Effect of c-fos overexpression on development and proliferation of peritoneal B cells
Satoshi Mori1,2,
Akemi Sakamoto1,
Kimihiro Yamashita1,
Lisa Fujimura1,
Masafumi Arima1,
Masahiko Hatano1,
Masaru Miyazaki2 and
Takeshi Tokuhisa1
1 Department of Developmental Genetics (H2) and 2 Department of General Surgery (F6), Graduate School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8670, Japan
Correspondence to: T. Tokuhisa; E-mail: tokuhisa{at}faculty.chiba-u.jp
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Abstract
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We examined effects of c-fos overexpression on the development and property of peritoneal B-1 cells using transgenic (H2-c-fos) mice carrying the c-fos gene under the control of the constitutive H-2Kb promoter. The number of B-1b cells in the peritoneal cavity of H2-c-fos mice was 4-fold larger than that in control littermates. Although the numbers of total peritoneal B cells and B-1a cells were similar between them, the peritoneal B-2 cell number in H2-c-fos mice was reduced to 50% of control littermates, suggesting the effect of c-fos overexpression on a balance of B-1b and B-2 cells in a peritoneal cavity. Adoptive transfer experiments with hematopoietic stem cells of H2-c-fos and control mice into irradiated H2-c-fos mice demonstrated that the augmentation of B-1b cells is due to the c-fos effect in B cells and the effect on environment of the peritoneal cavity of H2-c-fos mice. When peritoneal B cells were cultured with LPS in the presence or absence of IL-4, cell proliferation of B-1b cells was the highest among these peritoneal B cell subsets, and the proliferation of H2-c-fos B-1b cells was 3-fold higher than that of control B-1b cells. This augmentation is due to the c-fos effect in B cells. IgG1 production of B-1b cells in these cultures was slightly higher than those of B-1a and peritoneal B-2 cells. Thus, the c-fos overexpression augments development of B-1b cells in a peritoneal cavity and proliferation of peritoneal B-1b cells to LPS.
Keywords: B lymphocytes, lipopolysaccharide, cellular differentiation, transcription factors
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Introduction
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The proto-oncogene c-fos, which encodes a nuclear phospho-protein (c-Fos), is transiently induced in numerous cell types by many agents and conditions (14). c-Fos in a complex with products of another proto-oncogene c-jun (AP-1) regulates expression of AP-1 binding genes at transcriptional level (58). Thus, a function of c-Fos may be implicated in transduction of signals induced by growth and differentiation factors (911). To study biological effects of c-Fos on 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 antigens, the mice can produce primary IgM antibodies, but not IgG antibodies, and fail to generate memory B cells in the spleen. The co-culture experiments with H2-c-fos B cells and normal Th cells demonstrated that the abnormality is due to defects in B cells (13). Furthermore, splenic B cells from H2-c-fos mice cultured with larger amounts (>10 µg/ml) of LPS and IL-4 die by apoptosis and cannot differentiate into IgG1+ B cells (14), suggesting perturbation of IgG class-switching process in H2-c-fos B cells. However, serum IgG levels in H2-c-fos mice were similar to those of control littermates without production of IgG antibodies specific for immunized antigens (13).
Polyreactive serum IgM natural antibodies are mainly produced by B-1 cells (15). B-1 cells also give rise to the autoantibody-producing cells characteristic of several autoimmune disease states (16,17). B-1 cells are composed of two subsets, B-1a and B-1b cells, which can be distinguished only by the presence and absence of the CD5 antigen, respectively. Both B-1 cells produced high levels of IgM and IgG antibodies as measured in adoptive transfer experiments (18). The major difference is seen in the development of B-1a and B-1b cells from B220 progenitors in the adult bone marrow. Although B220 B-1a progenitors are rare in the adult bone marrow, progenitors for B-1b cells persist well into adulthood. A recent report (19) demonstrated that overproduction of interleukin (IL)-9 in transgenic mice generated a specific expansion of B-1b cells in the peritoneal and pleuropericardial cavities, although the IL-9 receptor was found to be expressed by B-1a and B-1b cells in normal mice. However, molecular mechanisms of development of these two distinct B-1 cells are largely unknown.
