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The Journal of Immunology, 2006, 176: 4690-4698.
Copyright © 2006 by The American Association of Immunologists

Phospholipase C{gamma}2 Dosage Is Critical for B Cell Development in the Absence of Adaptor Protein BLNK1

Shengli Xu*, Jianxin Huo*, Weng-Keong Chew*, Masaki Hikida{dagger}, Tomohiro Kurosaki{dagger} and Kong-Peng Lam2,*

* Laboratory of Immunology, Center for Molecular Medicine and Institute of Molecular and Cell Biology, Singapore; and {dagger} Laboratory of Lymphocyte Differentiation, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
B cell linker (BLNK) protein and phospholipase C{gamma}2 (PLC{gamma}2) are components of the BCR signalosome that activate calcium signaling in B cells. Mice lacking either molecule have a severe but incomplete block in B lymphopoiesis. In this study, we generated BLNK–/–PLC{gamma}2–/– mice to examine the effect of simultaneous disruption of both molecules on B cell development. We showed that BLNK–/–PLC{gamma}2–/– mice had compounded defects in B cell maturation compared with either single mutant, suggesting that these two molecules cooperatively or synergistically signaled B lymphopoiesis. However, Ig H chain allelic exclusion was maintained in single and double mutants, indicating that signals propagated by BLNK and PLC{gamma}2 were not involved in this process. Interestingly, in the absence of BLNK, B cell development was dependent on plc{gamma}2 gene dosage. This was evidenced by the proportionate decrease in splenic B cell population and increase in bone marrow surface pre-BCR+ cells in PLC{gamma}2-diploid, -haploid, and -null animals. Intracellular calcium signaling and ERK activation in response to BCR engagement were also proportionately decreased and delayed, respectively, with stepwise reduction of plc{gamma}2 dosage in a BLNKnull background. Thus, these data indicate the importance of BLNK not only as a conduit to specifically channel BCR-signaling pathways and as a scaffold for the assembling of macromolecular complex, but also as an efficient aggregator or concentrator of PLC{gamma}2 molecules to effect optimal signaling for B cell generation and activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Signals propagated by the BCR and its precursor, the pre-BCR, are critical for the development and activation of B lymphocytes (1, 2). The BCR is a multiprotein structure containing the signal-transducing Ig{alpha} and Igbeta subunits that are noncovalently linked to the membrane-bound Ig, which is itself composed of the rearranged Ig H and L chains (3). Engagement of the BCR is known to activate numerous intracellular-signaling pathways that ultimately lead to diverse gene expressions and different cellular responses (4).

One of the major downstream-signaling pathways triggered by BCR cross-linking is the Ca2+-signaling pathway in B lymphocytes (5). The activation of Ca2+ flux is envisioned to require the assembly of a BCR signalosome that comprises the spleen tyrosine kinase (Syk) and Bruton’s tyrosine kinase (Btk),3 the adaptor protein B cell linker (BLNK) and phospholipase {gamma}2 (PLC{gamma}2; Refs.6 and 7). The sequence of events in this model is as follow: BCR engagement activates Syk and Btk, and Syk phosphorylates BLNK, thereby enabling the latter to bind Btk and PLC{gamma}2. The recruited PLC{gamma}2 is subsequently activated by both Syk and Btk, and hydrolyzes phosphatidylinositol-4, 5-bisphosphate to yield inositol-1, 4, 5-triphosphate and diacylglycerol. Inositol-1, 4, 5-triphosphate and diacylglycerol then mediate the release of calcium from intracellular stores and the activation of protein kinase C, respectively.

Btk–/–, BLNK–/–, and PLC{gamma}2–/– mice all have a similar xid-like phenotype and this is in general support of the BCR signalosome hypothesis. These mutant mice manifested a partial block in bone marrow B lymphopoiesis, lacked CD5+ B-1 cells, and were unable to respond to T cell-independent type II Ags (8, 9, 10, 11, 12, 13, 14, 15). B cells from all three mutant mice had impaired Ca2+ signaling (10, 11, 15, 16), and were unable to activate the NF-{kappa}B-signaling pathway in response to BCR stimulation (17, 18, 19, 20). These data suggested that Btk, BLNK, and PLC{gamma}2 were likely to be important components of a common BCR-signaling pathway (7).

Despite the similar xid-like phenotype in the individual Btk, BLNK, PLC{gamma}2 single mutant mice, it remains to be determined whether these three molecules reside and function as a single signaling complex, or whether they have other functions independent of each other during B cell maturation. The recent characterization of BLNK–/–Btk–/– mice indicated that BLNK and Btk had synergistic roles in early B cell generation as the double mutant exhibited a more severe block in B lymphopoiesis (21). To determine whether other combinations of double mutant mice would have similar outcomes, we generated mice deficient in both BLNK and PLC{gamma}2. Here, we found that B cell development was also more severely blocked in the BLNK/PLC{gamma}2 double mutant mice, suggesting that BLNK and PLC{gamma}2 together synergistically signal B cell development. Moreover, in the absence of BLNK, the gene dosage of plc{gamma}2 became critical for B cell generation and activation, thus providing genetic evidence for the importance of BLNK as a "concentrator" of PLC{gamma}2 molecules in BCR signaling.


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

BLNK–/– (13), PLC{gamma}2–/– (15), and VHB1-8f (22) mice were generated previously and used at 6–12 wk of age according to institutional guidelines.

