Establishment and characterization of pro-B cell lines from motheaten mutant mouse defective in SHP-1 protein tyrosine phosphatase

Immunology Letters 63 (1998) 75 – 82

Establishment and characterization of pro-B cell lines from motheaten mutant mouse defective in SHP-1 protein tyrosine phosphatase Akitomo Miyamoto 1,a, Takahiro Kunisada a, Hidetoshi Yamazaki a, Kensuke Miyake b, Shin-Ichi Nishikawa c, Tetsuo Sudo d, Leonard D. Shultz e, Shin-Ichi Hayashi a,* a

Department of Immunology, School of Life Science, Faculty of Medicine, Tottori Uni6ersity, 86 Nishi-Machi, Yonago, Tottori, 683 -8503 Japan b Department of Immunology, Saga Medical School, Saga, 849 -0937 Japan c Department of Molecular Genetics, Faculty of Medicine, Kyoto Uni6ersity, Kyoto, 606 -8397 Japan d Basic Research Laboratories, Toray Industries Inc., Kamakura, 248 -0036 Japan e The Jackson Laboratory, Bar Harbor, ME 04609, USA Accepted 15 May 1998

Abstract Mice homozygous for the motheaten (Hcph me) mutation lack a functional SHP-1 protein tyrosine phosphatase, show severe immunologic dysregulation and die at an early age. Severe pneumonitis in me/me mice is associated with abnormal proliferation of macrophages and granulocytes. Overgrowth of macrophages in long term cultures of me/me bone marrow has prevented analyses of lymphopoiesis in vitro. To establish hematopoietic cell lines from me/me mice, we cultured me/me bone marrow with the PA6 stromal cell line in the presence of antagonistic antibody against the receptor (c-Fms) for macrophage colony stimulating factor (M-CSF). In these cultures, overgrowth of M-CSF-dependent macrophages was suppressed by the antagonistic antibody and other hemopoietic cell lineages were generated efficiently from me/me bone marrow. By using this culture system, we established me/me pro-B cell clones (MEBs) with rearranged DH-JH but not VH-DJH. The growth of MEB clones required IL-7 and c-Kit ligand, corresponding to normal pro-B cells which express SHP-1. MEB cells were sensitive to starvation by either IL-7 or c-Kit ligand, resulting in apoptotic death. The present culture system, which supports hematopoiesis of me/me bone marrow, provides useful tools for the determination of the role of SHP-1 in signal transduction of B lymphopoiesis. © 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: SHP-1; Protein tyrosine phosphatase; B lymphocyte; Immunodeficiency

1. Introduction Mice homozygous for the autosomal recessive mutation motheaten (me) lack functional SHP-1 protein tyrosine phosphatase (also known as hematopoietic cell phosphatase (Hcph), PTP1C, SHPTP1, SHP and PTPN6) and do not survive more than 3 weeks after * Corresponding author. Tel: + 81 859 348269; fax: + 81 859 348272; e-mail: [email protected] 1 Present address. Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, 812-8582 Japan.

birth [1–4]. SHP-1 is a member of the family of cytosolic protein tyrosine phosphatases (PTPs) which contain SH2 domains [5]. Since phosphorylation of proteins on tyrosine residues is a key regulatory event in cell survival, growth and differentiation, dephosphorylation catalyzed by PTPs provides critical regulatory mechanisms [6]. Systemic autoimmune and inflammatory diseases in me/me mice are caused by the aberrant balance between phosphorylation and dephosphorylation of signal transduction molecules that regulate the immune system [3,4]. Recently, it has been reported that SHP-1 plays critical roles in a variety of signal transduction path-

0165-2478/98/$19.00 © 1998 Published by Elsevier Science B.V. All rights reserved. PII S0165-2478(98)00058-3

