A Role for c-fos/Activator 1 in B Lymphocyte Terminal Differentiation Yusuke Ohkubo, Masafumi Arima, Eggi Arguni, Seiji Okada, Kimihiro Yamashita, Sadaki Asari, Shintaro Obata, This information is current as Akemi Sakamoto, Masahiko Hatano, Jiyang O-Wang, of September 26, 2021. Masaaki Ebara, Hiromitsu Saisho and Takeshi Tokuhisa J Immunol 2005; 174:7703-7710; ; doi: 10.4049/jimmunol.174.12.7703

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

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

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

Expression of B lymphocyte-induced maturation protein 1 (Blimp-1) factor is essential for promoting differ- entiation into plasma cells. However, a critical 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 Downloaded from 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 http://www.jimmunol.org/ 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 immu- nization. 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. The Journal of Immunology, 2005, 174: 7703–7710.

he transcriptional regulator B lymphocyte-induced matu- entiation of plasma cells and pre-plasma memory B cells (7). Thus, ration protein 1 (Blimp-1) 3 is a 98-kDa protein contain- these data support a model in which Blimp-1 expression is critical T ing five Kru¨ppel-type zinc fingers that confer sequence- for commitment to a plasma cell, rather than a memory cell, fate. specific DNA binding (1). Blimp-1 has been postulated to be a Since Bcl6 suppresses cytokine-driven differentiation of primary B master regulator of B cell terminal differentiation. In the BCL-1 cells to plasma cells by inhibiting STAT3-dependent transcrip- by guest on September 26, 2021 model of differentiation from a mature B cell to a tional events (6), STAT3 may be an important transcription factor plasma cell, ectopic expression of Blimp-1 is sufficient to cause for Blimp-1 expression. The recent report also demonstrated that terminal differentiation evidenced by loss of surface Igs, IgM se- Bcl6 is a potent repressor of transcriptional activity mediated by cretion, expression of CD138 (syndecan-1) on the cell surface, and AP-1 factors and that repression of AP-1 function by Bcl6 may be cessation of cell division (1–3). Blimp-1 was also found in a frac- a key mechanism for how Bcl6 regulates Blimp-1 expression (8). tion (4–15%) of germinal center B cells in murine spleen and The protooncogene c-fos encodes a nuclear phosphoprotein, c- ϩ human tonsils (4). In mice, Blimp-1 germinal center B cells ex- Fos. c-Fos in a complex with the product of another protooncogene press syndecan-1 and cytoplasmic Ig but not Bcl6 (4), and Blimp-1 c-jun (AP-1) regulates expression of the AP-1-binding at expression is repressed by Bcl6 (5, 6). The Blimp-1 conditional their transcriptional level (9–12). Since expression of c-fos and knockout mice demonstrated that Blimp-1 is required for differ- c-jun family genes is transiently induced in B cells stimulated with CD40 ligand (CD40L) and IL-4 as an immediate early , the function of c-Fos/AP-1 may be implicated in the transduction of Departments of *Developmental Genetics (H2) and †Medicine and Clinical Oncology (K1), Graduate School of Medicine, Chiba University, Chiba, Japan; ‡Division of signals induced by growth and differentiation factors (13, 14). We Hematopoiesis, Center for AIDS Research, Kumamoto University, Kumamoto, Ja- generated transgenic mice carrying the murine c-fos gene under the § pan; and Laboratory for B Lymphocyte Function, Research Center for Allergy and control of the murine MHC gene (H2-Kb) promoter (H2-c-fos) Immunology, RIKEN Yokohama Institute, Yokohama, Kanagawa 230-0045, Japan (15). Splenic T and B cells from the mice constitutively express a Received for publication January 20, 2004. Accepted for publication April 11, 2005. high level of the exogenous c-fos gene (16). When H2-c-fos mice 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 are immunized with T-dependent Ags, the mice can make the re- with 18 U.S.C. Section 1734 solely to indicate this fact. duced size of germinal centers and fail to generate memory B cells 1 This work was supported in part by Grants-in-Aid from the Ministry of Education, in the spleen (16, 17). These results suggest that the ectopic ex- Science, Technology, Sports and Culture of Japan and the Uehara Memorial pression of c-Fos may accelerate commitment of germinal center B Foundation. cells to a plasma cell fate. 2 Address correspondence and reprint requests to Dr. Takeshi Tokuhisa, Department of Developmental Genetics, Graduate School of Medicine (H2), Chiba University, 1-8-1 Engagement of CD40 on B cells by its ligand, gp39 or CD154, Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail address: [email protected] on activated T cells is a crucial component of cognate T cell help u.ac.jp (18). CD40 signaling in B cells triggers a potent proliferative re- 3 Abbreviations used in this paper: Blimp-1, B lymphocyte-induced maturation pro- sponse (19, 20), induces expression of costimulatory and adhesion tein 1; DIG, digoxigenin; AFC, Ab-forming cell; NP, (4-hydroxy-3-nitrophenyl)ac- etyl; CG, chicken ␥-globulin; PNA, peanut agglutinin; ChIP, chromatin molecules (21), and mediates clonal expansion and survival within immunoprecipitation. germinal centers (22). Mice deficient in either CD40 or CD40L are

