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The Journal of Immunology, 2007, 178: 2901-2907.
Copyright © 2007 by The American Association of Immunologists, Inc.

Augmentation of Signaling through BCR Containing IgE but not That Containing IgA Due to Lack of CD22-Mediated Signal Regulation1

Motohiko Sato*,{dagger}, Takahiro Adachi*,{dagger},{ddagger} and Takeshi Tsubata2,*,{dagger},{ddagger}

* Laboratory of Immunology, School of Biomedical Science, and {dagger} Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan; and {ddagger} Core Research for Engineering, Science, and Technology, Japan Science and Technology, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The B cell membrane molecules CD22 and CD72 contain ITIMs in their cytoplasmic portion, and negatively regulate signaling through BCR. Various lines of evidence suggest that ligation of BCR containing IgG (IgG-BCR) transmits augmented signaling due to lack of CD22-mediated signal regulation. However, the signaling capacities of BCR containing IgA and IgE remain largely undefined. In this study, we demonstrate that both IgE-BCR and IgG-BCR, but not IgA-BCR, transmit augmented signaling compared with IgM-BCR. Ligation of IgE-BCR does not induce signaling events required for CD22-mediated signal inhibition, and restoration of these signaling events by coligation of CD22 with BCR abrogates signal augmentation. Furthermore, the cytoplasmic portion of IgE but not that of IgA is sufficient for suppressing CD22-mediated signal inhibition. These findings strongly suggest that the cytoplasmic portion of IgE but not that of IgA reverses CD22-mediated signal inhibition, leading to augmentation of signaling through IgE-BCR but not IgA-BCR. Augmented IgE-BCR signaling appears to play a role in production of large amounts of IgE during helminth infection, whereas regulated signaling through IgA-BCR may be crucial for constitutive production of IgA for mucosal immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
B cell receptors composed of membrane-bound Ig and the signaling component Ig{alpha}/Igbeta play a crucial role in the development, activation, and tolerance of B cells. Ligation of BCR induces activation of the BCR-associated protein tyrosine kinases Lyn and Syk, which then phosphorylate signaling molecules such as SLP65/BLNK, leading to activation of various downstream signaling cascades including Ca2+ signaling and MAPK cascades (1, 2). BCR signaling is regulated either positively or negatively by various coreceptors including Fc{gamma}RIIB, CD19, CD22, and CD72 (3, 4, 5). Among them, Fc{gamma}RIIB, CD22, and CD72 contain ITIMs in the cytoplasmic region, and activate Src homology 2 domain-containing phosphatases such as Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1)3 and SHIP by recruiting them to phosphorylated ITIMs, resulting in down-modulation of BCR signaling. Phosphorylation of these coreceptors is mediated by the BCR-associated kinase Lyn, suggesting that activation of coreceptor-mediated signal inhibition requires association of these coreceptors with BCR (6, 7, 8, 9). Fc{gamma}RIIB is phosphorylated when BCR and Fc{gamma}RIIB are coligated by interaction with immune complexes consisting of IgG and Ags, leading to feedback regulation in which Ag-specific IgG down-modulates activation of B cells reactive to the responsible Ag (5). In contrast, CD22 and CD72 are phosphorylated, and negatively regulate BCR signaling upon stimulation with Ag alone, probably because these coreceptors constitutively associate with BCR (3). By regulating BCR signaling, CD22 and CD72 appear to set signaling thresholds for B cell activation.

Five distinct Ig classes, i.e., IgM, IgD, IgG, IgA, and IgE, are found in mammals (10). Naive B cells produce both IgM and IgD, whereas production of IgA, IgE, and IgG occurs after Ig class switching, which takes place in activated B cells. Membrane-bound Igs of all five classes associate with Ig{alpha}/Igbeta through which BCRs activate cytoplasmic signaling molecules, indicating that BCRs containing different Ig classes activate the same signaling cascades. However, various lines of evidence suggest that BCR containing IgG (IgG-BCR) transmits distinct signals from those transmitted by IgM-BCR and IgD-BCR (11, 12). We previously demonstrated that CD22 negatively regulates BCR signaling through IgM-BCR and IgD-BCR but not IgG-BCR signaling, suggesting that IgG-BCR transmits augmented signaling (11). This conclusion is supported by the observation of Martin and Goodnow (12) that IgG+ B cells proliferate much more strongly than IgM+ B cells in vivo upon Ag stimulation. Memory B cells express CD22 as well as naive B cells (11), although most of them express IgG-BCR resistant to CD22-mediated signal inhibition. Augmented signaling through IgG-BCR due to lack of CD22-mediated signal inhibition may play a role in rapid and robust Ab production in memory immune responses (13), which are crucial for host defenses against pathogens and mediate at least in part the effects of vaccination.

