa for technical assistance.

	Footnotes

 
¹ This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan; the Science and Technology Agency of Japan; the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research of Japan; Atsuko Ohuchi Memorial Research Fund; the Uehara Memorial Foundation; and the Mochida Memorial Foundation for Medical and Pharmaceutical Research.

² Address correspondence and reprint requests to Dr. Takeshi Tsubata, Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. E-mail address:

³ Abbreviations used in this paper: BCR, B cell Ag receptor; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-related kinase; FcRII, low-affinity Fc receptor for IgG; ITIM, immunoreceptor tyrosine-based inhibition motif; SHP-1, SH2-containing tyrosine phosphatase-1; SHIP, SH2-containing inositol 5-phosphatase; NP, (4-hydroxy-3-nitrophenyl) acetyl; NP-BSA, NP₁₅-coupled BSA; MBP, myelin basic protein.

Received for publication June 1, 1999. Accepted for publication November 16, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Reth, M., J. Wienands. 1997. Initiation and processing of signals from the B cell antigen receptor. Annu. Rev. Immunol. 15:453.[Medline]
2. Healy, J. I., C. C. Goodnow. 1998. Positive versus negative signaling by lymphocyte antigen receptors. Annu. Rev. Immunol. 16:645.[Medline]
3. Amigorena, S., C. Bonnerot, J. R. Drake, D. Choquet, W. Hunziker, J. G. Guillet, P. Webster, C. Sautes, I. Mellman, W. H. Fridman. 1992. Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes. Science 256:1808.[Abstract/Free Full Text]
4. Muta, T., T. Kurosaki, Z. Misulovin, M. Sanchez, M. C. Nussenzweig, J. V. Ravetch. 1994. A 13-amino-acid motif in the cytoplasmic domain of FcRIIB modulates B-cell receptor signalling. Nature 368:70.[Medline]
5. Ono, M., H. Okada, S. Bolland, S. Yanagi, T. Kurosaki, J. V. Ravetch. 1997. Deletion of SHIP or SHP-1 reveals two distinct pathways for inhibitory signaling. Cell 90:293.[Medline]
6. Doody, G. M., L. B. Justement, C. C. Delibrias, R. J. Matthews, J. Lin, M. L. Thomas, D. T. Fearon. 1995. A role in B cell activation for CD22 and the protein tyrosine phosphatase SHP. Science 269:242.[Abstract/Free Full Text]
7. Kubagawa, H., P. D. Burrows, M. D. Cooper. 1997. A novel pair of immunoglobulin-like receptors expressed by B cells and myeloid cells. Proc. Natl. Acad. Sci. USA 94:5261.[Abstract/Free Full Text]
8. Maeda, A., M. Kurosaki, M. Ono, T. Takai, T. Kurosaki. 1998. Requirement of SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 for paired immunoglobulin-like receptor B (PIR-B)-mediated inhibitory signal. J. Exp. Med. 187:1355.[Abstract/Free Full Text]
9. Yokoyama, W. M.. 1997. What goes up must come down: the emerging spectrum of inhibitory receptors. J. Exp. Med. 186:1803.[Free Full Text]
10. Lanier, L. L.. 1998. NK cell receptors. Annu. Rev. Immunol. 16:359.[Medline]
11. Chan, V. W., C. A. Lowell, A. L. DeFranco. 1998. Defective negative regulation of antigen receptor signaling in Lyn-deficient B lymphocytes. Curr. Biol. 8:545.[Medline]
12. Nishizumi, H., K. Horikawa, R. I. Mlinaric, T. Yamamoto. 1998. A double-edged kinase Lyn: a positive and negative regulator for antigen receptor-mediated signals. J. Exp. Med. 187:1343.[Abstract/Free Full Text]
13. Takai, T., M. Ono, M. Hikida, H. Ohmori, J. V. Ravetch. 1996. Augmented humoral and anaphylactic responses in FcRII-deficient mice. Nature 379:346.[Medline]
14. Schulte, R. J., M. A. Campbell, W. H. Fischer, B. M. Sefton. 1992. Tyrosine phosphorylation of CD22 during B cell activation. Science 258:1001.[Abstract/Free Full Text]
15. Leprince, C., K. E. Draves, R. L. Geahlen, J. A. Ledbetter, E. A. Clark. 1993. CD22 associates with the human surface IgM-B-cell antigen receptor complex. Proc. Natl. Acad. Sci. USA 90:3236.[Abstract/Free Full Text]