Here we examined properties of B-1 cells in the peritoneal cavity of H2-c-fos mice. We show that the number of B-1b cells but not that of B-1a cells in H2-c-fos mice was larger than that in control littermates. When peritoneal B cells were cultured with LPS, H2-c-fos B-1b cells proliferated more than control B-1b cells. We discuss the effect of c-fos overexpression on the development and properties of peritoneal B-1b cells.
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Methods
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Animals
C57BL/6CrSlc and C57BL/6 (B6-Ly5.1) mice were purchased from Japan SLC Co., Ltd (Hamamatsu, Japan) and Charles River Japan, Inc., respectively. Transgenic mice carrying the mouse c-fos gene under the control of the H-2Kb promoter (H2-c-fos) have been described previously (12). All mice were maintained under specific pathogen-free conditions in the animal center of the Graduate School of Medicine, Chiba University.
Antibodies and flow cytometry analysis
Peritoneal and spleen cells were stained with various monoclonal antibodies as follows (20). Cell suspensions were first blocked with unconjugated anti-CD32/16 (2.4G2, PharMingen, San Diego, CA) followed by incubation with biotinylated antibodies, and then incubation with directly conjugated antibodies and streptavidinperidinine chlorophyll protein (PerCP) (PharMingen). The following antibodies (PharMingen) were used for staining: phycoerythrin (PE)anti-B220, allophycocyanin (APC)anti-IgM, fluorescein isothiocyanate (FITC)anti-IgD, biotinanti-CD5, APCanti-B220, PEanti-CD23, FITCanti-IgM, FITCanti-Thy1.2, APCanti-IgG1. Cell suspensions were treated with lysis buffer (ACK; 0.155 M ammonium chloride, 0.1 M disodium EDTA, 0.01 M potassium bicarbonate) to lyse erythrocytes before staining. Single-cell suspensions were prepared in staining medium (PBS with 3% FCS and 0.1% sodium azide) and were stained with the antibodies described above. After 20 min incubation on ice, cells were washed twice with staining medium and resuspended in staining medium supplemented with propidium iodide (PI, 1 µg/ml) to exclude dead cells, and analyzed on a FACSCalibur (Becton Dickinson, San Jose, CA).
Preparation of peritoneal and splenic B cells
Peritoneal cells were obtained by washing the cavity with 10
15 ml of PBS. Peritoneal cells were stained with PEanti-B220, biotinanti-CD5, APCanti-IgM, FITCanti-IgD and streptavidinCyChrome. IgM+ IgDdull CD5+ B220dull (B-1a), IgM+ IgDdull CD5 B220+ (B-1b) and IgMdull IgD+ CD5 B220+ (B-2) cells were sorted separately by a FACSVantage (20). Splenic cells were staining with APCanti B220 and PEanti-CD23, and B220+ CD23+ cells (B-2 cells) were sorted. Purity of these FACS-sorted B cells was >98%.
Adaptive transfer of primitive hematopoietic stem cells
A primitive hematopoietic stem cell fraction in bone marrow cells was isolated by a previously described method (21). In brief, biotinylated antibodies to B220, Mac-1, Gr-1, CD4, CD8 and TER119 were purchased from PharMingen and used to detect lineage markers (Lin). FITCanti-Sca-1 and PEanti-c-kit antibodies were purchased from PharMingen. Biotinylated antibodies were visualized using streptavidinCyChrome (PharMingen). Bone marrow cells from H2-c-fos and control mice were stained with these antibodies, and Linc-kit±Sca-1± cells were sorted by a FACSVantage and used as a primitive hematopoietic stem cell fraction. H2-c-fos and control mice were lethally irradiated (8.5 Gy) 24 h prior to intravenous injection of these Linc-kit±Sca-1± cells (2 x 103) from H2-c-fos or control mice. For a competitive repopulation assay, Linc-kit+Sca-1+ cells in bone marrow cells from H2-c-fos (Ly5.2) and control (Ly5.1) mice were sorted by a FACSVantage. B6-Ly5.1 and H2-c-fos (Ly5.2) mice were lethally irradiated (8.5 Gy) 24 h prior to intravenous co-injection of these Linc-kit+Sca-1+ cells (2 x 103) from H2-c-fos and control mice. Peritoneal cells from the radiation chimeric mice were analyzed 8 weeks after transfer. These cells were stained with antibodies and analyzed on a FACSCalibur.
