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The Journal of Immunology, 2005, 174: 7703-7710.
Copyright © 2005 by The American Association of Immunologists

A Role for c-fos/Activator Protein 1 in B Lymphocyte Terminal Differentiation1

Yusuke Ohkubo*,{dagger}, Masafumi Arima*, Eggi Arguni*, Seiji Okada{ddagger}, Kimihiro Yamashita*, Sadaki Asari*, Shintaro Obata*, Akemi Sakamoto*, Masahiko Hatano*, Jiyang O-Wang§, Masaaki Ebara{dagger}, Hiromitsu Saisho{dagger} and Takeshi Tokuhisa2,*

Departments of* Developmental Genetics (H2) and {dagger} Medicine and Clinical Oncology (K1), Graduate School of Medicine, Chiba University, Chiba, Japan; {ddagger} Division of Hematopoiesis, Center for AIDS Research, Kumamoto University, Kumamoto, Japan; and § Laboratory for B Lymphocyte Function, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama, Kanagawa 230-0045, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Expression of B lymphocyte-induced maturation protein 1 (Blimp-1) transcription factor is essential for promoting B cell differentiation into plasma cells. However, a critical transcription factor for Blimp-1 expression in activated B cells is unclear. When splenic B cells were stimulated with CD40 ligand (CD40L) and IL-4, terminal differentiation was induced in the B cells from c-fos transgenic (H2-c-fos) mice but barely in those from control littermates and from c-fos-deficient mice. AP-1 family and Blimp-1 mRNAs were transiently induced in the control B cells, and overexpression of c-Fos induced a sufficient amount of Blimp-1 for terminal differentiation in the H2-c-fos B cells. When normal and c-fos-deficient B cells were stimulated with LPS, a sufficient amount of Blimp-1 for terminal differentiation was induced in those B cells. However, expression of c-fos/AP-1 family mRNAs in LPS-stimulated normal B cells was similar to that of normal B cells stimulated with CD40L and IL-4. EMSA and chromatin immunoprecipitation assays using the AP-1-binding DNA sequence in the murine Blimp-1 promoter region demonstrated that AP-1-binding activity in nuclear protein of LPS-stimulated normal B cells was prolonged more than that in normal B cells stimulated with CD40L and IL-4. Furthermore, the percentage of CD138+ B cells within germinal center B cells in the spleen and the number of Ab-forming cells in the bone marrow of H2-c-fos mice was larger than that of control mice 12 days after immunization. Thus, although c-Fos is not essential for Blimp-1 expression, c-Fos/AP-1 positively regulates Blimp-1 expression and terminal differentiation of activated B cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The transcriptional regulator B lymphocyte-induced maturation protein 1 (Blimp-1)3 is a 98-kDa protein containing five Krüppel-type zinc fingers that confer sequence-specific DNA binding (1). Blimp-1 has been postulated to be a master regulator of B cell terminal differentiation. In the BCL-1 lymphoma model of differentiation from a mature B cell to a plasma cell, ectopic expression of Blimp-1 is sufficient to cause terminal differentiation evidenced by loss of surface Igs, IgM secretion, expression of CD138 (syndecan-1) on the cell surface, and cessation of cell division (1, 2, 3). Blimp-1 was also found in a fraction (4–15%) of germinal center B cells in murine spleen and human tonsils (4). In mice, Blimp-1+ germinal center B cells express syndecan-1 and cytoplasmic Ig but not Bcl6 (4), and Blimp-1 expression is repressed by Bcl6 (5, 6). The Blimp-1 conditional knockout mice demonstrated that Blimp-1 is required for differentiation of plasma cells and pre-plasma memory B cells (7). Thus, these data support a model in which Blimp-1 expression is critical for commitment to a plasma cell, rather than a memory cell, fate. Since Bcl6 suppresses cytokine-driven differentiation of primary B cells to plasma cells by inhibiting STAT3-dependent transcriptional events (6), STAT3 may be an important transcription factor for Blimp-1 expression. The recent report also demonstrated that Bcl6 is a potent repressor of transcriptional activity mediated by AP-1 factors and that repression of AP-1 function by Bcl6 may be a key mechanism for how Bcl6 regulates Blimp-1 expression (8).

The protooncogene c-fos encodes a nuclear phosphoprotein, c-Fos. c-Fos in a complex with the product of another protooncogene c-jun (AP-1) regulates expression of the AP-1-binding genes at their transcriptional level (9, 10, 11, 12). Since expression of c-fos and c-jun family genes is transiently induced in B cells stimulated with CD40 ligand (CD40L) and IL-4 as an immediate early gene, the function of c-Fos/AP-1 may be implicated in the transduction of signals induced by growth and differentiation factors (13, 14). We generated transgenic mice carrying the murine c-fos gene under the control of the murine MHC gene (H2-Kb) promoter (H2-c-fos) (15). Splenic T and B cells from the mice constitutively express a high level of the exogenous c-fos gene (16). When H2-c-fos mice are immunized with T-dependent Ags, the mice can make the reduced size of germinal centers and fail to generate memory B cells in the spleen (16, 17). These results suggest that the ectopic expression of c-Fos may accelerate commitment of germinal center B cells to a plasma cell fate.

