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The Journal of Immunology, 2002, 168: 629-634.
Copyright © 2002 by The American Association of Immunologists

The Pre-B Cell Receptor Signaling for Apoptosis Is Negatively Regulated by Fc{gamma}RIIB1

Ibuki Kato*, Toshiyuki Takai{dagger} and Akira Kudo*

* Department of Life Science, Tokyo Institute of Technology, Yokohama, Japan; {dagger} Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Many studies have shown that Fc{gamma}RIIB is a negative regulator of B cell receptor signaling, and even though Fc{gamma}RIIB is expressed through all developmental stages of the B cell lineage, its involvement in pre-B cell receptor (pre-BCR) signaling has not been examined. To investigate Fc{gamma}RIIB function at the pre-B cell stage, we have established pre-BCR positive pre-B cell lines from normal mice and Fc{gamma}RIIB-deficient mice, named PreBR and Fc{gamma}-/-PreBR, respectively. These cell lines are able to differentiate into immature B cells in vitro by removal of IL-7. In PreBR, apoptosis was moderately induced by F(ab')2 anti-µ Ab, but not by intact anti-µ Ab. Phosphorylation of SH2-containing inositol 5-phosphatase (SHIP) and Dok, which are involved in Fc{gamma}RIIB signaling, was induced by anti-µ cross-linking in PreBR. In contrast, apoptosis was strongly induced by both the F(ab')2 and intact anti-µ Abs in Fc{gamma}-/-PreBR, and the level of phosphorylation of SHIP or Dok was much lower in Fc{gamma}-/-PreBR than those observed in PreBR. Restoration of Fc{gamma}RIIB to Fc{gamma}-/-PreBR followed by anti-µ cross-linking blocked severe apoptosis, and up-regulated SHIP and Dok phosphorylation. The results demonstrate that Fc{gamma}RIIB negatively regulates pre-BCR-mediated signaling for apoptosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Signaling through various B cell receptors plays an important role in several stages of B cell differentiation. 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 H and Ig L chain gene loci (1). The Ig H chain locus is usually rearranged before the Ig L chain locus. When a functional VHDHJH rearrangement occurs in a pre-B cell, this cell will express the pre-B cell receptor (pre-BCR)3 formed by the membrane-bound µH chain in complex with the surrogate L (SL) chain (2, 3).

In both mice and humans, the SL chain is composed of two proteins encoded by the pre-B cell specific genes, VpreB and {lambda}5 (4, 5, 6, 7). The analyses of bone marrow cells from {lambda}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 SL chain as markers showed that c-kit+CD25-SL+ pre-B/pre-BI cells were produced in normal numbers, whereas c-kit-CD25+SL+ large pre-BII cells and c-kit-CD25+SL- large and small pre-BII cells, as well as immature B cells, were at least 40-fold reduced. Components of the human SL chain also play an important role in B cell differentiation. Mutation in the human {lambda}5 gene markedly reduced the number of CD19+ B cells in the peripheral blood. There were almost no mature B cells in bone marrow, indicating that a more severe B cell deficiency is caused by loss of {lambda}5 expression in humans than in mice (9). Recently, we observed that the SL chain activated the {kappa}-chain rearrangement by restoration of {lambda}5 to {lambda}5-deficient pro-B cell lines (10). These results indicate that in mice and humans with {lambda}5 mutations, B cell differentiation is impaired at the transition from the pro-B/pre-BI to the pre-BII cell stage, during which Ig L chain gene rearrangement takes place.

In humans, 5% of bone marrow cells expressed the pre-BCR on the cell surface (11), however, in the mice, the expression of the pre-BCR was barely detectable on normal bone marrow cells (12). Salamero et al. (13) demonstrated that only 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. All published results indicate that there is relatively less pre-BCR expression on the surfaces of cell lines and normal pre-B cells. The pre-BCR is thought to transduce signals of proliferation, cell survival, and differentiation (14, 15, 16).