Peritoneal B cell culture with LPS and IL-4
Peritoneal or spleen cells (2 x 105/ml), or FACS-sorted peritoneal B-1a, B-1b, B-2 and splenic B-2 cells (1 x 105/well) were cultured in RPMI 1640 supplemented with 10% FCS (Intergen, New York, NY) with LPS (1100 µg/ml; Sigma) in the presence or absence of rIL-4 (104 U/ml) (22) for 37 days at 37°C in 5% CO2. These cultured cells were stained with PEanti-B220 and APCanti-IgG1 antibodies for 20 min on ice. These stained cells were analyzed on a FACSCalibur.
Determination of antibody titers in the culture
Cells were cultured with LPS in the presence or absence of rIL-4 for 7 days. Amounts of IgM and IgG1 antibodies in the culture supernatants were measured by ELISA, as described previously (13). Briefly, goat anti-mouse IgG+IgM (Caltag Laboratories Inc., South San Francisco, CA) were coated onto ELISA plates, and Igs on wells were developed with biotinylated goat anti-mouse IgM antibodies (Caltag), or anti-IgG1 antibodies (Caltag) followed by avidinperoxidase (Vector Laboratories, Burlingame, CA).
DNA synthesis
Cells were cultured with LPS in the presence or absence of rIL-4 for 3 days. These cultured cells were pulsed with 1 µCi of [3H]thymidine (Amersham International, Aylesbury, UK) for the last 8 h and [3H]thymidine uptake was measured in a liquid scintillation counter.
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Results
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Overexpression of c-fos in H2-c-fos mice modifies the balance between the numbers of B-1b and B-2 cells in the peritoneal cavity
B-1 and B-2 cells in the peritoneal cavity of 8-week-old H2-c-fos mice were analyzed on a FACS. The percentage of B-1 cells in H2-c-fos mice was 2-fold larger than that in control littermates (Fig. 1A). On the contrary, the percentage of B-2 cells in H2-c-fos mice was approximately half that of control mice. Further analysis of peritoneal B-1 cells demonstrated that the percentage (Fig. 1B) and the number (Fig. 2) of B-1b cells in H2-c-fos mice were larger than those in control mice. When we examined the number of B-1 cells in 4- and 8-week-old H2-c-fos mice, the numbers of B-1b cells in H2-c-fos mice were 34-fold larger than those in control littermates (Table 1). Since the number of B-1a cells in H2-c-fos mice was similar to that in control littermates, and the sum of B-1b and B-2 cell numbers was almost the same between H2-c-fos and control mice, B-1b cells may be dominant in the peritoneal cavity of H2-c-fos mice under a balance between the numbers of B-1b and B-2 cells.

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Fig. 1. Staining profiles of peritoneal cells of H2-c-fos mice. Peritoneal cells of H2-c-fos mice and control littermates 8 weeks of age were stained with various antibodies and analyzed using FACS. The numbers indicate the percentages of cells in each square. The percentages of B-1 cells (A) and B-1b cells (B) in H2-c-fos mice are larger than those in control mice.
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Fig. 2. A correlation between peritoneal B-1b and B-2 cell numbers. Peritoneal cells of H2-c-fos mice (closed circles) and control littermates (open circles) 8 weeks of age were stained with various antibodies and analyzed using FACS. Cell numbers of subsets of peritoneal cells were calculated from the percentages and total peritoneal cell numbers, and these cell numbers were compared.