IgE plays a crucial role in host defenses against helminth infection and the pathology of allergic diseases (14), whereas IgA is essential for mucosal immunity (15, 16). Signaling through IgE-BCR and IgA-BCR appears to play a role in these immune responses by regulating B cells expressing IgE and IgA, respectively. We recently demonstrated that BCR ligation by anti-Ig Abs is not necessarily equivalent to that due to Ags (17). Anti-Ig Abs reduce coreceptor-mediated signal inhibition, probably by disruption of association between coreceptors and BCR, by binding to the membrane-proximal part of BCR. To examine the signaling capacities of IgE-BCR and IgA-BCR, we established transfectants of B cell lines expressing BCR containing different Ig isotypes but the same Ig V region reactive to the hapten (4-hydroxy-3-nitrophenyl) acetyl (NP), and examined the BCR signaling induced by Ag stimulation. We demonstrate in this study that IgE-BCR transmits augmented signaling compared with signaling through IgM-BCR primarily due to lack of CD22-mediated signal inhibition, whereas IgA-BCR signaling is regulated by CD22 and is not augmented. There thus exist BCRs with high and low signaling capacity depending on Ig isotype, and CD22 plays a crucial role in distinguishing these two BCR groups. We discuss the biological significance of high and low signaling capacities of BCR with different Ig isotypes.


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

The retrovirus vectors pMx-Igµ, pMx-Ig{gamma}, and pMx-Ig{lambda} expressing the membrane-bound form of Ig µ H chain, the membrane-bound form of Ig {gamma}2a H chain, and Ig {lambda} L chain, respectively, were previously described (11). For generation of retrovirus vectors expressing membrane-bound forms of Ig{epsilon} and Ig{alpha} H chains, spleen cells and Peyer’s patches, respectively, were prepared from C57BL/6 mice. The cDNA for the membrane-bound form of the C region of the Ig{alpha} H chain (C{alpha}) was obtained from total RNA of Peyer’s patches by RT-PCR using specific primers 5'-AGT CTG CGA GAA ATC CCA CCA TCT ACC CA-3' and 5'-GGC CGC TCA GTA CTG GGG GAC CTC TTT GCT GCC AAA CGG GCC TCG AAC AGT-3'. The cDNA for the membrane-bound form of the C region of the Ig{epsilon} H chain (C{epsilon}) was obtained from total RNA of spleen cells treated with LPS and IL-4 by RT-PCR using specific primers 5'-ACA GCC TCT ATC AGG AAC CCT CAG CTC TAC-3' and 5'-AAG CGG CCG CCT ATG CCC TGG TCT GGA GGA T-3'. The cDNA for the V region of the IgH chain was obtained from total RNA of K46{gamma}2am{lambda} cells expressing NP-reactive Ig by RT-PCR using specific primers (11). These cDNA fragments were cloned into the pGEM-easy vector (Promega). Subsequently, the EcoRI-NotI fragments containing the C region of the membrane-bound form of Ig{epsilon} H chain and Ig{alpha} H chain were isolated and, together with the EcoRI fragment containing the V region obtained from K46{gamma}2am{lambda}, were inserted into pBlueScript, followed by deletion of the EcoRI site between the V and C regions of Ig by inverse PCR. The resulting cDNAs containing Ig{alpha} and Ig{epsilon} H chains were isolated by digestion with EcoRI and NotI and inserted into the retrovirus expression vector pMx (pMx-Ig{alpha} and pMx-Ig{epsilon}, respectively).

For construction of chimeric IgH chains containing the extracellular part of Igµ H chain and the cytoplasmic region of either Ig{epsilon} or Ig{alpha} H chains (IgM/E and IgM/A, respectively), cDNAs containing the cytoplasmic part of Ig{alpha} and Ig{epsilon} H chains were obtained by RT-PCR using RNA from IL-4/LPS-treated mouse spleen cells and Peyer’s patches, respectively. The cDNA containing the extracellular portion of Igµ H chain was obtained by PCR using pMx-Igµ as a template. The EcoRI-BglII fragment containing the extracellular portion of the IgH chain was cloned into pBlueScript together with the BglII-NotI fragment containing the cytoplasmic region of the Ig{epsilon} or Ig{alpha} H chains, followed by deletion of the recognition sequence BglII by inverse PCR. The EcoRI-NotI fragments containing the chimeric H chains were recovered and then cloned into the pMx vector (pMx-IgM/A and pMx-IgM/E). For construction of a retrovirus expression plasmid encoding the mutated IgH {gamma}2a chain in which amino acid residues constituting potential endosomal-lysosomal sorting signals were replaced by alanines (pMx-Ig{gamma}m), mutations were introduced by PCR-based site-directed mutagenesis using pMx-Ig{gamma} as a template. The PCR products were cloned into the pGEM-easy vector (Promega), and the EcoRI-NotI fragment was recovered and inserted into pMx.