Engagement of CD40 on B cells by its ligand, gp39 or CD154, on activated T cells is a crucial component of cognate T cell help (18). CD40 signaling in B cells triggers a potent proliferative response (19, 20), induces expression of costimulatory and adhesion molecules (21), and mediates clonal expansion and survival within germinal centers (22). Mice deficient in either CD40 or CD40L are unable to form germinal centers or to make high-affinity, class-switched Abs after immunization with T-dependent Ags (23, 24, 25). Several in vitro experiments have also suggested that CD40 signaling is important for promoting B cell differentiation and Ig secretion (26, 27, 28). However, stimulation of CD40 on B cells actively inhibits the B cells from differentiating into Ab-forming cells (AFCs) (29, 30, 31, 32). This arrest is manifested as a reduction in mRNA levels of secretary Ig gene products as well as the loss of Blimp-1 (32). Thus, a role for CD40 signaling in B cell terminal differentiation is controversial. In this study, we directly tested the role for the ectopic c-Fos in terminal differentiation of B cells activated with CD40L stimulation. When splenic B cells from H2-c-fos mice were cultured with CD40L and IL-4, cell proliferation of these B cells was augmented compared with that of normal control B cells. The H2-c-fos B cells but not the control B cells differentiated into AFCs within 2 days after stimulation. We discuss a critical role for c-Fos/AP-1 in terminal differentiation of B cells activated with CD40L stimulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals and immunization

C57BL/6CrSlc mice were purchased from Japan SLC. Transgenic mice carrying the mouse c-fos gene under the control of the H-2Kb promoter (H2-c-fos) (15), and c-fos-deficient mice (33) were maintained by heterozygous mating in our animal facilities. These mice were immunized i.p. with 100 µg of alum-precipitated DNP-OVA or with 50 µg of alum-precipitated (4-hydroxy-3-nitrophenyl)acetyl-chicken {gamma}-globulin (NP25-CG) (Biosearch Technologies).

Preparation of splenic B cells

Splenic B cells were enriched by depleting non-B lineage cells from spleen cells. In brief, spleen cells were incubated with PE-anti-CD43 Ab (BD Pharmingen). These cells were subsequently reacted with immunomagnetic beads coated with anti-PE Ab (Miltenyi Biotec). Labeled cells were removed by a MACS system (Miltenyi Biotec). The resulting B cell fraction contained >95% of B220+ B cells.

Cell culture and stimulation of lymphocytes

Purified B cells (1.5 x 105/ml) were cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% FCS (Intergen), 50 µM 2-ME, 100 µg/ml streptomycin (Wako Chemical), and 100 U/ml penicillin G/potassium (Banyu Pharmaceutical). For B cell activation, CD40L (culture supernatants from a myeloma cell line producing soluble CD40L-CD8 fusion protein) (34) in the presence or absence of rIL-4 (1000 U/ml) (35) or LPS (Sigma-Aldrich) were added to the cultures. These B cells were cultured in a humidified atmosphere at 37°C with 5% CO2.

mAbs and flow cytometry

Single-cell suspensions were prepared from cultured B cells or spleen cells, and then Fc receptors were blocked with 5 µg/ml anti-CD16/32 Ab (2.4G2; BD Pharmingen). Abs used were monoclonal FITC-anti-IgMb (BD Pharmingen), PE-anti-IgDb (Bioscience), allophycocyanin-anti-IgG1 (BD Pharmingen), PE-anti-CD19 (BD Pharmingen), PE-anti-B220 (BD Pharmingen), FITC-peanut agglutinin (PNA; Vector Laboratories), and biotinylated anti-syndecan-1 Abs (BD Pharmingen). Biotinylated Abs were detected by allophycocyanin-conjugated streptavidin (BD Pharmingen). CFSE labeling of B cells was performed as described previously (36). Cells were analyzed on a FACSCalibur (BD Biosciences) using CellQuest software (BD Biosciences). Dead cells were excluded on the basis of forward and side light scatter parameters and propidium iodide (2 µg/ml) staining.

Proliferation assay

Purified B cells (1 x 105/well) were cultured with various doses of CD40L with or without rIL-4 or various amounts of LPS in a 96-well microplate for 48 h. Cultured cells were pulsed with 1 µCi of [3H]thymidine (Amersham International) for the last 8 h, and [3H]thymidine uptake was measured in a liquid scintillation counter.

ELISPOT assay

AFCs producing IgM or IgG1 Abs were detected by ELISPOT assays as described elsewhere (37). Briefly, nitrocellulose filters were coated with 5 µg/ml goat anti-mouse IgG or IgM Abs in PBS at 4°C overnight and then blocked with 1% BSA in PBS. Serially diluted cells were incubated on the nitrocellulose filters at 37°C in 5% CO2. After a 2-h incubation, the filters were washed vigorously and spots were developed with alkaline phosphatase-conjugated anti-IgM or HRP-conjugated anti-IgG1 Abs. For detecting NP-specific AFCs, nitrocellulose filters were coated with NP-BSA (NP25-BSA; Biosearch Technologies).

