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J Immunol 2015; 194:3096-3101; Prepublished online 2
March 2015;
doi: 10.4049/jimmunol.1402352
http://www.jimmunol.org/content/194/7/3096
http://www.jimmunol.org/content/suppl/2015/03/02/jimmunol.140235
2.DCSupplemental
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Supplementary
Material
Rika Ouchida, Qing Lu, Jun Liu, Yingqian Li, Yiwei Chu,
Takeshi Tsubata and Ji-Yang Wang
The Journal of Immunology
FcmR Interacts and Cooperates with the B Cell Receptor To
Promote B Cell Survival
Rika Ouchida,*,1 Qing Lu,†,1 Jun Liu,† Yingqian Li,† Yiwei Chu,† Takeshi Tsubata,‡ and
Ji-Yang Wang†,‡,x,{
P
eripheral 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 Iga/Igb heterodimers. In vivo ablation of surface Ig (2) or inactivation of Iga (3)
causes rapid death of B cells, indicating that BCR transmits essential “tonic” survival signals in the absence of Ag ligands. Crosslinking 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-kB, and NFAT pathways.
Among these, NF-kB 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-kB pathway, which is
*Laboratory for Immune Diversity, Research Center for Allergy and Immunology,
RIKEN Yokohama Institute, Yokohama 230-0045, Japan; †Department of Immunology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China;
‡
Department of Immunology, Medical Research Institute, Tokyo Medical and Dental
University, Tokyo 113-8510, Japan; xBiotherapy Research Center, Fudan University,
Shanghai 200032, China; and {Immunobiology Institute, Fudan University, Shanghai
200032, China
1
R.O. and Q.L. contributed equally to this work.
Received for publication September 15, 2014. Accepted for publication January 21,
2015.
This work was supported in part by the Major National Scientific Research Projects
of China (2015CB943300 to J.-Y.W.), the National Natural Science Foundation of
China (81373129 to J.-Y.W.), a Grant-in-Aid for Scientific Research (C) from the
Japan Society for the Promotion of Science (25460604 to J.-Y.W.), and the National
Natural Science Foundation of China (81330080 to Q.L.).
Address correspondence and reprint requests to Dr. Ji-Yang Wang, Department of
Immunology, School of Basic Medical Sciences, Fudan University, 138 Yi Xue Yuan
Road, Shanghai 200032, China. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; B-CLL, B cell
chronic lymphocytic leukemia; GC, germinal center; IC, immune complex; MZ B,
marginal zone B; WT, wild-type.
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402352
characterized by the phosphorylation and ubiquitin-mediated
degradation of IkB inhibitory proteins, in particular IkBa. This
leads to the translocation of NF-kB1 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-kB pathway, which depends on the proteolytic processing of p100 to p52 to
generate p52/RelB (NF-kB2) 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-kB pathway substrate p100 for BAFFR-mediated
degradation (14–16).
The recently identified IgM FcR (FcmR) (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 FcmR regulates B cell development
and function. An intriguing clue came from the in vitro analysis,
which revealed a specific defect for FcmR2/2 B cells in anti-IgM–
induced survival and proliferation (19, 21). These observations
suggested a possible functional link between FcmR and BCR. In
the current study, we addressed the molecular mechanisms of FcmRmediated enhancement of anti-IgM–induced B cell survival. We show
that FcmR and BCR physically interact on the plasma membrane
of primary B cells and functionally cooperate to promote the activation of the noncanonical NF-kB pathway and BCL-xL expression. Importantly, FcmR alone in the absence of BCR signaling
had no effect on either B cell survival or NF-kB activation. These
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The IgM FcR (FcmR) promotes B cell survival, but the molecular mechanism remains largely unknown. We show using FcmR2/2
and wild-type mice that FcmR specifically enhanced B cell survival induced by BCR cross-linking with F(ab9)2-anti-IgM Abs while
having no effect on survival when the B cells were activated by CD40 ligation or LPS stimulation. FcmR expression was markedly
upregulated by anti-IgM stimulation, which may promote enhanced FcmR signaling in these cells. Immunofluorescence and
confocal microscopy analyses demonstrated that FcmR colocalized with the BCR on the plasma membrane of primary B cells.