Cells

The mouse B lymphoma lines WEHI-231 and BAL17 were previously described (18, 19). BAL17-IgM, BAL17-IgG, and BAL17-IgM/IgG cells expressing B1-8 IgM and the chimeric Ig containing the extracellular portion of IgM and cytoplasmic portion of IgG, respectively, were previously described (11). Cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 50 µM 2-ME, and 1 mM glutamine. To obtain retrovirus, plasmids were transfected with Plat-E cells by a method of calcium phosphate precipitation (20). Cells were infected with the retrovirus expressing IgH chain together with that expressing the {lambda} L chain. To purify cells expressing NP-reactive BCR, cells were stained with NP-conjugated PE and sorted by an autoMACS (Miltenyi Biotec) or Mo-flo (DakoCytomation).

Immunoprecipitation and Western blotting

For coligation of BCR and CD22, we incubated 0.2 µg/ml NP15-coupled BSA (NP-BSA) and either 10 µg/ml biotin-conjugated anti-mouse CD22 mAb Cy34.1 (BD Biosciences) or the same concentration of subclass-matched control mAb 4G10 (Upstate Biotechnology) with or without 3 µg/ml streptavidin for 10 min in medium. Cells were stimulated with this mixture, and lysed in Triton X-100 lysis buffer (1% Triton X-100, 10% glycerol, 150 mM NaCl, 2 mM EDTA, 0.02% NaN3, 10 µg/ml PMSF, and 1 mM Na3VO4). Lysates were immunoprecipitated with rabbit anti-mouse CD22 Ab (11) or anti-CD72 mAb K10.6 (21) together with protein G-Sepharose (Amersham Biosciences). Total cell lysates or immunoprecipitates were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were incubated with peroxidase-conjugated anti-phosphotyrosine mAb 4G10 (Upstate Biotechnology). Alternatively, membranes were reacted with rabbit anti-mouse CD72 Ab (22), anti-SHP-1 Ab (Santa Cruz Biotechnology), anti-phospho-ERK Ab (New England Biolabs), or anti-CD22 Ab followed by peroxidase-conjugated anti-rabbit IgG Ab (New England Biolabs), or anti-beta-tubulin mAb TUB 2.1 (Seikagaku Kogyo) followed by reaction with peroxidase-conjugated anti-mouse IgG Ab (Amersham Biosciences). Proteins were then visualized with an ECL system (Amersham Biosciences).

For precipitation of NP-reactive BCR, cyanogen bromide-activated Sepharose 4B beads (Amersham Biosciences) were conjugated with NP-BSA. Cell lysates were incubated with NP-BSA beads at room temperature. The precipitates were subjected to Western blot analysis using anti-CD22 Ab. The blots were reprobed with goat anti-mouse {lambda} Ab (Southern Biotechnology Associates), followed by reaction with peroxidase-conjugated anti-goat IgG Ab (Santa Cruz Biotechnology).

Flow cytometry

Cells were incubated with biotin-labeled anti-mouse CD72 mAb K10.6 or biotin-labeled anti-mouse CD22 mAb Cy34.1 (BD Biosciences), followed by reaction with FITC-labeled streptavidin (DakoCytomation). Alternatively, cells were stained with NP-conjugated PE. Cells were then analyzed by flow cytometry using a FACSCalibur (BD Biosciences).

Measurement of intracellular calcium mobilization

Cells were incubated in culture medium containing 5 mM fluo-4-AM (Molecular Probes) for 30 min. Cells were stimulated with NP-BSA and analyzed by flow cytometry using a FACSCalibur.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Augmentation of signaling through IgE-BCR but not IgA-BCR

To assess the signaling capacity of IgE-BCR and IgA-BCR, we constructed retrovirus vectors expressing the membrane-bound forms of Ig{epsilon} and Ig{alpha} H chains carrying the VHDJH complex derived from the NP-reactive hybridoma B1-8 (23). In combination with {lambda} L chain, this VHDJH complex reacts with NP. As controls, we constructed retrovirus vectors expressing membrane-bound forms of Igµ and Ig{gamma} H chains containing the same VHDJH complex. We transduced the B cell line WEHI-231 with these retrovirus vectors for IgH chains of different classes together with the retrovirus vector for {lambda} L chain to establish transfectants expressing NP-reactive IgM, IgG, IgA, or IgE (WEHI-IgM, WEHI-IgG, WEHI-IgA, and WEHI-IgE, respectively). The transfectants expressed similar levels of NP-reactive BCR (Fig. 1). We first examined Ag-induced Ca2+ mobilization. Upon stimulation with the Ag, NP15-coupled BSA (NP-BSA), WEHI-IgG cells exhibited higher intracellular Ca2+ concentration than WEHI-IgM cells (Fig. 2), demonstrating that ligation of IgG-BCR induces augmented Ca2+ signaling compared with IgM-BCR, in agreement with our previous finding (11). WEHI-IgE cells exhibited high Ca2+ response equivalent to that of WEHI-IgG cells. In contrast, WEHI-IgA cells exhibited Ca2+ mobilization comparable to that of WEHI-IgM cells. These findings indicated that ligation of IgE-BCR but not IgA-BCR generates augmented Ca2+ signaling in WEHI-231 cells.