16. Peaker, C. J., M. S. Neuberger. 1993. Association of CD22 with the B cell antigen receptor. Eur. J. Immunol. 23:1358.[Medline]
17. O’Keefe, T. L., G. T. Williams, S. L. Davies, M. S. Neuberger. 1996. Hyperresponsive B cells in CD22-deficient mice. Science 274:798.[Abstract/Free Full Text]
18. Otipoby, K. L., K. B. Andersson, K. E. Draves, S. J. Klaus, A. G. Farr, J. D. Kerner, R. M. Perlmutter, C. L. Law, E. A. Clark. 1996. CD22 regulates thymus-independent responses and the lifespan of B cells. Nature 384:634.[Medline]
19. Sato, S., A. S. Miller, M. Inaoki, C. B. Bock, P. J. Jansen, M. L. Tang, T. F. Tedder. 1996. CD22 is both a positive and negative regulator of B lymphocyte antigen receptor signal transduction: altered signaling in CD22-deficient mice. Immunity 5:551.[Medline]
20. Nitschke, L., R. Carsetti, B. Ocker, G. Kohler, M. C. Lamers. 1997. CD22 is a negative regulator of B-cell receptor signalling. Curr. Biol. 7:133.[Medline]
21. Tooze, R. M., G. M. Doody, D. T. Fearon. 1997. Counterregulation by the coreceptors CD19 and CD22 of MAP kinase activation by membrane immunoglobulin. Immunity 7:59.[Medline]
22. Cyster, J. G., C. C. Goodnow. 1997. Tuning antigen receptor signaling by CD22: integrating cues from antigens and the microenvironment. Immunity 6:509.[Medline]
23. O’Rourke, L., R. Tooze, D. T. Fearon. 1997. Co-receptors of B lymphocytes. Curr. Opin. Immunol. 9:324.[Medline]
24. Tedder, T. F., J. Tuscano, S. Sato, J. H. Kehrl. 1997. CD22, a B lymphocyte-specific adhesion molecule that regulates antigen receptor signaling. Annu. Rev. Immunol. 15:481.[Medline]
25. Sato, H., E. A. Boyes. 1976. A new alloantigen expressed selectively on B cells: the Lyb-2 system. Immunogenetics 3:565.
26. Nakayama, E., I. von Hoegen, J. R. Parnes. 1989. Sequence of the Lyb-2 B-cell differentiation antigen defines a gene superfamily of receptors with inverted membrane orientation. Proc. Natl. Acad. Sci. USA 86:1352.[Abstract/Free Full Text]
27. Robinson, W. H., M. M. Landolfi, H. Schafer, J. R. Parnes. 1993. Biochemical identity of the mouse Ly-19.2 and Ly-32.2 alloantigens with the B cell differentiation antigen Lyb-2/CD72. J. Immunol. 151:4764.[Abstract]
28. Adachi, T., H. Flaswinkel, H. Yakura, M. Reth, T. Tsubata. 1998. The B cell surface protein CD72 recruits the tyrosine phosphatase SHP-1 upon tyrosine phosphorylation. J. Immunol. 160:4662.[Abstract/Free Full Text]
29. Wu, Y., M. J. Nadler, L. A. Brennan, G. D. Gish, J. F. Timms, N. Fusaki, B. J. Jongstra, N. Tada, T. Pawson, J. Wither, et al 1998. The B-cell transmembrane protein CD72 binds to and is an in vivo substrate of the protein tyrosine phosphatase SHP-1. Curr. Biol. 8:1009.[Medline]
30. Nomura, T., H. Han, M. C. Howard, H. Yagita, H. Yakura, T. Honjo, T. Tsubata. 1996. Antigen receptor-mediated B cell death is blocked by signaling via CD72 or treatment with dextran sulfate and is defective in autoimmunity-prone mice. Int. Immunol. 8:867.[Abstract/Free Full Text]
31. Wienands, J., J. Hombach, A. Radbruch, C. Riesterer, M. Reth. 1990. Molecular components of the B cell antigen receptor complex of class IgD differ partly from those of IgM. EMBO J. 9:449.[Medline]
32. Yakura, H., I. Kawabata, T. Ashida, F. W. Shen, M. Katagiri. 1986. A role for Lyb-2 in B cell activation mediated by a B cell stimulatory factor. J. Immunol. 137:1475.[Abstract]
33. Reth, M., G. J. Hammerling, K. Rajewsky. 1978. Analysis of the repertoire of anti-NP antibodies in C57BL/6 mice by cell fusion. I. Characterization of antibody families in the primary and hyperimmune response. Eur. J. Immunol. 8:393.[Medline]
