FcμR Interacts and Cooperates with the B Cell Receptor To Promote B Cell Survival

Peripheral B cell survival relies on signals from the BCR and the BAFFR (1). The BCR is a heterotrimeric complex consisting of Ag binding Ig and the signaling Igα/Igβ heterodimers. In vivo ablation of surface Ig (2) or inactivation of Igα (3) causes rapid death of B cells, indicating that BCR transmits essential “tonic” survival signals in the absence of Ag ligands. Cross-linking the BCR on mature B cells with Ag or anti-IgM Abs initiates multiple intracellular signaling cascades, which eventually lead to the activation of ERK, NF-κB, and NFAT pathways. Among these, NF-κB appears to play a prominently protective role in the survival of Ag-stimulated B cells by inducing the expression of several antiapoptotic genes such as Bcl-2, Bcl-xL, and Bfl-1/A1 (4–6). BCR signaling activates the canonical NF-κB pathway, which is characterized by the phosphorylation and ubiquitin-mediated degradation of IκB inhibitory proteins, in particular IκBα. This leads to the translocation of NF-κB1 into the nucleus to activate target gene transcription. BAFFR is a member of the TNFR family. Deficiency of BAFF or BAFFR results in an almost complete loss of follicular and marginal zone (MZ) B cells (7–9), demonstrating a critical role for BAFFR-mediated signaling in B cell survival. In contrast to BCR, BAFFR activates the noncanonical NF-κB pathway, which depends on the proteolytic processing of p100 to p52 to generate p52/RelB (NF-κB2) nuclear complexes (10–13). Both BCR and BAFFR are required for the maintenance of peripheral B cell homeostasis. It has been shown that signals from the BCR and BAFFR cooperate to allow B cell survival at multiple stages of peripheral B cell differentiation and during immune responses. BCR promotes BAFFR-mediated signals through at least two mechanisms by upregulating the expression of BAFFR and by supplying the noncanonical NF-κB pathway substrate p100 for BAFFR-mediated degradation (14–16).

The recently identified IgM FcR (FcμR) (17, 18) has been shown to play a critical role in IgM homeostasis, B cell development and survival, germinal center formation, and humoral immune responses as well as in prevention of autoantibody production (19–22). It remains unclear, however, how FcμR regulates B cell development and function. An intriguing clue came from the in vitro analysis, which revealed a specific defect for FcμR^−/− B cells in anti-IgM–induced survival and proliferation (19, 21). These observations suggested a possible functional link between FcμR and BCR. In the current study, we addressed the molecular mechanisms of FcμR-mediated enhancement of anti-IgM–induced B cell survival. We show that FcμR and BCR physically interact on the plasma membrane of primary B cells and functionally cooperate to promote the activation of the noncanonical NF-κB pathway and BCL-xL expression. Importantly, FcμR alone in the absence of BCR signaling had no effect on either B cell survival or NF-κB activation. These results reveal a cross-talk downstream of FcμR and BCR signaling and provide mechanistic insight into FcμR-mediated enhancement of B cell survival after BCR stimulation.

C57BL/6 mice were purchased from CLEA Japan (Tokyo). FcμR-deficient mice have been described previously (19). The mice were maintained in specific pathogen-free conditions and all experimental procedures were approved by the Animal Experiment Committee of RIKEN.

Primary B cells were purified from the spleen of C57BL/6 and FcμR-deficient mice using an IMAG negative sorting kit (BD Biosciences). Purified B cells were cultured for 48 h under various conditions as described (19). The cells were stained with 7-aminoactinomycin D (7-AAD) and the percentages of viable (7-AAD^lowFSC^high) and dead (7-AAD^highFSC^low) cells were analyzed by FACS (BD Biosciences). The anti-mouse IgD^a Ab (clone AMS-9.1; catalog number 406108) and its isotype control (clone MG2b-57; catalog number 401212, LEAF purified) were purchased from BioLegend. The AMS-9.1 mAb was passed through a gel filtration column (PD MidiTrap G-25; GE Healthcare) to remove azide. The anti-mouse Igκ Abs (LE/AF goat F(ab′)₂ anti-mouse κ; catalog number 1052-14) were purchased from Southern Biotechnology Associates. Spleen B cells purified from BALB/c mice (a allotype) were used to investigate the effect of FcμR cross-linking on anti-IgD and anti-Igκ–induced B cell activation.