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Since progenitors of B-1b cells but not those of B-1a cells are maintained in the adult bone marrow (18,23,24), the effect of c-fos overexpression on development of B-1b cells was examined by co-transferring hematopoietic stem cells of H2-c-fos (Ly5.2) and control (Ly5.1) mice into lethally irradiated B6-Ly5.1 or H2-c-fos (Ly5.2) mice. As shown in Fig. 3(A), the percentage of Ly5.2+ (H2-c-fos) B-1 cells was similar to that of Ly5.1+ (control) B-1 cells in the peritoneal cavity of irradiated B6-Ly5.1 mice 8 weeks after transfer. The percentages of B-1 (15.7 ± 0.6%) and B-2 (76.3 ± 0.6%) cells derived from control mice were similar to those of B-1 (12.7 ± 5.0%) and B-2 (77.7 ± 4.7%) cells derived from H2-c-fos mice (n = 6). When hematopoietic stem cell mixture was transferred into irradiated H2-c-fos mice, the percentages of B-1 (65.0 ± 6.5%) and B-2 (32.0 ± 6.2%) cells derived from control mice were similar to those of B-1 (60.5 ± 6.0%) and B-2 (37.5 ± 5.5%) cells derived from H2-c-fos mice (n = 4). Both percentages of Ly5.2+ (H2-c-fos) B-1 cells and Ly5.1+ (control) B-1 cells were similar to those of H2-c-fos mice. These results suggest that the augmentation of B-1b cells in H2-c-fos mice is due to the effect of c-fos overexpression on the environment of the peritoneal cavity of H2-c-fos mice.

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Fig. 3. Repopulation analysis of peritoneal B-1b cells. Hematopoietic stem cells were transferred into lethally irradiated mice. Peritoneal cells of these recipient mice 8 weeks after transfer were stained with various antibodies and analyzed using FACS. (A) Hematopoietic stem cells of H2-c-fos (Ly5.2) and control (Ly5.1) mice were co-transferred into lethally irradiated B6-Ly5.1 or H2-c-fos (Ly5.2) mice. The numbers indicate the percentages of cells in each square. The data presented are representative of five independent experiments. (B) Hematopoietic stem cells of H2-c-fos or control mice were transferred into lethally irradiated H2-c-fos or control mice. The percentages of B-1b (closed bars) and B2 (open bars) cells in peritoneal B cells are shown. Results represent means and variations (SD) from three mice. The data presented are representative of two independent experiments.
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The effect of c-fos overexpression on development of B-1b cells was confirmed by adoptive transfer experiments. The percentage (40.2 ± 2.0%) of B-1b cells in irradiated H2-c-fos mice transferred with H2-c-fos hematopoietic stem cells was larger than that (20.4 ± 1.2%) in irradiated H2-c-fos mice transferred with control hematopoietic stem cells (Fig. 3B). Furthermore, the percentage (20.2 ± 1.5%) of B-1b cells in irradiated control mice transferred with H2-c-fos hematopoietic stem cells was larger than that (13.1 ± 1.3%) in irradiated control mice transferred with control hematopoietic stem cells. These results suggest that both c-fos overexpression in B cells and the environment of the peritoneal cavity of H2-c-fos mice affect the development of B-1b cells.
Overexpression of c-fos augments proliferation of B-1b cells stimulated with LPS
When peritoneal cells were stimulated with various doses of LPS and IL-4, proliferation of these cells was analyzed 3 days after stimulation. Proliferation of H2-c-fos peritoneal cells stimulated with 10 µg/ml of LPS in the presence or absence of IL-4 was 2-fold higher than that of control littermates (Fig. 4A). In order to identify the subset of proliferating peritoneal cells of H2-c-fos mice, peritoneal B-1a, B-1b and B-2 cells and splenic B-2 cells were isolated by FACS (Fig. 4B) and stimulated with 10 µg/ml of LPS in the presence or absence of IL-4. Peritoneal B-1b cells, but not B-1a and B-2 cells, from both H2-c-fos and control mice proliferated very well under both stimulations, and the proliferation of peritoneal B-1b cells was higher than that of splenic B-2 cells (Fig. 4C). Furthermore, the proliferation of H2-c-fos B-1b cells was 3-fold higher than that of control B-1b cells.