Cell preparation and culture

Bone marrow cells were obtained by injecting PBS containing 3% FCS and 0.1% NaN3 into the femurs and tibia of mice. Splenic cells were obtained by dissociating the tissue with a plastic mesh and rubber stopper. All cells were treated with RBC lysing solution (0.15 M NH4Cl, 1 mM KHCO3, and 0.1 mM Na2EDTA). For in vitro survival assay, cells were incubated in RPMI 1640 medium supplemented with 10% FCS.

Flow cytometry

Cells were stained with optimal amounts of FITC-, PE-, and biotin-conjugated mAbs for 10 min on ice and washed with PBS. Biotin-conjugated mAbs were revealed with streptavidin-CyChrome. FACS analyses were performed on a FACScan (BD Biosciences) using CellQuest software. The following mAbs were obtained from BD Pharmingen: anti-IgM (R6-60.2), anti-IgD (11-26c.2a), anti-B220 (RA3-6B2), anti-{kappa} (R5-240), anti-{lambda} (LS136), anti-pre-BCR (SL156), anti-CD25 (7D4), CD43 (S7), BP-1 (6C3), and MHC class II (M5/114.15.2).

B cell stimulation and immunoblotting

Splenic B cells from wild-type and mutant mice were purified by negative selection using an AUTOMACS cell sorter (Miltenyi Biotec) with anti-CD43 mAb-coupled magnetic microbeads. The purity of B cells obtained was constantly >90% as assessed by anti-B220 and anti-IgM mAb staining in FACS analysis. For B cell stimulation, 3 x 106 cells suspended in OPTI-MEM I (Invitrogen Life Technologies) were incubated with different doses of goat anti-mouse IgM F(ab')2 Ab (Jackson ImmunoResearch Laboratories) at 37°C for various periods of time. After washing with cold PBS, cells were lysed on ice for 30 min in a buffer (1% Nonidet P-40, 10 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA 0.2 mM Na3VO4) supplemented with protease inhibitors. The detergent-insoluble membrane (DIM) fraction was prepared as described (23). Immunoblotting was performed as described previously (19). Anti-phospho-ERK, anti-ERK, anti-PLC{gamma}1, anti-PLC{gamma}2 Abs were purchased from Santa Cruz Biotechnology. Anti-phospho-PLC{gamma}1 (Tyr783), anti-phospho-PLC{gamma}1 (Tyr771), anti-phospho-PLC{gamma}2 (Tyr759), and anti-phospho-PLC{gamma}2 (Tyr1217) Abs were obtained from Cell Signaling Technology.

Analyses of IgH gene rearrangements

Genomic DNA was obtained from CD19+ IgL– bone marrow B cells of wild-type and various mutant mice harboring a VHB1–8 IgH gene inserted into its physiological locus. PCR was performed as previously described to detect VHJ558 gene rearrangements and the amplified products were analyzed via Southern blotting using a 32P-labeled JH3-JH4 probe (24).

Measurement of calcium flux

Intracellular calcium concentration was measured using fura-2 acetoxymethyl ester (fura-2 AM) as described previously (25). Briefly, 5 x 106 purified B cells were suspended in loading buffer (20 mM HEPES, 5 mM glucose, 0.025% BSA, and 1 mM CaCl2, in PBS) and incubated with fura-2 AM (Molecular Probes) at 37°C for 45 min. Cells were washed twice and adjusted to 106 cells/ml with loading buffer. After 20 min of equilibration at room temperature, 2 x 106 cells were stimulated with 20 µg/ml goat anti-mouse IgM F(ab')2 Ab. Calcium flux was monitored using a spectrofluorometer (PerkinElmer LS-50B) at an emission wavelength of 515 nm and alternate excitation wavelengths of 340 and 380 nm. Calibration and calculation of calcium levels were performed as described (25).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
BLNK–/– mice have more severe B cell developmental defects than PLC{gamma}2–/– mice

Separate analyses of BLNK–/– or PLC{gamma}2–/– mice by various groups indicate that mice lacking either molecule have a similar xid-like phenotype, namely impaired B cell development, lack of CD5+ B-1 cells, and an inability to respond to T cell-independent type II Ags (10, 11, 12, 13, 14, 15). To determine whether BLNK–/– and PLC{gamma}2–/– mice have identical defects in B cell development, we directly compared the two mutant mice. As shown in Fig. 1A, BLNK–/– mice had significantly less fractions of immature and mature B cells in their bone marrow compared with wild-type or PLC{gamma}2–/– mice. In addition, both BLNK–/– and PLC{gamma}2–/– mice showed an increased fraction of the B220+ IgL– cells compared with control animals. The B220+ IgL– B cells comprise both pro-B and pre-B cells and as pro-B cells differentiate into large pre-B and later into small pre-B cells, they modify the expression of certain cell surface markers. In particular, they down-regulate the expression of sialoglycoprotein CD43 and the metallopeptidase BP-1, and up-regulate the expression of CD25 (IL-2R{alpha}) and MHC class II Ags (26). As shown in Fig. 1B, the majority of B220+ IgL– cells in wild-type mice were CD43–, BP-1–, CD25+ and MHC class II+ small pre-B cells. However, the B220+ IgL– cells in BLNK–/– mice expressed significantly higher amounts of CD43 and BP-1 and very little MHC class II Ags and CD25, suggesting that these cells were blocked at an early transition stage before the small pre-B cell stage, most likely at the late pro-B or large pre-B cell stage. However, the phenotype of B220+IgL– B cells in PLC{gamma}2–/– mice was intermediate between that of wild-type and BLNK–/– mice as they were CD43low and MHC class II+ like wild-type cells but were BP-1+ and CD25low like BLNK-deficient cells, indicating that PLC{gamma}2-deficient B cells were arrested in development compared with wild-type B cells but were less severely affected compared with BLNK-deficient B cells.