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ways including c-Kit, c-Fms, EpoR, IL-3R, FcgRIIb and sIgM in hematopoietic lineages [7 – 12]. Moreover, PTPs have been shown to regulate apoptosis in a variety of cell lineages [13 – 16]. A major biological in vivo result of SHP-1 deficiency in the growth of hematopoietic cells is severe pneumonitis associated with an abnormally increased number of macrophages in me/ me mice [17,18]. The higher proliferative capacity of macrophages observed in in vitro long term culture of me/me bone marrow cells might be related to the increase of macrophages in vivo. Practically, overgrowth of macrophages in vitro prevents the development of other cell lineages such as lymphocytes, granulocytes and erythrocytes [18 – 20]. The establishment of cell lines of these cell lineages from me/me mice has not been successful. We reasoned that if we could prevent the macrophage overgrowth in long term cultures of me/me bone marrow, it might be possible to establish cell lines of hematopoietic cell lineages of me/me mice which would help to investigate the function of SHP-1 in hematopoietic cells. To establish long term cultures of me/me hematopoietic cells, we grew bone marrow cultures of me/me mice with a stromal cell line in the presence of antagonistic antibody against the receptor (c-Fms) for macrophage-colony stimulating factor (M-CSF) [21,22]. In these cultures, the overgrowth of macrophages was suppressed and the growth of other hematopoietic cell lineages including granulocytes, B lymphocytes and mast cells was regularly observed. By using this culture system, we established pro-B cell clones with rearranged DH-JH, but not VH-DJH , from me/me bone marrow. The me/me pro-B cell clones (MEBs) were able to expand in vivo in immunodeficient hosts. Their growth in vitro was dependent on IL-7, c-Kit ligand and other unknown stromal cell molecule(s) and deprivation of IL-7 or c-Kit ligand promptly induced apoptotic cell death. Because there is little information about the signals of B lymphopoiesis before the antigen receptor-negative stage, MEBs established in this study may be useful for studies of the role of SHP-1 on signal transduction in the B cell lineage.

2. Materials and methods

2.1. Mice C3HeB/FeJ-a/a-Hcph me/Hcph me, hereafter termed me/me mice, (H-2k) were obtained by mating of me/ + heterozygous parents from the Jackson Laboratory (Bar Harbor, ME). 129/S6-RAG2 − / − (RAG2 − / − ) [23] and 129/S6 +/+ mice (H-2b) were the gifts of Dr K. Ikuta (Kyoto University, Japan). CB17-scid/scid (scid/ scid) mice (H-2d) were purchased from Clea Japan (Tokyo, Japan).

2.2. Establishment of B cell clones from me/me mice Bone marrow cells from 3-week old me/me homozygous mice were cultured on the PA6 stromal cell line [24,25] in RPMI-1640 (Gibco-BRL, Grand Island, NY) supplemented with 5% calf serum (CS) (Hyclone, Logan, UT), 5× 10 − 5 M 2-ME (Wako Pure Chemical Industries, Ltd., Osaka, Japan), 20 U/ml mouse recombinant IL-7 (rIL-7), 50 U/ml streptomycin, 50 mg/ml penicillin (Meiji Chem. Co. Ltd., Tokyo, Japan) and 10 mg/ml rat anti-mouse c-Fms antibody (AFS98) [20,21]. We refer to this as the ‘positive’ medium. The culture was supplemented with rIL-7 every 3 to 4 days and refed weekly by replacing 6 ml of fresh positive medium in a T-25 flask (Falcon c 3014; Becton Dickinson Labware, Lincoln Park, NJ). Every 2 months, hematopoietic cells were passaged onto freshly prepared PA6 under the same conditions. Six months after the initiation of culture, cells were harvested by pipetting, and isolated by limiting dilution to single cells per 200 ml of positive medium. Single cells were transferred into 96well plates previously seeded with PA6 stromal cells. Then, cloned cells were passaged into 24-well plates and then into T-25 flasks with PA6 stromal cells. All cultures were performed at 37°C with 5% CO2 in a humidified incubator.

2.3. Cultures Long term bone marrow cultures of B lineage cells from 129/S6 + /+ and 129/S6-RAG2 − / − mice were prepared according to the method described previously [26]. Briefly, 106 bone marrow cells were seeded onto PA6 stromal layers in positive medium without AFS98 in T-25 flasks and the medium was replaced twice a week. 70Z/3 pre-B cells and FDCP2 myeloid cells were maintained in RPMI-1640 containing 10% CS, 2-ME and antibiotics. In addition, mouse recombinant IL-3 (50 U/ml) was added to the FDCP2 cell cultures.