Figure 1
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  		FIGURE 1. Expression of NP-reactive BCR, CD22, and CD72 on transfectants of B cell lines WEHI-231 and BAL17. The indicated transfectants of WEHI-231 and BAL17 cells expressing NP-reactive BCRs of different Ig isotypes were stained with NP-conjugated PE. As a negative control, nontransfected WEHI-231 and BAL17 cells were analyzed in parallel. Alternatively, cells were stained with biotin-labeled anti-CD22 mAb Cy34.1 or biotin-labeled anti-CD72 mAb K10.6, followed by reaction with FITC-labeled streptavidin. Cells reacted with FITC-labeled streptavidin alone were used as a negative control (shaded histogram). Cells were analyzed by flow cytometry using a FACSCalibur.

 

Figure 2
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  		FIGURE 2. Ligation of IgE-BCR and IgG-BCR but not IgA-BCR generates augmented Ca2+ mobilization in WEHI-231 cells. The indicated WEHI-231 transfectants were loaded with fluo-4-AM, and intracellular free Ca2+ was measured by FACSCalibur. Cells had NP15-coupled BSA (top) or A23187 (bottom) added to a final concentration of 5 µg/ml or 10 µM, respectively, at 30 s (indicated by arrow), and measurement of free Ca2+ was continued for 180 s. Representative data for three experiments are shown.

 
We next assessed Ag-induced activation of ERK in WEHI-231 transfectants. When stimulated with NP-BSA, WEHI-IgE cells exhibited greater phosphorylation of ERK than WEHI-IgM cells (Fig. 3A). In contrast, Ag-induced phosphorylation of ERK in WEHI-IgA was almost equivalent to that in WEHI-IgM cells. Thus, both Ag-induced Ca2+ signaling and ERK activation were augmented in WEHI-IgE cells but not WEHI-IgA cells, suggesting that IgE-BCR possesses augmented signaling capacity compared with IgM-BCR and IgA-BCR.


Figure 3
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  		FIGURE 3. Ligation of IgE-BCR and IgG-BCR but not IgA-BCR induces augmented ERK phosphorylation in WEHI-231 and BAL17 cells. The indicated transfectants of WEHI-231 (A) and BAL17 (B) cells were stimulated with 0.2 µg/ml NP15-coupled BSA (NP-BSA) for the indicated durations of time at 37°C. Cells were lysed and were subjected to Western blot analysis using anti-phospho-ERK Ab followed by peroxidase-conjugated anti-rabbit IgG Ab. The same blots were reprobed with anti-beta-tubulin mAb TUB 2.1 to ensure equal loading. Representative data for at least three experiments are shown.

 
To determine whether differences in signaling capacity between IgE-BCR and IgA-BCR are restricted to WEHI-231 cells, we transduced BAL17, another mouse B lymphoma line, with retrovirus vectors for Igµ, {alpha}, {epsilon}, or {gamma} H chains together with that for {lambda} L chain, resulting in BAL17 cells expressing NP-reactive IgM-BCR, IgA-BCR, IgE-BCR, or IgG-BCR, respectively. We could not assess Ca2+ signaling in these transfectants because BAL17 has a defect in the machinery regulating transmembrane Ca2+ mobilization. When we examined Ag-induced phosphorylation of ERK, we obtained essentially the same results as for WEHI-231 cells. Upon stimulation with NP-BSA, phosphorylation of ERK in BAL17-IgA cells was equivalent to that in BAL17-IgM cells (Fig. 3B). In contrast, the level of phosphorylation of ERK in BAL17-IgE cells was higher than in BAL17-IgM and BAL17-IgA cells, and was similar to that in BAL17-IgG cells. Taken together, these findings indicate that IgE-BCR generates augmented signaling upon stimulation with Ags, whereas the signaling capacity of IgA-BCR does not differ from that in IgM-BCR.

Signaling through IgA-BCR, but not IgE-BCR, is negatively regulated by CD22

We previously demonstrated that CD22, an inhibitory BCR coreceptor, negatively regulates signaling through IgM-BCR and IgD-BCR but not signaling through IgG-BCR (11). Lack of CD22-mediated signal inhibition may be involved in augmentation of the signaling function of IgG-BCR. To assess the molecular mechanisms responsible for differences in signaling capacity between IgE-BCR and IgA-BCR, we examined whether signaling through IgE-BCR and IgA-BCR is regulated by CD22 and CD72, another inhibitory coreceptor functionally redundant with CD22. All transfectants of WEHI-231 and BAL17 expressed similar levels of both CD22 and CD72 (Fig. 1). Because both phosphorylation of CD22 and CD72 and their association with SHP-1 are required for signal inhibition mediated by these coreceptors (3), we examined these events in WEHI-231 transfectants. Upon stimulation with NP-BSA, WEHI-IgG cells exhibited only weak phosphorylation of CD22 and weak recruitment of SHP-1 to CD22, whereas CD22 exhibited marked phosphorylation and association with SHP-1 in WEHI-IgM cells (Fig. 4A). This finding indicated that ligation of IgG-BCR fails to activate CD22-mediated signal inhibition, as demonstrated previously (11). When stimulated with NP-BSA, CD22 was only weakly phosphorylated and only a small amount of SHP-1 was associated with CD22 in WEHI-IgE cells, although ERK was strongly phosphorylated (Fig. 4A). In contrast, WEHI-IgA cells exhibited marked phosphorylation of CD22 and association with SHP-1 as efficient as that of WEHI-IgM cells. Essentially the same result was obtained in BAL17 transfectants (Fig. 4B). These findings thus strongly suggested that signaling through IgA-BCR is negatively regulated by CD22, whereas ligation of IgE-BCR fails to induce CD22-mediated signal inhibition. In contrast, CD72 was strongly phosphorylated and efficiently coprecipitated SHP-1 upon Ag stimulation regardless of Ig isotype in both WEHI-231 and BAL17 transfectants (Fig. 4). Taken together, these findings suggest that CD22-mediated signal inhibition is activated upon BCR ligation in an Ig isotype-dependent manner, in that CD22 inhibits signaling through IgA-BCR but not that through IgE-BCR, whereas CD72 regulates BCR signaling regardless of Ig isotype.