Coimmunoprecipitation analysis further revealed that FcmR physically interacted with the BCR complex. Because NF-kB plays
a prominent role in B cell survival, we analyzed whether FcmR was involved in BCR-triggered NF-kB activation. FcmR did not
affect BCR-triggered IkBa phosphorylation characteristic of the canonical NF-kB activation pathway but promoted the production of the noncanonical NF-kB pathway component p52. Consistent with the elevated p52 levels, FcmR enhanced BCR-triggered
expression of the antiapoptotic protein BCL-xL. Importantly, FcmR stimulation alone in the absence of BCR signaling had no
effect on either IkBa phosphorylation or the expression of p52 and BCL-xL. Therefore, FcmR relied on the BCR signal to activate
the noncanonical NF-kB pathway and enhance B cell survival. These results reveal a cross-talk downstream of FcmR and BCR
signaling and provide mechanistic insight into FcmR-mediated enhancement of B cell survival after BCR stimulation. The
Journal of Immunology, 2015, 194: 3096–3101.
The Journal of Immunology
results reveal a cross-talk downstream of FcmR and BCR signaling
and provide mechanistic insight into FcmR-mediated enhancement
of B cell survival after BCR stimulation.
Materials and Methods
Mice
C57BL/6 mice were purchased from CLEA Japan (Tokyo). FcmR-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.
B cell survival assay
Analysis of FcmR expression after B cell activation
Purified spleen B cells were cultured in the presence of F(ab9)2 anti-IgM
Abs (5 mg/ml), soluble CD40L, or LPS (10 mg/ml) for 6, 24, and 48 h. The
cultured cells were first incubated with a rat IgG2b anti-mouse CD16/CD32
mAb (clone 2.4G2; BD Biosciences) to block FcgR and then stained with
either an anti-FcmR mAb (clone 4B5, rat IgG2a) or an isotype control Ab
(clone eBR2a; eBioscience). After washing, the cells were incubated with
PE-conjugated anti-rat IgG2a (clone RG7/1.30; BD Biosciences). The
mean fluorescence intensity of FcmR on purified spleen B cells before
culture was set as 1.
Immunofluorescence and confocal microscopy
Ten thousand wild-type (WT) B cells were seeded on poly-L-lysine (SigmaAldrich)–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 a-FcmR (original Ab, 1/500
dilution) together with one of the following Abs: FITC-rat IgG 2a antimouse IgM (clone R6-60.2; BD Biosciences), mouse IgG 1 antiCD79A (clone HM47, 1/500; Santa Cruz Biotechnology), or mouse
IgG2b 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 analysis
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-FcmR, an isotype control (rabbit IgG; Southern
Biotechnology Associates), goat a-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 SDSPAGE, and subjected to immunoblot. FcmR was detected with 4B5 rat antiFcmR, followed by HRP-conjugated goat anti-rat IgG (Jackson ImmunoResearch Laboratories); Iga was detected with mouse IgG1 anti-CD79A
(clone HM47), followed by HRP-conjugated goat anti-mouse IgG1 (cross
absorbed with mouse IgM, IgG2a, IgG2b, IgG3, and IgA, as well as pooled
human sera and purified human paraproteins; Southern Biotechnology
Associates); Igm was detected with HRP-conjugated goat anti-mouse IgM
(cross absorbed with mouse IgG1, IgG2a, IgG2b, IgG3, and IgA). The
following Abs were used to detect pIkBa, p100, p52, and BCL-xL: mouse
IgG1 anti–phospho-IkBa (clone 5A5; Cell Signaling Technology), rabbit
IgG anti–NF-kB2 p100/p52 (Cell Signaling Technology), rabbit IgG
a-Bcl-xL (BD Biosciences), and rabbit IgG a-actin (Sigma-Aldrich). HRPconjugated 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).
BCR Internalization assay
These experiments were performed as described previously (24).
Statistical analysis
Statistical significance was assessed by the unpaired t test.
