Inducible differentiation and apoptosis of the pre-B cell receptor-positive pre-B cell line
Ibuki Kato,
Takahiro Miyazaki,
Tetsuya Nakamura1 and
Akira Kudo
Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
1 Department of Infectious Diseases and Applied Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
Correspondence to:
A. Kudo
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Abstract
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The function of the pre-B cell receptor (pre-BCR) during B cell differentiation is not precisely defined. To investigate the pre-BCR receptor activity, we have established pre-BCR-positive pre-B cell lines that are able to differentiate into immature B cells in vitro. Antibody cross-linking of the pre-BCR induced apoptosis and differentiation accompanied with tyrosine phosphorylation. A specific tyrosine-phosphorylated 43 kDa protein (p43) was found down-stream of the pre-BCR. The results demonstrated the receptor function of pre-BCR, which indicates that a ligand-like molecule or a cross-linking structure on the cell surface is possibly present.
Keywords: apoptosis, gene rearrangement, pre-B cell receptor, surrogate light chain, tyrosine phosphorylation
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Introduction
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B cell differentiation from pluripotent stem cells to immature B cells in bone marrow is characterized by successive rearrangement of the gene segments of the Ig heavy and light chain gene loci (1). The Ig heavy chain locus is usually rearranged before the Ig light chain locus. When a functional VHDHJH rearrangement occurs in a pre-B cell, this cell will express the pre-B cell receptor (pre-BCR) formed by the membrane-bound µH chain in complex with the SL chain (2,3). In both mouse and human, the SL chain is composed of two proteins encoded by the pre-B cell specific genes, Vpre-B and
5/14.1 (47). The analyses of bone marrow cells from
5 gene targeted mice revealed that the number of CD43 small pre-B cells and of sIgM+ immature and mature B cells was drastically reduced, whereas that of CD43+ early precursor B cells was normal (8). Another analysis using c-kit, CD25 and the surrogate light chain (SL) as markers showed that c-kit+CD25SL+ pro-B/pre-B I cells were produced in normal numbers, whereas c-kitCD25+SL+ large pre-B-II cells and c-kit CD25+ SL large and small pre-B-II cells, as well as immature B cells, were at least 40-fold reduced. Mutation in the human
5 gene markedly reduced the number of CD19+ B cells in the peripheral blood and there were almost no mature B cells in bone marrow, indicating that a more severe B cell deficiency is caused by loss of
5 expression in humans than in mice (9). These results indicate that in mice and humans with
5 mutations, B cell differentiation is impaired at the transition from the pro-B/pre-B-I to the pre-B-II cell stage, during which Ig light chain gene rearrangement takes place.
In human, ~5% of bone marrow cells expressed the pre-BCR on the cell surface (10); however, in the mouse, the expression of the pre-BCR is barely detectable on normal bone marrow cells (11). Salamero et al. demonstrated that because ~2% of newly synthesized pre-BCR reached the cell surface in the human pre-B cell line, Nalm-6, the majority of pre-BCR remained in the cytoplasm (12). All published results indicate less pre-BCR expression on the surface of the cell line and normal cells.
Several hypotheses have been proposed regarding functions for the pre-BCR involving allelic exclusion, proliferation and differentiation, and induction of
chain gene rearrangement. Recently, another interesting function of the pre-BCR was also proposed, i.e. that SL chain serves as a folding template that tests the ability of µ chain to pair with conventional light chains (13).
It has been suggested that the pre-BCR functions in controlling rearrangement of heavy and light chain genes, possibly by affecting expression of Rag genes (14). Allelic exclusion at the IgH locus requires the expression of the pre-BCR (15) and the light chain locus is efficiently rearranged following appropriate signaling through the pre-BCR (16). Although in
5 knock-out mice, a low frequency of
chain rearrangement occurs in leaky B cells, the question remains whether pre-BCR actively up-regulates
chain rearrangement, so that when the pre-BCR appears during B cell differentiation, one of the first signals stimulates
chain rearrangement. Several reports showing coincident effects of heavy chain expression on germline transcription of the Ig
locus and its rearrangement support this possibility (1721). Recently, we also observed that the SL chain activated the
chain rearrangement by restoration of
5 into
5-deficient pro-B cell lines (22).
Because of the heterogeneity of bone marrow cells, a practical way to investigate the function of the pre-BCR is to establish a pre-B cell line expressing the pre-BCR on the surface. An in vitro B cell differentiation system has been established (23), in which pro-B cell lines were cloned from bone marrow cells, and cultured in the presence of a stromal cell line and IL-7. Removal of IL-7 or the stromal cells induces differentiation from pro-B to immature B cells. We have established pre-BCR+ pre-B cell lines (PreBR) by continuing the cultures in the presence of IL-7 after removal of the stromal cell line to induce the differentiation.
Removal of IL-7 from the PreBR cell cultures induces their differentiation into immature B cells and apoptosis. To examine the function of the pre-BCR, PreBR cells were treated with specific antibodies for cross-linking in the presence of IL-7. The results of the antibody treatment demonstrate that the pre-BCR delivers two signals that induce apoptosis and differentiation.