Figure 1
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  		FIGURE 1. Comparison of B cell development in wild-type, BLNK–/–, and PLC{gamma}2–/– mice. Mice of various genotypes were stained for (A) B220 and Ig{kappa} or {lambda}-expressing cells in the bone marrow, and (B) bone marrow B220+IgL– cells were further stained for their expression of the various differentiation markers, and (C) for the surface expression of the pre-BCR. D, Splenic cells of wild-type, BLNK–/–, and PLC{gamma}2–/– mice were stained for the expression of B220 and IgM; and (E) the absolute numbers of B cells present in the spleens of these mice were graphed. Numbers indicate percent of lymphocytes for A and D and percent of bone marrow B220+IgL– cells for C. Figures shown are representative of five independent analyses.

 
During development, B cell precursors express the pre-BCR, which is a critical regulator of early B lymphopoiesis (4). However, the cell surface expression of pre-BCR is difficult to detect on normal B cells. As shown previously (21) and confirmed here, a significant fraction of the B220+IgL– cells in BLNK–/– mice expressed the pre-BCR on their cell surfaces (Fig. 1C). In contrast, the surface pre-BCR-positive cells were a very minor population in PLC{gamma}2–/– mice and were hardly detectable in wild-type mice. These data again demonstrate a key difference in the effect of these two gene mutations on early B lymphopoiesis.

The more severe defect in B cell development in BLNK–/– mice was also evident by the much reduced B cell population in the spleen of these mice compared with that of PLC{gamma}2–/– mice (Fig. 1D). Compared with wild-type animals, PLC{gamma}2–/– and BLNK–/– mice had ~30 and 90% reduction in total splenic B cell number, respectively (Fig. 1E). Thus, the data indicate that both BLNK–/– and PLC{gamma}2–/– mice have a partial block in B cell development but with the arrest of B lymphopoiesis in BLNK–/– mice being more severe. In addition, BLNK but not the PLC{gamma}2 mutation resulted in the constitutive surface expression of the pre-BCR on B cell precursors. Thus, BLNK appears to play a more important role than PLC{gamma}2 in regulating early B cell development.

Normal PLC{gamma}1 activation in BLNK-deficient B cells

Although PLC{gamma}2 is the major isoform of PLC found in B cells, it has been shown that PLC{gamma}1, which is ubiquitously expressed and is the major isoform found in T cells, is also expressed and activated by BCR engagement in human B cells (27, 28). It is possible that BLNK is involved in the activation of PLC{gamma}1, in the same manner as it is involved in the activation of PLC{gamma}2 (19). This would provide an explanation to the more severe phenotype of BLNK–/– mice compared with PLC{gamma}2–/– mice. To assess this possibility, we examined PLC{gamma}1 expression and activation in B cells of wild-type and BLNK–/– mice.

As shown in Fig. 2A, PLC{gamma}1 was expressed in both wild-type and BLNK-deficient B cells. Next, we examined BCR-induced tyrosine phosphorylation of PLC{gamma}1 using site-specific Abs. Previous studies have established that the phosphorylation of Tyr783 is essential for the activity of PLC{gamma}1, whereas the phosphorylation of Tyr771 might play a negative role in the activation of PLC{gamma}1 (29). We found that the phosphorylation of PLC{gamma}1 on both tyrosines was comparable in wild-type and BLNK-deficient B cells in response to BCR stimulation (Fig. 2A), indicating that BLNK deficiency does not alter BCR-induced PLC{gamma}1 activation. Furthermore, we showed here that upon BCR engagement, there was an increased amount of PLC{gamma}1 in the lipid raft-containing DIM fractions obtained from both wild-type and BLNK–/– B cells (Fig. 2B), suggesting that PLC{gamma}1 is still able to be recruited to lipid raft upon BCR stimulation in the absence of BLNK. Recent studies have shown that linker for activation of T cells (LAT), a lipid raft-anchored adaptor protein that is mainly expressed in T cells and plays an essential role in TCR-induced PLC{gamma}1 activation (30), is also expressed in B cells and involves along with BLNK in pre-BCR signaling (31). Thus, we examined whether the membrane recruitment of PLC{gamma}1 could be via LAT. As shown in Fig. 2C, both BLNK and LAT associated with PLC{gamma}1 upon BCR engagement, indicating that both BLNK and LAT contribute to the lipid raft translocation and activation of PLC{gamma}1. Taken together, our data suggest that the more severe phenotype of BLNK–/– mice than that of PLC{gamma}2–/– mice is likely due to defects of other BLNK-mediated signaling pathways rather than the activation of PLC{gamma}1, which could still occur in the absence of BLNK.


