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Requirement for B Cell Linker Protein (BLNK) in B Cell Development

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Science  03 Dec 1999:
Vol. 286, Issue 5446, pp. 1949-1954
DOI: 10.1126/science.286.5446.1949

Abstract

Linker proteins function as molecular scaffolds to localize enzymes with substrates. In B cells, B cell linker protein (BLNK) links the B cell receptor (BCR)–activated Syk kinase to the phosphoinositide and mitogen-activated kinase pathways. To examine the in vivo role of BLNK, mice deficient in BLNK were generated. B cell development in BLNK −/− mice was blocked at the transition from B220+CD43+ progenitor B to B220+CD43 precursor B cells. Only a small percentage of immunoglobulin M++ (IgM++), but not mature IgMloIgDhi, B cells were detected in the periphery. Hence, BLNK is an essential component of BCR signaling pathways and is required to promote B cell development.

Engagement of the BCR activates distinct families of cytoplasmic protein tyrosine kinases (PTKs) to phosphorylate enzymes that are required for the generation of second messengers (1). In turn, the coordinate generation of second messengers is important for normal B cell function because disruption of selected signaling pathways is associated with B cell anergy (2). Linker or adapter molecules play integral roles in linking the BCR-activated PTKs with these enzymes. One such linker molecule, BLNK (also known as SLP-65, BASH, and BCA), is phosphorylated by Syk after BCR activation and interacts with enzymes, including phospholipase C–γ, Bruton's tyrosine kinase, and Vav (a guanine nucleotide exchanger for the Rho-GTPases), as well as the Grb2 and Nck linker proteins (3–5). An essential role for BLNK in BCR activation was demonstrated in a chickenBLNK −/− DT40 cell line that cannot increase the intracellular calcium concentration ([Ca2+]i) or efficiently activate the Erk-, JNK-, and p38-mediated signaling pathways (6).

To better define the expression pattern of BLNK, we developed an intracellular fluorescence-activated cell sorting (FACS) staining assay for BLNK. Consistent with earlier reports (3, 5), BLNK expression was detected in peripheral B, but not T, lymphocytes (7) (Fig. 1A). Analysis of bone marrow–derived cells showed the highest BLNK expression in early development, with progressively lower expression during B cell maturation (7, 8) (Fig. 1B). Hence, BLNK is expressed throughout B cell ontogeny and suggests a potential role for BLNK in B cell development, maturation, or function.

Figure 1

Expression of BLNK in lymphocyte development. (A) BLNK is expressed in murine B, but not T, cells. CD3+ (left panel) or B220+ (right panel) splenocytes isolated from C57BL/6 mice were analyzed by intracellular staining with an antiserum to BLNK (shadowed areas) or preimmune serum (solid line) (7). (B) BLNK expression during murine B cell development. Bone marrow– derived cells isolated from C57BL/6 mice were analyzed with four-color FACS analysis (8). Cells stained for B220, IgM, and CD43 (left panel) or for B220, IgM, and IgD (right panel) were analyzed as described above. Each developmental subset— B220+ CD43+IgM (pro-B cells; R1), B220+CD43IgM (pre-B cells; R2), B220+ IgM+ IgD(immature B cells; R3), and B220+IgM+IgDlo (mature B cells; R4)—was analyzed for BLNK expression (7).

To investigate the in vivo role of BLNK, we undertook a gene-targeting approach to generate and analyze BLNK −/−mutant mice. Because BLNK is a substrate of Syk andsyk −/− mice hemorrhage extensively in utero and die during the perinatal period (3, 9), we were concerned that BLNK −/− mice might suffer a similar fate. In addition, gene targeting of the BLNK homolog,SLP-76, results in mice that die from hemorrhage caused by a defect in collagen-induced platelet aggregation (10). To circumvent the embryonic lethality that may be encountered in germ line knockout mice, we also used the RAG2 −/−blastocyst complementation system to assay for BLNK function in lymphocytes (11). The data presented here represent analyses from both approaches.