The quality and magnitude of immune responses are determined by the summation of positive and negative signals at the cellular level. In mature B cells, the relative levels of stimulatory signaling and inhibitory signaling determine the amount and duration of Ab production. Mediators of signals at the B cell surface are stimulatory receptors, such as the B cell receptor and the inhibitory receptor, the low-affinity IgG receptor, Fc{gamma}RIIB. Fc{gamma}RIIB is widely expressed in hematopoietic cells, and Fc{gamma}RIIB inhibits c-kit-mediated proliferation in mast cells (17). Fc{gamma}RIIB-deficient mice displayed elevated Ig levels in response to both thymus dependent and independent Ags (18). In specific genetic backgrounds, deficiency of Fc{gamma}RIIB on B cells leads to autoimmune diseases (19).

A diverse group of inhibitory receptors, including Fc{gamma}RIIB, shares an immunoreceptor tyrosine-based inhibition motif (20). This motif, when tyrosine is phosphorylated, forms a docking site for the SH2-containing inositol 5-phosphatase (SHIP) (21), which is a major mechanism of Fc{gamma}RIIB-mediated inhibitory signaling (22). SHIP is widely expressed in hematopoietic cells, and it has been identified as a crucial negative regulator for B cell activation (23). SHIP contains an SH2 domain, three putative SH3-interacting motifs, and two potential binding sites for phosphotyrosine-binding (PTB) domain, allowing it to interact with membrane receptors (24), tyrosine kinases (25), and adapter proteins (26, 27). The noncatalytic carboxyl-terminal 190 aa of SHIP plays a critical role in SHIP function in B cells (28). Recent studies have shown that the RasGAP-binding protein, Dok, is a mediator of inhibitory Fc{gamma}RIIB signals in B cells (29) and T cells (30). The adapter protein, Dok, associates with RasGAP upon BCR aggregation (31, 32, 33). This response is correlated with SHIP phosphorylation and formation of a Dok-SHIP complex, mediated by interaction between phosphotyrosyl residues of SHIP and the PTB domain in Dok (29). There is no report on the function of Fc{gamma}RIIB at the pre-B cell stage. Nevertheless, some experiments were performed at the pro-B cells to show that Fc{gamma}RIIB is involved in proliferation (34).

Recently, we established pre-BCR positive pre-B cell lines that are able to differentiate into immature B cells in vitro. In these cell lines, Ab cross-linking of the pre-BCR induced apoptosis and differentiation accompanied with tyrosine phosphorylation (35). We show here that in a pre-BCR positive pre-B cell line from a Fc{gamma}RIIB-deficient mouse, a high level of apoptosis was induced by both intact or F(ab')2 anti-µ Ab cross-linking, and restoration of Fc{gamma}RIIB to Fc{gamma}RIIB-deficient cells blocked severe apoptosis following up-regulation of SHIP and Dok phosphorylation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and cell lines

Pro-B cells derived from bone marrow cells of BALB/c mice and Fc{gamma}RIIB-deficient mice were cultured with the mouse stromal cell line, ST2 (a gift of Dr. Nishikawa, Kyoto University, Kyoto Japan), in the presence of IL-7 as described previously (10). PreBR and Fc{gamma}-/-PreBR cell lines were established from pro-B cells in the presence of IL-7 after removal of ST2 as described previously (35). These cell lines were cultured in SF-03 medium (Sanko Jyunyaku, Tokyo, Japan) containing 5 x 10-5 M 2-ME, 1x nonessential amino acid solution (Life Technologies, Gaithersburg, MD), 0.03% primatone (Quest International, Naarden, The Netherlands), 2% FCS, and 100 U/ml rIL-7 (36) (a gift of Dr. Sudoh, Toray, Kamakura, Japan), as described previously (14).