Figure 2
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  		FIGURE 2. Normal expression and activation of PLC{gamma}1 in wild type and BLNK–/– B cells. A, Western blot analysis of PLC{gamma}1 expression and activation in wild-type and BLNK–/– B cells. Cells were stimulated for various periods of time with anti-IgM F(ab')2 Ab and whole cell lysates were probed with anti-PLC{gamma}1 or site-specific anti-phospho-PLC{gamma}1 Abs as indicated. B, Recruitment of PLC{gamma}1 to lipid raft-containing DIM fractions following BCR stimulation. DIM fractions from untreated and anti-IgM treated BLNK+/+ and BLNK–/– B cells were examined for the presence of PLC{gamma}1 molecules using anti-PLC{gamma}1 Ab. Anti-Lyn blot was included as loading control. C, Association of PLC{gamma}1 with BLNK and LAT following BCR stimulation in wild-type B cells. PLC{gamma}1 was immunoprecipitated from whole cell lysates of wild-type B cells that were untreated and stimulated with 20 µg/ml anti-IgM F(ab')2 Ab for 5 min. The blot was probed with anti-BLNK and anti-LAT Abs to examine the interaction of PLC{gamma}1 with BLNK and LAT. The results shown are representative of five independent analyses.

 
BLNK–/–PLC{gamma}2–/– mice exhibit compounded B cell developmental defects

The impaired BCR-induced PLC{gamma}2 phosphorylation in BLNK–/– B cells (19) together with the normal BLNK phosphorylation in PLC{gamma}2–/– B cells (14) would place PLC{gamma}2 downstream of BLNK in BCR signaling. The less severe phenotype of PLC{gamma}2–/– mice compared with BLNK–/– mice, as shown in Fig. 1, is also consistent with this notion. To further determine whether PLC{gamma}2 functions solely downstream of BLNK in BCR signaling or whether it also acts synergistically with BLNK to signal B cell development, we generated mice deficient for both BLNK and PLC{gamma}2.

As shown in Fig. 3, BLNK–/–PLC{gamma}2–/– mice exhibited compounded B cell developmental defects compared with either single mutant. The percentage of Ig+ B cells in the bone marrow of the double mutant was reduced 3- to 5-fold compared with BLNK–/– and PLC{gamma}2–/– mice, respectively (Fig. 3A). In addition, the fraction of splenic B cells in the double mutant was 20-and 10-fold reduced compared with wild-type and single BLNK or PLC{gamma}2 mutant mice (Fig. 3B). The B cell developmental defect in the double mutant was even more drastic compared with wild type or single mutant if one takes into account the absolute number of B lymphocytes present in these mice (Table I). The compounded defects in the double mutant suggest that PLC{gamma}2 and BLNK might not simply lie within a linear pathway and PLC{gamma}2 may have some functions independent of BLNK during B cell development.


Figure 3
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  		FIGURE 3. Compounded B cell developmental defects in mice lacking both BLNK and PLC{gamma}2 compared with single mutants. FACS analyses of B cell populations in (A) bone marrow and (B) spleen of wild-type, BLNK–/–, PLC{gamma}2–/–, and BLNK–/–PLC{gamma}2–/– mice. Numbers indicate percent of total lymphocytes and figures shown are representative of more than five independent analyses. C, Western blot analysis of PLC{gamma}2 phosphorylation in wild-type and BLNK–/– B cells. Cells were stimulated for various periods of time with anti-IgM F(ab')2 Ab and whole cell lysates were probed with anti-PLC{gamma}2 or site-specific anti-phospho-PLC{gamma}2 Abs as indicated. D, Recruitment of PLC{gamma}2 into lipid raft-containing DIM fractions following BCR stimulation. DIM fractions from untreated and anti-IgM treated BLNK+/+ and BLNK–/– B cells were examined for the presence of PLC{gamma}2 molecules using anti-PLC{gamma}2 Ab. Anti-Lyn blot was included as loading control. The results shown are representative of four independent analyses.

 

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  		Table I. Different B cell populations in bone marrow and spleena

 
We previously demonstrated that BCR-induced PLC{gamma}2 phosphorylation was impaired in BLNK-deficient B cells using anti-phosphotyrosine Ab that detect total phosphorylation of PLC{gamma}2 (19). In this study, we examined in detail the activation defect of PLC{gamma}2 in BLNK-deficient B cells using site-specific tyrosine phosphorylation Abs. Upon BCR stimulation, the phosphorylation of PLC{gamma}2 on Tyr759, which is the counterpart of Tyr783 of PLC{gamma}1 and is important for the phospholipase activity of PLC{gamma}2 (32), was significantly reduced in BLNK-deficient B cells compared with wild-type B cells (Fig. 3C). Consistent with the impaired PLC{gamma}2 phosphorylation on Tyr759, the BCR-induced translocation of PLC{gamma}2 into lipid raft-containing DIM fractions was also compromised in BLNK-deficient B cells compared with wild-type B cells (Fig. 3D), suggesting that BLNK is required for BCR-induced lipid raft recruitment and the enzymatic activation of PLC{gamma}2. However, the BCR-induced phosphorylation of PLC{gamma}2 on Tyr1217 was comparable in wild-type and BLNK-deficient B cells (Fig. 3C), suggesting the existence of BLNK-independent mechanism for the phosphorylation of PLC{gamma}2, which might also play a role in B cell development.

Ig H chain allelic exclusion is maintained in mice lacking BLNK and PLC{gamma}2

Each individual B lymphocyte expresses an Ab of a unique specificity comprising of one Ig H and L chain pair even though it possesses two H and four L chain gene alleles (3). This phenomenon is known as allelic exclusion and the process is thought to be regulated by pre-BCR and BCR signaling and to be dependent on the tyrosine kinases, Syk and Zap70 (33). We previously demonstrated that BLNK, a substrate of Syk, is not required for the allelic exclusion of IgH genes (24). However, it is not known whether PLC{gamma}2 is involved in signaling IgH allelic exclusion, and if the process is compromised in BLNK–/–PLC{gamma}2–/– mice, which exhibit compounded B cell developmental defects.

