Distinctive roles of Fyn and Lyn in IgD- and IgM-mediated signaling

Keisuke Horikawa1,2, Hirofumi Nishizumi1, Hisashi Umemori1, Shinichi Aizawa3, Kiyoshi Takatsu2 and Tadashi Yamamoto1

1 Departments of Oncology and
2 Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan
3 Department of Morphogenesis, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Kumamoto 860, Japan

Correspondence to: T. Yamamoto


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Src family kinases Fyn and Lyn associate with the B cell antigen receptor (BCR). Accumulating data show that Lyn plays important roles in BCR-mediated signaling, while the role of Fyn remains obscure. Here we dissected the role of Fyn and Lyn in BCR signaling using B cells from fyn–/–, lyn–/– and fyn/lyn double-deficient (fyn–/–lyn–/–) mice. In contrast to previous reports, fyn–/– B cells were slightly hyporeactive to both anti-IgM and anti-IgD–dextran. Although lyn–/– B cells were hyper-reactive to anti-IgM, anti-IgD-induced proliferation was impaired in lyn–/– B cells. Most of the other phenotypes of fyn–/–lyn–/– mice were similar to that of lyn–/– mice, except that proliferative responses of B cells to various stimuli, such as BCR cross-linking and lipopolysaccharide, were significantly lower in fyn–/–lyn–/– mice than in lyn–/– mice. Finally, immune responses to thymus-independent type 2 antigen were affected in these mutant mice. These observations suggest that Fyn and Lyn are involved in B cell functions, and play similar, but partly distinct, roles in BCR signaling.

Keywords: B cell receptor, B lymphocytes, knockout, signal transduction, Src family protein tyrosine kinase


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The immune system has to generate a diverse repertoire of antigen-recognizing receptors to respond to a vast array of pathogens. To do this, somatic rearrangements of Ig genes occur during lymphocyte development. Rearrangements of Ig heavy chain genes precede those in the light chain loci. Before the conventional light chain is produced, the rearranged µ heavy chain associates with the surrogate light chain consisting of Vpre-B and {lambda}5 proteins, forming the pre-BCR. After rearrangements of the light chains, immature B cells in the bone marrow express surface IgM (sIgM). In the periphery, mature B cells express sIgM and sIgD. These surface Ig act as antigen-recognizing receptors (1,2).

The BCR of mature B cells is composed of multiple protein subunits, including a membrane-bound Ig and heterodimers of the transmembrane polypeptides, Ig{alpha} and Igß (3). Stimulation of the BCR induces tyrosine phosphorylation of a series of cellular substrates including Ig{alpha} and Igß, phospholipase C-{gamma}, p21ras GTPase activating protein, a GTP exchange protein Vav, phosphatidylinositol 3-kinase, Shc, HS1, c-Cbl, and other unidentified proteins (48). BCR stimulation also induces tyrosine phosphorylation of CD22 and Fc{gamma}RII that are involved in negative regulation of the BCR signaling. These phosphorylation events are initiated by activation of Src family kinases, Fyn, Lyn, Blk, Lck and Fgr (912), which is followed by activation of Syk and Btk (13). The Src family kinases associate with the BCR of resting B cells via Ig{alpha} and the degree of association increases following BCR cross-linking (14). Like the mature BCR, the pre-BCR non-covalently associates with Ig{alpha}/Igß signaling subunits. This suggests that Src family kinases are activated in pre-BCR signaling and thereby involved in B cell development. Indeed, Fyn is activated following Ig{alpha} cross-linking in a pre-B cell line (15).

Src family tyrosine kinases are involved in various signaling pathways and their physiological importance has been demonstrated by gene disruption experiments. However, mutant mice lacking single Src family gene often showed phenotypes more limited than expected. For example, in spite of ubiquitous expression of src, fyn, and yes, src–/– mice showed only a bone-remodeling defect, osteopetrosis, due to the impairment of the function of osteoclasts, fyn–/– mice had mild effects in thymocytes and hippocampus, and yes–/– mice had no obvious phenotype (16). Disruption of the genes encoding hematopoietic cell-specific kinases Hck and Fgr produced no significant defect in the myeloid lineage, either (17). These results are most likely due to the functional redundancy of the overlapping Src family kinases. Supportingly, double mutant mice showed novel and severer abnormalities that were not obvious in single mutant mice. For example, combined deficiency of src, fyn and yes uncovered effects of yes deficiency, although yes–/– mice showed no apparent phenotype (18). hck–/–fgr–/– mice showed susceptibility to infection with Listeria monocytogenes that did not appear in hck–/– or fgr–/– mice (17). Furthermore, src–/–hck–/– mice manifested severer forms of osteopetrosis than src–/– mice (19).