Abs and flow cytometric analyses

FITC-conjugated mAb 1D3 (anti-mouse CD19) and biotin-conjugated mAb 2.4G2 (anti-mouse Fc{gamma}RII/III) were purchased from BD PharMingen (San Diego, CA). FITC-conjugated goat anti-mouse IgM (µ-chain specific), FITC-conjugated goat anti-mouse {kappa}-chain, unconjugated goat anti-mouse IgM (µ-chain specific) Ab, and F(ab')2 goat anti-mouse µ Ab were purchased from Southern Biotechnology Associates (Birmingham, AL). Rat mAb Vp245 (anti-mouse VpreB) and LM34 (anti-mouse {lambda}5) (37) were gifts from Dr. Karasuyama (Tokyo Medical and Dental University, Tokyo, Japan). FITC-conjugated streptavidin was purchased from Cosmo Bio (Tokyo, Japan). Flow cytometric analyses using the FACSCalibur (BD Biosciences, Mountain View, CA) were performed as described (10).

Detection of nuclear change (DNA fragmentation assay)

Cells were washed with PBS and resuspended in 400 µl of hypotonic buffer (0.15% Triton X-100 and 20 µg/ml RNase A) containing 50 µg/ml propidium iodide (PI), and analyzed by the flow cytometer, FACSCalibur.

Western blot analyses of poly(ADP-ribose) polymerase (PARP) cleavage

After treatment with Abs, 4 x 106 cells were dissolved in the SDS-PAGE sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-ME, 6 M urea, and 0.00125% bromphenol blue), and then sonicated for 15 min. Cell lysates were subjected to 7.5% SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schulell, Dassel, Germany). The filter was blocked with 5% nonfat dry milk in TTBS (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween 20) for 1 h and incubated with polyclonal rabbit anti-PARP Abs (BIOMOL, Plymouth Meeting, PA) at a dilution of 1/500 in 5% nonfat dry milk in TTBS for 2.5 h at room temperature. After three washes with TTBS, filters were incubated with a HRP-conjugated goat anti-rabbit IgG (Organon Teknika, Durham, NC) for 1 h and then detected by using the ECL kit (Amersham Pharmacia, Piscataway, NJ).

Retrovirus mediate gene transfer

The Fc{gamma}RIIB cDNA (a gift of Dr. Miyake, Saga Medical School, Saga, Japan) was recloned into a retroviral expression vector, pMX-puro, and the Fc{gamma}RIIB construct was transfected into {Psi}NX-Eco packaging cells (38). The cells were subsequently selected with 2 µg/ml puromycin (Nacalai Tesque, Kyoto, Japan). The virus infection was performed with a coculture of Fc{gamma}-/-PreBR cells and packaging cells. The stable transfectants were established by the selection of 1 µg/ml puromycin in the presence of IL-7.

Detection of intracellular Ca influx

PreBR cells were incubated in RPMI containing 5 µM Fluo-3/AM (Sigma, St. Louis, MO) and 0.02% pluronic F-127 (Sigma) at 37°C for 30 min. After washing, Fluo-3 fluorescence of cells was measured continuously by flow cytometry using the FACSCalibur (BD Biosciences).

Analysis of tyrosine-phosphorylated SHIP and Dok

Cells were treated with 20 µg/ml Abs, goat IgG, an intact anti-µ Ab, and a F(ab')2 anti-µ Ab for 60 s at 37°C. Afterward, the cold inhibition buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl2, 2 mM Na3VO4, 1 µg/ml aporotinin, and 1 µg/ml leupeptin) was added to samples. Then, cells were collected by centrifugation at 5000 rpm for 10 s and lysed with the inhibition buffer containing 1% Nonidet P-40. Cell lysates were immunoprecipitated with an anti-SHIP Ab (Santa Cruz Biotechnology, Santa Cruz, CA) or an anti-Dok Ab (a gift of Dr. Yamanashi, University of Tokyo, Tokyo, Japan), and subjected to SDS-PAGE and transferred to nitrocellulose membranes. The filters were then blocked with 5% nonfat dry milk in TTBS for 1 h and incubated with a HRP-conjugated anti-phosphotyrosine Ab (Santa Cruz Biotechnology), and then signals were detected using the ECL kit (Amersham Pharmacia).