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Methods
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Animals and cell lines
Pro-B cells derived from bone marrow cells of BALB/c mice were cultured with ST2 (a gift of Dr Nishikawa, Kyoto University) in the presence of IL-7 as described previously (22). PreBR cell lines were established from pro-B cells in the presence of IL-7 after removal of ST2. PreBR cells were cultured in SF-O3 medium (Sanko Jyunyaku, Tokyo, Japan) containing 5x105 M 2-mercaptoethanol, 1xnon-essential amino acids (Gibco/BRL, Gaithersburg, MD), 0.03% primatone (Quest International, Naarden, Netherlands), 2% FCS and 100 U/ml recombinant IL-7 (24) (a gift from Dr Sudoh, Toray, Kamakura, Japan). For in vitro differentiation, cells were washed to remove IL-7 and then cultured for 45 days at 5x105 to 1x106 cells/ml. The immature B cell line WEHI231 was cultured in RPMI 1640 containing 10% FCS. The VHDHJH structure of µH chain in PreBR1 and 2 cell lines was determined by PCR cloning and cDNA cloning. The common PCR primers for detection of VHJ558, VH7183 and VHQ52 families were prepared as described previously (25), and PCR was performed. The specific bands of VHJ558 for PreBR1 and VH7183 for PreBR2 respectively were found, and further DNA sequencing analyses of the recloning bands revealed that the productively rearranged VHDHJH structure of µ chain in the cell line is VHJ558DSP2JH2 for PreBR1 or VH7183DSP2JH3 for PreBR2. Further cDNA cloning of a µ chain in PreBR1 by using 5' RACE (Marathon cDNA amplification kit; Clontech, Palo Alto, CA) with the specific 3' primer located at the 5' side of Cµ sequences (26) was performed because this cell line was used for all experiments, which revealed the same VHJ558DSP2JH2 sequence. The results showed that PreBR1 and 2 are independent cell lines using different µ chains.
Antibodies and flow cytometric analyses
FITC-conjugated mAb 1D3 (anti-mouse CD19), biotin-conjugated mAb 7D4 (anti-mouse CD25) and hamster IgG were purchased from PharMingen (San Diego, CA). FITC-conjugated goat anti-mouse IgM (µ chain specific), FITC-conjugated goat anti-mouse
chain, unconjugated goat anti-mouse IgM (µ chain specific) antibody and F(ab')2 goat anti-mouse µ antibodies were purchased from Southern Biotechnology Associates (Birmingham, AL). Rat biotin-conjugated mAb, VP245 (anti-mouse Vpre-B) and LM34 (anti-mouse
5) (27) were gifted from Dr Karasuyama (Tokyo Metropolitan Institute of Medical Science, Tokyo). Rat mAb, A7R34 [anti-mouse IL-7 receptor (IL-7R)], was a gift from Dr Nishikawa (Kyoto University). HM-79-12 (hamster anti-mouse Igß) (28) was purified by Protein GSepharose (Pharmacia, Uppsala, Sweden) and biotin was conjugated by the company protocol (Pierce, Rockford, IL). FITC-conjugated streptavidin was purchased from Cosmo Bio (Tokyo, Japan) and FITC-conjugated goat anti-rat IgG was purchased from Cappel (Organon Technika, Durham, NC). Flow cytometric analyses using the FACSCalibur (Becton Dickinson, Mountain View, CA) were performed as described (22).
Cell surface labeling and immunoprecipitation
For surface labeling, cells were washed twice with biotinylation buffer (50 mM NaCl, 0.1 M HEPES, pH 8.0, 1 mM PMSF and 2 µg/ml leupeptin) and were incubated with 1.0 mg/ml of sulfo-NSH-biotin (Pierce) for 20 min at 4°C. After washing with cold PBS, aliquots of 107 cells were lysed in 300 µl of NP-40 or digitonin lysis buffer (1% NP-40 or 1% digitonin, 150 mM NaCl, 50 mM TrisHCl, pH 8.0, 50 mM iodoacetamide, 0.02% NaN3, 1 mM PMSF, 2 µg/ml aprotinin and 1 µg/ml pepstatin) for 30 min on ice. Precleared lysate was incubated with goat anti-mouse µ antibody or anti-Igß antibody at 4°C for 1 h and then with Protein GSepharose beads (Amersham Pharmacia, Little Chalfont, UK). Cell lysates were subjected to 13% SDSPAGE. Western blot analyses were performed by a standard protocol, and then surface biotinylated proteins were reacted with streptavidinhorseradish peroxidase (HRP) and detected by using the chemiluminescence ECL kit (Amersham Pharmacia). For the detection of Ig
, rabbit anti-mouse Ig
serum (29) was a kindly gift of Dr Jongstra (Toronto University). HRP-conjugated anti-rabbit IgG was purchased from Southern Biotechnology Associates.
RT-PCR analyses of Rag1 and 2 expression
Total RNA was extracted from cultured cells treated with mAb HM79-12 (anti-Igß) or hamster IgG and from cultured cells with IL-7 or without IL-7 by using the Glass MAX RNA Isolation Spin Cartridge System (Gibco/BRL) according to the recommended protocol of the manufacture. Then 1 µg of RNA was reverse transcribed in the supplied buffer using AMV reverse transcriptase (TaKaRa, Shiga, Japan) and 0.02 µg oligo(dT) primer. PCR was performed as described (30). Briefly, reaction mixtures for PCR amplification consisted of 1 µl from 50 µl cDNA mixtures, 200 nM each dNTPs, 500 nM each oligonucleotide, 10 mM TrisHCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 and 2.5 U of Taq polymerase (TaKaRa) in a volume of 50 µl. Reactions were cycled as follows: 30 cycles at 94°C for 1 min, at 60°C for 2 min and at 72°C for 1 min.
PCR primers were used for: RAG1, 5'-TGCAGACATTCTAGCACTCTGG-3', 5'-ACATCTGCCTTCACGTCGAT-3'; RAG2, 5'-CTTCTCTAGAGATTCCTGCTACCTCCCACC-3', 5'-TGTGGAATTCACTGCTGGGGTACCCAGGGG-3'; porphobilinogen deaminase (PBGD), 5'-TGTCCCGGTAACGGCGGCGCGGCCACAAC-3', 5'-GCCACCACAGTCTCGGTCTGTATGCGAGC-3'.
PCR analyses of Ig
gene rearrangement
Genomic DNA was extracted from cultured cells and PCR was performed as described (31). Briefly, reaction mixtures for PCR amplification consisted of 100200 ng of genomic DNAs, 200 nM each dNTPs, 500 nM each oligonucleotide, 10 mM TrisHCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 and 2.5 U of Taq polymerase (TaKaRa) in a volume of 50 µl. Reactions were cycled as follows: 28 cycles of 94°C for 30 s, 60°C for 1 min 30 s and 72°C for 1 min, increase in extention at 5 s/cycle.