2.4. DNA isolation and analysis of Ig gene rearrangement Cellular DNA from cultured cells was isolated by standard techniques [27]. Genomic DNA was digested by EcoRI, separated by 0.9% agarose gel electrophoresis and blotted onto nitrocellulose membranes (Hybond-N + ; Amersham, Buckinghamshire, England) by alkaline transfer. The filters were baked at 80°C for 2 h and hybridized with radio-labeled probes overnight at 65°C. To observe Ig gene rearrangements, we used a 1.5-kb HindIII-EcoRI fragment containing a JH4 gene segment as a probe for detection of DH-JH rearrangement and a 0.4-kb PstI fragment of the Dsp2 gene and a 0.6-kb BamHI fragment of the DFL16.1 gene for VH-DJH rearrangement. These probes were the gifts of Dr S. Takeda (Kyoto University, Japan).

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2.5. Western blot analysis

3. Results

Cell lysates were separated by 7.5% SDS-PAGE and blotted onto nitrocellulose membranes (ECL Hybond: Amersham). After they were washed in PBS containing 0.05% Tween-20 for 30 min, the membranes were incubated with 0.5 mg/ml mouse monoclonal anti-SHP-1 antibody (Clone c52: Transduction Laboratories, Lexington, KY) for 1 h, and then with HRP-antimouse IgG antibody for 1 h. Enhanced chemiluminescents were detected by using the ECL western blotting analysis system (Amersham).

3.1. Establishment of B lineage cell clones from me/me bone marrow

2.6. Flow cytometry Cells were incubated on ice with heat-inactivated normal rabbit serum for 20 min, then stained with biotinylated antibodies against mouse B220 (6B2: [28]), c-Kit (ACK4: [29]), IL-7Ra (A7R: [30]), Mac-1 (M1/70: [31]), CD5 (53–7.3: Pharmingen), HSA (J11D: [32]), CD43 (S7: [33]), BP-1 (6C3: Pharmingen), CD25 (3C7: Pharmingen), CD38 (CS/2: [34]), CD44 (KM201: [35]), Thy1.2 (30-H12: Gibco/BRL), IL-5Ra (H-7: a gift from Drs Y. Kikuchi and K. Takatsu [36]), l5 (LM34: a gift from Dr H. Karasuyama), integrins a4 (P/S-2: [37]), a5 (HMKa5.1: [38]), b1 (KMI-6: [39]), H-2Kk (11-4.1: a gift from Dr S. Ono), and IgM (ICN Pharmaceuticals, Inc., Costa Mesa, CA). The stained cells were further incubated with fluorescein isothocyanate (FITC)-conjugated streptavidin (Beckton Dickinson Immunocytometry System, San Jose, CA) and analyzed on EPICS-XL (Coulter Electronics Inc., Haleah, FL) flow cytometer.

In the bone marrow cultures from me/me mice, macrophages quickly overgrew [18–20], and other hematopoietic lineage cells could not be formed on PA6 stromal cells in the culture mediums originally described by Dexter et al. [40] or by Whitlock and Witte [41] (Fig. 1A). To inhibit the overgrowth of macrophages specifically, we added an antagonistic antibody, AFS98, directed against c-Fms, the receptor for M-CSF, into the culture. Addition of 10 mg/ml of the antibody to the culture medium allowed bone marrow cells from me/me mice to grow efficiently and differentiate into B-lineage cells on PA6 stromal cell layers in the presence of 20 U/ml IL-7 (Fig. 1B). After continuing

2.7. Cell cycle analysis Cells were harvested, washed 3 times with PBS and inoculated into 6-well plates previously seeded with PA6 in the presence of 20 U/ml IL-7 and/or 10 mg/ml antagonistic anti-c-Kit antibody, ACK2 [29]. After 6 or 24 h of incubation, harvested cells were fixed with 70% ethanol at 4°C. Cells were then digested with 100 mg/ml ribonuclease A in 192 mM phosphate-citrate buffer at room temperature. The amount of nuclear DNA stained by propidium iodide was measured by flow cytometry and the proportion of cells in each cell cycle stage was determined.

2.8. Growth of B cell clones in 6i6o To remove stromal cells, cultured cells were passed through a Sephadex G-10 column (Pharmacia). Nonadherent cells (1× 107) were injected into CB17-scid/ scid mice by intravenous or intraperitoneal routes. After 14 days, the presence of donor cells in bone marrow, spleen and peritoneal cavity of recipient mice was determined by using antibody recognizing donor H-2 haplotype.