Population study of bone marrow cells derived from Fc{gamma}RIIB-deficient mice

Bone marrow cells from Fc{gamma}RIIB-deficient mice (39) or littermate mice were treated with FITC-conjugated anti-B220 Ab (BD PharMingen) and PE-conjugated anti-µ specific Ab (Southern Biotechnology Associates), and analyzed by the flow cytometer, FACSCalibur (BD Biosciences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of the pre-BCR+ pre-B cell line, PreBR

We previously established pre-BCR+ pre-B cell lines (PreBR1 and PreBR2) (35). These cell lines had the large pre-BII phenotype, being positive for CD19, µlow, Ig{beta}, VpreB, {lambda}5, IL-7 receptor, and CD25, but negative for {kappa}, CD23, c-kit, and CD40 (Fig. 1Go and data not shown). In contrast, the immature B cell line, WEHI231, expressed the B cell receptor composed of µhigh and {kappa}. PreBR and WEHI231 cells expressed equal levels of Fc{gamma}RIIB, even though pre-BCR expression on PreBR was lower than BCR on WEHI231 (Fig. 1Go). Although the mAb 2.4G2 recognizes both mouse Fc{gamma}RIIB and Fc{gamma}RIII, PreBR cells from Fc{gamma}RIIB-deficient mice are negative for this Ab. Therefore, the positive staining by using 2.4G2 showed the expression of Fc{gamma}RIIB.



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  		FIGURE 1. Flow cytometric analyses of PreBR and WEHI231 cell lines. The surface Ag expression profiles of PreBR1, PreBR2, and WEHI231 were examined by flow cytometry using FITC-conjugated Ab: anti-CD19, anti-µ, anti-{kappa}, and biotin-conjugated anti-Fc{gamma}RIIB Ab followed by FITC-conjugated streptavidin. The solid line indicates staining with the designated Ab, the dotted line indicates staining with the no Ab for FITC-conjugated first Ab or FITC-conjugated streptavidin for biotin-conjugated first Ab. The result is a representative of three independent analyses.

 
Induction of apoptosis by a F(ab')2 anti-µ Ab, but not by an intact anti-µ Ab

To examine the function of the Fc{gamma}RIIB of PreBR, PreBR cells were treated with 5 µg/ml of an intact anti-µ Ab, a F(ab')2 anti-µ Ab, or a control goat IgG. After 2 days culture with Abs, apoptosis was analyzed by DNA fragmentation (Fig. 2GoA). Apoptosis of WEHI231 cells was induced by cross-linking with both the intact anti-µ Ab and the F(ab')2 anti-µ Ab. An increase of subdiploid cells in PreBR cells was observed following treatment with the F(ab')2 anti-µ Ab cross-linking; however, it was not detected by the intact anti-µ Ab cross-linking. 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 the intact anti-µ Ab and the F(ab')2 anti-µ Ab induced the production of 85 kDa PARP cleavage in WEHI231 cells, only the F(ab')2 anti-µ Ab-treated PreBR cells showed characteristic apoptosis-related 85 kDa fragments (Fig. 2GoB). Consistent with data shown in Fig. 2GoA–B, the results demonstrated that apoptosis of PreBR cells was preferentially induced by the F(ab')2 anti-µ Ab, but not by the intact anti-µ Ab.