Northern blot

Northern blotting was performed as described previously (38). Briefly, total RNA (10 µg) was electrophoresed through a 1.0% agarose gel containing formaldehyde and transferred to a nylon membrane (Roche Molecular Biochemicals). The filter was prehybridized for 1 h and hybridized overnight at 50°C in 50% formamide hybridization buffer with 0.5% SDS, 1% blocking reagent, and 15 ng/ml digoxigenin (DIG; Roche Molecular Biochemicals)-labeled probes. Following hybridization, the filter was washed twice for 15 min with 0.1x SSC and 0.1% SDS at 55°C. The DIG-labeled probe was detected with sheep anti-DIG Abs conjugated with alkaline phosphatase. The anti-DIG Ab detection reaction was performed using an enhanced chemiluminescent detection system (Roche Molecular Biochemicals). DIG-labeled probes (c-fos, c-jun, junB, and junD) were made as described previously (17, 39). Other probes (fosB, fra-1, fra-2, Blimp-1, XBP-1, and J chain) were made from the cDNAs, which were made by reverse-transcribed PCR with specific primers (40, 41, 42).

Isolation of nuclear proteins

Nuclear proteins were isolated from splenic B cells using the method as described previously (43), with slight modification. Briefly, splenic B cells (1 x 107) were resuspended in 400 µl of cold hypotonic buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.1% Triton X-100, 1 mM DTT, 100 mM PMSF, and 5 µg/ml aprotinin). Nuclei were collected by centrifugation and disrupted by sonication in 100 µl of immunoprecipitation buffer (1 mM HEPES (pH 7.5), 5 M NaCl, 2.5 mM EGTA, 1 mM EDTA, 0.1% Tween 20, 10% glycerol, 1 mM DTT, 100 mM PMSF, and 5 µg/ml aprotinin) at 4°C. Nuclear extracts were immediately stored at –80°C. The amount of protein was determined using the Bio-Rad protein assay (Bio-Rad).

EMSA

Double-stranded oligonucleotides corresponding to putative AP-1-binding sequences in the murine Blimp-1 gene (mAP1BS-1, 5'-TTATTAACCGATGCTGAGTCAGCACCACTATGGACAGAAA-3' and mAP1BS-2, 5'-TTTCTGTCCATAGTGGTGCTGACTCAGCATCGGTTAATAA-3', underlined; AP-1-binding sequence) were synthesized and biotinylated using the biotin 3'end-labeling kit (Pierce). Binding activity of AP-1 to the mAP1BS was determined using the LightShift chemiluminescent EMSA kit (Pierce) according to the manufacturer’s instruction. Competitive EMSA was done by adding 100-fold molar excess of unlabeled double-stranded oligonucleotides to the mixture. The sequences of the mutant oligonucleotides (two bases mismatch, underlined) were as follows: Mut-1, 5'-TTATTAACCGATGCCAAGTCAGCACCACTATGGACAGAAA-3' and Mut-2, 5'-TTTCTGTCCATAGTGGTGCTGACTTGGCATCGGTTAATAA-3'.

Chromatin immunoprecipitation (ChIP) assay

ChIP assays were performed as previously described (44). Briefly, formaldehyde solution (37%; Fisher Scientific), at a final concentration of 1%, was added directly to B cells (3 x 106) after each stimulation. Cross-linking of proteins on chromatin was allowed to occur at 4°C for 60 min, and the cells were lysed by SDS lysis buffer with protease inhibitors. Chromatin in the lysate was sonicated to an average length of 400–600 bp as determined by agarose gel electrophoresis. The suspension was precleared with salmon sperm DNA/protein A/agarose/50% slurry for 3 h at 4°C and incubated with 2 µg of rabbit polyclonal anti-c-Fos Abs (Santa Cruz Biotechnology) overnight. The immune complexes were incubated with salmon sperm DNA/protein A/agarose/50% slurry with mild shaking for 3 h at 4°C, washed, and eluted. After proteinase K treatment, DNA in samples was phenol extracted and resuspended in 50 µl of TE buffer (10 mM Tris-HCl (pH 7.5) and 1 mM EDTA). DNA solution (2 µl) was used for PCR amplification (28 cycles). PCR products were analyzed by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. The following primers were used: mAP1BS-1, 5'-GGAAAACAGAAGAACAAGCCTGTC-3' and 5'-ATAGCCAGCATCCCATCACAGC-3' and mAP1BS-2, 5'-AAGGAAAGCAGGGTAAACCGTG-3' and 5'-GTTAGCTTGCTCTTGTGCCAGG-3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
c-Fos augments proliferation of splenic B cells stimulated with CD40L and IL-4