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Fig. 4. Proliferation of peritoneal B cells stimulated with LPS and IL-4. Peritoneal cells (A) or FACS-isolated subsets of peritoneal B cells (B and C) of H2-c-fos mice (closed bars) and control littermates (open bars) 8 weeks of age, or FACS-isolated B-1b cells (D) from adoptive transfer experiments were stimulated with LPS in the presence or absence of IL-4. Cell proliferation was measured by [3H]thymidine uptake on day 3 of culture. Results (A and D) represent means and variations (SD) from triplicate cultures. (B) Purity of these FACS-isolated peritoneal B cells was demonstrated. The data presented are representative of three (AC) or two (D) independent experiments.
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We confirmed the effect of c-fos overexpression on proliferation of B-1b cells to LPS stimulation using FACS-isolated B-1b cells from the adoptive transfer experiments. As shown in Fig. 4(D), H2-c-fos B-1b cells developed in irradiated H2-c-fos or control mice proliferated more than control B-1b cells developed in irradiated H2-c-fos mice after stimulation with LPS in the presence or absence of IL-4. These results indicate that overexpression of c-fos in peritoneal B-1b cells augments proliferation capacity of B-1b cells stimulated with LPS.
Peritoneal B-1b cells stimulated with LPS and IL-4 efficiently differentiate to IgG1+ B cells and IgG1 producing cells
Differentiation of splenic B cells into IgG1+ B cells and IgG1-producing plasma cells after immunization with antigens can be mimicked by in vitro culture of splenic B cells with LPS and IL-4. In order to examine the effect of c-fos overexpression in peritoneal B cells on their differentiation process into IgG1-producing plasma cells, peritoneal cells from H2-c-fos mice were cultured with LPS (10 µg/ml) and IL-4. The percentage of IgG1+ B cells in these cultures was analyzed by cell surface staining with anti-B220 and anti-IgG1 antibodies on day 3 of culture. Since Ig class-switching is perturbed in splenic B-2 cells from H2-c-fos mice cultured with a large amount (>10 µg/ml) of LPS and IL-4 (13,14), the percentage of splenic B-2 cells of H2-c-fos mice was lower than that of control mice (Fig. 5A). Surprisingly, IgG1+ B cells was detected in the cultures of peritoneal cells of H2-c-fos mice although the percentage was lower than that in the culture of splenic B-2 cells of H2-c-fos and control mice. On the contrary, few IgG1+ B cells were developed in the control peritoneal cell culture. When we examined development of IgG1+ B cells from peritoneal cells stimulated with the various doses of LPS and IL-4, the percentages of IgG1+ B cells in H2-c-fos peritoneal cell cultures were larger than those in control peritoneal cell cultures at any dose (3100 µg/ml) of LPS examined (Fig. 5B).

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Fig. 5. Development of IgG1+ B cells in cultures of peritoneal cells stimulated with LPS and IL-4. Peritoneal and spleen cells of H2-c-fos mice (closed circles) and control littermates (open circles) 8 weeks of age were stimulated with various doses of LPS and IL-4. Surface IgG1+ B cells were detected on day 3 of culture by FACS. (A) Representative staining profiles of peritoneal and spleen cells stimulated with 10 µg/ml of LPS and IL-4. The numbers indicate the percentages of cells in each square. (B) The percentages of IgG1+ B cells in the culture of peritoneal and spleen cells stimulated with various doses of LPS and IL-4. The small bars indicate the means.