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 7704 Blimp-1 EXPRESSION BY c-fos/AP-1 unable to form germinal centers or to make high-affinity, class- ␮g/ml goat anti-mouse IgG or IgM Abs in PBS at 4°C overnight and then switched Abs after immunization with T-dependent Ags (23–25). blocked with 1% BSA in PBS. Serially diluted cells were incubated on the nitrocellulose filters at 37°C in 5% CO . After a 2-h incubation, the filters Several in vitro experiments have also suggested that CD40 sig- 2 were washed vigorously and spots were developed with alkaline phos- naling is important for promoting B cell differentiation and Ig se- phatase-conjugated anti-IgM or HRP-conjugated anti-IgG1 Abs. For de- cretion (26–28). However, stimulation of CD40 on B cells actively tecting NP-specific AFCs, nitrocellulose filters were coated with NP-BSA inhibits the B cells from differentiating into Ab-forming cells (NP25-BSA; Biosearch Technologies). (AFCs) (29–32). This arrest is manifested as a reduction in mRNA levels of secretary Ig gene products as well as the loss of Blimp-1 Northern blot (32). Thus, a role for CD40 signaling in B cell terminal differen- Northern blotting was performed as described previously (38). Briefly, to- tiation is controversial. In this study, we directly tested the role for tal RNA (10 ␮g) was electrophoresed through a 1.0% agarose gel contain- the ectopic c-Fos in terminal differentiation of B cells activated ing formaldehyde and transferred to a nylon membrane (Roche Molecular Biochemicals). The filter was prehybridized for 1 h and hybridized over- with CD40L stimulation. When splenic B cells from H2-c-fos mice night at 50°C in 50% formamide hybridization buffer with 0.5% SDS, 1% were cultured with CD40L and IL-4, cell proliferation of these B blocking reagent, and 15 ng/ml digoxigenin (DIG; Roche Molecular Bio- cells was augmented compared with that of normal control B cells. chemicals)-labeled probes. Following hybridization, the filter was washed ϫ The H2-c-fos B cells but not the control B cells differentiated into twice for 15 min with 0.1 SSC and 0.1% SDS at 55°C. The DIG-labeled probe was detected with sheep anti-DIG Abs conjugated with alkaline AFCs within 2 days after stimulation. We discuss a critical role for phosphatase. The anti-DIG Ab detection reaction was performed using an c-Fos/AP-1 in terminal differentiation of B cells activated with enhanced chemiluminescent detection system (Roche Molecular Bio- CD40L stimulation. chemicals). 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, Downloaded from Materials and Methods XBP-1, and J chain) were made from the cDNAs, which were made by reverse-transcribed PCR with specific primers (40–42). Animals and immunization C57BL/6CrSlc mice were purchased from Japan SLC. Transgenic mice Isolation of nuclear carrying the mouse c-fos gene under the control of the H-2Kb promoter Nuclear proteins were isolated from splenic B cells using the method as (H2-c-fos) (15), and c-fos-deficient mice (33) were maintained by het- described previously (43), with slight modification. Briefly, splenic B cells erozygous mating in our animal facilities. These mice were immunized i.p. (1 ϫ 107) were resuspended in 400 ␮l of cold hypotonic buffer (10 mM http://www.jimmunol.org/ with 100 ␮g of alum-precipitated DNP-OVA or with 50 ␮g of alum-pre- HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl , 0.1 mM EDTA, 0.1% cipitated (4-hydroxy-3-nitrophenyl)acetyl-chicken ␥-globulin (NP -CG) 2 25 Triton X-100, 1 mM DTT, 100 mM PMSF, and 5 ␮g/ml aprotinin). Nuclei (Biosearch Technologies). were collected by centrifugation and disrupted by sonication in 100 ␮lof Preparation of splenic B cells 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 Splenic B cells were enriched by depleting non-B lineage cells from spleen PMSF, and 5 ␮g/ml aprotinin) at 4°C. Nuclear extracts were immediately cells. In brief, spleen cells were incubated with PE-anti-CD43 Ab (BD stored at Ϫ80°C. The amount of protein was determined using the Bio-Rad Pharmingen). These cells were subsequently reacted with immunomagnetic protein assay (Bio-Rad). beads coated with anti-PE Ab (Miltenyi Biotec). Labeled cells were re- moved by a MACS system (Miltenyi Biotec). The resulting B cell