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The Journal of Immunology, 2007, 178: 926-935.
Copyright © 2007 by The American Association of Immunologists, Inc.

Redundancy in B Cell Developmental Pathways: c-Cbl Inactivation Rescues Early B Cell Development through a B Cell Linker Protein-Independent Pathway1

Haifeng Song*, Juan Zhang{dagger}, Y. Jeffrey Chiang*, Reuben P. Siraganian{dagger} and Richard J. Hodes2,*,{ddagger}

* Experimental Immunology Branch, National Cancer Institute, {dagger} Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, and {ddagger} National Institute on Aging, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Deficiency in the adaptor protein B cell linker protein (BLNK) results in a substantial but incomplete block in B cell development, suggesting that alternative pathways exist for B lineage differentiation. Another adaptor protein, c-Cbl, plays a negative regulatory role in several BCR-signaling pathways. We therefore investigated the role of c-Cbl during B cell development and addressed the possibility that redundancies in pathways for B cell differentiation could be further revealed by eliminating negative effects mediated by c-Cbl. Strikingly, c-Cbl inactivation reversed a number of the critical defects in early B cell differentiation that are seen in BLNK-deficient mice. c-Cbl–/–BLNK–/– mice exhibited normalized down-regulation of pre-BCR and CD43, up-regulation of MHC class II, and augmented L chain rearrangement, resulting in a successful transition from pre-B cells to immature B cells. c-Cbl inactivation also reversed the potentially tumor-predisposing hyperproliferative response of BLNK–/– pre-B cells to IL-7. Pre-BCR cross-linking induced enhanced and prolonged tyrosine phosphorylation in c-Cbl–/–BLNK–/– pre-BCR+ pre-B cells compared with c-Cbl+/–BLNK–/– cells, including elevated phosphorylation of Lyn, Syk, Btk, and phospholipase C-{gamma}2. Our studies suggest that some, but not all, pre-BCR-triggered developmental events can be mediated by BLNK-independent pathways that are negatively regulated by c-Cbl, and further suggest that different events during early B cell development require different strength or duration of pre-BCR signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Signaling from the BCR/precursor BCR (BCR/pre-BCR) plays a pivotal role in B cell development and function. Both BCR and pre-BCR signaling are initiated by activation of Src kinases, such as Lyn and Blk, upon receptor engagement. Activated Lyn phosphorylates tyrosines in the ITAM region of Ig{alpha} and Igbeta, and phosphorylated Ig{alpha} and Igbeta in turn recruit Syk kinase, which is then activated by Lyn (1, 2). Syk further activates several down-stream protein tyrosine kinases (PTK)3 and adaptor proteins. One of the substrates of Syk is B cell linker protein (BLNK; also termed SLP-65, or BASH). BLNK is an adaptor in bridging Syk to downstream signaling pathways by recruiting signaling molecules, such as Btk, phospholipase C (PLC)-{gamma}2, Vav, and Grb2 to the cell membrane to form a signalosome complex, where downstream events, including the PLC-{gamma}2 and Ca2+ mobilization pathway are activated (3, 4, 5). Pre-BCR signaling mediates important developmental events during pre-B cell transition, including early pre-B cell expansion, allelic exclusion, and modulation of cell surface molecules, as well as induction of cell cycle arrest and L chain rearrangement (6, 7, 8, 9). BCR signaling mediates positive and negative selection at the immature B cell stage and the development from transitional stage B cells (T1 and T2) to mature B cells in the spleen (10). In addition, BCR signaling is essential for the survival, maintenance, and functions of mature B cells (11). Although it has not been clearly demonstrated how the individual events that occur during B development are induced by specific signaling pathways, increasing evidence suggests that B cell-signaling pathways are redundant. One example of this apparent redundancy is the alteration in B cell development that occurs in BLNK-deficient mice. BLNK-deficient mice display a major block during the transition from early pre-B cells to small resting pre-B cell stage, manifested by the accumulation of large pre-B cells expressing surface pre-BCR and reduction in the number of small pre-B cells and immature B cells in the bone marrow (BM). Nevertheless, a small proportion of pre-B cells bypass the BLNK-dependent pathway and differentiate into immature B cells that migrate into the spleen, where most cells maintain an immature phenotype (12, 13, 14, 15). The incomplete block of B cell development in BLNK–/– mice suggests that there exists a BLNK-independent pathway(s) capable of supporting B cell development. The finding that B cell development is more severely defective in double knockout mice in which BLNK deficiency is combined with inactivation of linker for activation of T cells (LAT), Btk, or CD19 indicates that these molecules are involved in one or more BLNK-independent pathways of B cell development (16, 17, 18, 19).

In addition to triggering positive pathways for differentiation and activation, BCR/pre-BCR engagement also activates negative regulatory molecules capable of inhibiting these responses. One such negative signaling molecule is c-Cbl (Casitas B-lineage lymphoma proto-oncogene c). c-Cbl is an adaptor protein containing multiple functional domains. A phosphotyrosine-binding (PTB) region on its N terminus binds to the Src homology (SH) 2 region of PTKs; a conserved ring finger region has been demonstrated to play a negative regulatory role as a ubiquitin ligase E3 (20); a proline-rich region interacts with SH3 domain-containing proteins; and multiple tyrosine phosphorylation sites at the C terminus mediate interactions with SH2-domain-containing proteins (21, 22). Several molecules functioning in BCR-signaling pathways, including Syk, Lyn, and Vav, have been shown to be targets of c-Cbl for ubiquitination and consequent inactivation or degradation (23, 24, 25). B cell development and function in c-Cbl–/– mice are grossly normal, although a slightly altered distribution of B cell subsets was observed in peripheral B cells (26, 27). However, the function of c-Cbl during discrete stages of B cell development has not been investigated in detail.

In the current study, it was hypothesized that the deficiency of B cell development in BLNK–/– mice may be circumvented by enhancing BLNK-independent pathways. Our results demonstrated that c-Cbl inactivation substantially corrected the defect in B cell development in BLNK-deficient mice, and that rescue was most apparent in the pre-B cell transition of BM B cell development, indicating a previously unappreciated role for c-Cbl in inhibiting BLNK-independent B cell developmental pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Mice aged 8–12 wk were used in all experiments. BLNK-deficient mice and c-Cbl-deficient mice have been described previously (14, 28). Both BLNK–/– mice and c-Cbl–/– mice are of a mixed S129/C57BL/6 background. c-Cbl–/–BLNK–/– and control mice heterozygous (+/–) for c-Cbl or BLNK inactivation were generated by breeding c-Cbl–/– and BLNK–/– mice. All mice were maintained in the Bioqual facility.