To address these questions, a rearranged VHB1–8 transgene inserted into one of the two IgH loci by gene targeting (22) was introduced into BLNK–/–, PLC{gamma}2–/–, and double mutant animals. Genomic DNA were isolated from sorted CD19+IgL– bone marrow cells and subjected to PCR to detect endogenous DHJH and VH to DHJH rearrangements. The expression of an IgH transgene in pro-B cells leads to the early assembly of a pre-BCR that signals allelic exclusion by suppressing VH to DHJH rearrangement at the other IgH locus. As shown in Fig. 4, single or double mutant mice showed normal levels of DH to JH rearrangements at the other IgH locus, similar to wild-type mice. However, in contrast to wild-type mice where the rearrangements of VHJ558 gene family members to DH and downstream JH1, 2, 3 gene segments could be detected, such gene rearrangements at the other IgH locus were severely suppressed in wild-type, BLNK–/–, PLC{gamma}2–/–, and BLNK–/–PLC{gamma}2–/– mice bearing the IgH transgene. The lack of significant VH to DHJH gene rearrangements in these single or double mutant mice would suggest that the mechanism that maintains IgH allelic exclusion is largely intact in these animals. Thus, signals transduced by BLNK and PLC{gamma}2 are not essential for the maintenance of IgH allelic exclusion.


Figure 4
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  		FIGURE 4. Intact Ig H chain allelic exclusion in BLNK–/–PLC{gamma}2–/– mice. Genomic DNA extracted from bone marrow CD19+ IgL–cells of mice of various genotypes were serially diluted and subjected to PCR using primers to amplify DH to JH and VHJ558 to DHJH gene arrangements. The PCR products were analyzed by Southern blotting using a DNA fragment spanning JH3 and JH4 as probe. The housekeeping gene GADPH was amplified as a control for the amount of genomic DNA used in the PCR. The result is representative of two independent experiments.

 
Plc{gamma}2 dosage is critical for B cell development in the absence of BLNK

Interestingly, in the course of our analysis of the double mutant, we noticed that in the complete absence of blnk, B cell development was dependent on the copy number of plc{gamma}2 genes present in the mouse. As shown in Fig. 5A, when blnk was present in one or two copies, there was no obvious difference in the splenic B cell population regardless of whether the mice possess one or two copies of plc{gamma}2. B cell development was affected only when plc{gamma}2 was completely absent. Interestingly, when blnk was completely absent, there was a proportionate decrease in B cell population in mice that possessed either two, one, or zero copies of plc{gamma}2 as the splenic B cell population was reduced from 25.5% to 13.5% and finally to 4.1% of lymphocytes, respectively. The converse was not seen for plc{gamma}2null mice that possessed either two or one copies of blnk as these mice contained comparable fractions of B cells.


Figure 5
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  		FIGURE 5. Effect of plc{gamma}2 dosage on B cell development in the absence of BLNK. A, Flow cytometry analysis of B cell populations found in the spleens of mice of various genotypes. Cells were stained for IgM and IgD expression and numbers indicate the percent of lymphocytes present. B, Pre-BCR expression on B220+IgL– cells in the bone marrow of mice of various genotypes. Cells were stained for B220, pre-BCR and Ig{kappa}/{lambda} expression and numbers indicate percent of B220+IgL– cells present. Results shown are representative of five independent analyses. C, Splenic B cell numbers in mice of various genotypes. Each data point represents one single animal analyzed. D, Examination of the amount PLC{gamma}2 present in BLNK+/+ and BLNK–/– B cells harboring one or two copies of plc{gamma}2 gene. The whole cell lysates from B cells of different genotypes were probed with anti-PLC{gamma}2 Ab in Western blot analysis to detect the amount of PLC{gamma}2 molecules present. Anti-actin blot was included as control for the loading of cell lysates. The results shown are representative of three independent analyses.

 
This dosage effect could also be seen in the bone marrow of BLNK-deficient mice that harbored two, one, or zero copies of plc{gamma}2, in particular, by examining the fraction of surface pre-BCR+ cells. As seen in Fig. 5B, there was a proportionate increase in the fraction of surface pre-BCR+ cells when BLNK-deficient mice contained two, one, or zero copies of plc{gamma}2 gene. This suggested a more severe block in B cell development with incremental decrease in plc{gamma}2 gene copy number in a BLNKnull background. Again, this phenomenon was not seen as long as a single copy of blnk was present.

Enumeration of splenic B cells in BLNK-deficient mice that possessed two, one, or zero copies of plc{gamma}2 also revealed a dosage-dependent reduction in the total number of peripheral B cells in these animals (Fig. 5C). Taken together, the data suggest that B cell maturation is affected by the gene dosage of plc{gamma}2 when BLNK is completely absent.