PCR primers were used for: Vkcom, 5'-GGCTGCAGSTTCAGTGGCAGTGGRTCWGGRAC-3'; Jk5, 5'-TGCCACGTCAACTGATAATGAGCCCTCTC-3'; Ck, 5'-CCAAGGACGAGTATGAACGACATAACAGCTATAC-3', 5'-GTGTAATCTCACGGTATAGAGGTCCTTGAAG-3'.
Western blot analyses of poly(ADP-ribose)polymerase (PARP) cleavage
After treatment with antibodies, 4x106 cells were dissolved in SDSPAGE sample buffer (62.5 mM TrisHCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 6 M urea and 0.00125% bromphenol blue) and then sonicated for 15 min. Cell lysates were subjected to 7.5% SDSPAGE and transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany).The filters were blocked with 5% non-fat dry milk in TTBS (10 mM TrisHCl, pH 7.5, 150 mM NaCl and 0.05% Tween-20) for 1 h and incubated with polyclonal rabbit anti-PARP antibodies (BIOMOL, Plymouth Meeting, PA) at a dilution of 1:500 in 5% non-fat dry milk TTBS for 2.5 h at room temperature. After three washes with TTBS, filters were incubated with HRP-conjugated goat anti-rabbit IgG (Organon Teknika, Durham, NC) for 1 h and then detected by using the chemiluminescence ECL kit (Amersham Pharmacia).
Detection of nuclear change (DNA fragmentation assay)
Cells were washed with PBS and resuspended in 400 µl of a hypotonic buffer (0.15% Triton X-100 and 20 µg/ml RNase A) containing 50 µg/ml propidium iodide (PI) and analyzed by flow cytometry (FACSCalibur)
Plasma membrane phosphatidyl serine (PS) transition (Annexin V binding)
After antibody treatment of cultured cells, 200 µl of binding buffer (TaKaRa) was added to the cell suspension. Cells were incubated with FITC-conjugated Annexin V (TaKaRa) for 10 min at room temperature in the dark. After washing, cells were resuspended in binding buffer containing 1 µg/ml PI and analyzed by flow cytometry.
Analysis of tyrosine-phosphorylated proteins
PreBR and WEHI231 cells were incubated in both FCS and IL-7-free RPMI 1640 for 30 min. Cells were treated with 20 µg/ml of antibody, hamster IgG, anti-Igß mAb (HM79-12), goat IgG and F(ab')2 goat anti-mouse µ for 60 s at 37°C. For biotin-conjugated anti-Igß mAb and biotin-conjugated F(ab')2 goat anti-mouse µ antibodies (Southern Biotechnology Associates), cells were pretreated with 20 µg/ml of each antibody for 5 min on ice. After addition of streptavidin (0.5 mg/ml), cells were incubated at 37°C for 60 s. Afterwards, the cold inhibition buffer (10 mM TrisHCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl2, 2 mM Na3VO4, 1 µg/ml aprotinin and 1 µg/ml leupeptin) was added to all samples. To test for IL-7R signaling; PreBR cells were cultured in only IL-7-free SF-03 medium for 0, 0.5, 2 and 6 h respectively. In all samples, cells were collected by centrifugation at 5000 r.p.m. for 5 s and then lysed with the inhibition buffer containing 1% NP-40. The lysates were subjected to 7.5% SDSPAGE and transferred to nitrocellulose membranes. The filters were then blocked with 5% non-fat dry milk in TTBS for 1 h and incubated with HRP-conjugated anti-phosphorylated tyrosine antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or HRP-conjugated goat anti-mouse IgM (µ chain specific) (Southern Biotechnology Assocaites), and then detected by using the chemiluminescence ECL kit (Amersham Pharmacia).
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Results
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Establishment of pre-BCR+ pre-B cell lines (PreBR)
Pro-B cells derived from bone marrow cells were cultured in the presence of the stromal cell line, ST2, and IL-7. The pro-B cells did not express the pre-BCR consisting of µH chain and the SL chain. To obtain pre-BCR+ pre-B cell lines, we continued the culture in the presence of IL-7 after removal of ST2 to induce differentiation and then the cells were cloned by limiting dilution. Two independent cell lines, PreBR1 and 2, were established, in which VHDHJH sequences were analyzed as described in Methods, resulting in that they used different VHDHJH sequences which are productively rearranged. The phenotypes of the pre-BCR+ cell lines, PreBR1 and 2, were examined by flow cytometry (Fig. 1
). Both PreBR1 and 2 had the large pre-B-II phenotype, being positive for CD19, µlow, Igß, Vpre-B,
5, IL-7R and CD25, but negative for CD23, c-kit and CD40 (data not shown). The surface expression of the pre-BCR, consisting of the SL chain (Vpre-B and
5) and µH chain, was examined on PreBR cell lines at the protein level by using surface biotinylation and immunoprecipitation with goat anti-µ antibody (Fig. 2A
). The mouse immature B cell line, WEHI231, was used as a control. The result confirmed the presence of the pre-BCR complex on the surface of PreBR cells, while the BCR complex (µ and
chains) was found on WEHI231 cells. Furthermore, cell lysates were extracted from PreBR1 and WEHI231 cells by using two different detergents, digitonin and NP-40, and they were immunoprecipitated with goat anti-µ antibody or hamster anti-Igß antibody, then the filter was immunoblotted by rabbit anti-Ig
antibody, because BCR on B cells is known to be associated with Igß, which is found in digitonin treatment, and they dissociate in NP-40. The result showed that an evident signal of the 34 kDa of Ig
(shown by the arrow in Fig. 2B
) was present in the experiment in which anti-µ antibody immunoprecipitation of the PreBR1 cell lysate was performed in digitonin buffer but not in NP-40, while the same result was observed in WEHI231. A lower band showed a non-specific signal. The result demonstrated that the pre-BCR on PreBR cells was associated with the Ig
/Igß heterodimer in the similar manner to the BCR complex (Fig. 2B
).