Purified spleen B cells were cultured in the presence of F(ab′)₂ anti-IgM Abs (5 μg/ml), soluble CD40L, or LPS (10 μg/ml) for 6, 24, and 48 h. The cultured cells were first incubated with a rat IgG_2b anti-mouse CD16/CD32 mAb (clone 2.4G2; BD Biosciences) to block FcγR and then stained with either an anti-FcμR mAb (clone 4B5, rat IgG_2a) or an isotype control Ab (clone eBR2a; eBioscience). After washing, the cells were incubated with PE-conjugated anti-rat IgG_2a (clone RG7/1.30; BD Biosciences). The mean fluorescence intensity of FcμR on purified spleen B cells before culture was set as 1.

Ten thousand wild-type (WT) B cells were seeded on poly-l-lysine (Sigma-Aldrich)–treated coverslips and allowed to adhere for 15 min at 37°C. Cells were fixed for 15 min at room temperature in 3% paraformaldehyde (Electron Microscopy Sciences), washed with PBS and incubated in staining buffer (0.05% saponin, 10 mM glycine, 5% FBS, and PBS) for 15 min. Cells were incubated with rabbit IgG α-FcμR (original Ab, 1/500 dilution) together with one of the following Abs: FITC-rat IgG_2a anti-mouse IgM (clone R6-60.2; BD Biosciences), mouse IgG₁ anti-CD79A (clone HM47, 1/500; Santa Cruz Biotechnology), or mouse IgG_2b anti-CD79B (clone B29/123, 1/500; Santa Cruz Biotechnology) for 60 min at 37°C, and thereafter washed three times with ice-cold PBS. Cells were further stained with Alexa Fluor 488-goat anti-rabbit IgG (1/400; Molecular Probes) or Alexa Fluor 488-goat anti-rabbit IgG + Alexa Fluor 555-goat anti-mouse IgG (1/400; Molecular Probes) at room temperature for additional 30 min. Coverslips were washed twice and then mounted on slides with Fluoromount-G (Southern Biotechnology Associates). Images were acquired using a Leica TCS SP5 laser-scanning confocal microscope (LAS AF software) using the HCX PLAPO 363 objective (numerical aperture: 1.4).

Immunoprecipitation and immunoblot were performed as described previously (23). Briefly, spleen B cell lysates were precleared with protein G–Sepharose and then incubated overnight with protein G–Sepharose conjugated with rabbit anti-FcμR, an isotype control (rabbit IgG; Southern Biotechnology Associates), goat α-mouse IgM (Southern Biotechnology Associates), or an isotype control (Normal goat serum; Vector Labs). The precipitates were washed 10 times, resolved in a 4–20% gradient SDS-PAGE, and subjected to immunoblot. FcμR was detected with 4B5 rat anti-FcμR, followed by HRP-conjugated goat anti-rat IgG (Jackson ImmunoResearch Laboratories); Igα was detected with mouse IgG₁ anti-CD79A (clone HM47), followed by HRP-conjugated goat anti-mouse IgG₁ (cross absorbed with mouse IgM, IgG_2a, IgG_2b, IgG₃, and IgA, as well as pooled human sera and purified human paraproteins; Southern Biotechnology Associates); Igμ was detected with HRP-conjugated goat anti-mouse IgM (cross absorbed with mouse IgG₁, IgG_2a, IgG_2b, IgG₃, and IgA). The following Abs were used to detect pIκBα, p100, p52, and BCL-xL: mouse IgG₁ anti–phospho-IκBα (clone 5A5; Cell Signaling Technology), rabbit IgG anti–NF-κB2 p100/p52 (Cell Signaling Technology), rabbit IgG α-Bcl-xL (BD Biosciences), and rabbit IgG α-actin (Sigma-Aldrich). HRP-conjugated goat anti-mouse IgG₁ or anti-rabbit IgG was used as secondary Abs. Protein expression was analyzed with the Multi Gauge software of LAS-2000 luminescent Image Analyzer (Fuji film, Tokyo, Japan).

These experiments were performed as described previously (24).

Statistical significance was assessed by the unpaired t test.