The number of B220+ B cells in the spleen of H2-c-fos mice was similar to that of control littermates (17), and the amount of CD40 on H2-c-fos B cells was also similar to that on control B cells (data not shown). These B cells were stimulated with various doses of CD40L in the presence or absence of IL-4. Cell proliferation of the B cells was measured by DNA synthesis on day 2 of culture. The response of H2-c-fos B cells was markedly augmented compared with that of control B cells at any doses of CD40L examined (Fig. 1A). We have shown that H2-c-fos B cells stimulated with LPS proliferated more than LPS-activated normal B cells (45) and those results were repeated in Fig. 1B. The plateau responses of H2-c-fos and control B cells stimulated with CD40L and IL-4 were almost the same as those of H2-c-fos and control B cells stimulated with LPS, respectively. These results suggest that the amount of c-Fos may decide the intensity of signaling in activated B cells to proliferate.



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  		FIGURE 1. c-Fos augments cell proliferation of splenic B cells stimulated with CD40L. Splenic B cells from H2-c-fos (solid line) and wild-type control (broken line) mice were cultured with various concentrations of CD40L in the presence or absence of IL-4 (A) or with various doses of LPS (B). Cell proliferation was measured by [3H]thymidine uptake on day 2 of culture. Results represent means and variations (SD) from triplicate cultures. The data presented are representative of three independent experiments.

 
c-Fos augments terminal differentiation of B cells stimulated with CD40L and IL-4

CFSE-labeled H2-c-fos B cells were cultured with CD40L and IL-4 for 3 days, and cell surface expression of CFSE and several differentiation markers including CD138 (syndecan-1) on these B cells were analyzed on a FACS. As shown in Fig. 2A, the H2-c-fos B cells such as the control B cells divided up to five cell divisions within 3 days after stimulation, suggesting that the ectopic c-Fos does not accelerate cell cycle progression of B cells stimulated with CD40L and IL-4.



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  		FIGURE 2. c-Fos accelerates differentiation of splenic B cells stimulated with CD40L and IL-4. CFSE-labeled splenic B cells were stimulated with CD40L (1/8 dilution) and IL-4 for 3 days. A, Cell division of the activated H2-c-fos (c-fos) and wild-type control (WT) B cells was analyzed on FACS. The solid lines in the 0 division show the histograms of CFSE-labeled B cells without stimulation for 3 days. B, Expression of IgM, IgD, and syndecan-1 on the activated H2-c-fos (open histograms) and wild-type control (filled histograms) B cells was analyzed. The data presented are representative of two independent experiments.

 
Expression of IgM and IgD on the H2-c-fos and control B cells was reduced gradually as cell division progressed, and the reduction of surface IgM expression on the H2-c-fos B cells was slightly slower than that on the control B cells (Fig. 2B). However, the amount of IgD on the H2-c-fos B cells clearly reduced on division 1. Syndecan-1 expression was detected on some of the H2-c-fos B cells divided more than three cell divisions but not on the control B cells. Since the reduction of surface IgD expression and the induction of syndecan-1 expression on the H2-c-fos B cells were faster than those on the control B cells, the ectopic c-Fos might accelerate terminal differentiation of B cells stimulated with CD40L and IL-4.

Then we examined kinetics of syndecan-1 expression on H2-c-fos B cells after stimulation with CD40L and IL-4. Syndecan-1+ B cells were clearly detected in both H2-c-fos and control B cell cultures from day 2 of culture and the numbers in both cultures increased thereafter, although the percentages in the H2-c-fos culture were larger than those in the control culture (Fig. 3A). Kinetics of syndecan-1+ B cells developed in the H2-c-fos culture was compared with that in H2-c-fos B cell culture stimulated with LPS. The kinetics was almost similar between them and also similar to that in control B cell culture stimulated with LPS.



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  		FIGURE 3. c-Fos augments terminal differentiation of splenic B cells stimulated with CD40L and IL-4. Splenic B cells from H2-c-fos, c-fos-deficient, and wild-type control mice were cultured with CD40L (1/8 dilution) and IL-4 or with LPS (3 µg/ml) for 4 days. A, Expression of syndecan-1 on the activated B cells from H2-c-fos (c-fos) and wild-type control (WT) mice was analyzed on each day of culture. The numbers indicate the percentages of CD19dull and syndecan-1high cells in each oval. These results are representative of two independent experiments. B, IgM-AFCs in the B cell cultures from H2-c-fos ({blacksquare}) and wild-type control ({circ}) mice were detected by ELISPOT assays. Results represent means and variations (SD) from triplicate cultures. The data presented are representative of two independent experiments. C, Expression of syndecan-1 on the activated B cells from c-fos-deficient (c-fos-KO) and wild-type control (WT) mice was analyzed. The numbers indicate the percentages of CD19dull and syndecan-1high cells in each oval. These results are representative of two independent experiments.