In vitro differentiation of PreBR cell lines
Differentiation of PreBR1 and 2 was induced in vitro by removal of IL-7 from the culture (PreBR1 IL7 and PreBR2 IL7 in Fig. 1
); 8.0% of PreBR1 and 30.6% of PreBR2 became IgM+ (µhigh and
) immature B cells cultured for 5 days after removal of IL-7, the expression of Vpre-B and
5 decreased and that of CD25 increased. The low expression of Igß associated with the pre-BCR was down-regulated, while the high expression of it associated with the IgM receptor appeared after differentiation.
All cells were dead 6 days after removal of IL-7, because the growth of PreBR cells is completely dependent on IL-7 and the differentiated IgM+ cells from PreBR cells cannot survive under these culture conditions.
Induction of apoptosis by anti-µ but not anti-Igß antibody treatment.
To examine the function of the pre-BCR, PreBR cells were treated with anti-Igß antibody, F(ab')2 anti-µ antibody and control antibodies (hamster IgG and goat IgG) at 5 µg/ml. The immature B cell line, WEHI231, was used as a positive control because it undergoes apoptosis upon BCR cross-linking (32). After 2 days culture with antibodies, apoptosis was analyzed by DNA fragmentation (Fig. 3
) and Annexin V binding assays (Fig. 4
) respectively. Apoptosis of WEHI231 cells was induced by cross-linking with either anti-µ or anti-Igß antibody. An increase of subdiploid cells in PreBR cells (4050% in Fig. 3
) was found by anti-µ antibody cross-linking, which at a level comparable to that of WEHI231 cells (40%); however, it was not detected by anti-Igß cross-linking. In the Annexin V binding assays, anti-Igß cross-linking on PreBR cells also did not induce it; however, cross-linking by anti-µ antibody on PreBR cells induced more necrotic cell death, which is double positive of Annexin V and PI (41.36 and 32.52% for PreBR1 and 2 respectively in Fig. 4
), but did not significantly induce apoptotic cell death of Annexin V single-positive cells (2.16 and 3.30% for PreBR1 and 2 respectively). Cross-linking by anti-µ or anti-Igß antibody on WEHI231 cells induced both Annexin V single-positive cells (23.70 or 37.96%), and Annexin V and PI double-positive cells (54.36 or 35.88%), indicating standard apoptotic cell death. In the short time course experiments of anti-µ cross-linking for PreBR cells, i.e. 12, 18, 24, 30, 36 and 42 h, the majority of Annexin V-positive cells was Annexin V and PI double-positive cells. No significant population of Annexin V single-positive cells was observed (data not shown). From the result demonstrated by Annexin V staining it is not clear whether the cell death of PreBR cells by anti-µ cross-linking is apoptotic. However, the caspase inhibitor, zVAD-fmk, blocked the cell death of PreBR cells which was induced by anti-µ treatment (data not shown). Cleavage of PARP, an enzyme involved in DNA repair and gene maintenance, is thought to be a critical event that triggers nuclear DNA fragmentation by caspase, thus the presence of PARP cleavage activity shows direct evidence of apoptosis. Although both anti-µ and anti-Igß antibodies induced the 85 kDa PARP cleavage production in WEHI231 cells, only anti-µ antibody-treated PreBR cells showed characteristic apoptosis-related 85 kDa fragments (Fig. 5
). The results being consistent with data shown in Figs 3 and 4
, demonstrated that apoptosis of PreBR cells could be induced by anti-µ antibody but not anti-Igß antibody.

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Fig. 3. Induction of apoptosis by antibody cross-linking on PreBR cell line. PreBR1 and 2 cell lines and WEHI231 cells were used at 3x105 cells/ml. Control antibodies (hamster IgG and goat IgG), anti-Igß antibody and F(ab')2 anti-µ antibody were added at 5 µg/ml. After 2 days culture (in the presence of IL-7 for PreBR1 and 2), cells were washed in PBS, and resuspended in hypotonic buffer containing PI and 20 µg/ml of RNase A. DNA fragmentation was assayed by flow cytometry and the percentage indicates appearance of the cells with decreased DNA content in a subdiploid area. These histograms are representatives of four independent experiments.
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Fig. 4. Induction of apoptotic PS translocation by antibody cross-linking. PreBR1 and 2 cell lines and WEHI231 cells were used at 3x105 cells/ml. Control antibodies (hamster IgG and goat IgG), anti-Igß antibody and F(ab')2 anti-µ antibody were added at 5 µg/ml. After 2 days culture (in the presence of IL-7 for PreBR1 and 2), cells were washed with Annexin V binding buffer, stained with FITC-conjugated Annexin V and PI, and then analyzed by flow cytometry. These dot-plots are representatives of three independent experiments.
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Fig. 5. PARP cleavage in apoptosis-induced PreBR cell line. PreBR1 and WEHI231 cells were stimulated for 2 days with control antibodies (hamster IgG and goat IgG), anti-Igß antibody and F(ab')2 anti-µ antibody at 5 µg/ml (in the presence of IL-7 for PreBR1). After washing with PBS, cells were lysed and an equal amount of whole cell lysate was loaded onto each lane of a 7.5% SDSPAGE gel. After transfer to a nitrocellulose filter, the blots were incubated with polyclonal rabbit anti-PARP antibodies at a dilution of 1:500 in TTBS containing 5% non-fat dry milk for 2.5 h at room temperature. After three washes with TTBS, the filter was incubated with HRP-conjugated goat anti-rabbit IgG for 1 h and then detected by using the ECL chemiluminescence kit. The upper band corresponds to the 116 kDa whole PARP molecule, whereas the lower 85 kDa fragment is an apoptosis-specific degradation product.