In spite of Fyn association with the BCR and pre-BCR, fyn–/– mice reportedly showed little defects in B cell development and proliferation (20,21). This may be due to compensation of the function of Fyn by other Src family kinases, such as Lyn. Thus, we generated fyn–/–lyn–/– mice, and analyzed fyn–/–, lyn–/– and fyn–/–lyn–/– mice to elucidate the physiological roles of Fyn in BCR signaling. We show here that Fyn and Lyn distinctly participate in BCR-mediated signaling.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
fyn–/– and lyn–/– mice were generated and characterized as previously described (2224). fyn+/–lyn+/– heterozygotes obtained by crossing fyn–/–mice with lyn–/– mice were mated. Out of 141 offspring, eight were fyn–/–lyn–/– mice as expected. To increase the frequency of fyn–/–lyn–/– mice, fyn–/–lyn+/– heterozygotes, fyn+/–lyn–/– heterozygotes or fyn–/–lyn–/– homozygotes were mated with each other. fyn–/–lyn–/– mice obtained were viable, fertile and grew normally. The mice were maintained by brotherxsister matings. Genotypings of mice were performed at weaning by a PCR method using genomic DNA from punched-out ears. PCR for the fyn locus was performed as described (25). Primers used for distinguishing lyn+/+ mice from lyn+/– or lyn–/– mice were P1 (5'-GCTGCATTAAACACTCACTC-3') and P2 (5'-TGCCTTGGGAAAAGCGCCTC-3'). Amplification was for 35 cycles (60 s at 94°C, 60 s at 57°C and 120 s at 72°C). Primers used for distinguishing lyn–/– mice from lyn+/– or lyn+/+ mice were P1 and P3 (5'-CTGGACACACTTACTCTTCT-3'). Amplification was for 35 cycles (60 s at 94°C, 120 s at 57°C, and 180 s at 72°C). All mice were analyzed between 7 and 9 weeks of age, unless otherwise noted. All mice were bred under a specific pathogen-free condition in the laboratory animal research center at the Institute of Medical Science, University of Tokyo.

Flow cytometric analyses
Single-cell suspensions (5x105 cells) from each lymphoid tissues were incubated with antibodies for 30 min on ice in a staining buffer (PBS containing 2% FCS and 0.05% sodium azide). Stained cells were washed with the staining buffer twice. Data acquisition was performed with FACS (Becton Dickinson, Mountain View, CA) and gating and analysis were carried out using CellQuest programs (Becton Dickinson). Antibodies used were FITC-conjugated anti-B220 antibody (R3-6B2) (PharMingen, San Diego, CA), phycoerythrin (PE)-conjugated anti-Thy-1.2 antibody (30-H12) (PharMingen) and PE-conjugated goat F(ab')2 anti-IgM polyclonal antibody (Caltag, Burlingame, CA).

Immunization and ELISA
Mice were injected i.p. with 100 µg of DNP-conjugated keyhole limpet hemocyanin (DNP–KLH) or 100 µg of TNP-conjugated Ficoll (TNP–Ficoll). Levels of serum Ig, IgM, IgG1, IgG2a, IgG2b, IgG3 and IgA, and titers of DNP-specific and TNP-specific antibodies were determined by isotype-specific ELISA as described (24).

Isolation of splenic B cells and mitogenic response
Splenic B cells were isolated by using the Mini-MACS magnetic bead system (Milteny Biotec, Sunnyvale, CA) according to the manufacturer's instructions. The purity of the isolated cells was verified by flow cytometric analysis. The isolated cell preparations contained >95% B220+ cells.