To begin to understand the molecular basis for the effect of plc{gamma}2 dosage on the development of BLNK-deficient B cells, we examined the amount of PLC{gamma}2 molecules present in BLNK+/+ and BLNK–/– B cells that harbored one or two copies of plc{gamma}2. It is possible that the amount of PLC{gamma}2 molecules correlates with plc{gamma}2 gene copy numbers or that the absence of BLNK may affect PLC{gamma}2 expression and thereby, causing a difference in the physiological outcome as shown above. As seen in Fig. 5D, the amount of PLC{gamma}2 molecules present in B cells did correlate with plc{gamma}2 gene copy numbers. In both BLNK+/+ and BLNK–/– B cells, the amount of PLC{gamma}2 present was ~50% reduced when the cells possessed one less copy of plc{gamma}2. Furthermore, the complete absence of BLNK did not further affect the amount of PLC{gamma}2 molecules present in the cells as equivalent amount of PLC{gamma}2 molecule was found in both BLNK+/+PLC+/– and BLNK–/–PLC{gamma}2+/– B cells. Thus, the amount of PLC{gamma}2 molecules present in a B cell correlates with the number of copies of plc{gamma}2 gene present, and that the mere presence of BLNK molecules could ensure equivalent development of B cells in mice with either a single or two copies of plc{gamma}2 gene.

In vitro B cell survival is affected by plc{gamma}2 dosage in the absence of BLNK

As it was shown previously that BLNK and PLC{gamma}2 were both involved in transducing tonic survival signals to B cells (19, 20, 34), we next examined whether B cell survival would be affected by the gene dosage of plc{gamma}2 in the absence of BLNK. Primary B cells undergo apoptosis when cultured in vitro. In the presence of BLNK, B cells that possessed one or two copies of plc{gamma}2 exhibited similar rates of spontaneous cell death (Fig. 6). Only when two copies of plc{gamma}2 were lost did the cells become more susceptible to apoptosis. Consistent with the previous findings (19), the rate of spontaneous apoptosis was higher in BLNK-deficient B cells compared with wild-type B cells. Interestingly, when these BLNK-mutant B cells had one less copy of plc{gamma}2, the rate of apoptosis was further increased across the several time points tested, thus establishing a dosage effect of plc{gamma}2 on B cell survival under a BLNK-deficient condition.


Figure 6
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  		FIGURE 6. Effect of plc{gamma}2 dosage on B cell survival in the absence of BLNK. Wild-type and BLNK-deficient B cells that harbor two, one, or zero copies of plc{gamma}2 were cultured in triplicate for various periods of time as indicated and the fraction of live cells was quantified. Cell viability is expressed as the percentage of cells that were negative for propidium iodide and annexin V stainings. Data were presented as mean ± SD. Results shown are representative of three independent analyses for the various genotypes except for that of BLNK–/–PLC{gamma}2–/– which is representative for two independent analyses.

 
Effect of plc{gamma}2 dosage on Ca2+ signaling and ERK activation in BLNK-deficient B cells

Because in the absence of BLNK, one could detect a plc{gamma}2 dosage effect on B cell maturation and survival, we finally examined whether such a dosage effect of plc{gamma}2 could also be detected biochemically during B cell activation.

As the Btk-BLNK-PLC{gamma}2 complex was involved in triggering calcium flux in BCR-stimulated B cells (6), we examined whether the magnitude of intracellular calcium release was affected by plc{gamma}2 gene dosage in B cells lacking BLNK. As seen in Fig. 7A (left column), the engagement of the BCR in blnk wild-type B cells harboring one or two gene copies of plc{gamma}2 resulted in a similar magnitude of intracellular calcium release in these cells. However, in the absence of BLNK, the magnitude of calcium flux was severely impaired in cells that contain two copies of plc{gamma}2 (Fig. 7A, right column), and the magnitude of calcium flux was further reduced in cells that contain only a single copy of plc{gamma}2. Unfortunately, we could not obtain enough BLNK–/–PLC{gamma}2–/– B cells for calcium flux analysis due to the paucity of B cells in BLNK–/–PLC{gamma}2–/– mice (Fig. 3B). This situation is further exaggerated by the increased perinatal lethality associated with PLC{gamma}2 deficiency. PLC{gamma}2–/– mice suffered from a bleeding disorder (14) and were not derived in Mendelian ratio as crossing of BLNK+/–PLC{gamma}2+/– mice yielded BLNK–/–PLC{gamma}2–/– mice at a ratio of 1:40. Nevertheless, our data indicate that B cell activation, at least in terms of intracellular calcium flux, is also affected by the dosage of plc{gamma}2 in a BLNKnull background.


Figure 7
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  		FIGURE 7. Effect of plc{gamma}2 dosage on intracellular calcium signaling and ERK activation in the absence of BLNK. A, Examination of intracellular calcium flux in BLNK+/+ and BLNK–/– B cells harboring two or one copies of plc{gamma}2. Purified splenic B cells obtained from mice of various genotypes and were stimulated with 20 µg/ml anti-IgM F(ab')2 Ab. The concentration of intracellular calcium was quantified using a spectrofluorometer. B, Examination of BCR-induced ERK activation in B cells of various BLNK and PLC{gamma}2 genotypes. B cells from mice of different genotypes were treated with high (20 µg/ml), intermediate (2 µg/ml), and low (0.5 µg/ml) doses of anti-IgM F(ab')2 Ab. Equal amounts of whole cell lysates were probed using anti-phospho-ERK and anti-ERK Abs to detect the activation status of the ERK. The results shown are representative of at least three independent analyses. [Ca2+]i, Intracellular calcium concentration.