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Induction of differentiation by antibody treatment
PreBR cells differentiated into IgM+ immature B cell 5 days after removal of IL-7 (Fig. 1
). Since this process requires light chain gene rearrangement, Rag 1 and Rag 2 gene expression was up-regulated as expected within 23 days after removal of IL-7 (Fig. 6
). A similar up-regulation of Rag1 and Rag2 gene expression was observed when PreBR cells were treated with anti-Igß antibody for 13 h, and successively cultured for 1 day after removal of antibody (Fig. 7
). To further investigate the effect of Igß cross-linking, the induced rearrangement of
chain (V
to J
1) in PreBR1 was examined by PCR analyses (Fig. 8
). Anti-Igß antibody treatment resulted in induction of
chain gene rearrangement. On the other hand, the detected signal of anti-µ antibody-induced
rearrangement was greatly reduced after long exposure because the majority of cells died by apoptosis (data not shown). This result demonstrates that signaling from the pre-BCR can induce
chain gene rearrangement. We failed to detect
chain on the cell surface as a form of BCR by flow cytometry (data not shown). This can be explained as follows: the induction level of
chain may be too low to be detected, and anti-Igß and anti-µ antibodies may react to the formed BCR and induce cell death by apoptosis.

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Fig. 6. RT-PCR analyses of Rag1 and 2 expression in differentiated PreBR cell line in vitro. Total RNA (1 µg) for cDNA synthesis was extracted from PreBR1 and 2 cell lines cultured with IL-7 (0) or without IL-7 (14 days), and PCR was performed to detect Rag1 and 2 gene expression. PBGD was used as a positive control.
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Fig. 7. Up-regulation of Rag1 and 2 gene expression by anti-Igß antibody stimulation. PreBR1 and 2 cell lines cultured in the presence of IL-7 (medium) were treated with control hamster IgG or anti-Igß antibody for 13 h, and successively cultured for 1 day after removal of antibody. Total RNA (1 µg) was used for cDNA synthesis, and PCR was performed to detect Rag 1 and 2 gene expression.
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We examined next the pre-BCR signals generated after cross-linking by analysis of tyrosine phosphorylation. The cross-linking of pre-BCR on PreBR cells by antibody treatment for 60 s induced a rapid and transient tyrosine phosphorylation of multiple proteins (Fig. 9
), and the signal decreased after 10 min (data not shown). The phosphorylated proteins in PreBR1 induced by anti-Igß are similar to those by anti-µ, and the three major bands detected (~110, 78 and 72 kDa) are also similar to the phosphorylated proteins in WEHI231 cells induced by cross-linking of both anti-Igß and anti-µ antibodies. Control hamster and goat IgG antibody treatment did not elicit such phosphorylation, indicating that the phosphorylation was specific to pre-BCR. Super-cross-linking using streptavidin enhanced the signals of phosphorylated proteins. In addition, culture for 0.5 or 2 h after removal of IL-7 also induced the signal of tyrosine phosphorylation (Fig. 9
) and it decreased after 6 h (data not shown). A 43 kDa band of particular interest (indicated by the arrow in Fig. 9
) was prominently phosphorylated in PreBR cells by antibody treatment as well as the removal of IL-7; however, no detectable signal was found in BCR cross-linked WEHI231 cells. To identify this 43 kDa tyrosine-phosphorylated protein in PreBR cells, immunoprecipitation with anti-Igß antibody or immunoblotting with antibody against the MAP kinase, p38 or ERK1 and 2 was performed. However, these proteins were not candidates for p43 (data not shown). These results demonstrate that pre-BCR on PreBR cells transmits a signal leading to protein phosphorylation.

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Fig. 9. Induction of tyrosine phosphorylation by antibody treatment of PreBR cell line. PreBR1 and WEHI231 cells were preincubated with both FCS and IL-7-free RPMI 1640 for 30 min (medium), followed by the addition of 20 µg/ml of antibody, hamster IgG, anti-Igß antibody, goat IgG or F(ab')2 anti-µ antibody for 60 s at 37°C. For biotin-conjugated anti-Igß (Igß SA) or biotin-conjugated F(ab')2 anti-µ (µ F(ab')2 SA), cells were preincubated on the ice for 5 min with each antibody, then they were treated with streptavidin for 60 s at 37°C. All antibody-treated cells were lysed with NP-40 lysis buffer. The PreBR1 cell line was cultured in the absence of IL-7 (-IL-7) for 0, 0.5 and 2 h respectively, and then lysed with NP-40 lysis buffer. All lysates were subjected to SDSPAGE to detect tyrosine-phosphorylated protein, followed by Western blot analysis with anti-phosphorylated tyrosine antibody. The 43 kDa band (p43) is indicated by the arrow. To check the loading amount of cell lysate from equal cell numbers, Western blot analysis was performed by using HRP-conjugated goat anti-µ specific antibodies (µ) after reprobing the filter. The right panel was exposed longer than the left to detect a signal for p43.
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Discussion
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For some time, it was not established whether the pre-BCR actually behaves as a receptor itself, because no ligands were found and also several reports suggested that a ligand was unnecessary for function. In a previous study, we demonstrated that the SL chain, probably in the form of the pre-BCR, was able to activate
chain rearrangement (22), which suggested that pre-BCR gives rise to the signal for differentiation or the direct induction of rearrangement. To further investigate the receptor function of the pre-BCR, we established pre-BCR+ pre-B cell lines that are able to differentiate into immature B cells in vitro. This differentiation in vitro traced differentiation in vivo since expression of the pre-BCR was down-regulated while expression of IgM-BCR and CD25 was up-regulated. The results demonstrated that both removal of IL-7 and cross-linking by receptor-specific antibodies induced differentiation.