fraction EMSA contained Ͼ95% of B220ϩ B cells. by guest on September 26, 2021 Double-stranded oligonucleotides corresponding to putative AP-1-binding Cell culture and stimulation of lymphocytes sequences in the murine Blimp-1 gene (mAP1BS-1, 5Ј-TTATTAACC Ј ϫ 5 GATGCTGAGTCAGCACCACTATGGACAGAAA-3 and mAP1BS-2, Purified B cells (1.5 10 /ml) were cultured in RPMI 1640 medium (Sig- 5Ј-TTTCTGTCCATAGTGGTGCTGACTCAGCATCGGTTAATAA-3Ј, ma-Aldrich) supplemented with 10% FCS (Intergen), 50 ␮M 2-ME, 100 ␮ underlined; AP-1-binding sequence) were synthesized and biotinylated g/ml streptomycin (Wako Chemical), and 100 U/ml penicillin G/potas- using the biotin 3Јend-labeling kit (Pierce). Binding activity of AP-1 to the sium (Banyu Pharmaceutical). For B cell activation, CD40L (culture su- mAP1BS was determined using the LightShift chemiluminescent EMSA pernatants from a myeloma cell line producing soluble CD40L-CD8 fusion kit (Pierce) according to the manufacturer’s instruction. Competitive protein) (34) in the presence or absence of rIL-4 (1000 U/ml) (35) or LPS EMSA was done by adding 100-fold molar excess of unlabeled double- (Sigma-Aldrich) were added to the cultures. These B cells were cultured in stranded oligonucleotides to the mixture. The sequences of the mutant a humidified atmosphere at 37°C with 5% CO . 2 oligonucleotides (two bases mismatch, underlined) were as follows: Mut-1, mAbs and flow cytometry 5Ј-TTATTAACCGATGCCAAGTCAGCACCACTATGGACAGAAA-3Ј and Mut-2, 5Ј-TTTCTGTCCATAGTGGTGCTGACTTGGCATCGGTT Single-cell suspensions were prepared from cultured B cells or spleen cells, AATAA-3Ј. and then Fc receptors were blocked with 5 ␮g/ml anti-CD16/32 Ab (2.4G2; BD Pharmingen). Abs used were monoclonal FITC-anti-IgMb (BD Pharm- Chromatin immunoprecipitation (ChIP) assay ingen), PE-anti-IgDb (Bioscience), allophycocyanin-anti-IgG1 (BD Pharm- ingen), PE-anti-CD19 (BD Pharmingen), PE-anti-B220 (BD Pharmingen), ChIP assays were performed as previously described (44). Briefly, form- FITC-peanut agglutinin (PNA; Vector Laboratories), and biotinylated anti- aldehyde solution (37%; Fisher Scientific), at a final concentration of 1%, 6 syndecan-1 Abs (BD Pharmingen). Biotinylated Abs were detected by al- was added directly to B cells (3 ϫ 10 ) after each stimulation. Cross- lophycocyanin-conjugated streptavidin (BD Pharmingen). CFSE labeling linking of proteins on chromatin was allowed to occur at 4°C for 60 min, of B cells was performed as described previously (36). Cells were analyzed and the cells were lysed by SDS lysis buffer with protease inhibitors. Chro- on a FACSCalibur (BD Biosciences) using CellQuest software (BD Bio- matin in the lysate was sonicated to an average length of 400–600 bp as sciences). Dead cells were excluded on the basis of forward and side light determined by agarose gel electrophoresis. The suspension was precleared scatter parameters and propidium iodide (2 ␮g/ml) staining. with salmon sperm DNA/protein A/agarose/50% slurry for3hat4°Cand incubated with 2 ␮g of rabbit polyclonal anti-c-Fos Abs (Santa Cruz Bio- Proliferation assay technology) overnight. The immune complexes were incubated with salmon sperm DNA/protein A/agarose/50% slurry with mild shaking for ϫ 5 Purified B cells (1 10 /well) were cultured with various doses of CD40L 3 h at 4°C, washed, and eluted. After proteinase K treatment, DNA in with or without rIL-4 or various amounts of LPS in a 96-well microplate ␮ 3 samples was phenol extracted and resuspended in 50 l of TE buffer (10 for 48 h. Cultured cells were pulsed with 1 ␮Ci of [ H]thymidine (Amer- ␮ 3 mM Tris-HCl (pH 7.5) and 1 mM EDTA). DNA solution (2 l) was used sham International) for the last 8 h, and [ H]thymidine uptake was mea- for PCR amplification (28 cycles). PCR products were analyzed by elec- sured in a liquid scintillation counter. trophoresis on a 2% agarose gel and visualized by ethidium bromide stain- ELISPOT assay ing. The following primers were used: mAP1BS-1, 5Ј-GGAAAACAGAA GAACAAGCCTGTC-3Ј and 5Ј-ATAGCCAGCATCCCATCACAGC-3Ј AFCs producing IgM or IgG1 Abs were detected by ELISPOT assays as and mAP1BS-2, 5Ј-AAGGAAAGCAGGGTAAACCGTG-3Ј and 5Ј-GTT described elsewhere (37). Briefly, nitrocellulose filters were coated with 5 AGCTTGCTCTTGTGCCAGG-3Ј. The Journal of Immunology 7705

Results c-Fos augments proliferation of splenic B cells stimulated with CD40L and IL-4 The number of B220ϩ 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 Downloaded from proliferate. 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 FIGURE 2. c-Fos accelerates differentiation of splenic B cells stimu- IL-4 for 3 days, and cell surface expression of CFSE and several lated with CD40L and IL-4. CFSE-labeled splenic B cells were stimulated http://www.jimmunol.org/ differentiation markers including CD138 (syndecan-1) on these B with CD40L (1/8 dilution) and IL-4 for 3 days. A, Cell division of the cells were analyzed on a FACS. As shown in Fig. 2A, the H2-c-fos activated H2-c-fos (c-fos) and wild-type control (WT) B cells was analyzed B cells such as the control B cells divided up to five cell divisions 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, within 3 days after stimulation, suggesting that the ectopic c-Fos and syndecan-1 on the activated H2-c-fos (open histograms) and wild-type does not accelerate cell cycle progression of B cells stimulated control (filled histograms) B cells was analyzed. The data presented are with CD40L and IL-4. representative of two independent experiments.

Expression of IgM and IgD on the H2-c-fos and control B cells by guest on September 26, 2021 was reduced gradually as cell division progressed, and the reduc- tion 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-1ϩ 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 cul- ture were larger than those in the control culture (Fig. 3A). Kinetics of syndecan-1ϩ 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. We examined kinetics of IgM-AFCs developed in these B cell cultures. No IgM-AFC (Ͻ1of105 B cells) was detected in these B cell cultures before stimulation (Fig. 3B). More than 102 IgM- FIGURE 1. c-Fos augments cell proliferation of splenic B cells stimu- AFCs of 105 B cells were detected in H2-c-fos B cells stimulated lated with CD40L. Splenic B cells from H2-c-fos (solid line) and wild-type control (broken line) mice were cultured with various concentrations of with CD40L and IL-4 from day 2 after stimulation. The number in CD40L in the presence or absence of IL-4 (A) or with various doses of LPS the H2-c-fos culture increased and reached the plateau from day 3 (B). Cell proliferation was measured by [3H]thymidine uptake on day 2 of of culture. Although the number of IgM-AFCs in control B cell 2 culture. Results represent means and variations (SD) from triplicate cultures. culture stimulated with CD40L and IL-4 was not Ͼ10 IgM-AFCs The data presented are representative of three independent experiments. of 105 B cells within 4 days of culture, these small numbers were 7706 Blimp-1 EXPRESSION BY c-fos/AP-1

both c-fos-deficient and normal B cell cultures, although the per- centage 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-1ϩ 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 in- creased 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 Downloaded from 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 in- duced in both H2-c-fos and control B cells from 24 h after stim- ulation and increased thereafter.

Expression of AP-1 family genes was examined in normal B http://www.jimmunol.org/ cells stimulated with CD40L and IL-4 and compared with that of Blimp-1 and bcl6 in these B cells. Blimp-1 expression was tran- siently induced within 1 h after stimulation and the peak was be- tween 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 main- tained at the resting level in the B cells. Expression of c-fos, fosB, junB, and junD was transiently induced within 30 min after stim- FIGURE 3. c-Fos augments terminal differentiation of splenic B cells ulation and disappeared thereafter. However, expression of fra-1, by guest on September 26, 2021 stimulated with CD40L and IL-4. Splenic B cells from H2-c-fos, c-fos- fra-2, and c-jun was not detected in the B cells (data not shown). deficient, and wild-type control mice were cultured with CD40L (1/8 di- lution) and IL-4 or with LPS (3 ␮g/ml) for 4 days. A, Expression of syn- decan-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 (f) and wild-type control (E) mice were detected by ELISPOT assays. Results represent means and vari- ations (SD) from triplicate cultures. The data presented are representative of two independent experiments. C, Expression of syndecan-1 on the ac- tivated 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.

increasing from day 2 of culture and reached the plateau on day 3 of culture. Furthermore, ϳ10 IgM-AFCs of 105 B cells were de- tected 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 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 cells stimulated with CD40L and IL-4 was comparable to those in control (WT) mice were cultured with CD40L (1/8 dilution) and IL-4 (A)or H2-c-fos and control B cells stimulated with LPS. with LPS (3 ␮g/ml; B) for 3 days. Expression of Blimp-1 and other genes A role of c-Fos in terminal differentiation of B cells was further related with B cell terminal differentiation in the activated B cells was analyzed examined using c-fos-deficient mice. We analyzed syndecan-1 ex- on each day of culture by Northern blot. ␤-actin mRNA was used as an pression on c-fos-deficient B cells 4 days after stimulation with amount control of mRNA. The data presented are representative of two inde- ϩ CD40L and IL-4. Syndecan-1 B cells were clearly detected in pendent experiments. The Journal of Immunology 7707

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 Downloaded from with LPS (3 ␮g/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 su- FIGURE 5. AP-1 family is induced in control B cells pershift experiments were performed with various Abs (anti-c-Fos, anti- stimulated with CD40L and IL-4. Splenic B cells from wild-type mice were JunD, and control IgG). Nuclear proteins of activated B cells 6 h after cultured with CD40L (1/8 dilution) and IL-4 (A) or with LPS (3 ␮g/ml; B) stimulation were used for the cold competition and the Ab supershift ex- http://www.jimmunol.org/ for 3 days. Expression of the AP-1 family, Blimp-1, and bcl6 genes in the periments. These results are representative of two independent experi- activated B cells was analyzed using Northern blot. ␤-actin mRNA was ments. B, Binding of c-Fos to the mAP1BS-1 in nuclear proteins of these used as an amount control of mRNA. The data presented are representative activated B cells was analyzed by ChIP assays. PCR were performed on of two independent experiments. both input chromatin (total DNA) and immunoprecipitation products. The data presented are representative of two independent experiments.

When we examined expression of these genes in normal B cells H2-c-fos and control B cells stimulated with LPS until 24 h after stimulated with LPS, Blimp-1 expression was transiently induced stimulation. within 1 h after stimulation, down-regulated until 6 h after stim- The binding of c-Fos on the mAP1BS-1 and the AP1BS-2 was by guest on September 26, 2021 ulation, and then up-regulated from 12 h after stimulation and in- confirmed by ChIP assays with anti-c-Fos Abs. The mAP1BS-1 creased thereafter (Fig. 5B). Bcl6 expression was transiently in- was contained in the DNA complexes of H2-c-fos and control B duced in the B cells within 1 h after stimulation and the peak was cells 12 h after stimulation with CD40L and IL-4 (Fig. 6B). The between 3 and 6 h after stimulation and down-regulated thereafter. mAP1BS-1 was still contained in the DNA complex of the H2-c- These results suggest that AP-1 induces the transient Blimp-1 ex- fos B cells but not in that of the control B cells 24 h after stimu- pression in activated B cells within 3–6 h after stimulation. How- lation. The mAP1BS-2 was also contained in the DNA complexes ever, Blimp-1 expression in the LPS-stimulated B cells between 12 of H2-c-fos and control B cells (data not shown). The mAP1BS-1 and 72 h after stimulation cannot be explained by the mRNA ex- and the mAP1BS-2 (data not shown) were contained in the DNA pression of AP-1 family genes. complexes of H2-c-fos and control B cells stimulated with LPS until 24 h after stimulation. Since the binding activity of c-Fos/ c-Fos/AP-1 activity is prolonged in B cells activated with LPS AP-1 in nuclear proteins of these activated B cells to the mAP1BS The promoter region of murine Blimp-1 gene contains two putative correlated with Blimp-1 expression in these B cells, c-Fos/AP-1 AP-1-binding sequences (mAP1BS-1 and mAP1BS-2). Thus, positively regulates Blimp-1 expression in activated B cells. 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 c-Fos augments terminal differentiation of Ag-specific B cells in CD40L and IL-4 to the mAP1BS. As shown in Fig. 6A, a gel vivo retardation band to the mAP1BS-1 was observed using the nuclear Germinal center formation in the spleen of H2-c-fos mice is per- proteins of H2-c-fos and control B cells. This band from control B turbed and the perturbation could not be explained by apoptosis of cells was obviously removed by a nonlabeled probe with the same the germinal center B cells (17). These results prompted us to sequence (WT) as a cold competitor. In contrast, a mutated cold examine the incidence of terminal differentiation in germinal cen- probe (Mut) as a competitor did not inhibit formation of the gel ter B cells of H2-c-fos mice. Since a few of syndecan-1ϩ B cells retardation band. Furthermore, the band was removed by the ad- as plasma cell precursors are detected in germinal center B cells dition of anti-c-Fos Abs, partly by that of anti-JunD Abs, but not after immunization (4), the percentage of syndecan-1ϩ B cells in by that of control IgG Abs, indicating sequence-specific binding of germinal center B cells was analyzed in the spleen of H2-c-fos c-Fos/AP-1 to the mAP1BS-1. The band was still detected in nu- mice immunized with NP-CG in alum. Fig. 7A shows that germi- clear protein of the H2-c-fos B cells but not in that of the control nal center formation was perturbed in the spleen of H2-c-fos mice B cells 24 h after stimulation. The specific band to the mAP1BS-2 12 days after immunization. The percentage of PNAϩ B cells in was also detected in the nuclear proteins of H2-c-fos and control B spleen cells of H2-c-fos mice was less than that of control litter- cells (data not shown). The AP-1 band to the mAP1BS-1 and to the mates. The percentage of syndecan-1ϩ B cells in the PNAϩ B cells mAP1BS-2 (data not shown) was detected in nuclear proteins of of H2-c-fos mice was clearly larger than that of control mice (Fig. 7708 Blimp-1 EXPRESSION BY c-fos/AP-1

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 in- tensity 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 imme- diate early gene product is an amplifier of the initial signals. Fur- ther study is required to elucidate a target gene of c-Fos/AP-1 in the signal transduction pathway initiated by CD40L or LPS

stimulation. Downloaded from 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-defi- cient humans produce normal or elevated levels of serum IgM (47)

FIGURE 7. c-Fos augments terminal differentiation of Ag-specific B and that CD40 signaling directly prevents B cell terminal differ- http://www.jimmunol.org/ cells in H2-c-fos mice after immunization. H2-c-fos (c-fos) and wild-type entiation (29–32, 48). Thus, CD40 signaling is not only insuffi- control (WT) mice were immunized with NP-CG. A, Syndecan-1ϩ cells in cient to induce plasma cell formation, but actively arrests B cells germinal center (PNAϩ, B220ϩ) B cells in the spleen of mice 12 days after at a stage before terminal differentiation. We showed here that immunization were analyzed. The numbers indicate the percentages of ger- terminal differentiation was barely induced in control B cells stim- ϩ minal center B cells in total spleen cells and those of syndecan-1 cells in ulated with CD40L and IL-4 and that the ectopic c-Fos efficiently the germinal center B cells. B, Summary of the percentages from H2-c-fos induced terminal differentiation of the B cells. Indeed, AP-1 ac- f Ⅺ ( ) and wild-type control ( ) mice shown in A. Results represent means tivity was detected in the control B cells and the H2-c-fos B cells and variations (SD) from three mice per group. C, Numbers of AFCs pro- f until 12 and 24 h after stimulation, respectively. Although stimu- ducing IgM or IgG1 anti-NP Abs in the bone marrow of H2-c-fos ( ) and by guest on September 26, 2021 lation of control B cells with CD40L and IL-4 induced expression wild-type control (Ⅺ) mice 12 days after immunization were measured by of junB and junD genes until 24 h after stimulation, transcriptional ELISPOT assays with NP25-BSA. Results represent means and variations (SD) from three mice per group. 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 7B). These results were repeated in the spleen of H2-c-fos mice differentiation of the B cells. Thus, the effective AP-1 (c-Fos/Jun) immunized with DNA-OVA in alum (data not shown). may be a key transcription factor for terminal differentiation of B We have shown that large numbers of AFCs are detected in the cells stimulated with CD40L and IL-4. bone marrow of normal mice 14 days after immunization and that Blimp-1 expression was detected in H2-c-fos B cells but not in many of them are derived from germinal centers (37). To confirm control B cells stimulated with CD40L and IL-4 on days 1 and 2 the high incidence of terminal differentiation in germinal center B after stimulation. Since there are two AP-1-binding sites in the cells of H2-c-fos mice, numbers of IgM- and IgG1-AFCs were murine Blimp-1 promoter region and c-Fos binds to these sites in examined in the bone marrow of H2-c-fos mice 12 days after im- H2-c-fos B cells stimulated with CD40L and IL-4, effective AP-1 munization with NP-CG by ELISPOT assays. The number of IgM- composed of a c-Fos/Jun heterodimer may be enough to activate AFCs in the bone marrow of H2-c-fos mice was 5-fold larger than the Blimp-1 promoter in the B cells. Blimp-1 expression might be that of control mice (Fig. 7C). However, IgG1-AFCs were not negatively regulated by Bcl6 in control B cells stimulated with detected at all in the bone marrow of the H2-c-fos mice, repeating CD40L and IL-4 because overexpression of Bcl6 represses tran- the Ig-class switch abnormality of H2-c-fos mice (16, 17). These scription of the human Blimp-1 promoter almost entirely (8). How- results suggest that c-Fos/AP-1 positively regulates terminal dif- ever, Bcl6 is barely induced in either H2-c-fos or control B cells ferentiation of Ag-activated B cells. stimulated with CD40L and IL-4. Furthermore, splenic B cells from Bcl6-deficient mice stimulated with CD40L and IL-4 did not Discussion differentiate into IgM-AFCs and IgG1-AFCs (data not shown). Cell proliferation of H2-c-fos B cells stimulated with CD40L in These results suggest that little induction of Blimp-1 expression in the presence or absence of IL-4 was markedly augmented com- control B cells stimulated with CD40L and IL-4 is not due to the pared with that of control B cells. Since expression of the endog- negative regulatory effect of a transcriptional repressor, Bcl6, but enous AP-1 family genes and AP-1 activity were transiently in- due to the deficiency of an effective transcriptional factor, duced in control B cells stimulated with CD40L and IL-4, the c-Fos/AP-1. ectopic c-Fos augments a role for the endogenous AP-1 in cell Blimp-1 expression and AP-1 activity were detected in both H2- cycle progression of the B cells. However, the ectopic c-Fos did c-fos and control B cells stimulated with LPS from day 1 after not accelerate cell cycle progression of the B cells because both stimulation. Although mRNA expression of AP-1 family genes in H2-c-fos and control B cells divided five times within 3 days after control B cells stimulated with LPS was similar to that in control The Journal of Immunology 7709