Reagents and Abs

The following Abs and reagents were purchased from BD Pharmingen: biotinylated Abs specific for B220 (RA3-6B2), I-Ab, CD2, {lambda}5 (LM34), pre-BCR (SL156), CD19, IgM (II/41); FITC-conjugated Abs against B220 (RA3-6B2), IgM (II/41), BP-1, CD43 (S7), Ig{kappa} (187.7), CD5; PE-conjugated Abs against B220 (RA3-6B2), CD19, CD43, CD25, IL-7R; and allophycocyanin-labeled Abs against B220 (RA3-6B2), IgM (II/41), streptavidin-PerCP. Anti-IgD-biotin and anti-µ-FITC were purchased from Southern Biotechnology Associates. Streptavidin-Alexa Fluor 594 was purchased from Molecular Probes. Anti-Syk, anti-Lyn, and anti-PLC-{gamma}2 Abs were from Santa Cruz Biotechnology, anti-phosphotyrosine Ab (4G10) from Upstate Biotechnology anti-Btk from GenWay and the anti-Btk (pY551) phosphospecific Abs were from BD Biosciences.

Flow cytometry

A total of 1 x 106 cells were incubated with 2.4G2 Ab for 5 min on ice to block Fc{gamma}R, followed by addition of 50 µl of diluted biotinylated Abs and 20 min of incubation on ice. After washing with FACS buffer, the cells were stained for another 20 min in a combination of FITC-, PE-, allophycocyanin-conjugated Abs and streptavidin-PerCP. In staining for FACS sorting, streptavidin-Alexa Fluor 594 was used to replace streptavidin-PerCP. For intracellular {lambda}5 and µ-chain staining, the cells were first stained for cell surface markers, then fixed with Cyto/perm (BD Pharmingen), followed by staining with anti-{lambda}5 and anti-µ Abs. To stain intracellular Ig{kappa}, fresh BM cells were incubated with excess purified anti-Ig{kappa} mAb to block surface Ig{kappa}, followed by surface staining for other markers. Then cells were fixed and stained for intracellular {kappa} chain. Stained samples were analyzed using FACSCalibur with CellQuest software (BD Biosciences).

In vitro BM cell culture

BM B cell culture was performed with either IgM-depleted CD19+ BM B cells or with FACS-sorted CD19+CD43+IgM– pro-B cells. Briefly, BM suspensions were depleted of sIgM+ cells by negative selection using biotinylated anti-IgM and streptavidin microbeads (Miltenyi Biotec). Then, CD19+ cells were positively selected with CD19 microbeads (Miltenyi Biotec). CD19+CD43+IgM– pro-B cells were sorted with the FACS Vantage SE system (BD Biosciences). To measure the BM B cell response to IL-7 stimulation, 1–2.5 x 104 cells/well were cultured in 96-well flat-bottom plates with different concentration of IL-7 (R&D Systems) in Opti-Mem I medium (Invitrogen Life Technologies) supplemented with 10% FCS, 5 x 10–5M 2-ME, and penicillin/streptomycin for 4 and 6 days. The cultures were pulsed with 1 µCi/well of [3H]thymidine 8 h before harvest. [3H]Incorporation was measured using a beta counter system (1205 beta plates; PerkinElmer).

Establishment of pre-B cell lines

BM and fetal liver cells were cultured on S17 stromal cell layers in 96-well plate with 10 cells/well in IL-7 containing (3T3-IL-7 culture supernatant) Opti-Mem I medium. Ten days later, large cell colonies were observed and transferred to 6-well plates to expand the culture. The cultures were maintained for 5–8 mo with periodic analysis of surface markers. Colonies with similar phenotypes from each group were used for biochemical studies.

Biochemical studies

Pre-BCR+ pre-B cells were stimulated with anti-IgM F(ab')2 (µ-chain specific; The Jackson Laboratory) at 37°C and reaction was stopped at different time points by adding ice-cold PBS containing 5 mM EDTA, 2 mM Na3VO4, and protease inhibitors (2 mM PMSF, 90 mU/ml aprotinin, 50 µg/ml leupeptin, 50 µg/ml pepstatin). Cells were solubilized in lysis buffer (1% Triton X-100, 0.1% SDS, 50 mM Tris, pH 7.4, 50 mM NaCl, 50 mM NaF, plus protease inhibitors and Na3VO4), and postnuclear supernatants were immunoprecipitated with Abs bound to protein A-, or protein G-agarose beads. After rotation at 4°C for 1 h, the beads were washed four times with ice-cold lysis buffer and the proteins eluted by boiling for 10 min with SDS-PAGE sample buffer. In some experiments, total cell lysates were immunoprecipitated with anti-phosphotyrosine Ab 4G10 coupled to agarose beads, and after washing the bound proteins were eluted with 100 mM phenyl phosphate and analyzed by immunoblotting with the indicated Abs. Whole cell lysates or immunoprecipitated proteins were separated by SDS-PAGE and electrotransferred to polyvinylidene difluoride membranes (Millipore). The blots were probed with anti-phosphotyrosine or other specific Abs as indicated. Signals were revealed with ECL system (PerkinElmer). The signal intensity was analyzed with Imagequant program (Molecular Dynamics) and the phosphorylation signal was normalized based on total protein levels of the indicated molecules. Level of activation was expressed as ratio of normalized phosphorylation of stimulated samples vs unstimulated samples.