To investigate whether the pre-BCR transmits signals into cells, we tried to identify a pre-B cell-specific tyrosine-phosphorylated protein in the PreBR cell line in comparison with that from the IgM+ immature B cell line, WEHI231 by using anti-Igß or anti-µ antibody cross-linking because the association with Ig
/Igß heterodimer was shown to be essential for pre-BCR signaling for the progression of pro-B cells to the small pre-B stage (33). The results showed the presence of a pre-B cell-specific tyrosine-phosphorylated protein, the 43 kDa (p43), after cross-linking using anti-Igß antibody, although no significant 43 kDa signal was observed in WEHI231 after the same cross-linking. Although anti-µ treatment of PreBR cells also induced the phosphorylation of p43, it activated not only differentiation (
chain rearrangement) but also apoptosis. Cell degradation by strong apoptosis may make it difficult to find the rearranged band shown in Fig. 8
. These results suggest that the phosphorylation of p43 is specifically related to the differentiation from pre-B to immature B cells. Up to now, no reports have described the p43-related protein in B-lineage cells. Very interestingly, p43 also appeared when IL-7 was removed from the culture of the PreBR cell line to induce differentiation. It is pointed out how the IL-7R gives rise to the signal by the disappearance of IL-7. Physiologically, several different types of stromal cells are thought to be involved in B cell differentiation in bone marrow (34). When pre-B cells move to the next stromal cell for differentiation into immature B cells, the supply of IL-7 is shut off, implying a new concept of receptor signaling following ligand withdrawal for investigating the mechanism of differentiation. Marshall et al. (35) reported another mechanism of shutting off IL-7; the pre-BCR-expressing pre-B cells undergo a transient modulation of the IL-7 doseresponse threshold, followed by a complete loss of IL-7 responsiveness during differentiation to immature B cells. This result implies that the signals from the pre-BCR produce the shutdown of the IL-7 responsiveness. Other induced phosphorylated proteins are probably assigned to PI-3 kinase (110 kDa) and Syk (72 kDa), which sustains the phenotypes of knockout mice of PI-3 kinase (36) and Syk (37,38) attributed to the speculation that both proteins were located down-stream of the pre-BCR.
On bone marrow precursor B cells prepared ex vivo on ice (11), the expression of the pre-BCR was low and almost undetectable; however, incubation of the precursor cells for 1 h at 37°C was found to up-regulate the surface expression of the pre-BCR (39). This observation demonstrated that the pre-BCR can be expressed on the surface of mouse pre-B-II cells, which implies the feasibility of our study. Functionally, we noted that only cross-linking by anti-µ antibody but not anti-Igß antibody resulted in the strong signal for apoptosis, even though Igß is an essential component of the pre-BCR complex, and initial phosphorylation signals by antibody cross-linking are the same between anti-µ and anti-Igß antibodies. This phenomenon is explained by the different effect of cross-linking because anti-µ antibody is polyclonal and anti-Igß is monoclonal. Since the expression level of pre-BCR on preBR cells is lower than that of BCR on WEHI231 cells, the cross-linking effect by anti-Igß is too low to induce apoptosis. Consequently, a monoclonal anti-µ antibody, M41(40), did not induce apoptosis by cross-linking (data not shown).
The result from Annexin V staining demonstrated that the cell death of PreBR cells induced by anti-µ antibodies appears as more necrotic than apoptotic. However, other results, i.e. an increase of subdiploid cells and cleavage of PARP, clearly indicate that apoptosis is induced. Two explanations for not detecting significant Annexin V single-positive cells are raised. Firstly, the rapid movement of cells from Annexin V single-positive cells to Annexin V and PI double-positive cells happens. Koopman et al. (41) observed less Annexin V single-positive cells; when Namalwa cells were cultured in 1% FCS, the Annexin V single-positive population disappeared at 78 h, and only Annexin V and PI double-positive cells existed although many living cells (~50%) remained. Takeda et al. (42) also reported the similar phenomenon of apoptosis of splenic T cells. In their results, no significant Annexin V single-positive cells existed, and the majority were Annexin V and PI double-positive cells after short-term culturing for 5 h. Secondly, the dual signaling of the Fas receptor, i.e. apoptotic and necrotic, was recently observed (43), indicating the presence of regulatory necrotic control. Therefore, we could speculate the possibility that anti-µ treatment probably induces both signals, apoptotic and necrotic, into one cell. However, the caspase inhibitor, zVAD-fmk, blocked the cell death of PreBR1 cells induced by anti-µ treatment (data not shown), strongly suggesting that the major cell death mechanism of anti-µ-treated PreBR cells is apoptosis. Thus, the second explanation might not be appropriate. To conclude, the different mechanisms of cell death between PreBR cells and WEHI 231 cells in the case of Annexin V experiments may reflect the different properties of the complex.
Recently, ten Boekel et al. (44) reported that the pre-BCR utilizes only a specific portion of the VH gene repertoire, which specificity relates to the affinity of association between µ chain and the SL chain. Kline et al. (13) enhanced this function; µ chain is tested at the pre-BCR stage for its ability to pair with conventional light chain. The pre-BCR on PreBR cells is stably expressed on the surface, reflecting this specific combination of µ chain with the SL chain, which demonstrates the receptor function of pre-BCR, indicating that a ligand-like molecule or a cross-linking structure on the cell surface is possibly present.
Further experiments are necessary to examine the structure of the pre-BCR and the down-stream from the pre-BCR signaling.
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Acknowledgments
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We would like to thank Drs Tetsuo Sudoh, Hajime Karasuyama, Shin-ichi Nishikawa and Jan Jongstra for providing IL-7 and antibodies. We also thank Drs Pete Burrows and Takeshi Watanabe for critical review of the manuscript and suggestions for the apoptosis experiment. This work was supported by grants from the Ministry of Education, Science and Culture of Japan.