Purified splenic B cells were resuspended at 5x105 cells/ml in RPMI supplemented with 10% FCS (Gibco/BRL, Gaithersburg, MD), 2 mM L-glutamine, 50 µM 2-mercaptoethanol, 100 U/ml penicillin G and 100 µg/ml streptomycin, and dispensed in 200 µl aliquots into 96-well flat-bottom microtiter plates (Corning Glass Works, Corning, NY). Cells were stimulated with affinity-purified goat anti-mouse IgM polyclonal antibody (Sigma, St Louis, MO), goat affinity-purified F(ab')2 fragments to mouse IgM (ICN Pharmaceuticals, Aurora, OH), bacterial lipopolysaccharide (LPS; Difco, Detroit, MI) or 50 µl of culture supernatant from a myeloma cell line producing a soluble CD40 ligand (CD40L)–CD8 chimeric protein (26). Anti-IgD–dextran was a gift from Dr Clifford Snapper (Uniformed Services University of the Health Sciences, Bethesda, MD). After 42 h, cells were pulsed with 0.2 µCi/well of [3H]thymidine (Amersham, Little Chalfont, UK) and harvested onto glass fiber filters 6 h later. Incorporation of [3H]thymidine was measured by 3H-sensitive avalanche gas ionization methods using a Matrix 96 direct ß-counter (Packard, Meriden, CT). All data were means of triplicate wells. Results are expressed as the arithmetic mean ± SD of triplicate cultures.

In vitro kinase assay and Western blot analyses
Thymocytes or splenocytes (1x107 cells each) were lysed with TNE buffer (1% Nonidet P-40, 10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.2 mM sodium orthovanadate, 10 µg/ml of aprotinin and 10 µg/ml of leupeptin). Each lysate was precleared with Protein G–Sepharose. Anti-Fyn mAb Fyn 301 and anti-Lyn mAb Lyn 8 (both from Wako Pure Chemical, Osaka, Japan) were added into precleared lysates of thymocytes and splenocytes respectively. Immunoprecipitates were collected using Protein G–Sepharose, and washed 4 times with lysis buffer and once with kinase buffer (40 mM HEPES, pH 7.4, and 10 mM MgCl2). Each immune complex was suspended in 20 µl of kinase buffer with 1 µCi of [{gamma}-32P]ATP and incubated at 37°C for 30 min. The reaction was stopped by adding 20 µl of 3xsample buffer (195 mM Tris–HCl, pH 6.8, 9% SDS, 15% 2-mercaptoethanol and 30% glycerol). Then, the reaction mixtures were boiled for 5 min and fractionated on SDS–8.5% PAGE followed by autoradiography.

For protein tyrosine phosphorylation assay, purified splenic B cells (5x105 cells) were incubated with affinity-purified goat anti-mouse IgM polyclonal antibody (50 µg/ml). Cells were lysed with TNE buffer. Insoluble debris was removed by centrifugation and 50% volume of 3xsample buffer was added to each lysate. Mixtures were boiled for 5 min and resolved by SDS–8.5% PAGE. Following electrophoresis, proteins were transferred onto PVDF membranes (Millipore, Bedford, MA). The membranes were blocked by incubation with TBS buffer (20 mM Tris-HCl pH 7.4, and 150 mM NaCl) containing 5% BSA. Phosphotyrosine-containing proteins were detected by incubating the membrane first with biotinylated anti-phosphotyrosine mAb 4G10 and then with the horseradish peroxidase-conjugated streptavidin. Following extensive washing, the membranes were developed using the enhanced chemiluminescence detection system (NEN, Boston, MA).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Reduction of peripheral B cell population in lyn–/– and fyn–/–lyn–/– mice
Accumulating biochemical evidence indicates a physical interaction of the BCR with Fyn and Lyn, suggesting their involvement in BCR-mediated signaling (912). Indeed, lyn–/– mice showed various intriguing phenotypes, such as reduction of the number of peripheral B cells, marked elevation of serum IgM and IgA, deregulated proliferative response to BCR cross-linking, decreased tyrosine phosphorylation of cellular substrates following BCR cross-linking, and autoimmune phenomena (24,27,28). In contrast, previous studies using fyn–/– mice were unable to elucidate physiological roles of Fyn in signaling via the BCR (20,21). It was likely that deficiency of Fyn was complemented by Lyn. To examine this possibility, we generated fyn–/–lyn–/– mice and addressed possible contribution of Fyn to BCR signaling (Fig. 1Go).



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Fig. 1. Genotype of mice lacking the fyn and lyn genes. Representative PCR analysis of the fyn (A) and lyn (B) loci. (C) In vitro immune complex kinase assay of Fyn (upper panel) from thymocytes or Lyn (lower panel) from splenocytes of wild-type, fyn–/–, lyn–/– and fyn–/–lyn–/– mice.