Figure 4
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  		FIGURE 4. Ligation of IgA-BCR but not IgE-BCR induces efficient phosphorylation of CD22, and its association with SHP-1 is required for CD22-mediated signal inhibition in WEHI-231 and BAL17 cells. The indicated transfectants of WEHI-231 (A) and BAL17 (B) cells were stimulated with 0.2 µg/ml NP15-coupled BSA (NP-BSA) for the indicated durations of time at 37°C. Cells were lysed, and CD22 and CD72 were immunoprecipitated. Immunoprecipitates (IP) were subjected to Western blot analysis using anti-phosphotyrosine mAb 4G10 or anti-SHP-1 Ab. The same blots were reprobed with anti-CD22 Ab or anti-CD72 Ab to ensure equal loading. Representative data for at least three experiments are shown.

 
Neither IgG-BCR nor IgE-BCR associates with CD22

CD22 was shown to associate with IgM-BCR (24, 25), and this association appears to play a role in induction of CD22-mediated BCR regulation (3). We therefore examined whether CD22 associates with IgG-BCR and IgE-BCR. When BCR was precipitated from BAL17-IgM, BAL17-IgE, and BAL17-IgG using NP-BSA-conjugated Sepharose beads, CD22 was coprecipitated with IgM-BCR but not with IgE-BCR nor IgG-BCR (Fig. 5A). This finding indicates that CD22 is associated with IgM-BCR but not IgE-BCR nor IgG-BCR, and suggests that lack of association with CD22 plays a role in lack of CD22-mediated signal regulation in both IgG-BCR and IgE-BCR.


Figure 5
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  		FIGURE 5. Lack of CD22-mediated signal inhibition is responsible for augmentation of signaling through IgE-BCR. A, CD22 associates with IgM-BCR but not with IgG-BCR or IgE-BCR. The indicated BAL17 transfectants were lysed, and BCR was precipitated with NP15-coupled BSA (NP-BSA) beads. The precipitates were subjected to Western blot analysis using anti-CD22 Ab. The blot was reprobed with anti-{lambda} Ab to ensure equal loading. Representative data for at least three experiments are shown. B, Coligation of CD22 with IgE-BCR using biotin-conjugated anti-CD22 Ab and biotin-conjugated NP15-coupled BSA together with streptavidin. C, Restoration of CD22 phosphorylation and abrogation of signal augmentation by coligation of CD22 with BCR. WEHI-IgE cells (3 x 106) were stimulated with the mixture of indicated reagents for 3 min. As a control, 3 x 106 WEHI-IgM cells were incubated with 0.2 µg/ml biotin-conjugated NP15-coupled BSA for 3 min. Cells were then lysed, immunoprecipitated with anti-CD22 Ab, and subjected to Western blot analysis using anti-phosphotyrosine mAb 4G10 or anti-SHP-1 Ab. The same blot was reprobed with anti-CD22 Ab to ensure equal loading. Alternatively, total cell lysates were immunoblotted with anti-phospho-ERK Ab. The same blot was reprobed with anti-beta-tubulin Ab to ensure equal loading. Representative data for at least three experiments are shown.