Results
FcmR specifically enhances B cell survival induced by
anti-IgM stimulation
We recently reported that FcmR enhanced B cell survival induced
by anti-IgM but not LPS stimulation (19). To further analyze the
specificity of FcmR-mediated enhancement of B cell survival, we
cultured WT and FcmR2/2 splenic B cells in the presence of antiIgM F(ab9)2 fragment, CD40L, or LPS. Consistent with our previous findings (19), FcmR2/2 splenic B cells showed decreased
survival following a-IgM but not LPS stimulation (Fig. 1A, 1B).
In addition, we found that FcmR deficiency did not affect B cell
survival following CD40L ligation of CD40 (Fig. 1B). Moreover,
cross-linking FcmR on WT B cells with an anti-FcmR 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 FcmR 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 FcmR 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-Igk Abs (Supplemental Fig. 1C, 1D),
which cross-link both IgM and IgD. These observations collectively demonstrate that FcmR specifically enhances IgM BCR–
mediated B cell survival/activation.
Upregulation of FcmR expression on primary B cells by BCR
cross-linking
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 FcmR expression is regulated,
splenic B cells were cultured in the presence of F(ab9)2 anti-IgM
Abs, soluble CD40L, or LPS for different times and their FcmR
levels were compared with that before culture (0 h). As shown in
Fig. 2, FcmR 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 FcmR
expression by anti-IgM stimulation may in part contribute to the
FcmR-mediated enhancement of BCR-triggered B cell survival.
However, FcmR was also moderately upregulated by treatment with
CD40L or LPS without affecting B cell survival. Therefore, additional mechanisms likely exist to allow FcmR to specifically
enhance B cell survival induced by a-IgM stimulation. Although
we found that FcmR protein levels were upregulated upon B cell
activation, Choi et al. (21) found that transcript levels for FcmR
were reduced after stimulation with LPS or anti-CD40 or F(ab9)2
anti-IgM Abs. This discrepancy might be due to differential regulation of FcmR transcription and protein expression.
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Primary B cells were purified from the spleen of C57BL/6 and FcmRdeficient 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-AADlowFSChigh) and dead (7-AADhigh
FSClow) cells were analyzed by FACS (BD Biosciences). The anti-mouse
IgDa 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 antimouse Igk Abs (LE/AF goat F(ab9)2 anti-mouse k; 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 FcmR cross-linking on anti-IgD and anti-Igk–induced B
cell activation.
3097
3098
FcmR PROVIDES A SURVIVAL SIGNAL
non-Ig components of the BCR, the Iga/Igb signal transducing
molecules. As shown in Fig. 3A, FcmR indeed colocalized with
both Iga (middle panels) and Igb (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 FcmR and vice versa. It
appeared that .50% of the BCR was associated with FcmR on the
plasma membrane. To confirm that FcmR and BCR physically
interact, we immunoprecipitated FcmR under a mild detergent
condition and analyzed the coprecipitation of the BCR components. As shown in Fig. 3B, immunoprecipitation of FcmR from
splenic B cells pulled down IgH (Igm) and its associated Iga
(upper panel). Conversely, FcmR and Iga were coprecipitated with
IgM (lower panel). These results collectively indicate that FcmR
constitutively associates with the BCR on primary B cells.
Normal BCR internalization in FcmR-deficient B cells
FcmR physically associates with BCR on splenic B cells
The FcmR-mediated specific enhancement of BCR-mediated survival led us to hypothesize that there might be a physical interaction between FcmR and BCR. We first performed confocal
immunofluorescent staining. As shown in Fig. 3A, upper panels,
FcmR colocalized with IgM on the plasma membrane of splenic
B cells. However, our earlier findings suggested that FcmR is
likely occupied by IgM in vivo (19). In addition, the 4B5 a-FcmR
mAb binds to FcmR even after IgM binding (Supplemental Fig. 2).
Therefore, the colocalization between FcmR and IgM could simply be due to the binding of serum IgM to FcmR on splenic
B cells, rather than real colocalization of FcmR with membrane
BCR. We thereafter analyzed the colocalization of FcmR with the
FIGURE 2. FcmR is upregulated by BCR cross-linking. Purified spleen
B cells were cultured in the presence of F(ab9)2 anti-mouse IgM Abs,
soluble CD40L, or LPS for the indicated times and analyzed for cell
surface FcmR expression as described in Materials and Methods.