 
Macroscopically, fyn–/–lyn–/– mice did not show any apparent anomalies in non-lymphoid tissues. However, like in lyn–/– mice, the cellularities of the spleens in fyn–/–lyn–/– mice were about half of those in wild-type and fyn–/– mice (Fig. 2Go). The average number of total splenocytes was 8.1x107 cells in wild-type mice, 8.9x107 cells in fyn–/– mice, 5.0x107 cells in lyn–/– mice and 5.6x107 cells in fyn–/–lyn–/– mice (Fig. 2Go). Immunostaining of the splenocytes with anti-B220 antibody (pan B cell marker) and anti-Thy-1 antibody (pan-T cell marker) revealed that the population of B220+ cells in the spleen was 34–49% (mean 42%) of all nucleated cells in wild-type mice, 31–51% (mean 41%) in fyn–/– mice, 13–27% (mean 18%) in lyn–/– mice and 10–33% (mean 17%) in fyn–/–lyn–/– mice (representative cases are shown in Fig. 3Go, upper panel). The levels of IgM and IgD expression on the surface of B220+ splenocytes did not show much difference between the mutants and wild-type mice (data not shown). In contrast to the massive reduction of peripheral mature B cells in lyn–/– and fyn–/–lyn–/– mice, no significant anomalies were detected in the T cell population in the thymus and spleen of the mutant mice (data not shown).



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Fig. 2. Number of total splenocytes, splenic B cells and splenic T cells (A). Wild-type ({square}), fyn–/– ({blacksquare}), lyn–/– ({circ}) and fyn–/–lyn–/– (•).

 


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Fig. 3. Flow cytometric analyses. Cells from the spleen (upper panel) of wild-type and mutant mice were stained with FITC-conjugated anti-B220 antibody and PE-conjugated anti-Thy-1 antibody. Cells from the bone marrow (bottom panel) of wild-type and mutant mice were stained with FITC-conjugated anti-B220 antibody and PE-conjugated goat F(ab')2 anti-IgM polyclonal antibody. Stained cells were analyzed by two-color flow cytometry gated on lymphocytes. Numbers in quadrants indicate the percentage of the total lymphocyte population.

 
Ly-1+ B cells (B-1a cells) are characterized by surface phenotypes, distinct development and preferential distribution in the peritoneal cavity. Unlike conventional B cells (B-2 cell) in the spleen, no apparent difference was detected in the Ly-1+ B cell population among these mice, as was demonstrated by immunostaining of peritoneal cells with anti-B220 antibody and anti-CD5 antibody (data not shown).

B cell development in bone marrow of fyn–/–lyn–/– mice
We examined whether Fyn as well as Lyn is involved in signaling downstream of the pre-BCR during B cell development. Flow cytometric analyses of the bone marrow cells with anti-B220 antibody and anti-IgM antibody showed almost normal B cell development in fyn–/–lyn–/– mice (Fig. 3Go, bottom panel). However, although the proportion of immature B cells (B220lo/IgM+) was equivalent to that in controls, the population of recirculating B cells (B220hi/IgM+) was reduced as previously described in lyn–/– mice (24,27). To investigate the B cell precursor population more thoroughly, the B220+CD43+ cells from bone marrow of fyn–/–lyn–/– mice and age-matched controls were further subdivided by the expression of HSA and BP-1 according to Hardy's classification (29). The data suggested no significant blockage in transition from pro-B to pre-B cells even in fyn–/–lyn–/– mice (data not shown). Therefore, primitive B cells lacking both Fyn and Lyn differentiate and proliferate normally in the bone marrow.

Signaling through the BCR in the absence of Lyn and/or Fyn
We first examined the levels of protein tyrosine phosphorylation in splenic B cells after BCR cross-linking. Because of the massive decrease in total splenic B cells in lyn–/– and fyn–/–lyn–/– mice, we prepared lysates from the same number of splenic B cells of these mice. Immunoblotting experiments with an anti-phosphotyrosine antibody revealed little difference in the level of protein tyrosine phosphorylation between the wild-type B cells and fyn–/– B cells (Fig. 4Go, lanes 1–3 and 4–6). Protein tyrosine phosphorylation in lyn–/– B cells was delayed and reduced compared with that in B cells from wild-type mice (Fig. 4Go, lanes 1–3 and 7–9) (24). The level of protein tyrosine phosphorylation observed in fyn–/–lyn–/– B cells was almost equivalent to that found in lyn–/– B cells (Fig. 4Go, lanes 7–9 and 10–12).