Fig. 1. Suppression of macrophage overgrowth and induction of B lineage cells by addition of anti-c-Fms antibody in me/me bone marrow cultures. (A) Bone marrow cells (1 ×106) were cultured on PA6 for 7 days. In both cultures for myeloid cells (10% CS in a-MEM medium) and for B lymphocytes (5% CS, 2-ME and IL-7 in RPMI-1640 medium), only macrophages grew efficiently and no other lineage cells were observed. Scale bar indicates 100 mm. (B) me/me bone marrow cells were cultured on PA6 in RPMI-1640 medium supplemented with 5% CS, 2ME, IL-7 and 10 mg/ml antimouse c-Fms antibody (AFS98) for 7 days. B lineage cells grew efficiently in the culture. The photograph is shown at the same magnification as A.

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Fig. 2. Cell surface phenotypes of the MEB2 cell line. The MEB2 cells were analyzed for their cell surface antigens by flow cytometry. Cells were stained with various antibodies for cell surface antigens shown under each frame (thick line). Cells incubated with FITC-streptavidin only were included as controls (thin line).

this culture for 6 months, we cloned me/me B-lineage cells by limiting dilution on PA6 stromal cells plus IL-7 in the presence of anti-c-Fms antibody in 96-well plates. Four clones were obtained and maintained on PA6 in the presence of IL-7. We named to the established clones MEB1, MEB2, MEB3 and MEB4.

3.2. Characterization of surface molecules on MEB cells We assessed the expression of cell surface molecules of the four MEB clones using antibodies known to recognize B lineage cells. As shown in Fig. 2, these clones expressed cell surface B220, IL-7Ra, c-Kit, CD43, CD25, and HSA, while they were negative for BP-1, IL-5Ra, CD38, and IgM. As all MEB cells showed identical staining patterns that persisted for over one year, only the results of the MEB2 clone are presented. The staining pattern of MEB2 by the markers described above is consistent with that of pro-B cells, classified as fraction B according to Hardy’s criteria [42]. In addition, MEB cells were CD5-, Thy1.2-, integrin a4- and H-2Kk-positive. In contrast, the cells were negative for Mac1, CD34, CD44, integrin a5, integrin b1 and l5 (Fig. 2).

3.3. Analysis of Ig gene rearrangement of MEB clones As demonstrated above, the cell surface phenotypes of MEBs were comparable to those of conventional pro-B cells. A more direct assessment of the differentiation stage was carried out by Southern blot analysis for DH-JH and VH-DJH rearrangement. DH-JH gene rearrangement was observed in all four MEB cell lines using the 1.5 kb-HindIII-EcoRI fragment of plasmid

pJH as a probe (Fig. 3, left panel; multiple bands indicating gene rearrangement were detected except in RAG2 − / − B lineage cells and PA6 stromal cells). On the other hand, DFL16.1 and DSP2 probes did not detect any VH-DJH rearrangements in MEB cell clones (Fig. 3 middle and right panels, in which 70Z/3 pre-B cell DNA shows the VH-DJH rearrangement). As three (MEB1, MEB3 and MEB4) out of 4 clones revealed identical DH-JH fragments, they might have been derived from a single progenitor. MEB2 cells might have a different origin because they showed a different hybridization pattern. As a result, we have established two independent clones at similar differentiation stages. The pattern of Ig gene rearrangement of the MEBs confirmed that they are in an early pro-B cell stage.

3.4. IL-7 and c-Kit ligand regulate the cell cycle of MEB cells MEBs were maintained on PA6 stromal cells in the presence of exogenous recombinant IL-7. To assess the growth requirements of MEBs, we performed cell cycle analysis in the presence or absence of these growth factors. Deletion of IL-7 from the MEB2 cell culture on PA6 stromal cells caused cell cycle arrest at the G1 phase (60.5% G1 cells in Fig. 4A increased to 71.5% in Fig. 4C). When antagonistic anti-c-Kit antibody, ACK2 was added to the culture, accumulation at the G2/M phase was observed (13.1% G2-M cells in Fig. 4A increased to 17.2% in Fig. 4B), resulting from blocking the signal from the c-Kit ligand. Moreover, addition of anti-c-Kit antibody under IL-7 starvation induced MEB2 cells to undergo apoptosis within 24 h (15.3% apoptotic cells in Fig. 4C increased to 43.9% in Fig. 4D). The growth requirements of MEB2 cells

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Fig. 3. Detection of Ig rearrangement in MEB cells. Cellular DNA of MEBs, RAG2 − / − pro-B, 70Z/3, PA6, and 129/S6 B lineage cells was prepared as described in Section 2. Ig gene rearrangement for DH-JH was detected with pJH probes, and for VH-DJH was detected with DFL16.1 and DSP2 probes.