Immunofluorescence microscopy

Pre-BCR+ pre-B cells were settled on cover slides on ice for 40 min, warmed up at 37°C and stimulated with goat F(ab')2 Ab specific to mouse µ-chain. At different time points of the stimulation, cells were fixed with 4% paraformaldehyde for 15 min, quenched with 50 µM NH4Cl for 10 min, followed by 30 min of permeabilization with 0.05% saponin. The cells were then blocked with 5% goat serum in PBS for 30 min and incubated with rabbit anti-Syk or phospho-Syk (Y519/520-specific) Abs at 4°C for overnight. Staining was resolved with goat anti-rabbit IgG-Alexa 488 at room temperature for 40 min. The cells were imaged with a Zeiss LSM 510 META confocal microscope using a 1.4 oil planapochromat x63 objective. The mean fluorescence intensity (MFI) of phospho-Syk (Alexa 488 staining) from different time points was analyzed with Meta Morph software and the MFI was calculated based on the intensity of over 200 cells from five different view fields. The statistical confidence limit value was calculated by Excel software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
c-Cbl inactivation corrects the impaired early B cell development in BLNK-deficient mice

B cell development is deficient in BLNK–/– mice, manifested as a major block at the transition from late pro-B cells to pre-B cells, and a block in maturation from transitional B cells to mature B cells, with a resultant overall reduction in the number of peripheral B cells. This defect in B cell development, while profound, is not complete, suggesting that alternative pathways exist for B lineage differentiation. It has been demonstrated that c-Cbl plays a negative regulatory role in several BCR signaling pathways and that these signaling pathways are thus up-regulated upon c-Cbl-inactivation (21, 29, 30, 31). We therefore addressed the possibility that c-Cbl inhibits BLNK-independent B cell differentiation by assessing the effect of c-Cbl inactivation on the defect in B cell development in BLNK–/– mice. We first analyzed B cell developmental subpopulations in the BM from c-Cbl+/–BLNK+/– heterozygous control, c-Cbl–/–BLNK+/–, c-Cbl+/–BLNK–/– and double knockout c-Cbl–/–BLNK–/– mice. We found a similar total cellularity in the BM of all four genotypes (data not shown). As previously reported (14), the percentage of B220+ cells from BLNK–/– BM was significantly lower than in the control groups (Table I, c-Cbl+/–BLNK+/–, 50.1 ± 2.8%; c-Cbl–/–BLNK+/–, 53.5 ± 4.7%; c-Cbl+/–BLNK–/–, 34.4 ± 2.8%). In contrast, c-Cbl–/–BLNK–/– BM contained a nearly normal proportion of B220+ cells (46.6 ± 2.1%). Further analysis of B cell subsets by IgM and IgD costaining showed that the proportion of newly formed immature B cells (IgM+IgDlow/–) in c-Cbl+/–BLNK–/– mice was significantly reduced compared with heterozygous control and c-Cbl–/–BLNK+/– mice (Fig. 1a, Table I, c-Cbl+/–BLNK+/–, 10.1 ± 0.9%; c-Cbl–/–BLNK+/–, 10.6 ± 1.2%; c-Cbl+/–BLNK–/–, 5.8 ± 0.9%). However, normal proportions of immature B cells (11.1 ± 4.2%) were generated in c-Cbl–/–BLNK–/– mice, indicating that c-Cbl inactivation overcomes the developmental defect in BLNK-deficient B cells at a stage that precedes IgM+IgDlow/– immature B cells. The proportion of mature, presumably recirculating, B cells (IgMlowIgDhigh) in the BM of double knockout mice was lower than control mice, although significantly higher than that in c-Cbl+/–BLNK–/– mice (Fig. 1a and Table I), suggesting that a partial developmental block still exists in later stages of B cell development in the maturation of transitional B cells to mature B cells. The rescue of early B cell development in c-Cbl–/–BLNK–/– mice was also confirmed by B220, IgM costaining, which demonstrated that the proportion of B220lowIgM+ newly formed B cells in c-Cbl–/–BLNK–/– mice reached normal control level and was much higher than that in c-Cbl+/–BLNK–/– mice (Fig. 1b). Although the total proportion of B220high B cells in the double knockout mice was also increased, most of the cells maintained an immature transitional B cell (B220highIgMhigh) phenotype (Fig. 1b).


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  		Table I. Phenotypic analysis of BM B lineage populationsa

 

Figure 1
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  		FIGURE 1. Phenotypic characterization of B lineage cells in the BM. BM lymphoid cells were analyzed for stage of development by staining surface IgM and IgD (a) or B220 and IgM (b). The B220lowIgM– cell population was gated and analyzed for other developmental markers (c–g). Results are representative of six to eight experiments.

 
We next examined in more detail the effect of c-Cbl inactivation on early stages of B lineage development. The transition from pro-B to pre-B cells is an important checkpoint during B cell development. Following successful VDJ rearrangement, a mature µ chain is expressed, and together with a surrogate L chain (SLC) that consists of {lambda}5 and V-pre B proteins, forms the pre-BCR complex. The expression of pre-BCR on the cell surface elicits pre-BCR-signaling pathways, which integrate with IL-7R signaling to mediate early pre-B cell expansion. Pre-BCR signaling also regulates the expression level of many developmental markers, followed by the initiation of L chain rearrangement. It was demonstrated that BLNK plays an important role in these processes as BLNK–/– mice are markedly deficient in the transition from early large pre-B cells to small resting pre-B cells, resulting in accumulation of large numbers of surface pre-BCR-positive cells in the BM, in contrast to their nearly undetectable level in wild type mice (16, 17). In addition, BLNK has also been demonstrated to function as a tumor suppressor gene, because BLNK-deficient pre-BCR+ cells have enhanced proliferative capacity (16, 19, 32), and BLNK-deficient mice have a high incidence of pre-B cell lymphomas (16, 19, 32). To study the effect of c-Cbl inactivation at the pre-B cell stage, BM B220lowIgM– populations were gated and analyzed for B cell developmental markers. As previously reported (16), BLNK-deficient early pre-B cells failed to down-regulate surface pre-BCR and CD43 or to up-regulate MHC-II. However, pre-B cells from c-Cbl–/–BLNK–/– mice successfully did so (Fig. 1, c, d, and g), and the accumulation of pre-BCR+ cells in BLNK–/– BM was reversed to normal levels (Fig. 1c, Table I). In addition, while the proportion of pro-B cells (B220lowCD43+) was increased in c-Cbl+/–BLNK–/– BM, this population was reduced to normal level in c-Cbl–/–BLNK–/– mice (Table I). Furthermore, compared with the enhanced level of large cycling early pre-B cells in c-Cbl+/–BLNK–/– BM, the number of cells in cell cycle as assessed by flow cytometric light scatter is also reduced to normal level in c-Cbl–/–BLNK–/– BM (Fig. 1g). In contrast to the normalization of these parameters in c-Cbl–/–BLNK–/–mice, early pre-B cells in these double knockouts failed to down-regulate BP-1 or up-regulate CD2 and CD25 expression (Fig. 1, e and f). Intracellular {kappa} staining showed that c-Cbl-inactivation also partially corrected the impaired L chain rearrangement in BLNK–/– mice. Although ~4.5% of surface IgM–B220low B cells were positive for intracellular {kappa} staining in BLNK–/– mice, a higher proportion of intracellular {kappa}-positive cells (9.6%) were observed in c-Cbl–/–BLNK–/– mice, although this number was still lower than that from control mice (Fig. 2, Cbl+/–BLNK+/–, 17%; c-Cbl–/–BLNK+/–, 17%). In contrast to {kappa} staining, intracellular {lambda}5 protein, which is markedly elevated in BLNK–/– mice, was significantly down-regulated in c-Cbl–/–BLNK–/– mice (c-Cbl+/– BLNK+/–, 12%; c-Cbl–/–BLNK+/–, 17%; c-Cbl+/–BLNK–/–, 63%; c-Cbl–/–BLNK–/–, 34%). Overall, these data confirmed the accumulation of early pre-BCR+ pre-B cells in BLNK–/– BM and demonstrated that c-Cbl inactivation reduced the number of late pro-B/early pre-B cells, and enhanced the number of more mature small pre-B cells, consistent with an effect in selectively correcting some of the critical developmental events in BLNK–/– mice in the transition from large cycling to small resting pre-B cells.