34. Yamashita, M., M. Kimura, M. Kubo, C. Shimizu, T. Tada, R. M. Perlmutter, T. Nakayama. 1999. T cell antigen receptor-mediated activation of the Ras/mitogen-activated protein kinase pathway controls interleukin 4 receptor function and type-2 helper T cell differentiation. Proc. Natl. Acad. Sci. USA 96:1024.[Abstract/Free Full Text]
35. Bondada, S., D. E. Mosier. 1984. Activation of B lymphocytes by monovalent anti-Lyb-2 antibodies. J. Exp. Med. 159:1796.[Abstract/Free Full Text]
36. Pezzutto, A., B. Dorken, G. Moldenhauer, E. A. Clark. 1987. Amplification of human B cell activation by a monoclonal antibody to the B cell-specific antigen CD22, Bp 130/140. J. Immunol. 138:98.[Abstract]
37. Pezzutto, A., P. S. Rabinovitch, B. Dorken, G. Moldenhauer, E. A. Clark. 1988. Role of the CD22 human B cell antigen in B cell triggering by anti- immunoglobulin. J. Immunol. 140:1791.[Abstract/Free Full Text]
38. Venkataraman, C., N. Muthusamy, S. Muthukkumar, S. Bondada. 1998. Activation of lyn, blk, and btk but not syk in CD72-stimulated B lymphocytes. J. Immunol. 160:3322.[Abstract/Free Full Text]
39. Shultz, L. D., P. A. Schweitzer, T. V. Rajan, T. Yi, J. N. Ihle, R. J. Matthews, M. L. Thomas, D. R. Beier. 1993. Mutations at the murine motheaten locus are within the hematopoietic cell protein-tyrosine phosphatase (Hcph) gene. Cell 73:1445.[Medline]
40. Tsui, H. W., K. A. Siminovitch, L. de Souza, F. W. Tsui. 1993. Motheaten and viable motheaten mice have mutations in the haematopoietic cell phosphatase gene. Nat. Genet. 4:124.[Medline]
41. Hibbs, M. L., D. M. Tarlinton, J. Armes, D. Grail, G. Hodgson, R. Maglitto, S. A. Stacker, A. R. Dunn. 1995. Multiple defects in the immune system of Lyn-deficient mice, culminating in autoimmune disease. Cell 83:301.[Medline]
42. Nishizumi, H., I. Taniuchi, Y. Yamanashi, D. Kitamura, D. Ilic, S. Mori, T. Watanabe, T. Yamamoto. 1995. Impaired proliferation of peripheral B cells and indication of autoimmune disease in lyn-deficient mice. Immunity 3:549.[Medline]
43. Chan, V. W., F. Meng, P. Soriano, A. L. DeFranco, C. A. Lowell. 1997. Characterization of the B lymphocyte populations in Lyn-deficient mice and the role of Lyn in signal initiation and down-regulation. Immunity 7:69.[Medline]
44. Smith, K., D. M. Tarlinton, G. M. Doody, M. L. Hibbs, D. T. Fearon. 1998. Inhibition of the B cell by CD22: a requirement for Lyn. J. Exp. Med. 187:807.[Abstract/Free Full Text]
45. Cornall, R. J., J. G. Cyster, M. L. Hibbs, A. R. Dunn, K. L. Otipoby, E. A. Clark, C. C. Goodnow. 1998. Polygenic autoimmune traits: Lyn, CD22, and SHP-1 are limiting elements of a biochemical pathway regulating BCR signaling and selection. Immunity 8:497.[Medline]
46. Nadler, M., B. Chen, J. S. Anderson, H. H. Wortis, B. G. Neel. 1997. Protein-tyrosine phosphatase SHP-1 is dispensable for FcRIIB-mediated inhibition of B cell antigen receptor activation. J. Biol. Chem. 272:20038.[Abstract/Free Full Text]
47. Jamin, C., R. Le Corre, J. O. Pers, M. Dueymes, P. M. Lydyard, P. Youinou. 1997. Modulation of CD72 by ligation of B cell receptor complex molecules on CD5⁺ B cells. Int. Immunol. 9:1001.[Abstract/Free Full Text]
48. Van de Velde, H., I. von Hoegen, W. Luo, J. R. Parnes, K. Thielemans. 1991. The B-cell surface protein CD72/Lyb-2 is the ligand for CD5. Nature 351:662.[Medline]
49. Biancone, L., M. A. Bowen, A. Lim, A. Aruffo, G. Andres, I. Stamenkovic. 1996. Identification of a novel inducible cell-surface ligand of CD5 on activated lymphocytes. J. Exp. Med. 184:811.[Abstract/Free Full Text]
50. Bikah, G., F. M. Lynd, A. A. Aruffo, J. A. Ledbetter, S. Bondada. 1998. A role for CD5 in cognate interactions between T cells and B cells, and identification of a novel ligand for CD5. Int. Immunol. 10:1185.[Abstract/Free Full Text]