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Abbreviations
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BCR B cell receptor |
HRP horseradish peroxidase |
IL-7R IL-7 receptor |
PARP poly(ADP-ribose) polymerase |
PBGD porphobilinogen deaminase |
PI propidium iodide |
PS phosphatidyl serine |
SL surrogate light |
 |
Notes
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Transmitting editor: T. Watanabe
Received 18 October 1999,
accepted 18 November 1999.
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References
|
---|
-
Tonegawa, S. 1983. Somatic generation of antibody diversity. Nature 302:575.[ISI][Medline]
-
Karasuyama, H., Kudo, A. and Melchers, F. 1990. The proteins encoded by the Vpre-B and
5 pre-B cell-specific genes can associate with each other and with µ heavy chain. J. Exp. Med. 172:969.[Abstract]
-
Melchers, F., Karasuyama, H., Haasner, D., Bauer, S., Kudo, A., Sakaguchi, N., Jameson, B. and Rolink, A. 1993. The surrogate light chain in B-cell development. Immunol. Today 14:60.[ISI][Medline]
-
Kudo, A. and Melchers, F. 1987. A second gene, Vpre-B in the
5 locus of the mouse, which appears to be selectively expressed in pre-B lymphocytes. EMBO J. 6:2267.[Abstract]
-
Sakaguchi, N. and Melchers, F. 1986.
5, a new light-chain-related locus selectively expressed in pre-B lymphocytes. Nature 324:579.[ISI][Medline]
-
Kudo, A., Sakaguchi, N. and Melchers, F. 1987. Organization of the murine Ig-related
5 gene transcribed selectively in pre-B lymphocytes. EMBO J. 6:103.[Abstract]
-
Bauer, S. R., Kudo, A. and Melchers, F. 1988. Structure and pre-B lymphocyte restricted expression of the Vpre-B gene in humans and conservation of its structure in other mammalian species. EMBO J. 7:111.[Abstract]
-
Kitamura, D., Kudo, A., Schaal, S., Muller, W., Melchers, F. and Rajewsky, K. 1992. A critical role of
5 protein in B cell development. Cell 69:823.[ISI][Medline]
-
Minegishi, Y., Coustan-Smith, E., Wang Y.-H., Cooper, M. D., Campana, D. and Conley, M. E. 1998. Mutations in the human
5/14. 1 gene result in B cell deficiency and agammaglobulinemia. J. Exp. Med. 187:71.[Abstract/Free Full Text]
-
Lassoued, K., Nunez, C. A., Billips, L., Kubagawa, H., Monteiro, R. C., LeBien, T. W. and Cooper, M. D. 1993. Expression of surrogate light chain receptors is restricted to a late stage in pre-B cell differentiation. Cell 73:73.[ISI][Medline]
-
Karasuyama, H., Rolink, A., Shinkai, Y., Young, F., Alt, F. W. and Melchers, F. 1994. The expression of Vpre-B/
5 surrogate light chain in early bone marrow precursor B cells of normal and B cell-deficient mutant mice. Cell 77:133.[ISI][Medline]
-
Salamero, J., Fougereau, M. and Seckinger, P. 1995. Internalization of B cell and pre-B cell receptors is regulated by tyrosine kinase and phosphate activities. Eur. J. Immunol. 25:2757.[ISI][Medline]
-
Kline, G. H., Hartwell, L., Beck-Engeser, G. B., Keyna, U., Zaharevitz, S., Klinman, N. R. and Jack, H.-M. 1998. Pre-B cell receptor-mediated selection of pre-B cells synthesizing functional µ heavy chains. J. Immunol. 161:1608.[Abstract/Free Full Text]
-
Bauer, S. R. and Scheuermann, R. H. 1993. Expression of the Vpre-B/
5/µ pseudo-Ig complex correlates with downregulated RAG-1 expression and V(D)J type recombination: a mechanism for allelic exclusion at IgH locus. Transgene 1:33.
-
Loffert, D., Ehlich, A., Muller, W. and Rajewsky, K. 1996. Surrogate light chain expression is required to establish immunoglobulin heavy chain allelic exclusion during early B cell development. Immunity 4:133.[ISI][Medline]
-
Tsubata, T., Tsubata, R. and Reth, M. 1992. Cross-linking of the cell surface Ig (µ-surrogate light chains complex) on pre-B cells induces activation of V gene rearrangements at the Ig
locus. Int. Immunol. 4:637.[Abstract]
-
Stanhope-Baker, P., Hudson, K. M., Shaffer, A. L., Constantinescu, A. and Schlissel, M. S. 1996. Cell type-specific chromatin structure determines the targeting of V(D)J recombinase activity in vitro. Cell 85:887.[ISI][Medline]
-
Reth, M., Petrac, E., Wiese, P., Lobel, L. and Alt, F. W. 1987. Activation of V
gene rearrangemant in pre-B cells follows the expression of membrane-bound immunoglobulin heavy chains. EMBO J. 6:3299.[Abstract]
-
Iglesias, A., Kopf, M., Williams, G. S. Buhler, B. and Kohler, G. 1991. Molecular requirements for the µ-induced light chain gene rearrangement in pre-B cells. EMBO J. 10:2147.[Abstract]
-
Papavasiliou, F., Jankovic, M. and Nussenzweig, M. C. 1996. Surrogate or conventional light chains are required for membrane immunoglobulin mu to activate the precursor B cell transition. J. Exp. Med. 184:2025.[Abstract]
-
Young, F., Ardman B., Shinkai, Y., Lansford, R., Blackwell, T. K., Mendelsohn, M., Rolink, A., Melchers, F. and Alt, F. W. 1994. Influence of immunoglobulin heavy and light-chain expression on B-cell differentiation. Gene Dev. 8:1043.[Abstract]
-
Miyazaki, T., Kato, I., Takeshita, S., Karasuyama, H. and Kudo, A. 1999.