 
Finally, we examined BCR-induced ERK activation in BLNK-deficient B cells harboring different copies of plc{gamma}2. We previously showed that BLNK-deficient B cells could activate ERK signaling in response to BCR engagement (19). The BLNK–/– B cells harboring two copies of plc{gamma}2 responded similarly as wild-type B cells when they were treated with a high (20 µg/ml), intermediate (2 µg/ml), or low (0.5 µg/ml) dose of anti-IgM Ab (Fig. 7B). The BLNK-deficient B cells harboring only one copy of plc{gamma}2 also exhibited a kinetics of ERK activation that was indistinguishable from those of control cells when stimulated with a high dose of anti-IgM Ab. However, ERK activation in these cells was significantly delayed when they were treated with an intermediate (2 µg/ml) or low (0.5 µg/ml) dose of anti-IgM Ab. Again, this altered kinetics of ERK activation was not seen in BLNK+/+ cells that harbored one copy of plc{gamma}2 (data not shown). Thus, in the absence of BLNK, ERK activation in response to a low level of BCR engagement is also affected by the dosage of plc{gamma}2.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have shown here that BLNK and PLC{gamma}2 deficiencies affect B cell maturation at different stages of development. The BLNK mutation arrested B lymphopoiesis at an earlier stage compared with the PLC{gamma}2 mutation, and resulted in the presence of early B cell precursors harboring pre-BCR on their cell surfaces. BLNK deficiency also led to a more pronounced reduction in the peripheral B cell population compared with the PLC{gamma}2 mutation. These data are consistent with a model in which BLNK is upstream of PLC{gamma}2 in the signaling cascade, and therefore may participate in more branches of BCR-signaling pathways than PLC{gamma}2. Hence, the inactivation of BLNK would result in a more severe phenotype. In support of this argument, our previous analyses of BLNK-deficient B cells indicated that the activation of PLC{gamma}2 was impaired (19), whereas a similar analysis of PLC{gamma}2-deficient B cells showed that the activation of BLNK was intact (14). In addition, BLNK has been shown to bind Vav, Nck, and Grb2 (35) and to play a role in Vav localization in the lipid rafts (36). These molecules, however, have not been shown to interact with PLC{gamma}2. Our present study also indicated that PLC{gamma}1 was expressed in B lymphocytes and activated in response to BCR engagement but BLNK was apparently not essential for its activation (Fig. 2). Thus, the more severe phenotype of BLNK–/– mice is not due to a combined impairment of both the activation of PLC{gamma}1 and 2. The fact that PLC{gamma}1 is activated in BLNK–/– B cells also implies that it could not fully substitute for the role of PLC{gamma}2 in B cell development.

If PLC{gamma}2 functions solely downstream of BLNK, one would expect the phenotype of BLNK–/–PLC{gamma}2–/– mice to closely resemble that of the BLNK single mutant. This is, however, not the case. The double mutant in fact exhibited a far more drastic arrest of early B cell generation. The number of B cells in the periphery of the double mutant was also significantly reduced compared with either single mutant. The compounded phenotype of BLNK–/–PLC{gamma}2–/– mice may reflect both overlapping and distinct signaling pathways in which two molecules are involved. For example, and as mentioned above, other than PLC{gamma}2 and Btk, BLNK also interacts with Vav and Nck. In addition, although its activation is largely dependent on BLNK, PLC{gamma}2 may also participate in B cell development in a BLNK-independent manner, within or outside of BCR-signaling pathways. In support of this hypothesis, we found that the BCR-induced phosphorylation of PLC{gamma}2 on the C terminus Tyr1217 was not affected by BLNK deficiency. Interestingly, the phosphorylation of Tyr1217 and another C terminus tyrosine Tyr1197 of PLC{gamma}2 was also independent of Btk (32). These data indicate that BCR engagement can induce phosphorylation of PLC{gamma}2 on its C-terminal tyrosines in a BLNK- and Btk-independent manner, and it might contribute to the function of PLC{gamma}2 during B lymphopoiesis. Apart from the BCR-signaling pathway, the B cell activating factor belonging to the TNF family receptor (BAFF-R) signaling pathway also appears to use PLC{gamma}2 (20) but not BLNK (data not shown). However, this may explain the peripheral but not the bone marrow B cell maturation defects in the double mutant as BAFF-R apparently plays no role in early B cell generation (37). Nevertheless, our data indicate that BLNK and PLC{gamma}2 likely participate in both overlapping and distinct signaling pathways that influence B cell differentiation. The simultaneous collapse of all these pathways could have more than an additive effect on B cell generation.

In contrast to the compounded early B cell developmental defect, IgH chain allelic exclusion remained intact in BLNK–/–PLC{gamma}2–/– mice. It has been shown that Syk and Zap70 are together required for the maintenance of IgH allelic exclusion (33) and it was demonstrated recently that this process was severely compromised in PLC{gamma}2–/–PLC{gamma}1+/– mice (38). This would indicate that normal function of either PLC{gamma}1 or PLC{gamma}2, which are downstream of Zap70 and Syk, respectively, is sufficient to maintain IgH allelic exclusion. Thus, the intact IgH allelic exclusion in our BLNK–/–PLC{gamma}2–/– mice could be ascribed to the normal expression and activation of PLC{gamma}1, which are independent of BLNK (Fig. 2).

Perhaps the more interesting finding that emerged from our current study is the dependency of B cell generation on plc{gamma}2 gene dosage in the complete absence of BLNK. In a BLNKnull background, there was a proportionate decrease in B cell population in the periphery and an increase in pre-BCR+ cells in the bone marrow of plc{gamma}2-diploid, -haploid, and -null animals. Similarly, there was a proportionate decrease in B cell viability in vitro between BLNK-deficient B cells harboring two, one, and zero copies of plc{gamma}2. At the biochemical level, the reduction of one copy of plc{gamma}2 in BLNK-deficient B cells also resulted in a corresponding decrease in the magnitude of intracellular calcium signaling and delayed kinetics of ERK activation. Thus, one can detect a stepwise impairment of B cell generation, survival, and activation with a gradual reduction in plc{gamma}2 copy number when BLNK is absent but not vice versa.