We recently reported that FcμR enhanced B cell survival induced by anti-IgM but not LPS stimulation (19). To further analyze the specificity of FcμR-mediated enhancement of B cell survival, we cultured WT and FcμR^−/− splenic B cells in the presence of anti-IgM F(ab′)₂ fragment, CD40L, or LPS. Consistent with our previous findings (19), FcμR^−/− splenic B cells showed decreased survival following α-IgM but not LPS stimulation (Fig. 1A, 1B). In addition, we found that FcμR deficiency did not affect B cell survival following CD40L ligation of CD40 (Fig. 1B). Moreover, cross-linking FcμR on WT B cells with an anti-FcμR Ab enhanced anti-IgM– but not LPS- or CD40L-induced B cell survival (Fig. 1C). Mature B cells express both IgM and IgD on the cell surface. We further investigated whether cross-linking FcμR was able to enhance B cell survival or activation induced by anti-IgD Abs. The anti-IgD Ab AMS-9.1 induced B cell activation as reflected by the increased cell sizes and cell division (Supplemental Fig. 1A). However, cross-linking FcμR with the 4B5 mAb did not enhance the anti-IgD–mediated B cell activation (Supplemental Fig. 1B). In contrast, the 4B5 mAb was able to enhance B cell activation induced by anti-Igκ Abs (Supplemental Fig. 1C, 1D), which cross-link both IgM and IgD. These observations collectively demonstrate that FcμR specifically enhances IgM BCR–mediated B cell survival/activation.

BCR cross-linking upregulates BAFFR expression, which is one mechanism by which the BCR promotes BAFFR-mediated B cell survival (14–16). To analyze how FcμR expression is regulated, splenic B cells were cultured in the presence of F(ab′)₂ anti-IgM Abs, soluble CD40L, or LPS for different times and their FcμR levels were compared with that before culture (0 h). As shown in Fig. 2, FcμR cell surface expression was markedly upregulated after BCR cross-linking with anti-IgM Abs but only moderately increased by CD40L or LPS stimulation. The upregulation of FcμR expression by anti-IgM stimulation may in part contribute to the FcμR-mediated enhancement of BCR-triggered B cell survival. However, FcμR was also moderately upregulated by treatment with CD40L or LPS without affecting B cell survival. Therefore, additional mechanisms likely exist to allow FcμR to specifically enhance B cell survival induced by α-IgM stimulation. Although we found that FcμR protein levels were upregulated upon B cell activation, Choi et al. (21) found that transcript levels for FcμR were reduced after stimulation with LPS or anti-CD40 or F(ab′)₂ anti-IgM Abs. This discrepancy might be due to differential regulation of FcμR transcription and protein expression.

The FcμR-mediated specific enhancement of BCR-mediated survival led us to hypothesize that there might be a physical interaction between FcμR and BCR. We first performed confocal immunofluorescent staining. As shown in Fig. 3A, upper panels, FcμR colocalized with IgM on the plasma membrane of splenic B cells. However, our earlier findings suggested that FcμR is likely occupied by IgM in vivo (19). In addition, the 4B5 α-FcμR mAb binds to FcμR even after IgM binding (Supplemental Fig. 2). Therefore, the colocalization between FcμR and IgM could simply be due to the binding of serum IgM to FcμR on splenic B cells, rather than real colocalization of FcμR with membrane BCR. We thereafter analyzed the colocalization of FcμR with the non-Ig components of the BCR, the Igα/Igβ signal transducing molecules. As shown in Fig. 3A, FcμR indeed colocalized with both Igα (middle panels) and Igβ (lower panels) on the plasma membrane in resting B cells, with areas of coincident brighter staining. Visual analysis of merged images of more cells revealed that some BCR were not associated with FcμR and vice versa. It appeared that >50% of the BCR was associated with FcμR on the plasma membrane. To confirm that FcμR and BCR physically interact, we immunoprecipitated FcμR under a mild detergent condition and analyzed the coprecipitation of the BCR components. As shown in Fig. 3B, immunoprecipitation of FcμR from splenic B cells pulled down IgH (Igμ) and its associated Igα (upper panel). Conversely, FcμR and Igα were coprecipitated with IgM (lower panel). These results collectively indicate that FcμR constitutively associates with the BCR on primary B cells.

BCR is known to undergo constitutive and Ag-induced internalization, which serves as an important mechanism to regulate surface BCR levels and BCR signaling. The physical association of FcμR and BCR suggested that FcμR might affect BCR internalization and thereby regulate BCR signal strength. We first analyzed constitutive (Ag-independent) BCR internalization by incubating splenic B cells in the presence of brefeldin A, which inhibits the trafficking of the internalized BCR, or by using F(ab′) anti-IgM Abs, which are unable to trigger BCR signaling. In both cases, WT and FcμR^−/− B cells exhibited very similar kinetics of BCR internalization (Fig. 4, left and middle panels). We next analyzed ligand-dependent BCR internalization using F(ab′)₂ anti-IgM Abs, which initiate BCR signaling. F(ab′)₂ anti-IgM Abs induced a much more rapid BCR internalization (Fig. 4, right panel) compared with that induced by F(ab′) anti-IgM Abs (middle panel), but again, this ligand-dependent BCR internalization was unaffected by FcμR deficiency (Fig. 4, right panel). These observations complement our previous finding that FcμR does not contribute to the internalization of Ag and IgM/Ag immune complexes (IC) by B cells and the subsequent presentation on MHC class II molecules (19). Therefore, FcμR does not seem to regulate BCR signaling through modulating its internalization processes.