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  		FIGURE 2. Induction of apoptosis by pre-BCR cross-linking on PreBR cell lines. A, PreBR1, PreBR2, and WEHI231 cells were cultured at 2 x 105 cells/ml, and 5 µg/ml of a control Ab (goat IgG), an intact anti-µ Ab, or a F(ab')2 anti-µ Ab, were added. After 2 days culture, cells were washed in PBS and resuspended in hypotonic buffer containing PI. DNA content in the subdiploid area was examined and shown by percentage. Bars represent means ± SD of triplicate experiments. B, PreBR1 and WEHI231 cells were stimulated for 2 days with 5 µg/ml of a control Ab (goat IgG), an intact anti-µ Ab, or a F(ab')2 anti-µ Ab. After washing with PBS, cells were lysed, and an equal amount of whole cell lysate was loaded onto each lane of SDS-PAGE gel. After transfer to nitrocellulose filters, the blots were incubated with polyclonal rabbit anti-PARP Ab at a dilution of 1/500 in TTBS containing 5% nonfat dry milk for 2.5 h at room temperature. After three washes with TTBS, filters were incubated with a HRP-conjugated goat anti-rabbit IgG for 1 h, and then detected by using the ECL kit. The upper band corresponds to 116 kDa of the whole PARP molecule, whereas the lower 85-kDa fragment is an apoptosis-specific degradation product.

 
Establishment of pre-BCR+ pre-B cell lines from a Fc{gamma}RIIB-deficient mouse

Pro-B cells derived from bone marrow cells in a Fc{gamma}RIIB-deficient mouse were cultured in the presence of the stromal cell line, ST2, and IL-7. Initially, these cell lines did not express the pre-BCR consisting of µH chain and SL chain.

To obtain pre-BCR+ pre-B cell lines, we continued the cell culture in the presence of IL-7 after ST2 was removed to induce differentiation, and then the cells were cloned by limiting-dilution. The phenotypes of the pre-BCR+ pre-B cell lines from a Fc{gamma}RIIB-deficient mouse, Fc{gamma}-/-PreBR, were examined by flow cytometry (Fig. 3GoA). Fc{gamma}-/-PreBR had the large pre-BII phenotype, being positive for CD19, µlow, and VpreB, but negative for {kappa} and Fc{gamma}RIIB. Both the intact and the F(ab')2 anti-µ Ab treatment of Fc{gamma}-/-PreBR cells induced high levels of apoptosis (Fig. 3GoB).



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  		FIGURE 3. Phenotypes of the pre-B cell line from a Fc{gamma}RIIB-deficient mouse. A, The surface expressions of Fc{gamma}-/-PreBR cells were examined by flow cytometry using FITC-conjugated Ab: anti-CD19, anti-µ, and anti-{kappa}, and biotin-conjugated Ab: anti-VperB, anti-{lambda}5, and anti-Fc{gamma}RIIB followed by FITC-conjugated streptavidin. The solid line indicates staining with the designated Ab, the dotted line indicates staining with no Ab or FITC-conjugated streptavidin. B, Apoptosis in response to intact or F(ab')2 anti-µ Ab was assessed as in Fig. 2Go.

 
Restoration of the Fc{gamma}RIIB gene to Fc{gamma}-/-PreBR

To investigate the function of Fc{gamma}RIIB at the pre-B cell stage, restoration of the Fc{gamma}RIIB gene to Fc{gamma}-/-PreBR cells was performed. The mouse Fc{gamma}RIIB cDNA was inserted into an expression vector. Following that, the Fc{gamma}RIIB expression construct was transfected into the {Psi}NX-E cells. The resulting retrovirus particles were infected into Fc{gamma}-/-PreBR cells, and the stable transfectants, Fc{gamma}-/-PreBR(Fc{gamma})-3 and Fc{gamma}-/-PreBR(Fc{gamma})-67, were established by limiting-dilution. These cell lines generated expression of Fc{gamma}RIIB on the surface as shown in Fig. 4Go. Low and high cell surface expression levels of Fc{gamma}RIIB were observed on Fc{gamma}-/-PreBR(Fc{gamma})-3 and (Fc{gamma})-67 cell lines, respectively. In the control transfectant, phenotypes of Fc{gamma}-/-PreBR(mock) showed no differences from the original cell line, Fc{gamma}-/-PreBR.