 
We examined kinetics of IgM-AFCs developed in these B cell cultures. No IgM-AFC (<1 of 105 B cells) was detected in these B cell cultures before stimulation (Fig. 3B). More than 102 IgM-AFCs of 105 B cells were detected in H2-c-fos B cells stimulated with CD40L and IL-4 from day 2 after stimulation. The number in the H2-c-fos culture increased and reached the plateau from day 3 of culture. Although the number of IgM-AFCs in control B cell culture stimulated with CD40L and IL-4 was not >102 IgM-AFCs of 105 B cells within 4 days of culture, these small numbers were increasing from day 2 of culture and reached the plateau on day 3 of culture. Furthermore, ~10 IgM-AFCs of 105 B cells were detected in both H2-c-fos and control B cell cultures stimulated with LPS on day 1 of culture. These numbers increased and reached the plateau on day 2 of culture. Since the plateau numbers of IgM-AFCs in both H2-c-fos and control cultures stimulated with LPS were almost the same as those of H2-c-fos culture stimulated with CD40L and IL-4, the induction rate of IgM-AFCs in H2-c-fos B cells stimulated with CD40L and IL-4 was comparable to those in H2-c-fos and control B cells stimulated with LPS.

A role of c-Fos in terminal differentiation of B cells was further examined using c-fos-deficient mice. We analyzed syndecan-1 expression on c-fos-deficient B cells 4 days after stimulation with CD40L and IL-4. Syndecan-1+ B cells were clearly detected in both c-fos-deficient and normal B cell cultures, although the percentage in the c-fos-deficient B cell culture was smaller than that in the normal B cell culture (Fig. 3C). However, the percentages of syndecan-1+ B cells developed in B cell cultures stimulated with LPS were similar between c-fos-deficient and normal B cells.

Blimp-1 expression is induced in H2-c-fos B cells stimulated with CD40L and IL-4

Expression of bcl6 and Blimp-1 was examined in H2-c-fos and control B cells stimulated with CD40L and IL-4 by Northern blot. Expression of bcl6 mRNA was detected in H2-c-fos and control B cells at resting, and the expression was maintained at the resting level after stimulation (Fig. 4A). Blimp-1 expression was clearly induced in the H2-c-fos B cells 1 day after stimulation and increased thereafter. However, the expression was not induced in the control B cells within 2 days after stimulation and slightly up-regulated 3 days after stimulation. Expression of XBP-1 and J chain was induced in the H2-c-fos B cells but not in the control B cells within 2–3 days after stimulation. When H2-c-fos and control B cells were stimulated with LPS, bcl6 expression was not induced but was maintained at the resting level in the H2-c-fos and the control B cells (Fig. 4B). However, Blimp-1 expression was induced in both H2-c-fos and control B cells from 24 h after stimulation and increased thereafter.



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  		FIGURE 4. Blimp-1 expression is induced in H2-c-fos B cells stimulated with CD40L and IL-4. Splenic B cells from H2-c-fos (c-fos) and wild-type control (WT) mice were cultured with CD40L (1/8 dilution) and IL-4 (A) or with LPS (3 µg/ml; B) for 3 days. Expression of Blimp-1 and other genes related with B cell terminal differentiation in the activated B cells was analyzed on each day of culture by Northern blot. {beta}-actin mRNA was used as an amount control of mRNA. The data presented are representative of two independent experiments.

 
Expression of AP-1 family genes was examined in normal B cells stimulated with CD40L and IL-4 and compared with that of Blimp-1 and bcl6 in these B cells. Blimp-1 expression was transiently induced within 1 h after stimulation and the peak was between 3 and 6 h after stimulation (Fig. 5A). The expression was down-regulated thereafter and increased on day 3 after stimulation. However, bcl6 expression was not clearly induced but was maintained at the resting level in the B cells. Expression of c-fos, fosB, junB, and junD was transiently induced within 30 min after stimulation and disappeared thereafter. However, expression of fra-1, fra-2, and c-jun was not detected in the B cells (data not shown). When we examined expression of these genes in normal B cells stimulated with LPS, Blimp-1 expression was transiently induced within 1 h after stimulation, down-regulated until 6 h after stimulation, and then up-regulated from 12 h after stimulation and increased thereafter (Fig. 5B). Bcl6 expression was transiently induced in the B cells within 1 h after stimulation and the peak was between 3 and 6 h after stimulation and down-regulated thereafter. These results suggest that AP-1 induces the transient Blimp-1 expression in activated B cells within 3–6 h after stimulation. However, Blimp-1 expression in the LPS-stimulated B cells between 12 and 72 h after stimulation cannot be explained by the mRNA expression of AP-1 family genes.