Disruption of BLNK was accomplished by a targeted mutation of exon 1, which encodes amino acids 1 through 60, including the initiation codon (12) (Fig. 2A). For RAG2 −/−blastocyst complementation, the BLNK + allele in the BLNK +/− embryonic stem (ES) clone was further targeted with a puromycin selection cassette to generateBLNK −/− ES cells (13) (Fig. 2B). To generate germ line mutant mice, BLNK +/− ES cells were injected into B6 blastocysts to yield chimerae that were then crossed with wild-type B6 mice to generateBLNK +/− germ line mutants. The genotypes of the mature offspring from such crosses occurred at the expected Mendelian ratios and the BLNK −/− mutation did not incur any embryonic or perinatal lethality (14). Furthermore, mature BLNK −/− mice were healthy under specific pathogen-free conditions and did not display any evidence of gross hemorrhaging (14). To evaluate the developmental potential of BLNK −/− ES cells, we also injected these cells into RAG2 −/−blastocysts to generate chimerae (R2:BLNK −/−) for direct analysis (15).

Figure 2

Generation ofBLNK −/− mice. (A) Targeting ofBLNK. The genomic structure surrounding exon 1 ofBLNK (top), the targeting construct (middle), and the targeted allele (bottom) are depicted (12). Exon 1 includes amino acids 1 through 60 of the BLNK coding region. The correctly integrated construct converts the 14-kb wild type into 9- and 4.8-kb fragments when detected with the 5′ and 3′ probes, respectively. A GFP cDNA was also inserted into the targeting construct. However, GFP fluorescence was not detected in BLNK +/−splenocytes or bone marrow–derived cells, which was likely caused by transcriptional silencing of GFP by the PGK-neo cassette (12). (B) Targeting of the second BLNKallele. The genomic structure surrounding exon 1 of BLNK(top), the targeting construct (middle), and the targeted allele (bottom) are depicted (13). The correctly integrated construct converts the remaining 14-kb wild type into 9- and 4.3-kb fragments when detected with the 5′ and 3′ probes, respectively. (C) Southern (DNA) blot analysis ofBLNK +/+, BLNK +/−, andBLNK −/− mice. Bam HI–digested tail DNA was separated by electrophoresis and hybridized with the 5′ probe to detect the wild-type and mutant fragments (13). Blotting with the 3′ probe also revealed the predicted mutant 4.8-kb fragment inBLNK +/− and BLNK −/−mice (14). (D) Absence of BLNK protein inRAG2 −/− chimeric mice. B220+ bone marrow cells from 129 wild-type (Ly9.1+; left panels),RAG2 −/− (Ly9.1; middle panels), or R2:BLNK −/− chimeric (Ly9.1+; right panels) mice were analyzed by intracellular staining for BLNK as described in Fig. 1A (7). (E) Absence of BLNK protein in BLNK −/− bone marrow–derived cells. Bone marrow–derived cells from germ lineBLNK +/− (lane 1) andBLNK −/− (lane 2) littermates were immunoblotted with an antiserum to BLNK (17). Equal loading of cell lysates was confirmed by immunoblotting with an antiserum to actin (Sigma) (14).

To assess the effect of the mutation on BLNK expression, we used the Ly 9.1 surface marker to distinguish between bone marrow cells derived from the RAG 2 −/− blastocyst (Ly 9.1) and the BLNK −/− ES cells (Ly 9.1+) (16). Whereas the B220+Ly 9.1+ cells from wild-type 129 mice and B220+Ly9.1 cells fromRAG2 −/− mice expressed BLNK (Fig. 2D, left two bottom panels), no BLNK was detected in the B220+Ly9.1+ cells isolated from theR2:BLNK −/− chimerae, as assessed by intracellular staining (Fig. 2D, bottom right panel). Similar to the results from the R2:BLNK −/− chimerae, no BLNK protein was detected in cell lysates of total bone marrow from germlineBLNK −/− mice (17) (Fig. 2E).