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Fig. 4. The BCR cross-linking-induced protein tyrosine phosphorylation of splenic B cells from wild-type and mutant mice. Purified splenic B cells from each mouse were stimulated with affinity-purified goat anti-mouse IgM polyclonal antibody for the indicated time. Protein in cell lysates (5x105 splenic B cells per lane) were fractionated by 8.5% SDS–PAGE and transferred onto a PVDF membrane filter. This filter was probed with anti-phosphotyrosine mAb (4G10).

 
Unlike a previous report (20), B cell proliferation in response to anti-IgM stimulation with whole as well as F(ab')2 antibodies was slightly impaired in the absence of Fyn (Fig. 5A and BGo). On the contrary, proliferation of lyn–/– B cells in response to anti-IgM stimulation were augmented, as was reported by the others (28,30). The augmentation seen in the lyn–/– B cells was a little suppressed when Fyn was additionally absent. Therefore, the role of Fyn in the IgM-mediated signaling was different from that of Lyn.




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Fig. 5. Proliferative responses to various mitogens. Purified splenic B cells were stimulated with (A) affinity-purified goat anti-mouse IgM polyclonal antibody, (B) goat affinity-purified F(ab')2 fragments to mouse IgM, (C) anti-IgD–dextran, (D) bacterial LPS or (E) 50 µl of culture supernatant from a myeloma cell line producing a soluble CD40L–CD8 chimeric protein. After 42 h of incubation, cells were pulse-labeled with 0.2 mCi/well of [3H]thymidine for 6 h and harvested onto glass fiber filters. In (A) to (D), wild-type ({square}), fyn–/– ({blacksquare}), lyn–/– ({circ}) and fyn–/–lyn–/– (•). In (E), wild type ({square}), fyn ({blacksquare}), lyn–/– ({blacksquare}), fyn–/–lyn–/– ({blacksquare}).

 
Intriguingly, however, their roles were somewhat similar in the IgD-mediated signaling which was induced using a highly multivalent anti-IgD–dextran, an anti-IgD antibody covalently linked to dextran. Low concentration of anti-IgD–dextran can induce very high levels of B cell proliferation. Consequently, at concentration that can induce B cell proliferation, the level of tyrosine phosphorylation of cellular proteins is very low (31). In this study, we used anti-IgD–dextran at a low concentration (<3 ng/ml), which could induce B cell proliferation without a detectable increase in protein tyrosine phosphorylation. This system has been exploited as a model for B cell activation analogous to T cell-independent antigen type 2 (TI-2). As shown in Fig. 5(C)Go, the proliferative response of fyn–/– B cells to anti-IgD–dextran was moderately reduced as compared to that of control B cells. In contrast to results using anti-IgM antibody [whole and F(ab')2] as B cell activator, proliferation of lyn–/– B dells in response to anti-IgD–dextran was not enhanced but rather suppressed. Moreover, proliferation of fyn–/–lyn–/– B cells in response to anti-IgD–dextran was more severely impaired than that of lyn–/– B cells. Therefore, roles of Fyn and Lyn are similar in the IgD-mediated signaling.

Next, we examined proliferative responses of B cells to bacterial lipopolysaccharide (LPS) and CD40L (Fig. 5D and EGo). Compared to wild-type B cells, fyn–/– B cells showed slightly decreased proliferation in response to LPS and CD40L stimulation. In contrast, the proliferate responses were significantly impaired in lyn–/– and more severely in fyn–/–lyn–/– B cells. Thus, Lyn and Fyn are positively involved in LPS and CD40L signaling. This is consistent with previous findings that Fyn and Lyn are functionally associated with CD40 signaling (32,33).

Hyperimmunoglobulinemia and glomerulonephritis
In lyn–/– mice, serum levels of Ig are elevated, manifesting IgM and IgA gammopathy. In particular, the serum IgM level of lyn–/– mice is ~10-fold elevated over control values (24,27). The elevated Ig levels resulted in glomerulonephritis due to accumulation of immune complexes containing autoreactive antibodies in glomeruli. In fyn–/–lyn–/– mice, we found marked elevation of IgM and IgA, normal levels of IgG2a, and decreased levels of IgG1, IgG2b and IgG3 at the age of 2 months (data not shown). Levels of IgM and IgA were further elevated in 3-month-old mice (data not shown). The overproduction of IgM and IgA paralleled the plasmacytosis in lymphoid organs such as the bone marrow, the spleen, and the lymph node. Like lyn–/– mice, fyn–/–lyn–/– developed glomerulonephritis at their older ages (data not shown).