 
Lack of CD22-mediated signal inhibition is responsible for augmentation of signaling through IgE-BCR

The lack of association between IgE-BCR and CD22 suggests that CD22-mediated signal inhibition can be restored by coligating IgE-BCR with CD22. To examine whether lack of CD22-mediated signal inhibition is responsible for augmentation of signaling through IgE-BCR, we treated WEHI-IgE cells with biotinylated NP-BSA and biotinylated anti-CD22 mAb together with streptavidin so that IgE-BCR was ligated by NP-BSA and at the same time coligated with CD22 (Fig. 5B). With this treatment, CD22 was phosphorylated in WEHI-IgE cells to a level similar to that induced by stimulation with NP-BSA in WEHI-IgM cells (Fig. 5C). Moreover, in coligated WEHI-IgE cells, CD22 coprecipitated an amount of SHP-1 similar to that coprecipitated with CD22 in Ag-stimulated WEHI-IgM cells. These findings strongly suggested that, by coligating CD22 with BCR, CD22-mediated signal inhibition was activated in WEHI-IgE cells to a level similar to that in Ag-stimulated WEHI-IgM cells. In coligated WEHI-IgE cells, phosphorylation of ERK was reduced to a level almost equivalent to that in Ag-stimulated WEHI-IgM cells. Thus, the BCR signaling capacity of IgE-BCR became equivalent to that of IgM-BCR when CD22-mediated signal inhibition was activated to a level similar to that induced by IgM-BCR ligation, suggesting that lack of CD22-mediated signal inhibition plays a central role in augmentation of BCR signaling through IgE-BCR.

The cytoplasmic tail of IgE is responsible both for preventing CD22-mediated signal inhibition and generating augmented BCR signaling

We previously demonstrated that the cytoplasmic tail of the membrane form of IgG is necessary and sufficient for escape from CD22-mediated signal inhibition (11). To examine the roles of the cytoplasmic tails of IgE and IgA, we constructed retrovirus vectors expressing chimeric IgH chains containing the extracellular portion of the B1-8 Igµ H chain and the cytoplasmic tail of either Ig{alpha} or Ig{epsilon} H chain (IgM/A or IgM/E, respectively). We transduced BAL17 cells with these vectors together with the retrovirus encoding {lambda} L chain so that the transfectants expressed chimeric IgM/A-BCR or IgM/E-BCR reactive to NP (BAL17-IgM/A and BAL17-IgM/E). As controls, we examined BAL17-IgM and BAL17-IgM/G, the latter of which expresses the chimeric IgH chain containing the extracellular portion of B1-8 Igµ H chain and the cytoplasmic tail of IgG (11). All of these transfectants expressed similar levels of NP-reactive BCR (data not shown). When these transfectants were stimulated with NP-BSA, both BAL17-IgM and BAL17-IgM/A cells exhibited marked phosphorylation of CD22 and association of CD22 with SHP-1, whereas CD22 was weakly phosphorylated and weakly associated with SHP-1 in BAL17-IgM/G and BAL17-IgM/E cells (Fig. 6A) This finding indicated that the cytoplasmic tail of IgE is sufficient for abrogating CD22-mediated signal inhibition, as is the case for the cytoplasmic tail of IgG, whereas the cytoplasmic tail of IgA fails to reverse negative regulation of BCR signaling by CD22. Moreover, Ag-induced ERK phosphorylation was augmented in both BAL17-IgM/E and BAL17-IgM/G cells compared with BAL17-IgM and BAL17-IgM/A cells, indicating that the weak CD22 phosphorylation upon ligation of IgM/E and IgM/G is not due to weak signaling capacities of these chimeric BCRs, and that lack of CD22-mediated signal inhibition causes augmentation of signaling through IgM/E and IgM/G. Taken together, these findings indicate that the cytoplasmic tail of IgE but not that of IgA inhibits CD22-mediated signal inhibition, and thereby augments BCR signaling.


Figure 6
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  		FIGURE 6. The cytoplasmic tails of IgE and IgG abrogate CD22-mediated signal regulation by a pathway independent of potential sorting motifs. BAL17 transfectants expressing chimeric Ig containing the extracellular portion of IgM and the cytoplasmic tail of either IgA (BAL17-IgM/A) (A), IgE (BAL17-IgM/E) (A), or IgG (BAL17-IgM/IgG) (A), BAL17 transfectants expressing mutated IgG that lacks potential sorting motifs (BAL17-IgGm) (B), BAL17-IgM cells (A and B) and BAL17-IgG cells (B) were stimulated with 0.2 µg/ml NP15-coupled BSA for the indicated durations of time at 37°C. Cells were lysed, and CD22 and CD72 were immunoprecipitated. Immunoprecipitates (IP) were subjected to Western blot analysis using anti-phosphotyrosine mAb 4G10 or anti-SHP-1 Ab. The blots were reprobed with anti-CD22 Ab or anti-CD72 Ab to ensure equal loading. Alternatively, the level of phosphorylation of ERK was examined by Western blotting of total cell lysates using anti-phospho-ERK Ab. The same blots were reprobed with anti-beta-tubulin mAb to ensure equal loading. Representative data for at least three experiments are shown. The amino acid sequence of the cytoplasmic region of the mutated Ig {gamma}2a chain is shown below the blot (B). Amino acid residues constituting the potential dileucine motif and Yxx{phi} motif in the cytoplasmic tail of IgG2a were replaced by alanines.