FcmR cooperates with BCR to promote p52 and BCL-xL
expression
BCR signaling activates canonical NF-kB pathway to induce the
expression of anti-apoptotic genes and enhance B cell survival.
This prompted us to examine the effect of FcmR signaling on antiIgM–induced NF-kB activation. We first analyzed anti-IgM–induced
IkBa phosphorylation, which is known to correlate with NF-kB
activation in the canonical pathway (1). No difference was observed
in the magnitude and kinetics of pIkBa between WT B cells treated
with an isotype control or the 4B5 anti-FcmR Ab (Fig. 5A) or
between WT and FcmR2/2 B cells (Supplemental Fig. 3A), indicating that FcmR does not contribute to canonical NF-kB activation. We further analyzed the activation of the noncanonical NF-kB
pathway. BCR signaling does not activate the noncanonical NF-kB
pathway but produces the noncanonical NF-kB substrate p100.
Activation of the noncanonical NF-kB pathway results in the
processing of p100 to generate p52, which associates with RELB to
form NF-kB2 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-FcmR 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, antiFcmR Ab also enhanced the expression of BCL-xL at later points
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FIGURE 1. FcmR specifically enhances B cell survival after BCR crosslinking. (A) Representative FACS profiles showing viable (7-AADlow
FSChigh) cells. (B) WT and FcmR2/2 B cells were incubated with 30 mg/ml
F(ab9)2-anti-mouse IgM Abs, soluble CD40L, or 2 mg/ml LPS and analyzed for cell survival. (C) WT B cells were stimulated as in (B) in the presence
of either the 4B5 anti-FcmR (30 mg/ml) or an isotype control Ab. Mean 6
SD of four independent experiments is shown. *p , 0.05, **p , 0.01.
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 FcmR
and BCR suggested that FcmR 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(ab9) anti-IgM Abs, which
are unable to trigger BCR signaling. In both cases, WT and FcmR2/2
B cells exhibited very similar kinetics of BCR internalization
(Fig. 4, left and middle panels). We next analyzed ligand-dependent
BCR internalization using F(ab9)2 anti-IgM Abs, which initiate BCR
signaling. F(ab9)2 anti-IgM Abs induced a much more rapid BCR
internalization (Fig. 4, right panel) compared with that induced by
F(ab9) anti-IgM Abs (middle panel), but again, this ligand-dependent
BCR internalization was unaffected by FcmR deficiency (Fig. 4,
right panel). These observations complement our previous finding
that FcmR 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, FcmR does
not seem to regulate BCR signaling through modulating its internalization processes.
The Journal of Immunology
3099
FIGURE 3. FcmR physically associates
with the BCR complex on splenic B cells.
(A) FcmR colocalizes with the BCR complex as examined by confocal microscopy.
Colocalization of FcmR and IgM (upper
three panels), FcmR and Iga (middle three
panels), and FcmR and Igb (lower three
panels) are shown. (B) Coimmunoprecipitation of FcmR with the BCR complex.
Representative results of three independent
experiments are shown. Igm, Ig m H chain;
Input, immunoblotting of whole-cell lysates
without immunoprecipitation; N.S., nonspecific
band.
Discussion
In the current study, we have demonstrated that FcmR physically
associates with BCR in primary B cells and specifically enhances
B cell survival induced by anti-IgM but not CD40L or LPS stimulation. FcmR cooperates with BCR to promote the induction of
p52 and its target BCL-xL. Importantly, FcmR alone in the absence
of BCR signaling has no effect on either B cell survival or NF-kB
activation. In other words, FcmR relies on BCR signaling to elicit
its survival function.