Reduced immunological competence of Src family-deficient mice
Previous studies showed that fyn–/– mice had normal immune responses to thymus-dependent (TD) antigen (20). lyn–/– mice also showed virtually normal response to TD antigen, despite the lack of formation of germinal centers upon TD stimulation (24,27). However, as shown in Fig. 6Go, immunization of fyn–/–lyn–/– mice with TD antigen, DNP–KLH, resulted in reduced production of IgM and IgG1 (Fig. 6A and BGo). This may be due to the disabilities in the functional interaction between T cells and B cells or to the impaired signaling through CD40, which is required for thymus-dependent humoral immunity.




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Fig. 6. Humoral immune responses to exogenous antigens. Immune responses of wild-type and mutant mice to thymus-dependent antigen DNP–KLH (A and B) or thymus-independent antigen type 2, TNP–Ficoll (C and D). Mice were immunized i.p. with DNP–KLH (A and B) or TNP–Ficoll (C and D) on the days indicated by arrows. At the indicated day points, mice were bled and titers of DNP-specific IgM and IgG1 and TNP-specific IgM and IgG3 were measured by ELISA. (A and B) Wild-type ({circ}), mean of wild-type (solid line), fyn–/–lyn–/– (•) and mean of fyn–/–lyn–/– (dotted line). (C and D) Wild-type ({square}); fyn–/– ({blacklozenge}), lyn–/– ({circ}), fyn–/–lyn–/– (•), mean of wild-type (solid line), mean of fyn–/– (dotted line), mean of lyn–/– (broken line) and mean of fyn–/–lyn–/– (broken and dotted line).

 
Finally we examined immune responses of each mouse to the TI-2 antigen, TNP–Ficoll. As reported previously (20), fyn–/– mice showed mild reduction of IgG3 response to TI-2 antigen (Fig. 6C and DGo). However, unlike previous observation, fyn–/– mice also showed a small decrease in IgM production in response to TI-2 antigen (Fig. 6C and DGo). IgM and IgG3 responses of lyn–/– mice to TI-2 antigen were poorer than those of fyn–/– mice. Moreover, fyn–/–lyn–/– mice produced very low levels of IgM and almost no IgG3. These results indicate that the absence of Fyn or Lyn, or both, results in the impaired immune responses to TI-2 antigen.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Although current biochemical data imply involvement of Fyn in BCR signaling, studies with fyn–/– mice suggest that Fyn is dispensable in BCR signaling (20,21). The discrepancy was attributed to complementation of the Src family kinases in BCR signaling, which was supported by a study with the DT40 chicken B cell line in which Lyn is predominantly expressed among the family members. Introduction of not only Lyn but also Fyn or Lck into the DT40 cells in which the lyn gene was disrupted by gene targeting restored the otherwise abrogated protein tyrosine phosphorylation and Ca2+ influx (34). Then assuming that the roles of Fyn in BCR signaling become apparent in the absence of Lyn, we generated fyn–/–lyn–/– mice and examined additional effects of Fyn mutation. Unexpectedly, we could not find apparent phenotypes between lyn–/– and fyn–/–lyn–/– mice. The data suggest that Lyn plays a major role in the BCR signaling among the BCR-associated Src family kinases and Fyn an accessory role. However, intensive analysis of the B cells of these mutant mice as well as of fyn–/– mice disclosed different roles of Fyn and Lyn in sIgM-mediated signaling. Lyn has been shown to be involved in both positive and negative signaling downstream of sIgM. Our observation that fyn–/– B cells, unlike lyn–/– B cells, are slightly hyporeactive to sIgM stimulation suggest that Fyn may not be involved in the negative signaling. In support, we previously showed that Lyn but not Fyn is important in tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibition motif (ITIM) on the Fc{gamma}RIIb that plays a critical role in the negative signaling pathway (35).