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  		FIGURE 4. Restoration of Fc{gamma}RIIB to the Fc{gamma}RIIB-deficient pre-B cell line. The surface expressions of Fc{gamma}-/-PreBR (mock), (Fc{gamma})-3, (Fc{gamma})-67 cells were examined by flow cytometry using FITC-conjugated Ab: anti-CD19, anti-µ, and anti-{kappa}, and biotin-conjugated Ab: anti-VperB, anti-{lambda}5, and anti-Fc{gamma}RIIB followed by FITC-conjugated streptavidin. The solid line indicates staining with the designated Ab, the dotted line indicates staining with no Ab or FITC-conjugated streptavidin.

 
Fc{gamma}RIIB expression inhibits pre-BCR induced apoptosis

To determine whether transfectants, Fc{gamma}-/-PreBR(Fc{gamma})-3 and (Fc{gamma})-67, could restrain pre-BCR mediated apoptosis, these cell lines were treated with 5 µg/ml of the intact anti-µ Ab, the F(ab')2 anti-µ Ab or the control goat IgG, respectively. After 2 days culture with Abs, apoptosis was analyzed by DNA fragmentation (Fig. 5Go). In Fc{gamma}-/-PreBR(Fc{gamma})-67, the level of apoptosis was decreased ~20 or 30% with the intact or the F(ab')2 Ab cross-linking. We next evaluated the effect of Fc{gamma}RIIB expression on Ca2+ mobilization after Ab cross-linking. As shown in Fig. 6GoA, the pre-BCR-induced Ca2+ response was diminished in cell lines Fc{gamma}-/-PreBR(Fc{gamma})-3 and (Fc{gamma})-67 compared with Fc{gamma}-/-PreBR(mock). The peak of Ca2+ concentration in these cell lines decreased from 300 to 150 nM, which was a level similar to PreBR. In WEHI231, the peak of Ca2+ concentration was 550 nM, and the activation time was shorter than that in PreBR1. To study the mechanisms in the detail involved in the inhibition of pre-BCR-induced apoptosis, we examined the effect of Fc{gamma}RIIB expression on the activation of SHIP and Dok, which are known as adapter molecules down-stream of Fc{gamma}RIIB in mature B cells (Fig. 6GoB). In the Fc{gamma}RIIB restored cell line, Fc{gamma}-/-PreBR(Fc{gamma})-67, and PreBR1, SHIP and Dok were phosphorylated by anti-µ cross-linking. In contrast, in Fc{gamma}-/-PreBR(mock), the phosphorylation level of SHIP or Dok was diminished. These results demonstrated that restoration of Fc{gamma}RIIB to Fc{gamma}-/-PreBR cells followed by anti-µ cross-linking blocked high levels of apoptosis, and SHIP and Dok are situated downstream of Fc{gamma}RIIB activated by pre-BCR. To investigate the role of Fc{gamma}RIIB in vivo, bone marrow cells from Fc{gamma}RIIB-deficient mice were analyzed (Fig. 7Go). The number of pre-BI cells (B220+, µ-), pre-BII cells (B220+, µlow), or immature B cells (B220+, IgM+) in between Fc{gamma}RIIB-deficient mice (-/-) and wild-type littermates (+/+) was not changed.



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  		FIGURE 5. The effect of Fc{gamma}RIIB restoration in pre-BCR-mediated apoptosis. Fc{gamma}-/-PreBR (mock), (Fc{gamma})-3, or (Fc{gamma})-67 cells were cultured at 2 x 105 cells/ml, and 5 µg/ml of a control Ab (goat IgG), an intact anti-µ Ab or a F(ab')2 anti-µ Ab were added into the culture. After 2 days culture, cells were washed in PBS and resuspended in hypotonic buffer containing PI. Next, DNA content in a subdiploid area was examined. Bars represent means ± SD of triplicate experiments.