Figure 2
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  		FIGURE 2. Inactivation of c-Cbl in BLNK-deficient mice partially rescued {kappa} chain rearrangement. BM cells were incubated with excess of anti-Ig{kappa} mAb to block surface Ig{kappa}, followed by surface staining of IgM and B220, then fixed and permeabilized, followed by intracellular staining for {lambda}5 and {kappa}-chain. Data were analyzed and presented by gating on surface B220lowIgM– populations. The result shown is representative of three independent experiments.

 
c-Cbl inactivation corrects the impaired in vitro development of BLNK-deficient B cells

The observed effects of c-Cbl inactivation on B cell development in vivo could reflect cell autonomous effects on B lineage development and/or the effects of c-Cbl inactivation on stromal or other cells that influence B cell maturation. To determine whether c-Cbl inactivation directly affects the development of BLNK–/– B cells, we used an in vitro culture system for the IL-7-dependent differentiation of sorted CD19+IgM– BM cells. At day 4 of culture, 15.8 and 18.5% of total live cells became double positive for surface IgM and Ig{kappa} expression in control and c-Cbl–/–BLNK+/– groups, respectively; whereas only 2.6% of cells were IgM and Ig{kappa} positive in c-Cbl+/–BLNK–/– cultures, confirming their intrinsic deficiency in B cell development. In contrast, in c-Cbl–/–BLNK–/– cultures, 12.7% IgM, Ig{kappa} double-positive cells were detected (Fig. 3a). Enumeration of total cell number from each culture showed that the cells from c-Cbl+/–BLNK–/– culture increased ~12-fold from the cell number of initial culture, which was much higher than the increases in cell number of the c-Cbl+/– BLNK+/– and c-Cbl–/–BLNK+/– groups (Fig. 3b, 5.6-fold in c-Cbl+/–BLNK+/– group and 5.1-fold in c-Cbl–/–BLNK+/– group). The total cell number in day 4 culture from double knockout mice was similar to that from control and c-Cbl–/–BLNK+/– mice (Fig. 3b). When the total differentiated cell (IgM+Ig{kappa}+) number was calculated, c-Cbl+/–BLNK–/– culture gave ~3-fold fewer IgM+Ig{kappa}+ cells than the yield from control group and c-Cbl–/–BLNK+/– group. In contrast, the number of IgM+Ig{kappa}+ cells in the c-Cbl–/–BLNK–/– group was restored to control level (Fig. 3c). These data confirmed that BLNK–/– pro-B and pre-B cells are defective in in vitro differentiation and demonstrated that c-Cbl-inactivation substantially corrected this defect. Thus, in vitro cultures recapitulated the effect of c-Cbl inactivation observed in vivo in rescuing early B cell development.


Figure 3
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  		FIGURE 3. c-Cbl inactivation rescued B cell development in in vitro culture. FACS-sorted CD19+IgM– BM cells were cultured in IL-7-supplemented medium in duplicate wells (3.8 x 105 cells/well) in 24-well plates. At day 4 of culture, B cell differentiation was examined by staining for surface expression of IgM and Ig{kappa} (a). Total cell number of day 4 cultures was enumerated using optical microscopy and the increase in cell number was expressed as fold increase of total cell number of day 4 culture over the initial culture cell number (b). Differentiated IgM+Ig{kappa}+ B cell number was calculated by multiplying the percentages of IgM+Ig{kappa}+ B cells and total cell number (c). Results shown are representative of four independent experiments.

 
c-Cbl–/–BLNK–/– pre-B cells display normalized proliferation to IL-7 stimulation

Previous studies showed that BLNK–/– pre-B cells display an enhanced proliferative response to IL-7 stimulation and that this hyperresponsiveness depends on the surface expression of pre-BCR (32). It was also shown with pre-B cell lines that pre-BCR+ B cells have a lower threshold for proliferative response to IL-7 stimulation than do pre-BCR-negative pre-B cells (33). Enumeration of the total cell numbers from our in vitro cultures (Fig. 3b) suggests that the hyperproliferation of BLNK–/– pre-B cells to IL-7 stimulation was reversed to normal level in c-Cbl–/–BLNK–/– mice. To further assess this, CD19+IgM– BM B cells were cultured in IL-7-supplemented medium, and [3H]thymidine incorporation was measured. As expected, CD19+IgM– BM B cells from BLNK–/– mice had a much stronger proliferative response than c-Cbl+/–BLNK+/– heterozygous or c-Cbl–/–BLNK+/– controls at both days 4 and 7 of IL-7 stimulation. c-Cbl–/–BLNK–/– cultures showed a reduced level of proliferation that was similar to that seen in normal B cells. Because BLNK-deficient CD19+IgM– BM cells contain an increased proportion of pre-BCR+ cells, a subpopulation known to have a lower threshold for IL-7 stimulation, we purified CD43+CD19+pre-BCR– pro-B cells for further analysis in IL-7-driven culture, so that more equivalent cell populations could be compared. When the proliferative responses of these populations were assessed, BLNK-deficient pro-B cells again displayed a stronger response, while inactivation of c-Cbl in BLNK–/– mice corrected the IL-7 hyperresponsiveness (Fig. 4a).