The effects of BLNK deficiency on lymphocyte development in vivo was examined by analyzing cells isolated from primary and secondary lymphoid organs. Consistent with the absence of BLNK expression in T cells, T cell number, development, and function were normal in bothBLNK −/− germ line andR2:BLNK −/− chimeric mice (14,18–20). In contrast, an ∼65% reduction in splenocyte number was found in BLNK −/− mice as compared toBLNK +/+ or +/− mice (18). Because the development and function of peripheral T cells were normal (14, 19), we further investigated the nature of this defect by analyzing the B cell compartment in primary and secondary lymphoid organs. Although the numbers of cells recovered from the bone marrow ofBLNK +/+ and BLNK −/−mice were similar (18), bone marrow cells fromBLNK −/− germ line andR2:BLNK −/− chimeric mice displayed a profound block in B cell development. BLNK −/−mice accumulated B220+CD43+ progenitor B cells (pro-B cells) (21) (Fig. 3A). Consistent with the presence of pro-B cells, the levels of VH to DJH recombination were comparable inBLNK +/+ and BLNK −/−bone marrow–derived cells (14).BLNK −/− mice had CD43+ pro-B cells but failed to develop B220hiCD43 B cells, although a small percentage of B220+CD43 B cells was present (10.0 ± 8.7% forBLNK −/− versus 44.4 ± 14% forBLNK +/− or +/+, P < 0.001, n = 11) (20) (Fig. 3A). Because the transition from the CD43+ to CD43 stage is normally associated with a decrease in cell size, as measured by the forward scatter value (8), the B220+CD43 B cells isolated from BLNK −/− mice remained large, in contrast to the smaller B220+CD43 B cells from BLNK +/− mice (14). In addition, theBLNK −/− bone marrow (B lineage) cells failed to progress efficiently from the immature B220loIgMlo (immunoglobulin M, IgM) stage to transitional B220loIgMhi or mature B220hiIgM+ stages (1.3% ± 0.9% inBLNK −/− mice for the latter two stages versus 13.1 ± 5 in BLNK +/+ or +/− mice, P < 0.001, n = 10) (20) (Fig. 3A). The small percentage of IgMlo bone marrow B cells that develop in BLNK −/− mice express a mature surface BCR because many are also Igκ+ (14).

Figure 3

B cell development inBLNK −/− mice. Cells isolated from bone marrow (A), spleen (B), and lymph nodes (C) of 3- to 5-week-old animals were stained with the antibodies indicated in each figure and analyzed by FACS analysis (21). Data from both R2:BLNK −/− chimeric [left two panels for (A) through (C)] and germ line [right two panels for (A) through (C)] mice are shown. In the RAG2 −/−blastocyst complementation assay, 129 wild-type andRAG2 −/− age-matched mice were analyzed in parallel as controls (14). No differences were detected between BLNK +/+ andBLNK +/− mice (14). The percentages of gated cells are indicated. These analyses were representative of a minimum of five pairs each of RAG2 −/− chimeric and germ line animals. Experiments from both approaches produced similar results. (D) Peritoneal cells isolated from 6- to 13-week-old mice were stained with the antibodies indicated and analyzed by FACS analysis (21). Cell recoveries were comparable in yield from BLNK +/− andBLNK −/− mice [4.5 × 106 forBLNK +/− and (3.2 ± 0.6) × 106 for BLNK −/− mice, n = 5].