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  		FIGURE 5. AP-1 family gene expression is induced in control B cells stimulated with CD40L and IL-4. Splenic B cells from wild-type mice were cultured with CD40L (1/8 dilution) and IL-4 (A) or with LPS (3 µg/ml; B) for 3 days. Expression of the AP-1 family, Blimp-1, and bcl6 genes in the activated B cells was analyzed using Northern blot. {beta}-actin mRNA was used as an amount control of mRNA. The data presented are representative of two independent experiments.

 
c-Fos/AP-1 activity is prolonged in B cells activated with LPS

The promoter region of murine Blimp-1 gene contains two putative AP-1-binding sequences (mAP1BS-1 and mAP1BS-2). Thus, EMSA was performed to examine binding activity of c-Fos/AP-1 in nuclear proteins of H2-c-fos and control B cells stimulated with CD40L and IL-4 to the mAP1BS. As shown in Fig. 6A, a gel retardation band to the mAP1BS-1 was observed using the nuclear proteins of H2-c-fos and control B cells. This band from control B cells was obviously removed by a nonlabeled probe with the same sequence (WT) as a cold competitor. In contrast, a mutated cold probe (Mut) as a competitor did not inhibit formation of the gel retardation band. Furthermore, the band was removed by the addition of anti-c-Fos Abs, partly by that of anti-JunD Abs, but not by that of control IgG Abs, indicating sequence-specific binding of c-Fos/AP-1 to the mAP1BS-1. The band was still detected in nuclear protein of the H2-c-fos B cells but not in that of the control B cells 24 h after stimulation. The specific band to the mAP1BS-2 was also detected in the nuclear proteins of H2-c-fos and control B cells (data not shown). The AP-1 band to the mAP1BS-1 and to the mAP1BS-2 (data not shown) was detected in nuclear proteins of H2-c-fos and control B cells stimulated with LPS until 24 h after stimulation.



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  		FIGURE 6. Binding of nuclear proteins from activated B cells to the putative AP-1-binding sequence in the promoter region of the Blimp-1 gene (mAP1BS). Splenic B cells from H2-c-fos (transgenic (TG)) and wild-type control (WT) mice were cultured with CD40L (1/8 dilution) and IL-4 or with LPS (3 µg/ml) for 24 h. A, Nuclear proteins of these activated B cells were incubated with the mAP1BS-1 as a probe, and a retardation band was detected by EMSA. WT and Mut were used as cold competitors. Ab supershift experiments were performed with various Abs (anti-c-Fos, anti-JunD, and control IgG). Nuclear proteins of activated B cells 6 h after stimulation were used for the cold competition and the Ab supershift experiments. These results are representative of two independent experiments. B, Binding of c-Fos to the mAP1BS-1 in nuclear proteins of these activated B cells was analyzed by ChIP assays. PCR were performed on both input chromatin (total DNA) and immunoprecipitation products. The data presented are representative of two independent experiments.

 
The binding of c-Fos on the mAP1BS-1 and the AP1BS-2 was confirmed by ChIP assays with anti-c-Fos Abs. The mAP1BS-1 was contained in the DNA complexes of H2-c-fos and control B cells 12 h after stimulation with CD40L and IL-4 (Fig. 6B). The mAP1BS-1 was still contained in the DNA complex of the H2-c-fos B cells but not in that of the control B cells 24 h after stimulation. The mAP1BS-2 was also contained in the DNA complexes of H2-c-fos and control B cells (data not shown). The mAP1BS-1 and the mAP1BS-2 (data not shown) were contained in the DNA complexes of H2-c-fos and control B cells stimulated with LPS until 24 h after stimulation. Since the binding activity of c-Fos/AP-1 in nuclear proteins of these activated B cells to the mAP1BS correlated with Blimp-1 expression in these B cells, c-Fos/AP-1 positively regulates Blimp-1 expression in activated B cells.

c-Fos augments terminal differentiation of Ag-specific B cells in vivo

Germinal center formation in the spleen of H2-c-fos mice is perturbed and the perturbation could not be explained by apoptosis of the germinal center B cells (17). These results prompted us to examine the incidence of terminal differentiation in germinal center B cells of H2-c-fos mice. Since a few of syndecan-1+ B cells as plasma cell precursors are detected in germinal center B cells after immunization (4), the percentage of syndecan-1+ B cells in germinal center B cells was analyzed in the spleen of H2-c-fos mice immunized with NP-CG in alum. Fig. 7A shows that germinal center formation was perturbed in the spleen of H2-c-fos mice 12 days after immunization. The percentage of PNA+ B cells in spleen cells of H2-c-fos mice was less than that of control littermates. The percentage of syndecan-1+ B cells in the PNA+ B cells of H2-c-fos mice was clearly larger than that of control mice (Fig. 7B). These results were repeated in the spleen of H2-c-fos mice immunized with DNA-OVA in alum (data not shown).



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  		FIGURE 7. c-Fos augments terminal differentiation of Ag-specific B cells in H2-c-fos mice after immunization. H2-c-fos (c-fos) and wild-type control (WT) mice were immunized with NP-CG. A, Syndecan-1+ cells in germinal center (PNA+, B220+) B cells in the spleen of mice 12 days after immunization were analyzed. The numbers indicate the percentages of germinal center B cells in total spleen cells and those of syndecan-1+ cells in the germinal center B cells. B, Summary of the percentages from H2-c-fos ({blacksquare}) and wild-type control ({square}) mice shown in A. Results represent means and variations (SD) from three mice per group. C, Numbers of AFCs producing IgM or IgG1 anti-NP Abs in the bone marrow of H2-c-fos ({blacksquare}) and wild-type control ({square}) mice 12 days after immunization were measured by ELISPOT assays with NP25-BSA. Results represent means and variations (SD) from three mice per group.