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IgM and IgG1 antibody production in the supernatants of peritoneal cell cultures was analyzed 7 days after stimulation with 10 µg/ml of LPS in the absence and presence of IL-4, respectively. Although IgM antibody production by H2-c-fos peritoneal cells was similar to that by control peritoneal cells, IgG1 antibody production by H2-c-fos peritoneal cells was 2-fold more than that by control peritoneal cells (Fig. 6A). To identify the subset of peritoneal cells for producing IgM and IgG1 antibodies, peritoneal B-1a, B-1b and B-2 cells, and splenic B-2 cells were isolated by a FACS and stimulated with LPS for IgM antibody production or with LPS and IL-4 for IgG1 antibody production. The production of IgM and IgG1 antibodies by these B cell subsets was similar between H2-c-fos and control mice (Fig. 6B). IgM production by peritoneal B-1b and B-2 cells of H2-c-fos and control mice was 4-fold higher than that by peritoneal B-1a cells and splenic B-2 cells of H2-c-fos and control mice. IgG1 production by B-1b cells of H2-c-fos and control mice was slightly higher than that by B-1a or by peritoneal and splenic B-2 cells of H2-c-fos and control mice.

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Fig. 6. The amounts of IgM and IgG1 antibodies in the cultures of peritoneal B cells stimulated with LPS and IL-4. Peritoneal cells (A) or FACS-isolated subsets of peritoneal B cells and splenic B cells (B) of H2-c-fos mice (closed bars) and control littermates (open bars) 8 weeks of age were stimulated with LPS in the presence or absence of IL-4 for 7 days. The amounts of IgM and IgG1 antibodies in the culture supernatants were measured by ELISA. Results represent means and variations (SD) from triplicate cultures. The data presented are representative of three independent experiments.
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Discussion
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Overexpression of c-fos affects numbers of B-1b and B-2 cells in the peritoneal cavity of H2-c-fos mice. The number of peritoneal B-1b cells of H2-c-fos mice was 4-fold larger than that of control mice, and the number of peritoneal B-2 cells of H2-c-fos mice was 2-fold lower than that of control mice. Since the numbers of peritoneal B cells and B-1a cells were similar between H2-c-fos and control mice, the numbers of B-1b and B-2 cells may be balanced in a peritoneal cavity, and overexpression of c-fos may shift a balance to B-1b cells. B-1a cells differ from B-1b cells and splenic B-2 cells in their progenitors (18,23). The progenitors of B-1a cells are abundant in the fetal omentum and liver, but are missing in the bone marrow of adult mice (18,24). Our results of the numbers of B-1a cells in H2-c-fos and control mice support the hypothesis that the number of B-1a cells keeps constant in the peritoneal cavity by self-renewal (23,25). The progenitors of B-1b cells are present in the fetal omentum and liver as well as in the bone marrow of adult mice (18,23), and supply of B-1b cells from the bone marrow appears to be restricted by a negative feedback mechanism according to which the entrance of newly generated B cells into the peritoneal cavity is prevented by the presence of mature B cells (23). In the absence of a continuous supply of bone marrow-derived B-1b cells, the number of B-1b cells may be kept constant due to their self-renewal capacity such as B-1a cells (23,26). Our results of the numbers of peritoneal B-2 cells in H2-c-fos and control mice suggest that supply of peritoneal B-2 cells from the bone marrow is also restricted by a negative feedback mechanism according to total B cell numbers in a peritoneal cavity.
Overexpression of c-fos induced the dominancy of B-1b cells in a balance of B-1b and B-2 cells in the peritoneal cavity of H2-c-fos mice. The co-transfer experiment with hematopoietic stem cells of H2-c-fos and control mice demonstrated the dominancy of B-1 cells not in the peritoneal cavity of control hosts but in that of H2-c-fos hosts, and the percentages of both H2-c-fos and control B-1 cells were augmented in irradiated H2-c-fos hosts. Thus, the environment of the peritoneal cavity of H2-c-fos mice may be the cause of the dominancy. IL-5 (27,28) and IL-9 (19) are related to the generation and expansion of B-1 cells. IL-5 transgenic mice demonstrated expansion of the B-1 population with high levels of autoantibodies (27). In H2-c-fos mice, however, the number of B-1a cells is not expanded and there are no autoantibodies. Furthermore, IL-5 production is not augmented in splenic H2-c-fos T cells stimulated with anti-CD3 antibody (data not shown). Overproduction of IL-9 in transgenic mice stimulated the generation of B-1b cells but not B-1a cells (19) and did not stimulate the production of autoantibodies in vivo, strongly suggesting that overproduction of IL-9 is the cause of the B-1b dominancy in H2-c-fos mice. Since the promoter of the IL-9 gene contains AP-1-binding sequence (7) and IL-9 is a Th2 type cytokine (29), overexpression of c-fos/AP-1 in T cells might stimulate the production of IL-9 in H2-c-fos T cells. However, the augmentation of IL-9 mRNA expression was not detected in H2-c-fos T cells stimulated with anti-CD3 antibodies (data not shown). This is supported by the co-transfer experiments, because the mixed hematopoietic stem cell transfer in control hosts can generate H2-c-fos T cells that did not induce the dominance of B-1b cells.