Figure 4
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  		FIGURE 4. Inactivation of c-Cbl reversed the hyperproliferative response of BLNK-deficient BM B cells to IL-7 stimulation. a, CD43+CD19+IgM– pre-BCR– pro-B cells were sorted by FACS and cultured in medium containing different concentrations of IL-7 in 96-well flat-bottom plates with 104 cells/well. [3H]Thymidine incorporation was measured at day 4 of culture. b, BM cells were stained for surface B220, IgM, and IL-7R. Then the cells were fixed and permeabilized, followed by staining for intracellular µ-chain. B220lowIgM–cµ+ cells were gated and analyzed for surface IL-7R expression. The number given is MFI. Result is representative of three independent experiments.

 
It was shown previously that pre-BCR signaling down-regulates IL-7R expression (34). We therefore asked whether a change in IL-7R expression could contribute to the reversal of IL-7 response in c-Cbl–/–BLNK–/– pre-B cells. c-Cbl–/–BLNK–/– BM pro-B/pre-B cells were indeed found to express a lower level of cell surface IL-7R{alpha} than BLNK–/– mice, although it was still slightly higher than that from control and c-Cbl–/– BM (Fig. 4b). Thus, differential expression of IL-7R may contribute to the observed differences in IL-7 responsiveness.

c-Cbl inactivation partially rescues conventional B2 B cell, but not B1 B cell development in BLNK–/– mice

We next assessed the effect of c-Cbl inactivation on development of peripheral B cell populations. Consistent with previous reports, the proportion of B220-positive cells in the spleen is significantly reduced in BLNK-deficient mice. This defect was normalized by c-Cbl inactivation (Table II; c-Cbl+/–BLNK+/–, 53.4 ± 1.9%; c-Cbl–/–BLNK+/–, 53.4 ± 4.3%; c-Cbl+/–BLNK–/–, 21.2 ± 3.1%; c-Cbl–/–BLNK–/–, 47.7 ± 3.2%). However, because the absolute lymphocyte numbers in the spleens of both c-Cbl+/–BLNK–/– and c-Cbl–/–BLNK–/– mice were lower than control mice, the total B cell number in the spleen of c-Cbl–/–BLNK–/– mice was still ~3-fold lower than the heterozygous control, although 3- to 4-fold higher than c-Cbl+/–BLNK–/– mice (Table II; c-Cbl+/–BLNK+/–, 4.3 ± 0.2 x 107; c-Cbl–/–BLNK+/–, 3.8 ± 0.2 x 107; c-Cbl+/–BLNK–/–, 0.5 ± 0.12 x 107; c-Cbl–/–BLNK–/–, 1.7 ± 0.4 x 107). Examination of B cell subsets by IgM and IgD costaining for T1, T2, and mature follicular (FO) B cells (Fig. 5a), and CD21 combined with CD23 staining for marginal zone (MZ) and FO B cells (Fig. 5b) showed that all subsets were, to different extents, increased in c-Cbl–/–BLNK–/– mice compared with c-Cbl+/–BLNK–/– mice (Table II). It is noteworthy that the proportion of T2 B cells (IgMhighIgDhigh) in c-Cbl+/–BLNK–/– mice was lower than that in control mice, while this population in c-Cbl–/–BLNK–/– mice reached normal levels (Fig. 5a, Table II: c-Cbl+/–BLNK+/–, 10.0 ± 0.6%; c-Cbl–/–BLNK+/–, 10.3 ± 1.6%; c-Cbl+/–BLNK–/–, 5.5 ± 0.5%; c-Cbl–/–BLNK–/–, 12.3 ± 2.0%). The splenic IgMhighIgDlow/– population contains T1 immature B cells and mature MZ B cells. When the IgMhighIgDlow/– population was analyzed with AA4.1 staining to distinguish immature (AA4.1+) and mature (AA4.1–) B cells, it was found that the percentages of both T1 and MZ B cells in the double knockout mice were normalized to control levels (data not shown). Although the proportions of T1, T2, and MZ B cells from c-Cbl–/–BLNK–/– mouse spleen were normalized, the percentage of mature FO B cells (IgMlowIgDhigh) was still significantly lower than control group (Fig. 5a and Table II), indicating that a developmental block still exists at the transition from T2 to mature FO B cells, but not to MZ B cells


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  		Table II. B cell subpopulations in the spleena

 

Figure 5
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  		FIGURE 5. Phenotypic analysis of peripheral B cells. a, IgM and IgD costaining was performed to distinguish different developmental stages of splenic B cells. b, MZ B cells were distinguished from FO B cells by CD21 and CD23 costaining. c, Peritoneal B1 B cells were revealed by CD5 and IgM staining. All analysis was based on lymphocyte-gated cells. The result from one of five independent experiments is shown.