B cells of lyn–/– mice were initially reported to be hypoproliferative to BCR stimulation (24,27). Later, however, the lyn–/– B cells were shown to be hyper-reactive to the BCR stimulation (28,30). The discrepancy was attributed to the purity of the B cells analyzed. Because of the abundance of unusually differentiated B220+/Mac-1+ lymphocytes in the spleen of lyn–/– B cells, the Mac-1+ cells could be easily contaminated in the B cell preparation. This would lead to the misinterpretation of the B cell reactivity, provided that the Mac-1+ cells were hyporeactive to the BCR stimulation. Since the spleen of young lyn–/– mice contained less Mac-1+ lymphocytes, B cells were highly purified from <9-week-old lyn–/– mice. As expected, these lyn–/– B cells were hypersensitive to anti-IgM stimulation. In contrast, the same preparation of lyn–/– B cells to anti-IgD–dextran was rather hyporeactive, as compared to the control B cells. Our results suggest that the role of Lyn in the signaling downstream of sIgM is different from that of sIgD. Distinct biological and biochemical effects were reported between sIgD- and sIgM-mediated signaling (3638), though the notion that the function of sIgD is different from that of sIgM is still controversial. The distinguishable proliferative responses of lyn–/– B cells to sIgM and sIgD cross-linking could be due to the difference of the molecules associated with sIgM and sIgD (39). Recent observations revealed that Lyn is involved in tyrosine phosphorylation of both immunoreceptor tyrosine-based activation motif (ITAM) on Ig{alpha}/Igß and ITIM on Fc{gamma}RIIb and CD22, which respectively leads activation of positive and negative signaling downstream of sIgM. It is interesting to hypothesize that Lyn-mediated ITIM phosphorylation is poor in sIgD signaling.

We showed here that the number of peripheral mature B cells was greatly reduced and the response to TI-2 antigen was impaired in lyn–/– and fyn–/–lyn–/– mice. Similar phenotypes are observed in mice lacking the cytoplasmic domain of Ig{alpha} (mb-1{Delta}c/{Delta}c mice). The peripheral B cell population of mb-1{Delta}c/{Delta}c mice is 1/100 of normal mice (40). Furthermore, recent reports using the technique of inducible gene targeting showed that mature peripheral B cells still depend on the BCR for their survival and persistence. These data indicated that signaling mediated by the BCR, whether B cell autonomous or low level, is required to establish the mature B cell pool. Since both Lyn is constitutively associated with Ig{alpha} and functionally involved in BCR signaling, lack of Lyn would cause the impairment of the signaling for B cell survival required for the maintenance of the mature B cell pool.

Similarly, the number of peripheral B cells in xid mice carrying mutations in the btk gene (41) is about half of that of normal mice and xid mice fail to respond to TI-2 antigen. Taken together, these observations suggest that Lyn mediates signal transduction from Ig{alpha} to Btk activation. Consistently, Src family kinases activate Btk (42). This signaling to Btk activation is required for the potency of B cells to respond to TI-2 antigen and for maintaining, at least in part, the size of the mature B cell pool. Moreover, xid B cells are unable to proliferate satisfactory upon CD40 ligation and LPS stimulation (41,43). We previously showed that lyn–/– B cells exhibited impaired proliferative responses to CD40 ligation and LPS stimulation (24), and showed here that responses of fyn–/–lyn–/– B cells were even more impaired. These results suggest that Fyn and Lyn functionally associate with Btk in CD40 and LPS signaling as well as in BCR signaling. Consistently, tyrosine phosphorylation of Btk following BCR cross-linking was impaired in lyn–/– and fyn–/–lyn–/– B cells (44).