 
The cytoplasmic tails of IgG and IgE but not those of other isotypes, contain the potential dileucine-based and YXX{phi} ({phi}; L, I, M, F, V) motifs, both of which are involved in endosomal-lysosomal sorting (26). These motifs are conserved among IgG subtypes and between humans and mice (27, 28). To examine whether these motifs are involved in the suppression of CD22-mediated BCR regulation, we constructed a retrovirus vector encoding the mutated IgH {gamma} chain in which the amino acid residues constituting the potential endosomal-lysosomal sorting signals were replaced by alanines (Fig. 6B). We transduced BAL17 cells with this retrovirus vector together with the retrovirus vector expressing the {lambda} L chain. When stimulated with NP-BSA, the transfectant BAL17-IgGm exhibited only weak phosphorylation of CD22 and recruitment of a small amount of SHP-1 to CD22, as was the case for BAL17-IgG cells (Fig. 6B). The level of ERK phosphorylation in BAL17-IgGm cells was similar to that in BAL17-IgG cells, and stronger than that in BAL17-IgM cells. Taken together, these findings indicate that the cytoplasmic regions of both IgG and IgE are crucial for augmentation of signaling through these BCRs by suppression of CD22-mediated signal inhibition, although the conserved dileucine and Yxx{phi} motifs in the cytoplasmic tail do not participate in this augmentation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
By examining transfectants of two B cell lines, WEHI-231 and BAL17, expressing BCR with the same Ig V region but different isotypes, we have demonstrated in this study that ligation of IgE-BCR but not IgA-BCR generates augmentation of signaling, including both Ca2+ signaling and ERK activation, compared with signaling through IgM-BCR. The signaling capacities of BCRs with different Ig isotypes appear to depend on the presence or absence of CD22-mediated signal inhibition. Indeed, signaling events required for CD22-mediated signal inhibition, such as phosphorylation of CD22 and association of CD22 with SHP-1, were efficiently induced by ligation of IgM-BCR or IgA-BCR but not ligation of IgE-BCR. Restoration of these signaling events by coligation of CD22 with IgE-BCR eliminated the augmentation of IgE-BCR signaling. Thus, lack of CD22-mediated negative regulation of BCR signaling is responsible for augmentation of through IgE-BCR. We further demonstrated that substitution of the cytoplasmic tail of IgM by that of IgE abrogates CD22-mediated signal inhibition and augments BCR signaling, suggesting that the cytoplasmic tail of IgE is sufficient for augmentation of BCR signaling through IgE-BCR. In contrast, the cytoplasmic tail of IgA does not participate in this augmentation. Taken together, these findings indicate that the cytoplasmic tail of IgE protects BCR signaling from CD22-mediated signal inhibition, leading to an augmentation of through IgE-BCR, whereas the signaling capacity of IgA-BCR is similar to that of IgM-BCR in the presence of CD22-mediated negative regulation of BCR signaling.

BCRs with different Ig isotypes use Ig{alpha}/Igbeta to transmit transmembrane signaling and thus activate the same signaling cascades through Ig{alpha}/Igbeta. However, the hypothesis that the signaling function of BCR depends on Ig isotype was first suggested by our previous finding that CD22 negatively regulates signaling through IgM or IgD but not IgG (11). Augmentation of signaling of IgG-BCR was directly demonstrated in the present study (Figs. 2 and 3). We also demonstrated that IgE-BCR but not IgA-BCR transmits this augmentation of signaling. Thus, BCRs of different Ig isotypes can be divided into two groups based on their signaling function. Both IgG-BCR and IgE-BCR transmit augmented signaling compared with signaling through IgM-BCR, IgD-BCR, or IgA-BCR. Each of these low-signaling BCRs but none of the high-signaling BCRs are negatively regulated by CD22, and IgE-BCR transmits augmented signaling primarily due to lack of CD22-mediated signal inhibition. CD22 thus plays a major role in determining the signaling capacity of BCR signaling depending on Ig isotype.

Memory B cells generated in response to various Ags such as viruses and bacteria mostly express IgG-BCR, and rapidly produce large amounts of Abs when they interact with the Ags (13). During memory responses to helminths including Nippostrongylus brasiliensis and Schistosoma mansoni (S. mansoni), large amounts of IgE are rapidly produced (29, 30). Thus, IgE+ memory B cells as well as IgG+ memory B cells efficiently respond to Ag stimulation during memory responses. Although memory B cells express CD22 as well as naive B cells (11), expression of IgG-BCR and IgE-BCR in memory B cells appears to result in augmentation of BCR signaling due to resistance of these BCRs to CD22-mediated signal inhibition. Thus, expression of IgG-BCR and IgE-BCR may be involved in rapid and robust Ab production during memory responses through augmentation of BCR signaling. Indeed, Martin and Goodnow (12) demonstrated previously that expression of IgG-BCR on naive B cells induces robust expansion of these B cells upon Ag stimulation and production of large amounts of Abs, suggesting that functional difference between IgG/IgE-BCR and IgM/IgD-BCR is sufficient for induction of efficient responses to B cells. We demonstrate in this study that lack of CD22-mediated signal inhibition plays a major role in differences in signaling between IgE-BCR and IgM-BCR. Thus, lack of CD22-mediated signal regulation of IgG/IgE-BCR may play a crucial role in robust B cell activation during memory responses, although this does not exclude the involvement of other factors.