 
As previously reported (14), peritoneal B1 B cells (CD5+IgM+) are completely absent in BLNK–/– mice. We found that there was no rescue of B1 B cell development by c-Cbl-inactivation (Fig. 5c). Taken together, these findings indicate that c-Cbl-deficiency partially rescued conventional B2 B cell, but not B1 B cell development.

c-Cbl inactivation induces elevated and prolonged pre-BCR signaling

In B cell lines, c-Cbl has been shown to negatively regulate several signaling molecules downstream of BCR, including Syk and Lyn (31, 35, 36). However, studies of c-Cbl-deficient splenic B cells have given different results: c-Cbl positively regulates Syk, Btk, and ERK activation and Ca2+ mobilization, but negatively regulates Lyn, PI3K, and Akt (27). To determine how c-Cbl modulates pre-BCR-signaling in BLNK-deficient pre-B cells, pre-BCR+ pre-B cells derived from c-Cbl+/–BLNK–/– and c-Cbl–/–BLNK–/– fetal liver were stimulated by pre-BCR cross-linking with anti-µ F(ab')2 Ab, and total tyrosine phosphorylation was analyzed by immunoblotting with a phosphotyrosine-specific Ab (4G10). Pre-B cells from Cbl+/–BLNK+/– and Cbl–/–BLNK+/– mice could not be included in this analysis due to difficulty in generating pre-BCR+ cell lines from these genotypes. Pre-B cells from Cbl+/–BLNK–/– and Cbl–/–BLNK–/– mice expressed equivalent cell surface levels of pre-BCR/{lambda}5 and CD19 (data not shown). In both Cbl+/–BLNK–/– and Cbl–/–BLNK–/– pre-B cells, pre-BCR engagement consistently induced tyrosine phosphorylation of multiple bands (Fig. 6a). For both groups, the peak intensity of overall phosphorylation signaling was 2 min after stimulation. However, pre-BCR signaling in c-Cbl–/–BLNK–/– pre-B cells was more sustained (at 5, 10, and 20 min) than that in BLNK–/– pre-B cells. One exception was a band at ~110 kDa that appears prominently in BLNK–/– and is absent in c-Cbl–/–BLNK–/– pre-B cells. Immunoprecipitation with anti-c-Cbl Ab demonstrated that this band was c-Cbl (data not shown).


Figure 6
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  		FIGURE 6. Pre-BCR cross-linking in c-Cbl–/–BLNK–/– compared with BLNK–/– pre-B cells induced increased and prolonged tyrosine-phosphorylation signaling. A total of 2 x 107 pre-BCR+ pre-B cells of 5–8 mo of culture from c-Cbl–/–BLNK–/– and c-Cbl+/–BLNK–/– mice were incubated at 37°C for the indicated time with F(ab')2 Abs specific for mouse µ chain. The cells were then washed and lysed, and a portion of the total cell lysates analyzed for tyrosine phosphorylation by immunoblotting with mAb 4G10 (a). For specific measurement of Btk, total cell lysates were blotted with phosphospecific anti-Btk (pY551) and anti-Btk Abs (d). For measurements of Syk and PLC-{gamma}2, lysates were precipitated with Abs specific for Syk or PLC-{gamma}2, and the precipitates were analyzed by immunoblotting with anti-phosphotyrosine mAb 4G10, anti-Syk or anti-PLC-{gamma}2 as indicated (b and e). For measurement of tyrosine phosphorylated Lyn, lysates were precipitated with anti-phosphotyrosine mAb 4G10 and immunoblotted with anti-Lyn Ab (c). The signal intensity was analyzed with ImageQuant program (Molecular Dynamics) and the phosphorylation signals of Syk, Btk, and PLC-{gamma}2 were normalized based on total protein level of the molecules. Level of activation was expressed as ratio of stimulated samples vs unstimulated samples. In the experiment shown in c, the intensity of blotting of the total cell lysates with anti-Lyn was 1.3 times in the c-Cbl–/–BLNK–/– compared with the c-Cbl+/–BLNK–/– cells (data not shown). Results shown are representative of two independent experiments.

 
Immunoprecipitation was then used to assess tyrosine phosphorylation of specific molecules in response to pre-BCR engagement. Phosphorylation of Syk, a critical mediator of pre-BCR function was first assessed by precipitation of lysates with anti-Syk Ab followed by immunoblotting with anti-phosphotyrosine mAb 4G10. Pre-BCR cross-linking triggered stronger and more sustained phosphorylation of Syk in c-Cbl–/–BLNK–/– compared with the response of BLNK–/– pre-B cells (Fig. 6b). The enhanced and prolonged phosphorylation of Syk in c-Cbl–/–BLNK–/– pre-B cells was confirmed by confocal microscopy (Fig. 7) and by flow cytometry (data not shown). These data indicate that Cbl negatively regulates tyrosine phosphorylated Syk in response to pre-BCR signaling. Because Syk phosphorylation can be regulated by Lyn kinase, Lyn activation was analyzed by immunoprecipitation with anti-phosphotyrosine mAb 4G10, followed by immunoblotting for Lyn. Both basal and the receptor stimulated phosphorylation of Lyn were enhanced in c-Cbl–/–BLNK–/– pre-B cells, indicating an increase in this very proximal component of pre-BCR signaling (Fig. 6c).


Figure 7
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  		FIGURE 7. Pre-BCR cross-linking in c-Cbl–/–BLNK–/– pre-B cells induced a stronger and more prolonged Syk-phosphorylation than BLNK–/– pre-B cells by confocal microscopic study. Pre-BCR+ pre-B cells were allowed to settle on cover slides on ice for 40 min, warmed to 37°C, and stimulated as described in Fig. 6. At the indicated time points after stimulation, cells were fixed with 4% paraformaldehyde for 15 min, quenched with 50 µM NH4Cl for 10 min, followed by 30 min of permeabilization with 0.05% saponin. The cells were then blocked with 5% goat serum in PBS for 30 min and incubated overnight at 4°C with rabbit anti-Syk or anti-phospho-Syk (Y519/520-specific) Abs. Staining was resolved with goat anti-rabbit IgG-Alexa 488 at RT for 40 min. The cells were imaged with a Zeiss LSM 510 META confocal microscope. a-d, Representative images at 5 min of stimulation. e, The MFI of phosphor-Syk (Alexa 488 staining) from different time points was analyzed with MetaMorph software and the MFI was calculated based on the intensity of over 200 cells from five different view fields. Results are expressed as mean ± SE as calculated with Excel software.