shown here are comparable to those of normal pro-B cells that express IL-7R and c-Kit [43]. In the presence of IL-7 and 1 mg/ml of recombinant mouse c-Kit ligand but not PA6 stromal cells, MEB2 cells survived for only 2 days (data not shown). These results indicated that both IL-7 and c-Kit ligand are required to support MEB cell proliferation and other unknown molecule(s) from stromal cells are also likely to be essential. SHP-1 is known to be closely associated with the signaling pathway of c-Kit [7], and is an important regulator of apoptosis [13–16]. However, MEB cells underwent apoptosis in the absence of IL-7 and c-Kit ligand.

3.5. Presence of SHP-1 in B progenitor cells In the previous section, we could not detect any effect of SHP-1 deficiency upon growth factor requirements for cell cycle progression and apoptosis of me/me pro-B cells. It might be possible that B precursors corresponding to the stage of MEB cells do not express SHP-1, and B lymphopoiesis in me/me mice is normal in the pro-B stage. None of the MEB cells expressed the SHP-1 68kD protein, whereas pro-B cells isolated from RAG2 − / − mice that had not rearranged the DH-JH genes produced high levels of SHP-1. SHP-1 was also present in 70Z/3 pre-B cells and FDCP2 myeloid cells (Fig. 5). As we confirmed that SHP-1, is expressed from pro-B to pre-B stages, any defects in MEB cells might be caused by the lack of SHP-1 which is expressed in normal B-lineage cells.

3.6. MEB cells are able to grow following engraftment in scid/scid mice Next, growth and differentiation of MEB cells was

tested in vivo. MEB2 cells (1× 107) were injected intravenously into scid/scid mice. After 2 weeks, the presence of donor MEB2 cells in bone marrow, spleen and peritoneal cavity was determined by donor-specific H2Kk expression. About 8.9% of bone marrow cells (Fig. 6A, left histogram) and 0.8% of spleen cells (Fig. 6B, left histogram) were derived from MEB2 cells. Detectable levels of H-2Kk expressing cells were not recovered from the peritoneal cavity (data not shown). Initial surface phenotype of MEB2 cells (B220 + IgM-) showed no changes in scid/scid hosts either in bone marrow (Fig. 6A, middle and right histograms) or in spleen (Fig. 6B, middle and right histograms). When MEB2 cells were injected intraperitoneally, we observed similar results to those shown above with intravenous injection (data not shown). In conclusion, MEB2 cells can proliferate without apparent differentiation into more mature B cells in vivo. They preferentially reside in bone marrow irrespective of the site of injection.

4. Discussion In this report, we have devised a culture system that supports the generation of hematopoietic cells from me/me bone marrow progenitors. Addition of anti-cFms antagonistic antibody into the culture of bone marrow cells effectively suppressed the overgrowth of macrophages. Using this culture system, pro-B cell clones were established from me/me bone marrow. It is well documented that generation of macrophages depends on granulocyte-macrophage colony stimulating factor as well as M-CSF. In the me/me bone marrow culture, blockage of M-CSF/c-Fms signaling did not

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Fig. 4. Cell cycle regulation by IL-7R and c-Kit signaling in the MEB cells. MEB2 cells (0.5× 106 /ml) were cultured in the presence (+ ) or absence ( −) of IL-7 and/or anti-c-Kit antagonistic antibody (ACK2). After 24 h, cells were harvested and lysed, and then DNA was stained with propidium iodide. DNA content corresponding to apoptotic(a), G1(b), S(c), and G2/M(d) nuclei was quantified and the proportion of gated cells was represented.

impede generation of other hemopoietic cell lineages. This is consistent with our previous observation in vivo [44,45]. Moreover, in vitro, the number of macrophages generated on the fibroblasts of M-CSF-deficient op/op mice was less than 5% of the number generated on normal fibroblasts [46]. Predominance of macrophages in me/me mice could be explained by prolongation and/or extension of M-CSF/c-Fms signaling due to the lack of SHP-1 activity [3,4]. Using mature peripheral me/me B, T, and NK cells, SHP-1 has been shown to be not only a functional molecule in the antigen receptor complex but an important regulator for delivering apoptosis signals in lymphoid cells [13–16]. The requirement of MEB cells for IL-7 and c-Kit ligand was comparable to that of conventional pro-B cells described by Yasunaga et al. [43]. MEB cells were sensitive to starvation of either of these, which resulted in induction of apoptosis. We showed that SHP-1 is expressed in B cells from the pro-B to pre-B stages. Thus, it is possible that only restricted stages of B cell differentiation require SHP-1 activity to induce apoptotic death. This suggests the possibility that SHP-1 may have other unknown functions in B cell progenitors. As shown Fig. 1, signalling via c-Fms is essential for overgrowth of macrophages