Rapid and robust immune responses are an important part of host defenses against viruses and bacteria because many of these pathogens induce acute infectious disease. However, it appears that rapid immune responses also play crucial roles in host defenses against helminth infection. Indeed, S. mansoni cercariae immediately become schistosomula upon infection though skin, and in a few days reach the lungs, where they become resistant to immune responses (31). During infection with Trichinella spiralis, infective larvae lay around 1500 eggs, and newborn larvae move to muscles where they induce symptoms (32). Newborn larvae but not muscle larvae nor adult worms are sensitive to eosinophil-mediated cytotoxicity (33, 34). Thus, rapid IgE production is required to generate immune responses to helminth infections at the stage at which they are sensitive to immune responses, leading to efficient host protection, although helminth infection is usually more chronic than infection with many viruses and bacteria.

Although IgA is produced after class switching, as is the case for IgG and IgE, signaling through IgA is regulated by CD22 and is not augmented compared with IgM-BCR signaling. This finding is in agreement with the report by Tedder and colleagues (35) that CD22-deficient mice exhibit high IgA titer. Large amounts of IgA, as much as 40–60 mg/day in humans, are secreted every day into the gut lumen (36). A sizable proportion of IgA-producing cells in the gut appear to be derived from B-1 cells, and secretory IgA contains large proportions of polyreactive Abs that bind to various Ags including self-Ags and bacterial Ags (15, 16). Although mucosal Ag stimulation induces both production of specific IgA and generation of IgA+ memory B cells (37), a large amount of polyreactive IgA appears to be produced in constitutive fashion, as is the case for IgM, and thereby functions as a first line of defense against pathogens in the mucosa. CD22-mediated signal inhibition may play a role in constitutive production of IgA by regulating inducible IgA production, thereby maintaining the daily amount of IgA secretion and preventing production of excess IgA autoantibody.

CD22-mediated BCR regulation requires phosphorylation of CD22 by the kinase Lyn (6, 7, 8). Because Lyn is associated with BCR and is activated upon BCR ligation (38), phosphorylation of CD22 appears to require colocalization of CD22 with BCR (6, 7, 8). We have demonstrated in this study that CD22 associates with IgM-BCR but not IgG-BCR or IgE-BCR. In the absence of association with BCR, CD22 may not be phosphorylated by the BCR-associated kinase Lyn upon ligation of IgG-BCR or IgE-BCR, resulting in lack of CD22-mediated regulation of signaling through these BCRs. We previously showed that the cytoplasmic tail of IgG is sufficient for abrogation of CD22-mediated signal inhibition (11). We have demonstrated that the cytoplasmic tail of IgE has the same activity. The cytoplasmic tails of IgG and IgE thus augment BCR signaling, probably by inducing dissociation of BCR from CD22, although how this occurs is not yet known. The cytoplasmic tails of IgG and IgE have 28 aa residues each, and contain the potential dileucine and Yxx{phi} motifs, both of which are involved in endocytosis (26). These motifs are conserved between humans and mice, and among IgG subclasses (27), but do not play a role in inactivation of CD22-mediated BCR regulation (Fig. 6B). Nonetheless, the signal-augmenting function of these cytoplasmic tails may play a role in their support of Ab production, as suggested by the observation that knock-in mice lacking cytoplasmic tails of IgG or IgE exhibit reduced production of IgG or IgE, respectively (39, 40). Further studies are required to elucidate the molecular mechanisms by which IgG-BCR and IgE-BCR become resistant to CD22-mediated signal inhibition.

Besides immunity to infection, both IgG and IgE play important roles in autoimmune diseases and allergy. B cells producing IgG autoantibodies and IgE Abs to allergens do not appear to be regulated by inhibitory coreceptors, suggesting that regulation of these pathological B cells may be difficult. Elucidation of the molecular mechanisms of resistance of IgG-BCR and IgE-BCR to coreceptor-mediated signal regulation may thus be crucial for the development of new strategies for controlling autoimmune diseases and allergy as well as those for augmentation of immunity to infection.


   Acknowledgments
 
We thank Drs. N. Tada (Tokai University, Isehara, Japan) and T. Kitamura (The University of Tokyo, Japan) for providing CD72 mAb (K10.6) and Plat-E cells, respectively, and K. Mizuno and A. Yoshino for technical assistance.


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


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

1 This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Japan Society for the Promotion of Science, and the Ichiro Kanehara Foundation. Back

2 Address correspondence and reprint requests to Dr. Takeshi Tsubata, Laboratory of Immunology, School of Biomedical Science, and Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. E-mail address: tsubata.imm{at}mri.tmd.ac.jp Back

3 Abbreviations used in this paper: SHP-1, Src homology 2 domain-containing protein tyrosine phosphatase-1; NP, (4-hydroxy-3-nitrophenyl) acetyl. Back

Received for publication February 22, 2006. Accepted for publication December 13, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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