 


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  		FIGURE 6. Decrease of apoptosis signaling in Fc{gamma}RIIB-restored cell lines. A, PreBR, Fc{gamma}-/-PreBR (mock), (Fc{gamma})-3, and (Fc{gamma})-67 cells were incubated with Fluo-3/AM at 37°C for 30 min, and stimulated with 20 µg/ml of an intact anti-µ Ab. The data is shown as the mean of intracellular free Ca2+ concentration. B, Following stimulation with a control Ab (goat IgG), an intact anti-µ Ab or a F(ab')2 anti-µ Ab at 37°C for 1 min, cells were solubilized. These lysates were immunoprecipitated with an Ab against SHIP or Dok, and an immunoblot was performed using an HRP-conjugated anti-phosphotyrosine Ab. The blots were stripped to perform another immunoblot with an anti-SHIP Ab or an anti-Dok Ab, respectively.

 


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  		FIGURE 7. Population study of bone marrow cells from Fc{gamma}RIIB-deficient mice. Bone marrow cells from Fc{gamma}RIIB-deficient mice (-/-) and littermate mice (+/+) were examined by flow cytometry using FITC-conjugated anti-B220 Ab and PE-conjugated anti-µ specific Ab. The numbers indicate percentages of cells falling within the lymphocyte gates. The representative FACS profiles are shown, and similar results were obtained from five different wild-type and the mutant littermates, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previously, we revealed that Ab cross-linking of the pre-BCR on the PreBR cell-line induced apoptosis and differentiation accompanied with tyrosine phosphorylation (35), while the immature B cell line, WEHI231, also undergoes apoptosis upon BCR cross-linking (40). Although PreBR cells express µlow and WEHI231 cells express µhigh, the expression levels of Fc{gamma}RIIB on both cell lines are equal. Thus, we have supposed that Fc{gamma}RIIB on PreBR has an important role for pre-BCR signaling. To confirm the function of the Fc{gamma}RIIB with pre-BCR, PreBR cells were treated with the anti-µ Ab. Apoptosis of PreBR cells was induced by pre-BCR cross-linking with only the F(ab')2 Ab, while the intact anti-µ Ab could not induce apoptosis in PreBR cells. Furthermore, in Fc{gamma}-/-PreBR, both the intact and the F(ab')2 anti-µ Abs induced high levels of apoptosis, demonstrating that Fc{gamma}RIIB on PreBR negatively regulates pre-BCR signaling.

To investigate the role of Fc{gamma}RIIB in B cell populations in vivo, pre-B cells in bone marrow from Fc{gamma}RIIB-deficient mice were analyzed. Against our expectation, we could not detect the alteration in B cell population in Fc{gamma}RIIB-deficient mice. Because few pre-BCR positive pre-B cells are present in bone marrow, it is difficult to find the difference between mice when the alteration shows a minor level (12). To exclude the possibility that the observed phenomena are due to the intrinsic properties of established cell lines, we restored Fc{gamma}RIIB to the Fc{gamma}-/-PreBR cell lines. In these restored cells, the level of Ca2+ influx became diminished, and the phosphorylation of SHIP or Dok induced by pre-BCR cross-linking was a similar level to that of control PreBR cells. However, in this restoration, the level of apoptosis was not completely recovered, even though restoring Fc{gamma}RIIB decreased the apoptosis level. Thus it may be important to gain an adequate expression level of Fc{gamma}RIIB, because Fc{gamma}RIIB keeps the balance between positive and negative signals.

Calcium signaling is important for cell death or differentiation of immune cells. Several Ca2+-sensitive transcriptional regulators, including NF-{kappa}B, JNK, and NFAT, participate in various combinations to promote the expression of genes that underlie these responses. Dolmetsch et al. (41) demonstrated that NF-{kappa}B and JNK were selectively activated by a large transient Ca2+ rise, whereas NFAT was activated by a low sustained Ca2+ plateau. Our results showed that in PreBR cells, Ca2+ influx was a sustained plateau for over 3 min at a low level. In contrast, in WEHI231 cells, the high level of Ca2+ mobilization was transiently induced. Thus the pre-BCR signaling by Ab cross-linking on PreBR cells may activate NFAT, and the BCR signaling in WEHI231 cells may activate NF-{kappa}B and JNK.