Analysis of splenocytes revealed a substantial decrease in the numbers of IgM+ peripheral B cells (2.4 ± 2.6% forBLNK −/− versus 30.7 ± 6.2% forBLNK +/+ or +/−, P < 0.001, n = 10) (20) (Fig. 3B). As in the bone marrow, the fewBLNK −/− IgM+ B cells found in the spleen were also larger in size than IgMhi BLNK +/+ B cells (14). Concomitant with the profound decrease in peripheral B cells in the spleen, IgM+ B cells were also reduced in the lymph node (Fig. 3C). Hence, the absence of BLNK results in a developmental block that leads to reduced numbers of IgM+ cells in the periphery. Older BLNK −/− mice (8 to 13 weeks old) showed increased numbers of B220+IgM+ B cells [(1.4 ± 1.2) × 106B220+IgM+ cells, n = 8] as compared to younger BLNK −/− mice [3 to 6 weeks old; (0.58 ± 0.31) × 106, P < 0.001, n = 9]. In spite of this accumulation, these older BLNK −/− mice still have more than 10 times fewer B220+IgM+ B cells than their age-matched BLNK +/+ or +/− counterparts [(19 ± 6.7) × 106B220+IgM+ cells in olderBLNK +/+ or +/− mice, n = 6, versus (1.4 ± 1.2) × 106B220+IgM+ cells in olderBLNK −/− mice, P < 0.001, n = 8] (20).

Analysis for mature B cells revealed a marked reduction of B220hiIgM+ cells (<1%) in the bone marrow of young and old BLNK −/− mice (Fig. 3A) (14). Mature IgMloIgDhicells were similarly reduced (<1%) in the periphery of young and oldBLNK −/− mice (Fig. 4, A and B). Staining with CD21 revealed the presence of CD21+IgMhi T2 transitional B cells and a reduction of CD21+IgMlo mature B cells (<1%) in BLNK −/− mice (22) (Fig. 4B). Consistent with the decrease in mature B cells, serum Ig in older BLNK −/− mice was significantly reduced as compared to the amount in wild-type mice (23) (Fig. 4C).

Figure 4

Decreased maturation of B cell development and function in BLNK −/− mice. (A) Splenocytes isolated from 3- to 5-week-oldBLNK +/+ or BLNK −/− mice were analyzed by FACS staining for IgM and IgD (21). (B) Splenocytes from older BLNK +/+ orBLNK −/− mice (8 to 13 weeks old) were stained with the antibodies indicated in each figure and analyzed by FACS analysis (21). The percentages of gated cells are indicated.BLNK −/− spleens were reduced in cell number by ∼70% as compared to BLNK +/+ spleens (14, 18) (see text for absolute numbers of cells recovered). (C) Serum immunoglobulin levels of 8- to 13-week-old wild-type, germ line BLNK −/− andR2:BLNK −/− mice were determined by ELISA (23). Each diamond represents the value derived from an individual mouse. The black bar denotes the mean of each group. (D) B220+ B cells from olderBLNK +/+ (solid line) andBLNK −/− (dotted line) mice were analyzed by FACS analysis for increases in [Ca2+]i after BCR cross-linking with antibody to IgM F(ab′)2 fragments (20 and 40 μg/ml) (26). Antibody to IgM F(ab′)2 was added at time 0 at the indicated concentrations. Ionomycin was added at 5 min to ensure proper loading of cells with Fluo-4. Diamond,BLNK +/+ splenocytes; square, BLNK −/− splenocytes. (E) B220+ B cells from older BLNK +/+(top) and BLNK −/− (bottom) mice were analyzed by FACS analysis for up-regulation of CD69 and CD86 expression after BCR cross-linking (27). The dotted line represents cells treated with media. The solid line represents cells treated with antibody to IgM F(ab′)2 (10 μg/ml).

The BLNK −/− B cells that accumulated in the periphery of older mice further revealed a maturation defect in these cells. In contrast to BLNK +/+ mice, in which transitional B220+IgM++IgD+ B cells develop into mature B220hiIgMloIgDhi B cells, and in contrast to the B cells that accumulate in the λ5 −/− mice (24),BLNK −/− splenic B cells are larger in size and express higher membrane IgM (Fig. 4B) (14). These IgMhi cells may represent B cells that have matured through the pro- to precursor B cell (pre-B cell) transition but are blocked in IgM signaling and, therefore accumulate as large IgMhicells. Alternatively, these cells may result from a selection bias in which B cells can bypass the absence of BLNK by increasing membrane Ig expression and decreasing the signaling threshold. AsCD45 −/− immature B cells expressing a transgenic BCR can be rescued from death by chronic exposure to antigen (25), heightened BCR signaling may bypass the requirement for CD45. Similarly, BLNK deficiency may abolish development and result in the death of most B cells, except those that express very large amounts of IgM, which partially compensates for the signaling defect incurred by BLNK deficiency. The IgM++ BLNK −/− B cells could increase free cytoplasmic calcium after BCR cross-linking, albeit less efficiently than wild-type cells, despite expressing 10- to 50-fold more membrane IgM than BLNK +/+ B cells (26) (Fig. 4D). In addition, BLNK −/− B cells also up-regulate CD69 and CD86 cell surface activation markers after BCR cross-linking (27) (Fig. 4E). Hence, these large IgM++ BLNK −/− B cells are capable of some BCR-mediated signaling functions.

We also analyzed the development of a distinct subset of B cells known as B-1a cells. These cells are distinguished from conventional B-2 cells by their expression of CD5 and their capacity for self-renewal (28). Whereas BLNK +/+and BLNK +/− mice had comparable numbers of peritoneal cells, BLNK −/− mice had a substantial decrease in the CD5+IgM+ B-1a B cell population (<1%) in the peritoneum in young and old mice (Fig. 3D) (14). In addition, CD5CD11b+IgM+ B-1b B cells were also absent (<0.5%) in the peritoneum and the spleen ofBLNK −/− mice (14, 29). Hence, BLNK is also required for development of B-1 cells.

Because signals from both the pre-B and IgM BCRs are required for normal B cell development (1, 30), these studies showed the critical role of BLNK in the development of IgM+ cells. Similar to syk −/− mice (9), the absence of BLNK also compromises pre-BCR function to affect the development of B220+CD43 B cells that, in turn, limit their differentiation into B220hiIgM+ B cells in the bone marrow. As a result, few B cells are present in the periphery. However, whereas the IgM+ B cells that develop insyk −/− mice express little membrane IgM (9), the B cells that accumulate inBLNK −/− mice express large amounts of membrane IgM (Fig. 4B). This difference suggests that additional substrates of Syk might exist to partially transduce pre-BCR signals in the absence of BLNK. In accordance with this, the IgM++ B cells that accumulate in the periphery of older BLNK −/−mice can generate second messengers after BCR activation. Additional studies aimed at comparing syk −/− andBLNK −/− mice will be required to assess this possibility.

Finally, the developmental block at the pro- to pre-B cell transition observed in a BLNK-deficient patient is similar, though not identical, to the phenotype observed inBLNK −/− mice (31). Although IgMhi B cells accumulate in the periphery ofBLNK −/− mice, no peripheral B cells were detected in this adult patient. Similar discordance in phenotypes has been observed in immunodeficiencies involving Btk and λ5 in which the human phenotype appears to be more severe than the murine phenotype (32). These differences may reflect a greater dependence on pre-BCR function in human B cell development, a species-specific difference in the regulation of signaling molecules that dictate activation thresholds, or both. Such species-specific differences have been observed in T cell development in which Syk is more highly expressed in developing human CD4+ T cells than in murine CD4+ T cells and may provide a mechanism to explain the phenotypic differences observed between ZAP-70–deficient mice and humans (33). Additional investigation is required to determine whether species-specific differences in the regulation of BLNK or other regulators of B cell development may account for the differences observed between human and murine BLNK deficiencies. However, the present studies in a human and in mice demonstrate a central role for BLNK in relaying signals in the pre-BCR and BCR signaling pathways.

  • * To whom correspondence should be addressed. E-mail: achan{at}im.wustl.edu

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