 
We have shown that large numbers of AFCs are detected in the bone marrow of normal mice 14 days after immunization and that many of them are derived from germinal centers (37). To confirm the high incidence of terminal differentiation in germinal center B cells of H2-c-fos mice, numbers of IgM- and IgG1-AFCs were examined in the bone marrow of H2-c-fos mice 12 days after immunization with NP-CG by ELISPOT assays. The number of IgM-AFCs in the bone marrow of H2-c-fos mice was 5-fold larger than that of control mice (Fig. 7C). However, IgG1-AFCs were not detected at all in the bone marrow of the H2-c-fos mice, repeating the Ig-class switch abnormality of H2-c-fos mice (16, 17). These results suggest that c-Fos/AP-1 positively regulates terminal differentiation of Ag-activated B cells.


   Discussion
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 Abstract
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Disclosures
 References
 
Cell proliferation of H2-c-fos B cells stimulated with CD40L in the presence or absence of IL-4 was markedly augmented compared with that of control B cells. Since expression of the endogenous AP-1 family genes and AP-1 activity were transiently induced in control B cells stimulated with CD40L and IL-4, the ectopic c-Fos augments a role for the endogenous AP-1 in cell cycle progression of the B cells. However, the ectopic c-Fos did not accelerate cell cycle progression of the B cells because both H2-c-fos and control B cells divided five times within 3 days after stimulation. The cell numbers of H2-c-fos B cells divided more than four divisions were larger than those of control B cells, and cell proliferation of H2-c-fos B cells stimulated with CD40L in the absence of IL-4 was higher than that of control B cells at any doses of CD40L examined. These results suggest that H2-c-fos B cells are more sensitive to CD40L stimulation and that the amount of c-Fos/AP-1 in B cells stimulated with CD40L may decide the intensity of CD40 signaling to proliferate. The ectopic c-Fos also augmented cell proliferation of naive B cells stimulated with LPS (45). AP-1 activity was detected in both control and H2-c-fos B cells 24 h after LPS stimulation, suggesting that the augmentation of cell proliferation is not due to the prolonged activation of AP-1 in the B cells. These results also suggest that the amount of c-Fos/AP-1 decides the intensity of LPS signaling to proliferate. Thus, a function of c-Fos/AP-1 induced in activated B cells as an immediate early gene product is an amplifier of the initial signals. Further study is required to elucidate a target gene of c-Fos/AP-1 in the signal transduction pathway initiated by CD40L or LPS stimulation.

Many investigators have suggested that CD40 signaling induces not only B cell activation and proliferation but also B cell terminal differentiation, particularly in combination with cytokines (20, 28, 46). In contrast, several reports have suggested that CD40L-deficient humans produce normal or elevated levels of serum IgM (47) and that CD40 signaling directly prevents B cell terminal differentiation (29, 30, 31, 32, 48). Thus, CD40 signaling is not only insufficient to induce plasma cell formation, but actively arrests B cells at a stage before terminal differentiation. We showed here that terminal differentiation was barely induced in control B cells stimulated with CD40L and IL-4 and that the ectopic c-Fos efficiently induced terminal differentiation of the B cells. Indeed, AP-1 activity was detected in the control B cells and the H2-c-fos B cells until 12 and 24 h after stimulation, respectively. Although stimulation of control B cells with CD40L and IL-4 induced expression of junB and junD genes until 24 h after stimulation, transcriptional activity of AP-1 composed of a Fos/Jun heterodimer is 25-fold more effective than that of a Jun/Jun homodimer (49), and AP-1 composed of JunB/JunD may not be effective to induce terminal differentiation of the B cells. Thus, the effective AP-1 (c-Fos/Jun) may be a key transcription factor for terminal differentiation of B cells stimulated with CD40L and IL-4.

Blimp-1 expression was detected in H2-c-fos B cells but not in control B cells stimulated with CD40L and IL-4 on days 1 and 2 after stimulation. Since there are two AP-1-binding sites in the murine Blimp-1 promoter region and c-Fos binds to these sites in H2-c-fos B cells stimulated with CD40L and IL-4, effective AP-1 composed of a c-Fos/Jun heterodimer may be enough to activate the Blimp-1 promoter in the B cells. Blimp-1 expression might be negatively regulated by Bcl6 in control B cells stimulated with CD40L and IL-4 because overexpression of Bcl6 represses transcription of the human Blimp-1 promoter almost entirely (8). However, Bcl6 is barely induced in either H2-c-fos or control B cells stimulated with CD40L and IL-4. Furthermore, splenic B cells from Bcl6-deficient mice stimulated with CD40L and IL-4 did not differentiate into IgM-AFCs and IgG1-AFCs (data not shown). These results suggest that little induction of Blimp-1 expression in control B cells stimulated with CD40L and IL-4 is not due to the negative regulatory effect of a transcriptional repressor, Bcl6, but due to the deficiency of an effective transcriptional factor, c-Fos/AP-1.