During lymphocyte development, somatic rearrangements of Ig genes are achieved in an orderly fashion. Genetic approaches using targeted disruption of gene encoding surface antigen receptors and antigen receptor-associated molecules have shown that intracellular molecules that mediate signaling through the receptors are involved in lymphocyte development. For example, the Src family kinase, Lck, is essential for T cell development. Accordingly, lck–/– mice show thymic atrophy and a dramatic reduction of CD4+CD8+ cells (45). Other observations indicate pivotal roles of this kinase in pre-TCR signaling (46). As pre-TCR and pre-BCR similarly use signal transduction components for lymphocyte development, Src family kinases might also play crucial roles in B cell development at the pre-B cell stage. This idea is also supported by biochemical data that Fyn is involved in pre-BCR signaling (15). Upon stimulation of the pre-BCR by yet unidentified signals, Src family kinases associated with the pre-BCR may be activated to initiate intracellular biochemical events. However, disruption of the individual gene encoding Fyn, Lck, Fgr, Blk, Hck or Lyn kinase did not cause any perturbation of B cell development in the bone marrow (16). These unexpected results have been explained by the functional redundancy of the Src family kinases. Supporting this explanation is the observation that T cell development is more severely blocked in fyn–/–lck–/– mice than in lck–/– mice, while fyn–/– mice show no perturbation in T cell development (47,48). Thus, we scrutinized early B cell development in fyn–/–lyn–/– mice. Contrary to our expectations, combined disruption of Fyn and Lyn did not perturb early B cell development in the bone marrow. Similarly, combined disruptions of fyn/yes and fgr/hck did not alter B cell development in the bone marrow (16). Therefore, in pre BCR signaling, Src family kinases might have highly overlapping functions, allowing compensation of the disrupted kinases by the remaining kinases. An alternative possibility is that signaling through the pre-BCR might be different from that of the mature BCR; the former may be more dependent on Syk than the latter. This idea is consistent with the data that B cell development is greatly impaired in syk–/– mice (49,50).

The mechanism of splenomegaly in lyn–/– mice remains unclear. We previously showed accumulation of Mac-1+ cells in the enlarged spleen of old lyn–/– mice (24). However, splenomegaly cannot be explained only by accumulation of the cells. As described by Chan et al. (28), the enlarged spleen contains large number of immature myeloid cells, fully differentiated neutrophils, lymphoblasts and plasma cells. How these cells accumulate is not known and would be a future problem. It is reasonable to assume that splenomegaly induced in fyn–/–lyn–/– mice is due to the unusual accumulation of these cells.

The causes of hyperglobulinemia (IgM and IgA) and autoantibody production also remains unclear. lyn–/– B cells are activated at least in part due to defects in the negative signaling pathway. As a result, BCR cross-linking-induced activation of the intracellular signaling molecules, such as MAP kinase (28) and protein kinase C (51), is elevated in lyn–/– mice. The enhanced signaling could contribute to development of hyperglobulinemia and autoantibody production. Our previous data suggest that Fyn does not contribute much to Fc{gamma}RIIb signaling, one of the negative signaling pathway (35). However, fyn–/–lyn–/– mice also show hyperglobulinemia because of the lack of Lyn. We think that the splenomegaly is mostly due to the unusual accumulation of myeloid cells and therefore is not much related to hyperglobulinemia. Although B cells are hyperactive in the absence of Lyn or both Lyn and Fyn, immune responses of lyn–/– mice to TD antigen are normal (24), whereas the responses of fyn–/–lyn–/– mice were low. Therefore, we think that Fyn deficiency contributes to impaired B cell responses to TD antigen. Apparently, TCR signaling is partly affected in the absence of Fyn. However, it remains to be addressed how the lack of Fyn in T cells affects TD response.

It is also intriguing that mice lacking Fyn, Lyn or both have compromised immune responses to TI-2 antigen, which mimics a repetitive and prolonged BCR signaling (52). fyn–/– mice had a mild defect in the immune response; lyn–/– and fyn–/–lyn–/– mice showed more severely reduced responses. The results are consistent with reduced proliferative responses to anti-IgD–dextran. Taken together, these results imply that there is an order in functional significance of the Src family kinases in many aspects of BCR signaling, Lyn being a primary component of BCR signaling; Fyn and other kinases are secondary. It is likely that this order depends on the expression level of each kinase and/or the preference of each kinase for downstream effectors.


    Acknowledgments
 
We thank T. Ishida for technical suggestions, H. Baba and S. Uehara for helpful discussions, and H. Onoda and T. Wakabayashi for histology preparation. This work was supported by a grant from the Ministry of Education, Science, Sports and Culture of Japan.


    Abbreviations
 
BCRB cell receptor
CD40LCD40 ligand
ITAMimmunoreceptor tyrosine-based activation motif
ITIMimmunoreceptor tyrosine-based inhibition motif
KLHkeyhole limpet hemocyanin
LPSlipopolysaccharide
PEphycoerythrin
sIgMsurface IgM
sIgDsurface IgD
TDthymus-dependent
TIthymus-independent

    Notes
 
Transmitting editor: S. Nishikawa

Received 8 February 1999, accepted 18 May 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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