The cooperation between FcmR 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, FcmR is upregulated
by BCR cross-linking, which likely contributes to the FcmR-mediated
enhancement of BCR-triggered cell survival. However, one critical
difference between BAFFR and FcmR 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 FcmR
alone in the absence of BCR cross-linking is unable to activate
either the canonical or the noncanonical NF-kB pathway to induce
B cell survival. This difference predicts that BAFFR and FcmR
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 FcmR2/2 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 FcmR function on BCR signaling allows FcmR to specifically enhance the survival of Ag-stimulated B cells. Although
we have shown that cross-linking FcmR with the 4B5 anti-FcmR
mAb could enhance B cell survival induced by F(ab9)2 anti-IgM
Abs, it remains to be investigated whether FcmR 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
FcmR with soluble IgM.
It is intriguing to note that the MZ B cell population was significantly reduced in FcmR2/2 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
FIGURE 4. FcmR does not affect BCR internalization. Constitutive (ligand-independent) BCR internalization analyzed by using brefeldin A (left panel)
or F(ab9) anti-IgM Abs (middle panel). Right panel, Ligand-dependent BCR internalization analyzed with F(ab9)2 anti-IgM Abs. The experiments were
performed as described previously (13).
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of anti-IgM stimulation (Fig. 5B, 5C). Conversely, FcmR2/2
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 FcmR in promoting BCL-xL expression. However, it should be noted that the decreased BCL-xL expression in
FcmR2/2 B cells also could be due to the differences in B cell
subsets and maturation status between WT and FcmR2/2 mice.
Intriguingly, although FcmR promoted p52 accumulation and
BCL-xL expression after BCR stimulation, cross-linking FcmR
alone in the absence of BCR signaling had no detectable effect on
either IkBa phosphorylation (Fig. 5D) or the induction of p100,
p52, or BCL-xL (Fig. 5E). These observations suggest that FcmR
by itself is unable to activate NF-kB1 or NF-kB2 but relies on BCR
signaling to promote NF-kB2 activation.
3100
by cooperating with the FcmR-mediated signal. Absence of FcmR
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 FcmRdeficient 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 FcmR- and CD19-deficient mice suggests that, similar to CD21/CD19 coreceptor complex, FcmR may
function as a positive regulator in B cell responses to IgM-ICs in
GCs. Indeed, similar to CD19, the presence of FcmR reduced the
dose of BCR stimulation needed for sustaining B cell viability
(19). Our present findings also indicate that integration of BCR
and FcmR signaling at the level of BCL-xL upregulation by IgMICs 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 FcmR-deficient B cells, we
found that FcmR did not affect ligand-dependent and -independent
BCR internalization. Therefore, although FcmR associates with
BCR, it does not elicit its function through modulating BCR
internalization. It remains to be investigated how signals downstream of BCR and FcmR 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 FcmR are
phosphorylated upon ligand binding (17). Given the physical association between FcmR 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 BCRmediated signal cascades.
FcmR-deficient mice produce elevated IgG autoantibodies as they
age (19–22), suggesting that FcmR is required for maintaining selftolerance. The results of the current study demonstrate that FcmR
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 FcmR 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 FcmR 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 FcmR 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 FcmR 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 FcmR 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.
Acknowledgments
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.
Disclosures
The authors have no financial conflicts of interest.
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
FIGURE 5. FcmR cooperates with BCR to activate the noncanonical
NF-kB pathway. (A–C) WT B cells were stimulated for the indicated times
with anti-IgM (10 mg/ml) in the presence of anti-FcmR (30 mg/ml) or an
isotype control Ab. (A) IkBa phosphorylation. (B) p100, p52, and BCL-xL
protein expression. b-actin was used as a loading control. (C) Quantification of p100, p52, and BCL-xL protein expression relative to b-actin.
The expression at time 0 in WT B cells stimulated with anti-IgM + isotype
was set as 1. Mean 6 SD of three independent experiments is shown.
*p , 0.05. (D and E) Cross-linking FcmR alone in the absence of BCR
stimulation has no detectable effect on NF-kB activation. Splenic B cells
were stimulated for the indicated durations with anti-FcmR (30 mg/ml) or
an isotype control Ab. (D) IkBa phosphorylation. (E) p100, p52, and BCLxL protein expression.
FcmR PROVIDES A SURVIVAL SIGNAL
The Journal of Immunology
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