Blimp-1 expression and AP-1 activity were detected in both H2-c-fos and control B cells stimulated with LPS from day 1 after stimulation. Although mRNA expression of AP-1 family genes in control B cells stimulated with LPS was similar to that in control B cells stimulated with CD40L and IL-4, DNA-binding activity of AP-1 in the LPS-stimulated B cells was prolonged more than that in the CD40L-stimulated B cells, suggesting that AP-1 is unstable in the CD40L-stimulated B cells. c-Fos/AP-1 may be dephosphorylated and/or denatured in control B cells stimulated with CD40L and IL-4 within 24 h after stimulation. These results support that c-Fos/AP-1 is a key transcription factor for terminal differentiation of B cells stimulated with CD40L and IL-4. However, terminal differentiation of B cells was induced in c-fos-deficient B cells stimulated with CD40L and IL-4 or with LPS, suggesting the functional redundancy of c-Fos family proteins. Thus, the effective AP-1, including Fos family proteins, may be a key transcription factor for Blimp-1 expression and terminal differentiation of activated B cells.

Functionally effective memory B cells are developed in germinal centers (37), and signaling through CD40 drives germinal center B cells toward a memory cell phenotype by preventing terminal differentiation of these B cells (29, 31). However, the in vitro studies shown here may not support the negative effect of CD40 signaling on terminal differentiation of germinal center B cells, since overexpression of c-Fos-induced terminal differentiation of B cells stimulated with CD40L and IL-4. Furthermore, the in vivo results may also deny the negative effect of CD40 signaling. CD40 signaling is essential for activated B cells to differentiate into germinal center B cells (22, 23, 24, 25), indicating that germinal center B cells are stimulated with CD40L on helper T cells. The germinal center formation is perturbed in H2-c-fos mice after immunization (17) and the percentage of plasma cell precursors (syndecan-1+ germinal center B cells) in the spleen of H2-c-fos mice was 2- to 3-fold larger than that of control mice after immunization. Thus, the ectopic c-Fos may compose c-Fos/AP-1 in germinal center B cells and induce Blimp-1 expression in these B cells at an earlier stage, leading to premature generation of plasma cells, which emigrate from the germinal centers. Indeed, the number of IgM-AFCs in the bone marrow of H2-c-fos mice was 5-fold larger than that of control mice after immunization. These in vitro and in vivo results suggest that CD40 signaling in germinal center B cells may not actively prevent their terminal differentiation.

Bcl6, which is highly expressed in germinal center B cells (50, 51), is also essential for development of germinal center B cells (52) and plays a critical role in maintaining these B cells at the germinal center stage by preventing expression of Blimp-1 (5, 6). Since syndecan-1+ B cells in germinal centers emigrate from the germinal centers, this higher incidence of terminal differentiation in germinal center B cells of H2-c-fos mice may explain the reduced size of germinal centers in H2-c-fos mice (17). The repression of Blimp-1 expression by Bcl6 may be due to inhibit the transcriptional activity of AP-1 by its direct binding to Jun family but not Fos family proteins (8), although the binding of Bcl6 to Jun proteins may be a relatively low affinity interaction, a transient interaction, or a complex interaction that requires other proteins to stabilize the interaction in vivo. Furthermore, the in vitro studies shown here suggest that c-Fos/AP-1 is a key transcription factor for Blimp-1 expression in activated B cells. Thus, despite the presence of large amounts of Bcl6, the ectopic c-Fos efficiently binds to Jun family proteins to make effective c-Fos/AP-1 to induce Blimp-1 expression in germinal center B cells. Regardless of the nature of the interaction between Bcl6 and Jun proteins, c-Fos/AP-1 may be a critical factor for Blimp-1 expression in germinal center B cells.


   Acknowledgments
 
We thank Dr. H. Zhang for discussion, H. Satake for technical assistance, and K. Yagyu and N. Kakinuma for secretarial assistance.


   Disclosures
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 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-in-Aid from the Ministry of Education, Science, Technology, Sports and Culture of Japan and the Uehara Memorial Foundation. Back

2 Address correspondence and reprint requests to Dr. Takeshi Tokuhisa, Department of Developmental Genetics, Graduate School of Medicine (H2), Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail address: tokuhisa{at}med.m.chiba-u.ac.jp Back

3 Abbreviations used in this paper: Blimp-1, B lymphocyte-induced maturation protein 1; DIG, digoxigenin; AFC, Ab-forming cell; NP, (4-hydroxy-3-nitrophenyl)acetyl; CG, chicken {gamma}-globulin; PNA, peanut agglutinin; ChIP, chromatin immunoprecipitation. Back

Received for publication January 20, 2004. Accepted for publication April 11, 2005.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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