Fig. 5. Absence of SHP-1 protein in MEB cells. Cell lysates (1 ×106 cells) of MEB (1 – 4), RAG2 − / − pro-B, 70Z/3, and FDCP2 cells were electrophoresed on acrylamide gels and blotted onto nitrocellulose membranes. Anti-SHP-1 antibody was used to detect the 68 kD SHP-1 protein using the ECL system.

even in the SHP-1-deficient culture. Paulson et al. [16] have reported that W v/W v mice which are defective c-Kit and exhibit a mast cell deficiency, crossed by me/me (W 6/W 6, me/me double mutants) were observed mast cell growth. W 6/W 6 mutants do not absolutely loose c-Kit activity, but reduce it. So, when MEB cells are comlpetely blocked a signal to c-Kit by an antagonistic antibody, c-Kit-starved apoptosis may be not

Fig. 6. Recovery of MEB cells in vivo. MEB2 cells (1 × 107) were injected into scid/scid mice intravenously. After 14 days, donor cells in recipient bone marrow (A) and spleen (B) were detected by H-2KK expression. Anti-B220 and anti-IgM antibodies were used for the detection of B cell lineages. Injection with or without MEB2 cells is indicated as (MEB2) or ( − ), respectively.

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significantly affected by loss of SHP-1. The roles of SHP-1 in early B cell progenitors may be revealed by using MEB cells. Nearly all B cells in homozygous me/me mice have been shown to be CD5 + cells (B-1a cells in Herzenberg’s classification) [47] which are derived from fetal liver but not from adult bone marrow in normal mice [48]. The relationship between SHP-1 activity and CD5 expression in the B cell lineage is not clear. A mouse deficient for CD22, which recruits SHP-1 to the antigen receptor complex and regulates the antigen receptor signaling negatively [49,50], was reported to harbor hyperresponsive B cells and expanded numbers of peritoneal CD5 + B cells [51]. Additionally, mRNA expression of CD5 is induced by anti-Ig crosslinking [52]. It seems likely that CD5 expression is correlated with CD22 and SHP-1 as a negative regulator of antigen receptor signaling. MEB clones are derived from me/me bone marrow, and express CD5 but not antigen receptors. This means that the expression of CD5 on B cell lineages occurs prior to that of antigen receptors. CD5 may have a role distinct from signaling via the antigen receptor complex. Further analysis of MEB cells transfected with SHP-1 or Ig genes will be needed. In this study, we established B progenitor clones from SHP-1 deficient me/me bone marrow. These clones were shown to be useful for the analysis of roles of the SHP-1 signaling pathway. We are willing to distribute these clones widely for studies to clarify mechanisms of B lymphopoiesis. Currently, we are planning experiments on in vivo treatments to rescue me/me mice. It is possible that anti-c-Fms antibody administration may cure the severe inflammatory disease caused by macrophages, because a humoral autoimmune response is not directly responsible for morbidity and mortality of the me/me mice [53].

Acknowledgements We are grateful to Dr E. Hashimoto (Tottori University), Drs K. Ikuta and S. Takeda (Kyoto University) for technical supervision, mice and probes, respectively. We thank Drs M. Ogawa (Basel Institute for Immunology), H. Karasuyama (Tokyo Metropolitan Institute of Medical Science), Y. Kikuchi and K. Takatsu (Tokyo University) and S. Ono (Osaka University) for antibodies. We also thank Dr E. Nanba (Gene Research Center, Tottori University) and Dr T. Shibahara (Laboratory Animal Research Center, Faculty of Medicine, Tottori University) for their help and Ms. T. Shinohara for her secretarial assistance. This work was supported by grants from the Ministry of Education, Science, and Culture in Japan, from the Ministry of Science and Technology of Japan, from National Institutes of Health (CA20408) and from the Cellular Tech-

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