This article has been cited by other articles:

				H. Gary-Gouy, A. Sainz-Perez, J.-B. Marteau, A. Marfaing-Koka, J. Delic, H. Merle-Beral, P. Galanaud, and A. Dalloul Natural Phosphorylation of CD5 in Chronic Lymphocytic Leukemia B Cells and Analysis of CD5-Regulated Genes in a B Cell Line Suggest a Role for CD5 in Malignant Phenotype J. Immunol., October 1, 2007; 179(7): 4335 - 4344. [Abstract] [Full Text] [PDF]

				N. Fujiwara, S. Hidano, H. Mamada, K. Ogasawara, D. Kitamura, M. D Cooper, N. Hozumi, C.-l. H Chen, and R. Goitsuka A novel avian homologue of CD72, chB1r, down modulates BCR-mediated activation signals Int. Immunol., May 1, 2006; 18(5): 775 - 783. [Abstract] [Full Text] [PDF]

				D. H. Li, J. W. Tung, I. H. Tarner, A. L. Snow, T. Yukinari, R. Ngernmaneepothong, O. M. Martinez, and J. R. Parnes CD72 Down-Modulates BCR-Induced Signal Transduction and Diminishes Survival in Primary Mature B Lymphocytes J. Immunol., May 1, 2006; 176(9): 5321 - 5328. [Abstract] [Full Text] [PDF]

				M-C Zhang, N Misu, H Furukawa, Y Watanabe, M Terada, H Komori, T Miyazaki, M Nose, and M Ono An epistatic effect of the female specific loci on the development of autoimmune vasculitis and antinuclear autoantibody in murine lupus Ann Rheum Dis, April 1, 2006; 65(4): 495 - 500. [Abstract] [Full Text] [PDF]

				Y. Hitomi, N. Tsuchiya, A. Kawasaki, J. Ohashi, T. Suzuki, C. Kyogoku, T. Fukazawa, S. Bejrachandra, U. Siriboonrit, D. Chandanayingyong, et al. CD72 polymorphisms associated with alternative splicing modify susceptibility to human systemic lupus erythematosus through epistatic interaction with FCGR2B Hum. Mol. Genet., December 1, 2004; 13(23): 2907 - 2917. [Abstract] [Full Text] [PDF]

				M. Ogimoto, G. Ichinowatari, N. Watanabe, N. Tada, K. Mizuno, and H. Yakura Impairment of B cell receptor-mediated Ca2+ influx, activation of mitogen-activated protein kinases and growth inhibition in CD72-deficient BAL-17 cells Int. Immunol., July 1, 2004; 16(7): 971 - 982. [Abstract] [Full Text] [PDF]

				Y. Hokazono, T. Adachi, M. Wabl, N. Tada, T. Amagasa, and T. Tsubata Inhibitory Coreceptors Activated by Antigens But Not by Anti-Ig Heavy Chain Antibodies Install Requirement of Costimulation Through CD40 for Survival and Proliferation of B Cells J. Immunol., August 15, 2003; 171(4): 1835 - 1843. [Abstract] [Full Text] [PDF]

				C. Wakabayashi, T. Adachi, J. Wienands, and T. Tsubata A Distinct Signaling Pathway Used by the IgG-Containing B Cell Antigen Receptor Science, December 20, 2002; 298(5602): 2392 - 2395. [Abstract] [Full Text] [PDF]

				C. H. Nielsen and R. G. Q. Leslie Complement's participation in acquired immunity J. Leukoc. Biol., August 1, 2002; 72(2): 249 - 261. [Abstract] [Full Text] [PDF]

				H.-J. Wu, C. Venkataraman, S. Estus, C. Dong, R. J. Davis, R. A. Flavell, and S. Bondada Positive Signaling Through CD72 Induces Mitogen-Activated Protein Kinase Activation and Synergizes with B Cell Receptor Signals to Induce X-Linked Immunodeficiency B Cell Proliferation J. Immunol., August 1, 2001; 167(3): 1263 - 1273. [Abstract] [Full Text] [PDF]

				R. Goitsuka, H. Mamada, D. Kitamura, M. D. Cooper, and C.-l. H. Chen2 Genomic Structure and Transcriptional Regulation of the Early B Cell Gene chB1 J. Immunol., August 1, 2001; 167(3): 1454 - 1460. [Abstract] [Full Text] [PDF]

				T. Adachi, J. Wienands, C. Wakabayashi, H. Yakura, M. Reth, and T. Tsubata SHP-1 Requires Inhibitory Co-receptors to Down-modulate B Cell Antigen Receptor-mediated Phosphorylation of Cellular Substrates J. Biol. Chem., July 6, 2001; 276(28): 26648 - 26655. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles