We examined the role of c-Fos in the differentiation of mature B cells into IgG-producing cells using transgenic mice carrying the c-fos gene under the control of the IFN-α/β-inducible Mx promoter (Mx-c-fos) or the constitutive H-2Kb promoter (H2-c-fos). Splenic B cells from Mx-c-fos mice were cultured with LPS and rIL-4, and IgG1+ B cells were developed in the culture after day 3. When IFN-α/β was added to the culture from day 2, development of IgG1+ B cells was perturbed, and the number of apoptotic cells increased within 24 h, suggesting that c-Fos induces apoptosis in Ig class-switching B cells. To confirm the effect of c-Fos on B cell differentiation in vivo, H2-c-fos mice were immunized with DNP-OVA. The mice produced primary IgM, but not IgG, anti-DNP Ab in serum and failed to generate germinal centers in spleen. The perturbation of germinal center formation in H2-c-fos mice was rescued by mating them with transgenic mice carrying the bcl-2 gene with the Ig promoter. However, primary IgG1 anti-DNP Ab production was still suppressed in doubly transgenic mice, suggesting that Bcl-2 can delay the time of c-Fos-induced apoptosis in Ig class-switching B cells but cannot rescue the death. Since c-Fos is induced in mature B cells reacted with Ags, and clonal deletion of self-reactive B cells in germinal centers is insensitive to Bcl-2, these results suggest that c-Fos plays a causal role in clonal deletion of germinal center B cells.

The proto-oncogene c-fos, which encodes a nuclear phosphoprotein (c-Fos), is transiently induced in numerous cell types by many agents and conditions (1, 2, 3, 4). c-Fos in a complex with products of another proto-oncogene, c-jun (AP-1), regulates the expression of AP-1 binding genes at the transcriptional level (5, 6, 7, 8). Thus, a function of c-Fos may be implicated in the transduction of signals induced by growth and differentiation factors (9, 10, 11). To study the biologic effects of c-Fos in the differentiation of lymphocytes, we generated transgenic mice carrying the murine c-fos gene under the control of the murine MHC gene (H2-Kb) promoter (H2-c-fos) (12). Splenic T and B cells from H2-c-fos mice constitutively express a high level of the exogenous c-fos gene. When H2-c-fos mice were immunized with Ags, the mice can produce primary IgM Ab, but not IgG Ab, and fail to generate memory B cells in spleen. Since the activity of Th cells and APCs from H2-c-fos mice was normal, the abnormality is due to defects in B cells (13). Furthermore, splenic B cells from H2-c-fos mice cultured with LPS (>5 μg/ml) and rIL-4 cannot differentiate into IgG1+ B cells (14), suggesting a perturbation of the IgG class-switching process in B cell differentiation.

When mice are immunized with T cell-dependent Ag, Ag-reactive B cells in spleen are activated by interaction with Th cells in periarteriolar lymphoid sheaths (PALS)4 (15, 16). These activated B cells generate Ab-producing foci in PALS to produce the majority of primary IgM and IgG Abs (17) or migrate into follicles to form germinal center (GC) that is identified by binding capacity to peanut agglutinin (PNA). Nascent GC B cells undergo massive clonal expansion to form a dark zone occupied by surface Ig (sIg)-negative centroblasts (18, 19). Somatic hypermutations of the Ig gene occur in centroblasts (20, 21, 22). These cells further differentiate into sIg-positive centrocytes at a light zone in the GC. Those mutated centrocytes with higher affinity to self Ags or with lower affinity to immunized Ag undergo selective apoptosis (23, 24, 25, 26, 27), and those with higher affinity to the Ag differentiate into memory B cells or Ab-forming cells (28, 29). In the differentiation process, an Ig class switch occurs in B cells, mainly in centrocytes (30). Since perturbation of IgG production in H2-c-fos mice (13, 14) was suggested to be due to an impairment of the IgG class-switching process in B cells, the perturbation can be histologically confirmed in spleen from H2-c-fos mice. However, GC formation in spleen from immunized H2-c-fos mice has never been examined.

c-Fos-induced perturbation of B cell differentiation has been demonstrated in early B cell development (31, 32). Our recent study using fetal liver cells from transgenic mice carrying the IFN-α/β-inducible c-fos gene (Mx-c-fos) demonstrated that exogenous c-Fos induces apoptosis in pro-B (B220+, CD43+) cells (33). These results strongly suggested that the perturbation of IgG production in H2-c-fos mice was due to apoptosis in IgG class-switching B cells. Apoptosis is a physiologic type of cell death (34), and some apoptosis in B cells can be rescued by overexpression of Bcl-2 (35, 36, 37). Here we provide evidence that overexpression of c-Fos induces apoptosis in Ig class-switching B cells in vitro and perturbs GC formation and primary IgG production in vivo. Overexpression of Bcl-2 in H2-c-fos B cells can rescue GC formation but not IgG production. Since c-Fos is induced in mature B cells reacted with Ags (38) and clonal deletion of self-reactive B cells in GCs is insensitive to overexpression of Bcl-2 (39), c-Fos-induced apoptosis may mimic clonal deletion in GCs. We discuss a possible physiologic role of c-Fos in GC B cells.

C57BL/6CrSlc mice were purchased from Japan SLC (Hamamatsu, Japan). Transgenic mice carrying the mouse c-fos gene under the control of the H-2Kb promoter (H2-c-fos) or the Mx gene promoter (Mx-c-fos) have been described previously (12, 40). Transgenic mice carrying the mouse bcl-2 gene fused to the Ig promoter/enhancer (Ig-bcl-2) have been described previously (41).

DNP-OVA was prepared by coupling OVA (Sigma, St. Louis, MO) with 2,4-dinitrophenylbenzensulfonic acid under alkaline conditions (13). Mice were immunized i.p. with 100 μg of alum-precipitated DNP-OVA. Sera were collected from the mice on day 12 after immunization. The amount of DNP-specific Ab in serum was measured by ELISA as described previously (13). Briefly, DNP-BSA was coated onto ELISA plates, and Igs on wells were developed with biotinylated goat anti-mouse IgM Ab (Caltag, South San Francisco, CA) or with anti-IgG1 Ab (Caltag) followed by avidin-peroxidase (Vector, Burlingame, CA). Pooled sera from C57BL/6 mice immunized with related Ag were used as a standard. Ab titers of the 1/500 diluted standard sera were arbitrarily taken as 1 U/ml.

Spleens were isolated from mice on day 12 after immunization. One-third of the spleen was used for flow cytometric analysis, and two-thirds were embedded in OCT compound (Miles, Elkhart, IN) and frozen in liquid nitrogen. GC formation in spleen was examined by histologic analysis as previously described (42). Briefly, serial frozen sections (6 μm) were fixed in cold acetone, and the activity of endogenous peroxidase was quenched with 3% H2O2 in methanol. Sections were stained with PNA coupled to horseradish peroxidase (HRP; EY Laboratory, San Mateo, CA) or with biotinylated anti-B220 mAb (RA3-6B2; PharMingen, San Diego, CA) followed by HRP-streptavidin (Nichirei, Tokyo, Japan). Bound HRP activity was visualized with the diaminobenzidene kit (Nichirei).

Spleen cells were stained with FITC-labeled PNA (Vector) and biotinylated anti-B220 mAb for 15 min on ice (33). After washing, the cells were stained with phycoerythrin (PE)-streptavidin (PharMingen) for 15 min on ice. For three-color analysis, the cells were stained with FITC-labeled PNA, PE-conjugated anti-B220 mAb (PharMingen), and biotinylated anti-IgG1 mAb (G1-6.5; PharMingen) followed by allophycocyanin-streptavidin (PharMingen). After washing, the cells were finally suspended in 3% FCS/PBS with 1 μg/ml of propidium iodide to exclude dead cells and were analyzed by FACSCalibur (Becton Dickinson, San Jose, CA).

Splenic B cells were prepared by treatment of spleen cells with anti-Thy-1 Ab and complement, as described previously (43). Briefly, 2 × 107 cells in 1 ml of RPMI 1640 medium (Life Technologies, Grand Island, NY) were treated with 1 μg of rat anti-Thy-1.2 mAb (Cedarlane Laboratories, Ontario, Canada) at room temperature for 30 min. These treated cells were mixed with 1/20 diluted guinea pig complement (Low Tox, Cedarlane) and incubated at 37°C for 30 min. Viable cells were isolated by centrifugation through a Lympholyte M density gradient (Cedarlane). The resulting B cell fraction contained >80% B220+ cells.

Splenic B cells were cultured in RPMI 1640 supplemented with 10% FCS (Intergen, New York, NY) at 2.5 × 105/ml with LPS (5 μg/ml; Sigma) and rIL-4 (104 U/ml) (44) in the presence or the absence of mouse IFN-α/β (200 U/ml; Sigma) for 6 days at 37°C in 5% CO2. These cultured cells were stained with FITC-labeled anti-B220 mAb and biotinylated anti-IgG1 mAb for 30 min, followed by PE-streptavidin for 15 min on ice, then cells were incubated with PE-labeled anti-B220 mAb for 30 min on ice. These stained cells were analyzed by FACSCalibur.

Apoptotic cells were detected in B220+ cells cultured with LPS and rIL-4 by the annexin V staining method and the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) method (45) with some modifications. Cells (1 × 106) were assessed for binding of annexin V using the annexin V-FITC kit (Bender MedSystems, Vienna, Austria) following the manufacturer’s instructions. Briefly, cells were incubated with allophycocyanin-conjugated anti-B220 mAb (PharMingen) for 20 min on ice and washed twice with 3% FCS/PBS. Then, the cells were stained with FITC-labeled annexin V for 10 min at room temperature, washed once, resuspended with 3% FCS/PBS with 1 μg/ml of propidium iodide, and analyzed on FACSCalibur using CellQuest software (Becton Dickinson).

For the TUNEL method, cells (1 × 106) were stained with biotinylated anti-B220 mAb for 15 min on ice followed by PE-streptavidin. After washing, the cells were permeabilized and fixed in 200 μl of ice-cold 70% ethanol and stored at 4°C overnight. The cells were washed twice, and then the TUNEL reaction was conducted by incubating the cells for 1 h at 37°C in 50 μl of a reaction solution containing 0.3 nM FITC-12-dUTP (Boehringer Mannheim, Mannheim, Germany), 2 μl of 25 mM CoCl2, 25 U of TdT (Boehringer Mannheim), and TdT buffer (30 mM Tris, pH 7.2, and 140 mM sodium cacodylate). The reaction was stopped by adding 2 μl of 0.5 M EDTA to the reaction mixture. After washing twice, the cells were analyzed by FACSCalibur.

The amount of c-fos mRNA was determined by Northern blot analysis as previously described (43). Briefly, total RNA (10 μg) was electrophoresed through a 1.0% agarose gel containing formaldehyde and transferred to a nylon membrane (Boehringer Mannheim). The filter was prehybridized for 3 h and hybridized overnight at 50°C in 50% formamide hybridization buffer with 0.5% SDS, 1% blocking reagent, and 15 ng/ml of the c-fos probe. Following hybridization, the filter was washed twice for 5 min each time with 2× SSC and 0.1% SDS at room temperature and twice for 15 min each time with 0.1× SSC and 0.1% SDS at 50°C. The digoxigenin-labeled c-fos probe was detected with sheep anti-digoxigenin Ab conjugated with alkaline phosphatase. The anti-digoxigenin Ab detection reaction was performed using an enhanced chemiluminescent detection system (Boehringer Mannheim). A 846-bp SalI-PvuII fragment (exon 4) of the murine c-fos genomic DNA subcloned in a pGEM-4Z vector was labeled by digoxigenin (Boehringer Mannheim) using PCR with T7 and SP6 primers and then was used as a probe.

Differentiation of mature B cells into IgG1-producing plasma cells in spleen from immunized mice can be mimicked by in vitro culture of splenic B cells with LPS and IL-4 (46). To examine the effect of c-Fos on the differentiation process, splenic B cells from Mx-c-fos mice were cultured with LPS and rIL-4 in the presence or the absence of IFN-α/β. The inducibility of c-fos mRNA expression in those B cells was analyzed by Northern blot (Fig. 1). c-fos mRNA was undetectable in control B cells stimulated with LPS and rIL-4 even after the addition of IFN-α/β on day 2 of culture, although c-fos mRNA was detected in those B cells by reverse transcribed PCR (data not shown). The mRNA was induced in Mx-c-fos B cells stimulated with LPS and rIL-4 until day 2 of culture in the absence of IFN-α/β and became undetectable after day 4 of culture. This c-fos induction may be due to IFN-α/β produced by remaining macrophages in the B cell fraction activated with LPS in the culture (47). When IFN-α/β was added to the culture on day 2, large amounts of c-fos mRNA were continuously detected in Mx-c-fos B cells until day 6 of culture. c-fos mRNA was also induced in Mx-c-fos B cells on day 5 of culture after the addition of IFN-α/β on day 4.

FIGURE 1.

Expression of the exogenous c-fos gene is induced in Mx-c-fos B cells cultured with LPS and rIL-4. Splenic B cells from Mx-c-fos mice were cultured with LPS and rIL-4. IFN-α/β was added to the culture on day 2 or day 4. Levels of c-fos mRNA in B cells were analyzed by Northern blot.

FIGURE 1.

Expression of the exogenous c-fos gene is induced in Mx-c-fos B cells cultured with LPS and rIL-4. Splenic B cells from Mx-c-fos mice were cultured with LPS and rIL-4. IFN-α/β was added to the culture on day 2 or day 4. Levels of c-fos mRNA in B cells were analyzed by Northern blot.

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Splenic B cells from Mx-c-fos mice were cultured with LPS and rIL-4 for 5 days by the addition of IFN-α/β on day 2 of culture. The number of viable B cells and IgG1+ B cells in those cultures was analyzed by cell surface staining with anti-B220 and anti-IgG1 mAb and by FACS (Fig. 2). The number of viable B cells was increasing and reached a plateau level by day 3 in control cultures. The addition of IFN-α/β did not affect the number of viable B cells in control cultures. The viable B cell number in Mx-c-fos B cell culture without the addition of IFN-α/β was twofold higher than that in control B cell culture or that in Mx-c-fos B cell culture with the addition of IFN-α/β on day 2. This augmentation of B cell proliferation may be due to the effect of c-Fos on acceleration of cell cycle progression of B cells activated with LPS (48). The number of IgG1+ B cells became detectable in control B cell culture after day 3 and increased until day 5 of culture. The addition of IFN-α/β on day 2 of culture did not modify the kinetics of generation of IgG1+ B cells in control B cell culture. The number of IgG1+ B cells in Mx-c-fos B cell culture without the addition of IFN-α/β reached a plateau level by day 4 and showed slightly earlier kinetics than those of controls. However, the number of IgG1+ B cells did not increase in Mx-c-fos B cell culture after the addition of IFN-α/β on day 2. These results suggest that overexpression of c-Fos in Ig class-switching B cells perturbs further differentiation of B cells.

FIGURE 2.

Differentiation of IgG1+ B cells is perturbed in Mx-c-fos B cell culture by the addition of IFN-α/β. Splenic B cells from Mx-c-fos (closed symbols) and littermate control (open symbols) mice were cultured with LPS and rIL-4 for 5 days. IFN-α/β was added to the culture on day 2. The cultured cells were stained with anti-B220 and anti-IgG1 mAb. The number of viable B cells and IgG1+ B cells in those cultured cells was calculated by FACS analysis. Results represent the means and variations (SD) of triplicate cultures. The data presented are representative of two independent experiments.

FIGURE 2.

Differentiation of IgG1+ B cells is perturbed in Mx-c-fos B cell culture by the addition of IFN-α/β. Splenic B cells from Mx-c-fos (closed symbols) and littermate control (open symbols) mice were cultured with LPS and rIL-4 for 5 days. IFN-α/β was added to the culture on day 2. The cultured cells were stained with anti-B220 and anti-IgG1 mAb. The number of viable B cells and IgG1+ B cells in those cultured cells was calculated by FACS analysis. Results represent the means and variations (SD) of triplicate cultures. The data presented are representative of two independent experiments.

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The effect of c-Fos on differentiating B cells was further examined in Mx-c-fos B cell culture with LPS and rIL-4 by an addition of IFN-α/β on day 2 or day 4 of culture. Generation of IgG1+ B cells in the culture was examined on day 5 of culture by staining those cultured cells with anti-IgG1 and anti-B220 mAb and analysis by FACS. Figure 3 shows that the number (14 ± 1.6 × 104/ml) of IgG1+ B cells in Mx-c-fos B cell culture without the addition of IFN-α/β was similar to that (16 ± 0.9 × 104/ml) in control B cell culture. The number (1.0 ± 0.6 × 104/ml) in Mx-c-fos B cell culture by the addition of IFN-α/β on day 2 was distinctly lower than that (16 ± 2.6 × 104/ml) in control B cell culture after the addition of IFN-α/β on day 2. IgG1+ B cells were still detected in Mx-c-fos B cell culture by the addition of IFN-α/β on day 4, although the number (8.5 ± 1.6 × 104/ml) was lower than that (14 ± 1.6 × 104/ml) in Mx-c-fos B cell culture without the addition of IFN-α/β. Since a larger number of dead cells was detected in Mx-c-fos B cell culture by the addition of IFN-α/β (data not shown), the results shown in Figure 3 suggested that c-Fos induced cell death in Ig class-switching B cells and in nascent surface IgG1+ B cells.

FIGURE 3.

Differentiation of IgG1+ B cells is perturbed in Mx-c-fos B cell culture by the addition of IFN-α/β on day 2 or day 4. Splenic B cells from Mx-c-fos mice were cultured with LPS and rIL-4 for 5 days. IFN-α/β was added to the culture on day 2 or day 4. The cultured cells were stained with anti-B220 and anti-IgG1 mAb. The number of IgG1+ B cells in those cultured cells was calculated by FACS analysis. Results represent the means and variations (SD) of triplicate cultures. The data presented are representative of three independent experiments.

FIGURE 3.

Differentiation of IgG1+ B cells is perturbed in Mx-c-fos B cell culture by the addition of IFN-α/β on day 2 or day 4. Splenic B cells from Mx-c-fos mice were cultured with LPS and rIL-4 for 5 days. IFN-α/β was added to the culture on day 2 or day 4. The cultured cells were stained with anti-B220 and anti-IgG1 mAb. The number of IgG1+ B cells in those cultured cells was calculated by FACS analysis. Results represent the means and variations (SD) of triplicate cultures. The data presented are representative of three independent experiments.

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To examine the effect of c-Fos on cell death (apoptosis) in mature B cells differentiating into IgG1+ B cells, Mx-c-fos B cells were cultured with LPS and rIL-4, and IFN-α/β was added to the culture on day 2. Apoptotic cells were analyzed in those B cells on day 3 of culture using the annexin V staining method and the TUNEL method. Figure 4A reveals that the percentage (11.9%) of annexin V+ B cells in Mx-c-fos B cell culture without the addition of IFN-α/β was similar to that (12.9%) in control B cell culture. The percentage (28.8%) in Mx-c-fos B cell culture after the addition of IFN-α/β was distinctly higher than that (12.8%) in control B cell culture after the addition of IFN-α/β. Furthermore, the percentage of TUNEL+ B cells increased in Mx-c-fos B cell culture, but not in control B cell culture after the addition of IFN-α/β (Fig. 4 B). These results suggest that overexpression of c-Fos increases apoptosis in Ig class-switching B cells.

FIGURE 4.

Some of activated B cells are apoptotic in Mx-c-fos B cell culture by the addition of IFN-α/β. Splenic B cells from Mx-c-fos mice were cultured with LPS and rIL-4 for 3 days. IFN-α/β was added to the culture on day 2. Apoptotic B (B220+) cells were detected by FACS with the annexin V staining method (A) and the TUNEL method (B). The dUTP histograms of B cells are shown.

FIGURE 4.

Some of activated B cells are apoptotic in Mx-c-fos B cell culture by the addition of IFN-α/β. Splenic B cells from Mx-c-fos mice were cultured with LPS and rIL-4 for 3 days. IFN-α/β was added to the culture on day 2. Apoptotic B (B220+) cells were detected by FACS with the annexin V staining method (A) and the TUNEL method (B). The dUTP histograms of B cells are shown.

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Since Ag-specific IgG Abs and memory B cells cannot be generated in immunized H2-c-fos mice (13), the structure of B cell follicles and the development of GC B cells were analyzed in spleen from H2-c-fos mice immunized with DNP-OVA. Figure 5,A shows that primary B cell follicles, marginal zones, and PALS were histologically identified in spleen from H2-c-fos mice. The develop-ment of GC B (PNA+, B220+) cells was analyzed by cell surface staining of spleen cells with PNA and anti-B220 mAb (Fig. 5 B). The development of GC B cells was very poor in spleen from H2-c-fos mice, although PNA-binding GC B cells clearly developed in control littermates from day 10 after immunization.

FIGURE 5.

GC formation is impaired in spleen from H2-c-fos mice. H2-c-fos mice were immunized with DNP-OVA. A, Sections of spleen from immunized H2-c-fos mice and control littermates on day 12 after immunization were stained with anti-B220 mAb. Hematoxylin counterstain was used. B, Splenocytes from H2-c-fos mice and control littermates on days 7, 10, and 14 after immunization were stained with PNA and anti-B220 mAb. Those stained GC B (PNA+, B220+) cells were analyzed by FACS. The numbers indicate the percentages of cells within boxed regions based on the total number of cells analyzed.

FIGURE 5.

GC formation is impaired in spleen from H2-c-fos mice. H2-c-fos mice were immunized with DNP-OVA. A, Sections of spleen from immunized H2-c-fos mice and control littermates on day 12 after immunization were stained with anti-B220 mAb. Hematoxylin counterstain was used. B, Splenocytes from H2-c-fos mice and control littermates on days 7, 10, and 14 after immunization were stained with PNA and anti-B220 mAb. Those stained GC B (PNA+, B220+) cells were analyzed by FACS. The numbers indicate the percentages of cells within boxed regions based on the total number of cells analyzed.

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The in vitro results shown in Figures 1 through 41–4 strongly suggested that perturbation of GC formation in H2-c-fos spleen was due to cell death of Ig class-switching B cells. However, we could not detect larger numbers of TUNEL+ B cells in GC from H2-c-fos spleen (data not shown) because of poor formation of GCs. Since overexpression of Bcl-2 can rescue some apoptosis in B cells (35, 36, 37), H2-c-fos mice were mated with Ig-bcl-2 mice. F1 progeny was immunized with DNP-OVA, and GC formation was histologically analyzed in spleens on day 12 after immunization (Fig. 6). PNA-binding GC B cells were detected in spleens from Ig-bcl-2 mice, and the number of PNA-binding B cells in each GC was larger than that in control mice. Although GC formation was slightly detected in spleen from H2-c-fos mice, PNA-binding GC B cells were clearly identified in spleen from doubly transgenic mice.

FIGURE 6.

GC formation impaired in spleen from H2-c-fos mice is rescued by overexpression of Bcl-2. H2-c-fos mice were mated with Ig-bcl-2 mice. F1 progeny was immunized with DNP-OVA. Sections of spleen from immunized mice on day 12 after immunization were stained with PNA (brown). Hematoxylin counterstain was used.

FIGURE 6.

GC formation impaired in spleen from H2-c-fos mice is rescued by overexpression of Bcl-2. H2-c-fos mice were mated with Ig-bcl-2 mice. F1 progeny was immunized with DNP-OVA. Sections of spleen from immunized mice on day 12 after immunization were stained with PNA (brown). Hematoxylin counterstain was used.

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These results were confirmed by cell surface staining of spleen cells from immunized mice with PNA and anti-B220 mAb (Fig. 7 A). The percentage (0.9 ± 0.5%) of PNA+ B cells in spleen cells from H2-c-fos mice (n = 5) was less than that (2.5 ± 0.9%) in spleen cells from control mice (n = 5). The percentage (5.1 ± 4.7%) from Ig-bcl-2 mice (n = 4) was augmented, and that (2.9 ± 0.7%) from doubly transgenic mice (n = 4) was more than that from H2-c-fos mice.

FIGURE 7.

IgG1+ B cells are developed in GC B (PNA+, B220+) cells of spleen from immunized (H2-c-fos × Ig-bcl-2), doubly transgenic mice. H2-c-fos mice were mated with Ig-bcl-2 mice. F1 progeny was immunized with DNP-OVA. Splenocytes from F1 progeny on day 12 after immunization were stained with PNA, anti-B220, and anti-IgG1 mAb. Those stained GC B (PNA+, B220+) cells (A) and IgG1+ B cells in GC B (PNA+, B220+) cells (B) were analyzed by FACS. The numbers indicates the percentages of cells within boxed regions based on the total number of cells analyzed.

FIGURE 7.

IgG1+ B cells are developed in GC B (PNA+, B220+) cells of spleen from immunized (H2-c-fos × Ig-bcl-2), doubly transgenic mice. H2-c-fos mice were mated with Ig-bcl-2 mice. F1 progeny was immunized with DNP-OVA. Splenocytes from F1 progeny on day 12 after immunization were stained with PNA, anti-B220, and anti-IgG1 mAb. Those stained GC B (PNA+, B220+) cells (A) and IgG1+ B cells in GC B (PNA+, B220+) cells (B) were analyzed by FACS. The numbers indicates the percentages of cells within boxed regions based on the total number of cells analyzed.

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Development of Ig class-switched B cells in GCs from spleen of F1 progeny was further examined by three-color analysis with FACS (Fig. 7 B). IgG1+ B cells (49%) were identified in PNA-binding GC B (PNA+, B200+) cells (2.4%) from control littermates. Although perturbation of GC formation (0.7%) was confirmed in spleen from H2-c-fos mice, IgG1+ B cells (42%) could be detected in PNA-binding B cells from H2-c-fos spleen. IgG1+ B cells (32%) were also detected in PNA-binding B cells (3.0%) from doubly transgenic mice, indicating that the exogenous Bcl-2 can rescue perturbation of GC formation, including the Ig class-switching process, in H2-c-fos mice.

We then examined the amount of primary IgG1 anti-DNP Ab in serum from doubly transgenic mice, since perturbation of mature B cell differentiation into GC B cells was rescued. Titers of primary IgM and IgG1 anti-DNP Ab in sera from F1 progeny on day 12 after immunization were measured by ELISA (Fig. 8). All F1 progeny produced primary IgM Ab at a comparable level. However, the amounts of IgG1 anti-DNP Ab in H2-c-fos and doubly transgenic mice were lower than those in control and Ig-bcl-2 mice, although the amount in doubly transgenic mice was about fivefold higher than that in H2-c-fos mice. This lower production of IgG1 Ab was maintained in doubly transgenic mice until day 28 after immunization (data not shown). These results suggest that the exogenous Bcl-2 can delay the time of cell death in B cells during differentiation in GCs, but cannot rescue perturbation of mature B cell differentiation into IgG-producing cells in H2-c-fos mice.

FIGURE 8.

Production of the primary IgG1 Ab is suppressed in immunized (H2-c-fos × Ig-bcl-2) doubly transgenic mice. H2-c-fos mice were mated with Ig-bcl-2 mice. F1 progeny was immunized with DNP-OVA and bled on day 12 after immunization. IgM and IgG1 anti-DNP titers in sera were measured by ELISA. A circle indicates an individual mouse. Titers of IgM and IgG1 anti-DNP Ab in sera from preimmune control mice were <10 and 1 U/ml, respectively.

FIGURE 8.

Production of the primary IgG1 Ab is suppressed in immunized (H2-c-fos × Ig-bcl-2) doubly transgenic mice. H2-c-fos mice were mated with Ig-bcl-2 mice. F1 progeny was immunized with DNP-OVA and bled on day 12 after immunization. IgM and IgG1 anti-DNP titers in sera were measured by ELISA. A circle indicates an individual mouse. Titers of IgM and IgG1 anti-DNP Ab in sera from preimmune control mice were <10 and 1 U/ml, respectively.

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Differentiation of mature B cells into IgG-producing cells is perturbed by overexpression of c-Fos (13). Furthermore, GC formation of B cells in spleen from H2-c-fos mice was perturbed (Fig. 1). Since the Ig class switch mainly occurs in GC B cells (20, 21, 22, 30), perturbation of the differentiation may occur in B cells at the Ig class-switching step. This is supported by in vitro studies using Mx-c-fos B cells. Ig class-switched (IgG1+) B cells became detectable in Mx-c-fos B cell culture with LPS and rIL-4 after day 3 of culture, and overexpression of c-Fos in the B cells after day 2, but not until day 2, of culture perturbed development of IgG1+ B cells (Figs. 2 and 3). This perturbation is specific for IgG1+ B cells because the viable B cell number in those cultures was not affected by c-Fos. As IgG1+ B cells were detected in control B cell culture from day 3, the IgG1 class-switch recombination begins to be processed in B cells before day 3 of culture. Therefore, the results shown in Figures 2 and 3 strongly suggest that differentiation of H2-c-fos B cells into IgG-producing cells is perturbed at the Ig class-switching step.

Those Ig class-switching B cells with overexpression of c-Fos may die by apoptosis, since the number of activated B cells that died by apoptosis increased in Mx-c-fos B cell culture within 1 day after the addition of IFN-α/β (Fig. 4). This cell death was not due to the apoptotic effect of IFN-α/β (>1,000 U/ml) as previously described (49), because the lower dose of IFN-α/β (200 U/ml) used in our experiments did not increase the number of apoptotic cells in control B cell culture ( Figs. 2–4). The exogenous c-fos gene was induced in Mx-c-fos B cells as early as 1 h after IFN-α/β stimulation (43), indicating that the onset of c-fos expression precedes apoptosis in the B cells. This c-Fos-induced apoptosis may also occur in nascent surface IgG+ B cells, since IgG1+ B cells were developed in GCs from immunized H2-c-fos and doubly transgenic mice (Fig. 7,B) without the production of primary IgG1 Ab in sera (Fig. 8). Furthermore, the number of IgG1+ B cells in Mx-c-fos B cell culture on day 5 after the addition of IFN-α/β on day 4 was about half that in Mx-c-fos B cell culture without the addition of IFN-α/β (Fig. 3), although the number in Mx-c-fos B cell culture without IFN-α/β reached a plateau level after day 4 of culture (Fig. 2).

We have recently demonstrated that pro-B (B220+, CD43+) cells developed in fetal liver cell culture from Mx-c-fos mice were selectively killed by apoptosis after c-Fos induction (33). Therefore, c-Fos may play a role as a potent inducer of apoptosis in pro-B cells and Ig class-switching B cells. c-Fos-induced apoptosis is additionally supported by findings that induction of c-fos expression is an early event in many instances of mammalian apoptosis (50, 51, 52, 53, 54) and that reduction of c-Fos activity by antisense oligonucleotides can prevent growth factor-deprived lymphoid cells from undergoing apoptosis (51). c-Fos, which can compose AP-1 with jun gene products to regulate the expression of target genes (5, 6, 7, 8), may be a required component of the gene regulatory pathway that leads to cell death in certain cell types (53). Indeed, overexpression of c-Jun is sufficient to trigger apoptosis in fibroblasts (55). Since detectable amounts of Jun family proteins are produced in mature B cells (48) and in early B-lineage cells (56), c-Fos from the exogenous gene can effectively form AP-1 molecules with those Juns. We have demonstrated that the amount of c-Fos correlates with the activity of AP-1 in mature B cells (48) and in early B-lineage cells (32) from Mx-c-fos mice. Thus, apoptosis in those B cells may be due to an excess of c-Fos/AP-1.

Induction mechanisms of apoptosis in Ig class-switching B cells initiated by c-Fos could be accounted for in several ways. First, c-Fos might perturb Ig class-switch recombination machinery in B cells. The initial event of the IgG1 class-switch process is to induce the expression of IgG1 germline transcript in activated B cells within 1 day after rIL-4 stimulation (14), and the expression is essential to the switching process (57). However, overexpression of c-Fos in Mx-c-fos B cells until day 2 of culture did not affect the development of IgG1+ B cells (Fig. 2), indicating that expression of the IgG1 germline transcript is not impaired by c-Fos. Indeed, the expression is induced in H2-c-fos B cells stimulated with rIL-4 within 1 day after stimulation (14). Furthermore, IgG1+ B cells can develop in immunized H2-c-fos spleens (Fig. 7 B) and in H2-c-fos B cells cultured with lower doses of LPS (<2.5 μg/ml) and rIL-4 (14). Therefore, it is unlikely that IgG1 class-switch machinery is impaired by c-Fos.

Second, since the c-fos gene is transiently induced in cells treated with DNA-damaging agents (58, 59, 60), such as ionizing irradiation (59) or UV (60), c-Fos may have a protective function, including DNA repair, against harmful consequences of agents. DNA recombination occurs in Ig class-switching B cells (61, 62), and DNA repair may be required for B cells to differentiate into IgG-producing cells, because DNA repair enzyme is detected in B cells with IgG class-switch recombination (63). Therefore, overexpression of c-Fos may perturb DNA repair functions in Ig class-switching B cells. This may explain c-Fos-induced apoptosis in pro-B cells, since the V(D)J recombination has to be repaired in pro-B cells to differentiate into pre-B cells (64). However, repair of UV-induced DNA lesions was not affected in fibroblasts lacking c-fos, although survival of the fibroblasts was drastically reduced by UV irradiation (65), suggesting that c-Fos may not perturb DNA repair functions in B cells.

Third, we have previously shown that prolonged overexpression of c-Fos perturbs the cell cycle progression of mature B cells by sIg cross-linking (43). The c-fos gene is transiently induced in B cells, and the prolonged overexpression inhibits B cells from entering the S phase of the cell cycle. This perturbation of cell cycle progression is due to poor degradation of the cyclin kinase inhibitor p27kip1 in the G1 phase of the cell cycle. Since cell cycle arrest is required for the process of DNA repair, overexpression of c-Fos may continuously inhibit cell cycle progression of Ig class-switching B cells even after DNA repair is completed. Those arrested B cells will result in cell death. Further study is required to elucidate the mechanisms of apoptosis by c-Fos.

In B cell development at the GC stage, deletion by apoptosis occurs at the transition from centroblasts to centrocytes. Centrocytes with higher affinity to self Ags undergo selective apoptosis (23, 24, 25, 26, 27). This apoptosis in GC B cells is insensitive to Bcl-2 (39). c-Fos-induced apoptosis in Ig class-switching B cells may also be insensitive to Bcl-2, since overexpression of Bcl-2 cannot rescue the production of primary IgG1 Ab in doubly transgenic mice at control levels. Indeed, c-fos mRNA was detected in GC B cells from immunized normal spleen by reverse transcribed PCR (data not shown). Therefore, deregulation of c-Fos may augment signal transduction to induce apoptosis in GC B cells. Since nascent surface IgG1+ B cells seem to be sensitive to c-Fos-induced apoptosis (Fig. 3), the c-Fos-induced apoptosis may mimic the deletion of self-reactive B cells in GCs. This idea is supported by the evidence that this selective apoptosis in GC B cells requires prolonged sIg cross-linking (66). The sIg cross-linking induces transient expression of c-Fos in mature B cells (38), and the prolonged sIg cross-linking may induce prolonged overexpression of c-Fos. In that case, c-Fos may play a causal role in the deletion of GC B cells with sIg receptors for self Ags. Thus, c-fos transgenic models will provide a unique opportunity to investigate molecular mechanisms of apoptosis of self-reactive B cells developed in GCs.

We thank Drs. T. Takemori, L. Hu, T. Fukuda, and K. Kobayashi for helpful discussion and collaboration; Drs. M. Katsumata and Y. Tsujimoto for Ig-bcl-2 transgenic mice; Ms. Y. Iwata for technical assistance; and Ms. N. Fujita for secretarial assistance.

1

This work was supported in part by grants-in-aid for Cancer Research from the Ministry of Education, Science, Sports, and Culture of Japan; grants from the Ministry of Health and Welfare of Japan; and special coordination funds for the promotion of science and technology from the Science and Technology Agency of Japan.

4

Abbreviations used in this paper: PALS, periarteriolar lymphoid sheaths; GC, germinal center; PNA, peanut agglutinin; sIg, surface Ig; HRP, horseradish peroxidase; PE, phycoerythrin; TUNEL, TdT-mediated deoxyuridine triphosphate-biotin nick end labeling.

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