 
It has been proposed that, in addition to a BLNK-dependent mechanism for pre-BCR signaling, there exists a distinct BLNK-independent but Btk-mediated pathway (37). We therefore examined the possibility that the ability of c-Cbl inactivation to restore pre-BCR signaling in BLNK-deficient cells might be mediated through Btk activation. Baseline phosphorylation of Btk, before in vitro activation, was elevated in c-Cbl–/–BLNK–/– pre-B cells relative to BLNK–/– pre-B cells. Cross-linking of the pre-BCR resulted in increased tyrosine phosphorylation of Btk, and the strength of this response was increased in c-Cbl–/–BLNK–/– pre-B cells over the response in BLNK–/– pre-B cells, consistent with a role for Btk in mediating the effect of c-Cbl inactivation (Fig. 6d). Phosphorylation of PLC-{gamma}2, proposed to be involved in both BLNK-dependent and BLNK-independent pathways of pre-BCR signaling (37), was also increased in c-Cbl–/–BLNK–/– pre-B cells (Fig. 6e).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Previous studies of c-Cbl-deficient mice suggested that c-Cbl may affect B cell development (27). However, it has not previously been determined at which stage and how c-Cbl carries out its role in regulating this process. In the current study, we analyzed B cell development in c-Cbl–/–BLNK–/– mice, and found that c-Cbl most strongly affects early B cell development in the BM. Specifically, c-Cbl inactivation corrected several components of the impaired B cell developmental process in BLNK–/– mice, including: 1) down-regulation of the expression of pre-BCR and CD43 molecules on early pre-B cell surface; 2) up-regulation of cell surface MHC class II expression on pre-B cells; 3) initiation of {kappa}-chain rearrangement in small pre-B cells; and 4) transition of pre-B cells to surface IgM+ immature B cells. However, c-Cbl appears to have less effect on other developmental events, such as down-regulation of surface BP-1 and up-regulation of CD25 and CD2 expression. In addition, the transition from T2 B cells to mature B cells was not rescued in c-Cbl–/–BLNK–/– mice. c-Cbl thus differentially regulates distinct B cell development events.

The expression of pre-BCR on the surface of early pre-B cells elicits signals that are essential for B cell development. A critical proximal event in pre-BCR signaling is mediated by the tyrosine kinase Syk, as evidenced by the essentially complete abrogation of B cell development that results from inactivation of Syk. However, it appears that at least two independent pathways exist downstream of Syk, each of which is able to support a significant though reduced level of B cell development (37, 38). One of these pathways is BLNK dependent, and its function is reflected in the substantial though incomplete block in B cell development observed in BLNK-deficient mice. The existence of a second, BLNK-independent, pathway(s) is suggested by the incomplete nature of this developmental block, and is further supported by analysis of compound genetic ablations. It has been demonstrated that Btk deficiency results in a partial developmental defect, but that inactivation of both BLNK and Btk results in a profound and essentially complete arrest, resembling that which results from Syk deficiency. The effect of c-Cbl inactivation presented here can be assessed in the context of these multiple pathways of pre-BCR signaling. The observation that c-Cbl inactivation enhances tyrosine phosphorylation of Syk and Lyn in BLNK-deficient pre-B cells suggests a proximal effect on strength of signaling that could enhance early B cell development through multiple downstream pathways and signaling events. The inactivation of Cbl could remove a negative feedback loop thereby increasing the activation of Lyn and the molecules that are to its downstream such as Syk, Btk and PLC-{gamma}2. In addition, however, the finding of increased Btk phosphorylation suggests that enhancement of a Btk-dependent pathway might be a specific mechanism by which c-Cbl inactivation affects signaling and B cell development. The enhancement of Btk response could reflect a direct interaction of c-Cbl and Btk and/or an indirect consequence of enhanced proximal events. Additional evidence supporting a critical role of Btk in the restoration of B cell development by c-Cbl inactivation has been generated in our recent finding that the ability of c-Cbl inactivation to restore B cell development in BLNK-deficient mice is completely abrogated by inactivation of Btk (data not shown).

Two independent pre-BCR signaling pathways downstream of Syk have been proposed to mediate specific events important in B cell development (37, 38). A BLNK-independent pathway exists, which converges with IL-7R signaling to activate ERK and thus confers on pre-BCR+ B cells a lower threshold for proliferative response to IL-7 stimulation than that observed in pre-BCR-negative pre-B cells (33). A Syk-dependent, BLNK-independent pathway also mediates allelic exclusion (19, 39). In contrast, it has been proposed that a second signaling pathway downstream of Syk is BLNK-dependent and that this pathway is essential for down-regulation of pre-BCR expression, and thus for suppression of pre-B cell expansion (16, 32, 38, 40). BLNK-dependent pathways have also been shown to induce the modulation of other cell surface molecules and to stimulate L chain rearrangement (16, 34, 38, 41, 42). In BLNK–/– mice, pre-BCR down-regulation is blocked, leading to a prominent accumulation of pre-BCR+ pre-B cells in the BM (32). Our studies revealed that c-Cbl inactivation corrects the deficiency in down-regulation of pre-BCR in BLNK–/– mice, indicating that surface pre-BCR expression can be regulated not only by a BLNK-dependent pathway, but also by a BLNK-independent, c-Cbl-modulated pathway. Similarly, the expression of CD43 and MHC-II, as well as the induction of L chain rearrangement, another critical step in B cell differentiation, are also modulated by BLNK-independent, c-Cbl-modulated pathways, whereas the expression of CD25, CD2, and BP-1 is relatively unaffected by c-Cbl expression. The results of our studies reinforce the differential effects of specific signaling pathways on distinct aspects of differentiation and indicate the complex plasticity of signaling regulation during B cell development. When BLNK is present, the role of c-Cbl in early B cell development is not apparent. However, in BLNK-deficient mice, c-Cbl-modulated signaling pathways can regulate critical B cell development events.

It has been suggested that BLNK functions as a tumor suppressor, as BLNK-deficient pre-B cells display a hyperproliferative response to IL-7 signaling, and pre-B cell lymphomas appear at increased frequency in BLNK-deficient mice (32). Furthermore, BLNK mutations have also been observed in ~50% of the human pre-B cell lymphomas, further supporting the conclusion that BLNK acts as a tumor suppressor gene (43). Because inactivation of c-Cbl reversed the hyperproliferative responses of BLNK-deficient pre-B cells to IL-7 stimulation in our studies, it can be postulated that the pre-B cell tumors induced by BLNK-deficiency would be prevented by c-Cbl-inactivation. In addition, pre-B cell tumors have also been observed in BLNK–/–Btk–/– mice at a frequency higher than that in BLNK–/– mice, suggesting Btk may cooperate with BLNK in mediating tumor suppressor function (16). Studies on possible protective effects of c-Cbl inactivation on pre-B cell tumors induced by impaired BCR signaling in those mice are in progress. A number of mechanisms may explain the reversal of IL-7 hyperresponsiveness of BLNK–/– pro-B/pre-B cells by c-Cbl inactivation. The decreased proportion of pre-BCR+ cells in c-Cbl–/–BLNK–/– BM could reduce the frequency of cells that are hyperresponsive to IL-7. The reduced density of cell surface IL-7R{alpha} that we have observed on c-Cbl–/–BLNK–/– BM B cells relative to that expressed in BLNK–/– mice may also contribute to the decreased response.