BCR signaling activates canonical NF-κB pathway to induce the expression of anti-apoptotic genes and enhance B cell survival. This prompted us to examine the effect of FcμR signaling on anti-IgM–induced NF-κB activation. We first analyzed anti-IgM–induced IκBα phosphorylation, which is known to correlate with NF-κB activation in the canonical pathway (1). No difference was observed in the magnitude and kinetics of pIκBα between WT B cells treated with an isotype control or the 4B5 anti-FcμR Ab (Fig. 5A) or between WT and FcμR^−/− B cells (Supplemental Fig. 3A), indicating that FcμR does not contribute to canonical NF-κB activation. We further analyzed the activation of the noncanonical NF-κB pathway. BCR signaling does not activate the noncanonical NF-κB pathway but produces the noncanonical NF-κB substrate p100. Activation of the noncanonical NF-κB pathway results in the processing of p100 to generate p52, which associates with RELB to form NF-κB2 and activates the expression of antiapoptotic proteins such as BCL-xL (14–16). Intriguingly, we found that p52 levels were elevated at later time points in WT B cells stimulated with both anti-IgM and anti-FcμR as compared with those stimulated with anti-IgM and an isotype control Ab (Fig. 5B, 5C, Supplemental Fig. 3D). Moreover, consistent with the elevated levels of p52, anti-FcμR Ab also enhanced the expression of BCL-xL at later points of anti-IgM stimulation (Fig. 5B, 5C). Conversely, FcμR^−/− B cells showed a reduction in BCR-triggered BCL-xL expression compared with WT B cells (Supplemental Fig. 3B, 3C), which supports a role for FcμR in promoting BCL-xL expression. However, it should be noted that the decreased BCL-xL expression in FcμR^−/− B cells also could be due to the differences in B cell subsets and maturation status between WT and FcμR^−/− mice.

Intriguingly, although FcμR promoted p52 accumulation and BCL-xL expression after BCR stimulation, cross-linking FcμR alone in the absence of BCR signaling had no detectable effect on either IκBα phosphorylation (Fig. 5D) or the induction of p100, p52, or BCL-xL (Fig. 5E). These observations suggest that FcμR by itself is unable to activate NF-κB1 or NF-κB2 but relies on BCR signaling to promote NF-κB2 activation.

In the current study, we have demonstrated that FcμR physically associates with BCR in primary B cells and specifically enhances B cell survival induced by anti-IgM but not CD40L or LPS stimulation. FcμR cooperates with BCR to promote the induction of p52 and its target BCL-xL. Importantly, FcμR alone in the absence of BCR signaling has no effect on either B cell survival or NF-κB activation. In other words, FcμR relies on BCR signaling to elicit its survival function.

The cooperation between FcμR and BCR in enhancing B cell survival to some extent resembles the relationship between BAFFR and BCR (14–16). As is the case for BAFFR, FcμR is upregulated by BCR cross-linking, which likely contributes to the FcμR-mediated enhancement of BCR-triggered cell survival. However, one critical difference between BAFFR and FcμR is that BAFFR signaling by itself is able to generate p52 and promote BCL-xL expression in B cells by collaborating with BCR “tonic” signals, whereas FcμR alone in the absence of BCR cross-linking is unable to activate either the canonical or the noncanonical NF-κB pathway to induce B cell survival. This difference predicts that BAFFR and FcμR contribute to Ag-independent and -dependent B cell survival, respectively. In agreement with this prediction, mice lacking BAFF/BAFFR have almost a complete loss of mature B cells (7–9), whereas FcμR^−/− mice have relatively normal sizes of the follicular B cell pool but show reduced B cell survival after BCR stimulation and impaired germinal center (GC) formation and Ab production against a T-dependent Ag (19). Therefore, the dependence of FcμR function on BCR signaling allows FcμR to specifically enhance the survival of Ag-stimulated B cells. Although we have shown that cross-linking FcμR with the 4B5 anti-FcμR mAb could enhance B cell survival induced by F(ab′)₂ anti-IgM Abs, it remains to be investigated whether FcμR signaling by its bona fide ligand soluble IgM has the same effect. Further studies are required to clarify this issue by using BCR-transgenic B cells in which one can simultaneously cross-link BCR with specific Ag and FcμR with soluble IgM.