Some of B-1a cells were maintained in the peritoneal cavity of irradiated hosts transferred with bone marrow derived stem cells and the percentage of B-1b cells in the competitive repopulation analysis could not be identified by our FACS because of the five color staining. Thus, we confirmed the dominancy of B-1b cells in the peritoneal cavity of H2-c-fos mice by adoptive transfer experiments. The effect of the peritoneal cavity environment of H2-c-fos mice on the dominancy of B-1b cells was repeated. Furthermore, the c-fos overexpression in B cells also affects the dominancy of B-1b cells. Since the proliferation of H2-c-fos B-1b cells to LPS stimulation was augmented, and given the known ability of peritoneal B-1 cells to be activated by LPS in vivo (30,31), the dominancy of B-1b cells may be partly due to the augmented proliferation capacity of H2-c-fos B-1b cells to LPS. Further study is required to elucidate the mechanism of the B-1b dominancy in H2-c-fos mice.
B-1 cells can produce large quantities of multireactive IgM, IgG3 and IgA (23). Since the ablation of B-1 cells is accompanied by a drastic reduction of serum antibody titers (32), the maintenance of B-1 cells at a stable level might be necessary to control the level of serum antibody titers. Antibody production by B-1 cells could be induced by some multivalent T cell-independent antigens, especially in connection with the production of autoreactive and anti-bacterial specificities (23,24). Thus, B-1 cells are considered carriers of natural immunity (33). However, the exact origin of naturally occurring antigens which stimulate the differentiation of B-1 cells into antibody-producing cells remains elusive. Murakami et al. (30) demonstrated the critical role of the microbial microenvironment in antibody production by B-1 cells. Thus, it seems likely that antibody production by B-1 cells is induced polyclonally by bacteria-derived LPS. When splenic B cells from H2-c-fos mice were cultured with LPS (>10 µg/ml) and IL-4, these B cells could not differentiate into IgG1+ B cells (13,14), suggesting perturbation of the IgG class-switching process in B cell differentiation. These Ig class-switching B cells with c-fos overexpression may die by apoptosis (34). This c-fos-induced apoptosis may also occur in nascent surface IgG+ B cells since IgG1+ B cells were developed in germinal centers from immunized H2-c-fos mice without production of primary IgG1 antibody in sera (34). However, H2-c-fos B-1b cells were less sensitive to apoptosis induced in class-switching B cells with LPS and IL-4 stimulation. Furthermore, proliferation of peritoneal B-1b cells to LPS stimulation was the highest among the peritoneal B cell subsets, and the c-fos overexpression in B-1b cells augmented the proliferation. Thus, the H2-c-fos mouse model will provide a unique opportunity to investigate the development of B-1b cells in the peritoneal cavity, and roles of B-1b cells activated with polyclonal stimulations.
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Acknowledgements
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We would like to extend our thanks to Dr S. Okada for helpful discussion and collaboration, H. Satake for technical assistance and N. Kakinuma for secretarial assistance. This work was supported in part by the Grants-in-Aid for Cancer Research from the Ministry of Education, Science, Sports, and Culture of Japan.
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Abbreviations
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c-Fos | c-fos gene product |
Lin | lineage markers |
PerCP | peridinine chlorophyll protein |
PI | propidium iodide |
WT | wild-type control |
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Notes
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Transmitting editor: K. Okumura
Received 11 November 2003,
accepted 25 July 2004.
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