In mature B cells, BCR-Fc{gamma}RIIB coaggregation leads to the tyrosine phosphorylation of Fc{gamma}RIIB on its immunoreceptor tyrosine-based inhibition motif, which in turn leads to the recruitment and subsequent tyrosine phosphorylation of SHIP. This phosphorylation creates the binding site for the PTB domain of Dok (30). Once Dok is found in proximity to the BCR, it becomes tyrosine phosphorylated, presumably caused by BCR-activated Src-family kinases (42). The phosphorylated-Dok recruits RasGAP, which catalyzes the intrinsic GTPase activity of Ras, converting Ras-GTP to Ras-GDP. This suggests that a similar system functions in the pre-B cell stage.

It was reported that Fc{gamma}RIIB down-regulated cell growth in the pro-B cell stage or in the mature B cell stage (35), and our results demonstrate that, in pre-B cell stage, Fc{gamma}RIIB diminishes the signals for apoptosis. Thus, Fc{gamma}RIIB negatively regulates response through the BCR at each stage in B cell differentiation. To examine the possibility of serum IgG as a ligand for cross-linking, PreBR1 cells were treated with F(ab')2 anti-µ Ab together with mouse aggregated IgG (data not shown). The result showed that a high dosage of aggregated IgG slightly inhibited F(ab')2 anti-µ Ab-induced apoptosis, suggesting that it is difficult to conclude from our experiments that serum IgG is possibly a candidate of ligand for cross-linking. Even though inhibition occurs in in-vitro, we cannot expect the cross-linking of Fc{gamma}RIIB by IgG in vivo at the pre B cell stage because a quite low level of IgG in bone marrow exists.

As shown in Fig. 5Go, in the Fc{gamma}RIIB restored cell line, the level of apoptosis was also decreased by F(ab')2 anti-µ Ab cross-linking, and SHIP and Dok were phosphorylated at the time. Therefore, Fc{gamma}RIIB may transmit negative signals intracellularly in the pre B cell stage without ligand binding to Fc{gamma}RIIB. In the human pre B cell line, the fraction of pre-BCR localized to lipid raft before receptor cross-linking, and receptor engagement enhanced this association (43). Moreover, in the human B cell line, BCR stimulation induced rapid and transient translocation of SHIP into lipid raft (44). Recently, in the 11th International Congress of Immnology, Aman et al. (45) found basal localization of Fc{gamma}RIIB at lipid raft in the B cell line. Thus, Fc{gamma}RIIB and pre-BCR are possibly co-localized at lipid raft, which is the major functional compartment for negative signal in PreBR cells, and transduce the same signals as treatment of intact Ab for cross-linking. The function of Fc{gamma}RIIB in the pre-B cell stage is important for blocking apoptosis, because cell survival in the pre-B cell stage in vivo is critical for expanding Ab repertoire.


   Acknowledgments
 
We thank Drs. S. Nishikawa, T. Sudoh, H. Karasuyama, K. Miyake, and Y. Yamanashi for providing ST2, IL-7, VpreB, and {lambda}5 Abs, the mouse Fc{gamma}RIIB cDNA, and Dok Ab, respectively. We thank Dr. S. Bauer for critical reading of the manuscript.


   Footnotes
 
1 This work was supported in part by grants-in-aid from the Ministry of Education, Sports, Science and Technology, and from Ministry of Agriculture, Forestry and Fisheries. I.K. is a research fellow of the Japan Society for the Promotion of Science. Back

2 Address correspondence and reprint requests to Dr. Akira Kudo, Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 Japan. E-mail address: akudo{at}bio.titech.ac.jp<./> Back

3 Abbreviations used in this paper: pre-BCR, pre-B cell receptor; PreBR, pre-BCR positive pre-B cell line; SL, surrogate light; PARP, poly(ADP-ribose) polymerase; PI, propidium iodide; SHIP, SH2-containing inositol 5-phosphatase; PTB, phosphotyrosine binding.<./> Back

Received for publication June 29, 2001. Accepted for publication November 6, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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