It is intriguing to note that the MZ B cell population was significantly reduced in FcμR^−/− mice (19, 20). It has been suggested that self-reactive B cells may be driven to become MZ B cells (25). An interesting hypothesis would be that MZ B cells may be stimulated by self-Ag to generate a relatively strong survival signal by cooperating with the FcμR-mediated signal. Absence of FcμR would thus result in a reduced self-Ag–triggered BCR signal in MZ B cells that is required for maintaining their survival.

During an immune response, Ag-specific B cells are activated in the B cell follicles of the secondary lymphoid organs in response to IC bound to follicular dendritic cells. In this way, the complement receptor (CD21/CD19 complex) coclusters with BCR upon interaction with Ags bearing complement C3d, resulting in efficiently lowering the activation threshold of B cells in comparison of stimulation by BCR alone (26). Notably, the phenotype of FcμR-deficient mice has a marked similarity to that of CD19-deficient mice in terms of decreased MZ B cells, impaired GC formation, reduced Ab production to T-independent and T-dependent Ags, and impaired memory responses (27, 28). The close correspondence in the phenotype of FcμR- and CD19-deficient mice suggests that, similar to CD21/CD19 coreceptor complex, FcμR may function as a positive regulator in B cell responses to IgM-ICs in GCs. Indeed, similar to CD19, the presence of FcμR reduced the dose of BCR stimulation needed for sustaining B cell viability (19). Our present findings also indicate that integration of BCR and FcμR signaling at the level of BCL-xL upregulation by IgM-ICs may help overcome anergy- or apoptosis-inducing effects of the BCR alone and promote the survival and expansion of B cells to initiate GC reactions.

Engagement of the BCR initiates two concurrent processes, signaling and receptor internalization. The latter is an important mechanism to regulate the BCR signal strength and prevent excessive B cell activation. Using WT and FcμR-deficient B cells, we found that FcμR did not affect ligand-dependent and -independent BCR internalization. Therefore, although FcμR associates with BCR, it does not elicit its function through modulating BCR internalization. It remains to be investigated how signals downstream of BCR and FcμR cross-talk to promote p52 induction and BCL-xL expression. Earlier studies have shown that multiple tyrosine and serine residues in the cytoplasmic tail of FcμR are phosphorylated upon ligand binding (17). Given the physical association between FcμR and BCR, one possible scenario is that after BCR stimulation, these residues might be phosphorylated by BCR-activated protein tyrosine kinases and thereby recruit more signaling molecules, participating in and amplifying the BCR-mediated signal cascades.

FcμR-deficient mice produce elevated IgG autoantibodies as they age (19–22), suggesting that FcμR is required for maintaining self-tolerance. The results of the current study demonstrate that FcμR promotes BCR-triggered survival of mature B cells. BCR ligation in different contexts can lead to different biological outcomes, and immature B cells in the bone marrow have been shown to undergo apoptosis upon BCR cross-linking. A reduction in BCR signaling due to the absence of FcμR may lead to insufficient elimination of autoreactive immature B cells in the BM. In addition, autoreactive B cells can be generated in the GC by Ig gene somatic hypermutation (29), and some autoreactive GC B cells might escape the deletion mechanism because of reduced BCR signaling. Studies are in progress to investigate the role of FcμR in the deletion of autoreactive B cells in the BM and during the GC reaction.

BCR signaling also plays an important role in neoplasia. Malignant B cells from patients with chronic lymphocytic leukemia (B-CLL) express much higher levels of FcμR than normal B cells from healthy donors (30, 31). Antigenic stimulation through the BCR is thought to promote the outgrowth of B-CLL (32, 33), and our results suggest that elevated FcμR expression may enhance a BCR-triggered survival signal and contribute to the pathogenesis of B-CLL. Further elucidation of the precise molecular details by which FcμR cooperates with BCR to regulate B cell survival should accelerate our understanding of the etiology of immunological disorders and B cell malignancies associated with altered BCR signals.

We thank Hiroshi Ohno and Hiromi Kubagawa for helpful advice, Hiromi Mori for excellent technical support, and the Animal Facility of RIKEN Center for Integrative Medical Sciences for maintaining and breeding the mice.

The authors have no financial conflicts of interest.