5 is required for rearrangement of the Ig
light chain gene in pro-B cell lines. Int. Immunol. 11:1195.[Abstract/Free Full Text]
-
Rolink, A., Kudo, A., Karasuyama, H., Kikuchi, Y. and Melchers, F. 1991. Long-term proliferating early pre B cell lines and clones with the potential to develop to surface Ig-positive, mitogen reactive B cells in vitro and in vivo. EMBO J. 10:327.[Abstract]
-
Sudo, T., Ito, M., Ogawa, W., Iizuka, M., Kodama, H., Kunisada, T., Hayashi, S.-I., Ogawa, M., Sakai, K. and Nishikawa, S.-I. 1989. Interleukin 7 production and function in stromal cell-dependent B cell development. J. Exp. Med. 170:333.[Abstract]
-
Lefkovits, I., ed. 1997. Immunology Methods Manual. Academic Press, San Diego, CA.
-
Kottmann, A. H., Zevnik, B., Welte, M., Nielsen, P. J. and Kohler, G. 1994. A second promoter and enhancer element within the immunoglobulin heavy chain locus. Eur. J. Immunol. 24:817.[ISI][Medline]
-
Karasuyama, H., Rolink, A. and Melchers, F. 1993. A complex of glycoproteins is associated with Vpre-B/
5 surrogate light chain on the surface of µ heavy chain-negative early precursor B cell lines. J. Exp. Med. 178:469.[Abstract]
-
Koyama, M., Ishihara, K., Karasuyama, H., Cordell, J. L., Iwamoto, A. and Nakamura, T. 1997. CD79
/CD79ß heterodimers are expressed on pro-B cell surfaces without associated µ heavy chain. Int. Immunol. 9:1767.[Abstract]
-
Jugloff, L. S. and Jongstra-Bilen, J. 1997. Cross-linking of the IgM receptor rapid translocation of IgM-associated Ig
, Lyn, and Syk tyrosine kinases to the membrane skeleton. J. Immunol. 159:1096.[Abstract]
-
Grawunder, U., Leu, T. M. J., Schatz, D. G., Werner, A., Rolink, A. G., Melchers, F. and Winkler, T. H. 1995. Down-regulation of Rag1 and Rag2 gene expression in pre-B cells after functional immunoglobulin heavy chain rearrangement. Immunity 3:601.[ISI][Medline]
-
Pennycook, J. L. M. H., Marshall, A. J. and Wu, G. E. 1997. PCR assays for endogenous Ig gene rearrangement. In Lefkovits, I., ed., Immunology Methods Manual, vol. 1, p. 237. Academic Press, San Diego, CA.
-
Doi, T., Motoyama, N., Yokunaga, A. and Watanabe, T. 1999. Death signals from the B cell antigen receptor target mitochondria, activating necrotic and apoptotic death cascades in a murine B cell line, WEHI-231. Int. Immunol. 6:933.
-
Papavasiliou, F., Misulovin, Z., Suh, H. and Nussenzweig, M. C. 1995. The role of Igß in precursor B cell transition and allelic exclusion. Science 268:408.[ISI][Medline]
-
Jacobsen, K. and Osmond, D. G. 1990. Microenvironmental organization and stromal cell associations of B lymphocyte precursor cells in mouse bone marrow. Eur. J. Immunol. 20:2395.[ISI][Medline]
-
Marshall, A. J., Fleming, H. E., Wu, G. E. and Paige, C. J. 1998. Modulation of the IL-7 dose-response threshold during pro-B cell differentiation is dependent on pre-B cell receptor expression. J. Immunol. 161:6038.[Abstract/Free Full Text]
-
Suzuki, H., Terauchi, Y., Fujiwara, M., Aizawa, S., Yazaki, Y., Kadowaki, T. and Koyasu, S. 1999. Xid-like immunodeficiency in mice with disruption of the p85
subunit of phosphoinositide 3-kinase. Science 283:390.[Abstract/Free Full Text]
-
Turner, M., Mee, P. J., Costello, P. S., Williams, O., Price, A. A., Duddy, L. P., Furlong, M. T., Geahlen, R. L. and Tybulewicz, V. L. J. 1995. Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk. Nature 378:298.[ISI][Medline]
-
Cheng, A. M., Rowly, B., Pao, W., Hayday, A., Bolen, J. B. and Pawson, T. 1995. Syk tyrosine kinase required for mouse viability and B-cell development. Nature 378:303.[ISI][Medline]
-
Winkler, T. H., Rolink, A., Melchers, F. and Karasuyama, H. 1995. Precursor B cells of mouse bone marrow express two different complexes with the surrogate light chain on the surface. Eur. J. Immunol. 25:446.[ISI][Medline]
-
Leptin, M., Postash, M., J., Grutzmann, R., Heusser, C., Shulman, M., Kohler, G and Melchers, F. 1984. Monoclonal antibodies specific for murine IgM I. Characterization of antigenic determinants on the four constant domains of the mu heavy chain. Eur. J. Immunol. 14:534.[ISI][Medline]
-
Koopman, G., Reutelingsperger, C. P. M., Kuijten, G. A. M., Keehnen, R. M. J., Pals, S. T. and van Oers, M. H. J. 1994. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84:1415.[Abstract/Free Full Text]
-
Takeda, K., Kaisho, T., Yoshida, N., Takeda, J., Kishimoto, T. and Akira, S. 1998. Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis: generation and characterization of T cell-specific Stat-3 deficient mice. J. Immunol. 161:4652.[Abstract/Free Full Text]
-
Vercammen, D., Brouckaert, G., Denecker, G., de Craen, M. V., Declercq, W., Fiers, W. and Vandenabeele, P. 1998. Dual signaling of the Fas receptor: initiation of both apoptotic and necrotic cell death pathways. J. Exp. Med. 188:919.[Abstract/Free Full Text]
-
ten Boekel, E., Melchers, F. and Rolink, A. G. 1997. Changes in the VH gene repertoire of developing precursor B lymphocytes in mouse bone marrow mediated by the pre-B cell receptor. Immunity 7:357.[ISI][Medline]