B cells stimulated with CD40L and IL-4, DNA-binding activity of Disclosures AP-1 in the LPS-stimulated B cells was prolonged more than that The authors have no financial conflict of interest. in the CD40L-stimulated B cells, suggesting that AP-1 is unstable in the CD40L-stimulated B cells. c-Fos/AP-1 may be dephospho- References rylated and/or denatured in control B cells stimulated with CD40L 1. Turner, C. A. Jr., D. H. Mack, and M. Davis. 1994. Blimp-1, a novel zinc finger- and IL-4 within 24 h after stimulation. These results support that containing protein that can drive the maturation of B lymphocytes into immu- noglobulin-secreting cells. Cell 77: 297–306. c-Fos/AP-1 is a key transcription factor for terminal differentiation 2. Lin, Y., K. Wong, and K. Calame. 1997. Repression of c- transcription by of B cells stimulated with CD40L and IL-4. However, terminal Blimp-1, an inducer of terminal B cell differentiation. Science 276: 596–599. differentiation of B cells was induced in c-fos-deficient B cells 3. Angelin-Duclos, C., G. Cattoretti, D. Chang, K. Lin, Y. Lin, J. Yu, and K. Calame. 1999. Role of B lymphocyte induced maturation protein-1 in terminal stimulated with CD40L and IL-4 or with LPS, suggesting the func- differentiation of B cells and other cell lineages. Cold Spring Harbor Symp. tional redundancy of c-Fos family proteins. Thus, the effective Quant. Biol. 64: 61–70. AP-1, including Fos family proteins, may be a key transcription 4. Angelin-Duclos, C., G. Cattoretti, K.-I. Lin, and K. Calame. 2000. Commitment of B lymphocytes to a plasma cell fate is associated with blimp-1 expression in factor for Blimp-1 expression and terminal differentiation of acti- vivo. J. Immunol. 165: 5462–5471. vated B cells. 5. Shaffer, A. L., X. Yu, Y. He, J. Boldrick, E. P. Chan, and L. M. Staudt. 2000. BCL-6 represses genes that function in lymphocyte differentiation, inflammation, Functionally effective memory B cells are developed in germi- and cell cycle control. Immunity 13: 199–212. nal centers (37), and signaling through CD40 drives germinal cen- 6. Reljic, R., S. D. Wagner, L. J. Peakman, and D. T. Fearon. 2000. Suppression of ter B cells toward a memory cell phenotype by preventing terminal signal transducer and activator of transcription 3-dependent B lymphocyte ter- minal differentiation by BCL-6. J. Exp. Med. 192: 1841–1848. differentiation of these B cells (29, 31). However, the in vitro stud- 7. Shapiro-Shelef, M., K. I. Lin, L. J. McHeyzer-Williams, J. Liao, ies shown here may not support the negative effect of CD40 sig- M. G. McHeyzer-Williams, and K. Calame. 2003. Blimp-1 is required for the Downloaded from naling on terminal differentiation of germinal center B cells, since formation of immunoglobulin secreting plasma cells and pre-plasma memory B cells. Immunity 19: 607–620. overexpression of c-Fos-induced terminal differentiation of B cells 8. Vasanwala, F. H., S. Kusam, L. M. Toney, and A. L. Dent. 2002. Repression of stimulated with CD40L and IL-4. Furthermore, the in vivo results AP-1 function: a mechanism for the regulation of Blimp-1 expression and B lymphocyte differentiation by the B cell lymphoma-6 protooncogene. J. Immunol. may also deny the negative effect of CD40 signaling. CD40 sig- 169: 1922–1929. naling is essential for activated B cells to differentiate into germi- 9. Chiu, R., W. J. Boyle, J. Moek, T. Smeal, T. Hunter, and M. Karin. 1988. The c-fos protein interacts with c-jun/AP-1 to stimulate transcription of AP-1 respon- nal center B cells (22–25), indicating that germinal center B cells http://www.jimmunol.org/ sive genes. Cell 54: 541–552. are stimulated with CD40L on helper T cells. The germinal center 10. Franza, B. R., F. J. Rausher, S. F. Josephs, and T. Curran. 1988. The fos complex formation is perturbed in H2-c-fos mice after immunization (17) and fos-related antigens recognize sequence elements that contain AP-1 binding and the percentage of plasma cell precursors (syndecan-1ϩ germi- sites. Science 239: 1150–1153. 11. Rauscher 3rd, F. J., L. C. Sambucetti, T. Curran, R. J. Distel, and nal center B cells) in the spleen of H2-c-fos mice was 2- to 3-fold B. M. Spiegelman. 1988. Common DNA binding site for Fos protein complexes larger than that of control mice after immunization. Thus, the ec- and transcription factor AP-1. Cell 52: 471–480. 12. Sassone-Corsi, P., W. W. Lamph, M. Kamps, and I. M. Verma. 1988. Fos asso- topic c-Fos may compose c-Fos/AP-1 in germinal center B cells ciated cellular P39 is related to nuclear transcription factor AP-1. Cell 54: and induce Blimp-1 expression in these B cells at an earlier stage, 553–560. leading to premature generation of plasma cells, which emigrate 13. Holt, J. T., T. V. Gopal, A. D. Moulton, and A. W. Nienhuis. 1986. Inducible

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