The dependency of B lymphopoiesis and activation on plc{gamma}2 dosage in the absence of BLNK emphasizes the important role of BLNK as a scaffold protein within the BCR-signaling complex. Following "tonic" or activating signaling, BLNK brings together Btk and PLC{gamma}2, leading to the activation of the latter to further effect efficient downstream signaling. In the absence of BLNK, PLC{gamma}2 can apparently still translocate to the plasma membrane through the binding of its pleckstrin homology domain to PI(3,4,5)P3 and be activated by Btk, which can also localize to the membrane in a similar manner. This could explain the leaky phenotype of BLNK-deficient mice and the attenuated calcium signaling in BLNK-deficient B cells (10, 11). However, the coming together of Btk and PLC{gamma}2 might be a chance or fortuitous event and presumably effect inefficient signaling without the proper "anchor" BLNK, as the process is subject to the cellular localization of these molecules and the number of these molecules present in a cell. This situation is further compounded if the amount of PLC{gamma}2 in a B cell is reduced, which will lead to a decreased chance encounter of Btk and PLC{gamma}2 and result in less PLC{gamma}2 being activated. Indeed, we found that the amount of PLC{gamma}2 molecules present in a B cell is influenced by its gene copy number (Fig. 5D). Therefore, as an adaptor protein, BLNK plays a scaffolding role of bringing Btk and PLC{gamma}2 molecules together and stabilizing the formation of the macromolecular complex. This could partially explain the dependency of B cell development on plc{gamma}2 gene dosage in the absence of BLNK.

In addition, BLNK can play a role as an aggregator of PLC{gamma}2 molecules other than as a scaffold protein. A single molecule of BLNK binds a single molecule of Btk but three molecules of PLC{gamma}2, enabling three molecules of PLC{gamma}2 to be activated simultaneously by a single "cis-"-associated Btk (39). Therefore, by binding three molecules of PLC{gamma}2 concurrently, BLNK serves as an amplifier of PLC{gamma}2 signaling. When the "concentrator" BLNK is present, presumably enough PLC{gamma}2 molecules are efficiently concentrated and become activated simultaneously by BLNK-bound Btk (40, 41) to ensure optimal signaling, so one does not detect a physiological difference in BLNK+/+ or BLNK+/– mice bearing two or one copies of plc{gamma}2 (Fig. 5–7). When BLNK is completely lacking, the stoichiometry of PLC{gamma}2 activation is changed as the chance of enough numbers of PLC{gamma}2 molecules being stabilized at the plasma membrane and activated to effect downstream signaling is dramatically reduced. In this situation, the activation of PLC{gamma}2 becomes critically dependent on the total amount of PLC{gamma}2 molecules available in the cell. Therefore, one could see more severe defects if the number of PLC{gamma}2 molecules in a B cell is halved as in the case of the heterozygous animals. This provides further explanation for the quantitative aspect of B cell generation that is influenced by the copy number of plc{gamma}2 in a BLNKnull background. Thus, by "concentrating" PLC{gamma}2 molecules, BLNK makes the PLC{gamma}2 signaling more efficient and malleable.

A recent publication also alluded to the possibility that Btk could also be a limiting component of the BCR signalosome (42). Using mice that express levels of Btk lower than that found in wild-type mice, the author showed that the concurrent haploinsufficiency of BLNK could affect BCR signaling. This is consistent with of our data and the finding that one molecule of BLNK interacts with one molecule of Btk but three molecules of PLC{gamma}2 (39). Thus, the decreased expression of Btk and BLNK reduces the strength of the signal emanating from the BCR signalosome, which could influence the activation of B cells.

In conclusion, our current study suggests that within the BCR-signaling pathway, BLNK and PLC{gamma}2 might not simply lie linearly and they may also have synergistic and other independent functions in B cell development. Our data also provide the genetic and physiological evidence of the importance of BLNK as a scaffold protein and aggregator of PLC{gamma}2 molecules. It would be interesting to determine whether BLNK’s role as an aggregator and concentrator could also be extended to other signaling molecules such as Vav and Nck, and whether in the absence of BLNK, one would also see a dosage effect of these molecules on B cell generation. Our data also suggest that B cell generation as a whole is influenced by the quantitative aspect of pre-BCR and BCR signaling. Last but not least, systematic mutation of individual or a combination of the three tyrosine residues involved in BLNK-PLC{gamma}2 interaction may further shed light on BLNK’s role as an "amplifier" of BCR signaling during B cell differentiation.


   Acknowledgments
 
We thank members of the Lam laboratory for helpful discussion.


   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 Biomedical Research Council of the Agency for Science, Technology, and Research (A*STAR). Back

2 Address correspondence and reprint requests to Dr. Kong-Peng Lam, Laboratory of Immunology, Center for Molecular Medicine, 61 Biopolis Drive, Lab 6-15, Proteos, Singapore 138673. E-mail address: mcblamkp{at}imcb.a-star.edu.sg Back

3 Abbreviations used in this paper: Btk, Bruton’s tyrosine kinase; BLNK, B cell linker; PLC, phospholipase C; DIM, detergent-insoluble membrane; LAT, linker for activation of T cells. Back

Received for publication June 16, 2005. Accepted for publication February 8, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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