c-Cbl is an adaptor protein with multiple functions. One major role of c-Cbl is to mediate the ubiquitination and subsequent degradation of its target proteins. In B cells, several signaling proteins including Syk and Lyn have been described as targets of c-Cbl. Syk-c-Cbl association is induced by BCR ligation and c-Cbl-phosphorylation (31). c-Cbl is itself a substrate for phosphorylation by Syk and Lyn (2, 25, 44), and it is conceivable that activated Syk and Lyn induce a feedback down-regulation of their own protein levels by mediating c-Cbl phosphorylation and in turn enhancing c-Cbl-dependent degradation of target molecules. Our studies showed that tyrosine phosphorylation of the proximal signaling molecules Lyn and Syk, and their downstream substrates, Btk and PLC-{gamma}2, together with additional unidentified proteins, was elevated in c-Cbl–/–BLNK–/– pre-B cells compared with c-Cbl+/–BLNK–/– pre-B cells. As noted above, it is possible that a Btk-PLC-{gamma}2 pathway mediates the corrected phenotypes that we observed in c-Cbl–/–BLNK–/– mice, a possibility supported by our recent finding that inactivation of Btk abrogates the ability of c-Cbl inactivation to restore B cell development in BLNK-deficient mice (data not shown). These results suggest that different events downstream of pre-BCR signaling have different thresholds in the strength and duration of signaling and that elevated and/or prolonged Lyn and Syk activation resulting from c-Cbl inactivation can bypass the BLNK-dependent signaling pathway to induce critical differentiation events including the down-regulation of pre-BCR and up-regulation of MHC-II, as well as initiation of L chain rearrangement. In contrast, activation of BLNK-dependent pathways may be essential for the up-regulation of CD25 and CD2, and down-regulation of BP-1. It has been widely proposed that BCR signaling strength determines peripheral B cell fate, specifically that B1 B cell development requires the strongest BCR signaling; that an intermediate-high level of BCR signaling favors follicular B cell development; and that weak BCR signaling directs B cell development to marginal zone B cells (10, 45, 46). We propose that pre-BCR signaling strength and duration similarly determine distinct downstream events during pre-B cell transition.

Studies using genetic approaches suggest that pre-BCR signaling pathways may be particularly redundant. Many molecules, such as Zap70, LAT, and SLP-76, that are expressed in T cells, but not in mature B cells, are expressed in pre-B cells; and it appears that these molecules can partially replace their B lineage counterparts in mediating signaling transduction during pre-B cell transition (18, 47, 48, 49, 50). Therefore, a finely tuned and redundant signaling network may be particularly important in pre-BCR signaling and early B cell development, and it is possible that c-Cbl plays a more important role in this early signaling network than in mature B cells.

Although c-Cbl inactivation selectively corrected some critical events in the early stages of BM B cell development in BLNK–/– mice, it did not correct the block from T2 B cells to mature B cells in the spleen of those mice. Splenic B cell development takes place in discrete steps from transitional T1 stage to T2 stage, then to the mature B cell stage (10). The sequential progression from T1 to T2 to mature B cells may require an increasingly higher threshold level of BCR signaling. T1 cells require a low-threshold "tonic BCR signal" to generate T2. In contrast, a relatively higher level of BCR signaling is required for the transition from T2 B cells to mature B cells (10). Thus, the failure of c-Cbl inactivation to rescue development of mature B cells in BLNK–/– mice may reflect a failure to achieve the strength of BCR-dependent signaling required for this transition. c-Cbl deficiency enhances the positive selection of TCR-transgenic CD4+ T cells (28), probably due to elevated Lck and Zap70 signaling. The role of c-Cbl in CD4+ T cell development and selection is also strongly supported by studies of c-Cbl–/–SLP-76–/– mice, in which the lethality as well as defective development of CD4+ T cells observed in SLP-76–/– mice is rescued. In contrast, inactivation of another close member of the c-Cbl family, Cbl-b, did not rescue CD4+ T cell development (51). In parallel to what has been observed in early T cell differentiation, we show here that c-Cbl regulates important events in early B cell development, whereas we have observed that Cbl-b inactivation did not correct the impaired B cell development in BLNK–/– mice (our unpublished data). Thus, we conclude that c-Cbl, but not Cbl-b, regulates B cell development, prominently in a BLNK-independent manner during early stages of B cell development.


   Acknowledgments
 
We thank Genevieve Sanchez and staff at Bioqual for excellent animal care; Susan Sharrow, Larry Granger, and Tony Adams for expert assistance with FACS sorting; and Drs. Rudi W. Hendriks, Susan Pierce, and Jan Cerny for critical reading and comments on the manuscript. We also thank Michael Kruhlak for assistance with the Zeiss confocal laser scanning microscope manipulation and data analysis; Steve Bauer and Craig Milne for advice on culture of pre-B cell lines; Hae Won Sohn for advice on immunofluorescence staining; and Mary Robinson for technical assistance with biochemistry studies.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Intramural Research Program of the National Cancer Institute, Center for Cancer Research and the National Institute of Dental Research, National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Richard J. Hodes, National Institute on Aging, National Institutes of Health, Building 10, Room 4B10, 9000 Rockville Pike, Bethesda, MD 20892. E-mail address: hodesr{at}31.nia.nih.gov Back

3 Abbreviations used in this paper: PTK, protein tyrosine kinase; BLNK, B cell linker protein; PLC, phospholipase C; LAT, linker for activation of T cells; SH, Src homology; BM, bone marrow; MFI, mean fluorescence intensity; MZ, marginal zone; FO, follicular; pre-BCR, precursor BCR; c-Cbl, Casitas B-lineage lymphoma proto-oncogene c; SLC, surrogate L chain. Back

Received for publication May 22, 2006. Accepted for publication November 8, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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