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Department of Laboratory Medicine, University of California San Francisco, San Francisco, California 94143; the Howard Hughes Medical Institute, The Children's Hospital, Boston, Massachusetts 02115; the || Unit of Applied Cell and Molecular Biology, Umea University, S-901 87 Umea, Sweden; and the ¶ Howard Hughes Medical Institute, University of California Los Angeles, Los Angeles, California 90095
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25% of endogenous levels (Btklo) was crossed onto Btk–/– and Btk–/–lyn–/– backgrounds to demonstrate that Btk is limiting for BCR signaling in the presence but not in the absence of Lyn. These observations indicate that the net outcome of Lyn function in vivo is to inhibit Btk-dependent pathways in B and myeloid cells, and that Btklo mice are a useful sensitized system to identify regulatory components of Btk signaling pathways.
Key Words: B cell receptor • B cell development • Src family kinases • transgenic mice • immunodeficiency
Abbreviations used: ALPH, alkaline phosphatase; BBS, borate-buffered saline; BCR, B cell antigen receptor; BrdU, bromodeoxyuridine; Btk, Bruton's tyrosine kinase; Btklo, mice lacking the endogenous Btk gene and expressing 25% of endogenous levels of Btk in B cells from an Ig heavy chain enhancer/promoter–driven transgene; KLH, keyhole limpet hemocyanin; TNP, 2,4,6-trinitrophenyl; me, motheaten; wt, wild-type; xid, X-linked immunodeficiency; XLA, X-linked agammaglobulinemia.
The development of a diverse repertoire of B cells and the maintenance of self-tolerance depend on signals transduced by the B cell antigen receptor (BCR).1 The outcome of BCR engagement varies from proliferation and differentiation to deletion depending on the developmental stage of the B cell, concurrent signals, and the degree of BCR cross-linking (for review see reference 1). A complex signaling network translates BCR-mediated signals into the appropriate response given the context in which they are received. One of the initial biochemical consequences of BCR engagement is the sequential activation of a cascade of tyrosine kinases belonging to the Src, Btk/Tec, and Syk/ Zap70 families. The phosphorylation of multiple substrates by these kinases leads to signaling events which include stimulation of the Ras/mitogen-activated protein kinase (MAPK) pathway, phosphoinositide hydrolysis, Ca2+ flux, and the activation of PI3-kinase
Src family kinases, including Lyn, Blk, Fyn, Lck, and Fgr, are activated rapidly upon BCR cross-linking (2). Among Src family kinases, only mutations in Lyn have been described as affecting BCR signaling (12–16, 20). Intriguingly, Lyn appears to be involved in both the initiation of BCR signals and their subsequent downregulation (14, 20). Anti-IgM-mediated cross-linking of the BCR results in slightly delayed and reduced tyrosine phosphorylation of Ig
Mutations in Lyn also affect B cell development. The frequency of peripheral B cells is reduced approximately twofold in lyn–/– mice (12–14, 20). The remaining cells have an immature cell surface phenotype and a shorter life span than do wild-type B cells (14). Serum IgM and IgA levels are increased (12, 13). Aged lyn–/– animals develop autoantibodies and exhibit splenomegaly due to extramedullary hematopoiesis and the expansion of IgM-secreting B lymphoblasts (12–14). The phenotype of lyn–/– mice is strikingly similar to that of motheaten (me) mice (21), which are deficient in the negative regulator of BCR signaling SH2-containing protein tyrosine phosphatase 1 (SHP1). This suggests that other Src kinases cannot compensate for Lyn in the termination of BCR signals.
Several lines of evidence indicate that Btk is downstream of Src family kinases in a BCR signaling pathway. Coexpression of Btk and Lyn in fibroblasts leads to the transphosphorylation of Btk on Y551 and activation of Btk kinase activity (22, 23). Btk is also phosphorylated on Y551 in response to BCR cross-linking (22, 24). The ability of an activated form of Btk to transform fibroblasts is dependent on both the activity of Src family kinases and the presence of Y551 (25, 26). Mutation of Y551 also prevents Btk from mediating BCR-induced Ca2+ flux in B cells (27, 28). These combined observations indicate that transphosphorylation by Src kinases is critical for Btk function.
Mutations in Btk result in the B cell immunodeficiencies X-linked agammaglobulinemia (XLA) in humans (29, 30) and X-linked immunodeficiency (xid) in mice (31, 32). XLA patients have a block at the preB stage of development, resulting in a severe deficit of circulating B cells and serum Ig (for review see reference 33). Both xid and Btk–/– (9–11) mice have a more subtle phenotype (for review see reference 33). They have a 30–50% decrease in the number of peripheral B cells, with the most profound reduction in the mature IgMloIgDhi subset. xid mice have reduced levels of serum IgM and IgG3 and do not respond to type II T cell–independent antigens. They also lack B1 cells. Responses to the engagement of several cell surface receptors including BCR, IL-5R, IL-10R, and CD38 are impaired in the absence of Btk. B cells expressing reduced levels of Btk are hyposensitive to anti-IgM (34), suggesting that Btk is limiting for the transmission of signals from the BCR.
Despite the biochemical evidence that Lyn and Btk operate sequentially in common signaling pathways, the different phenotypes of Btk–/– and lyn–/– mice (low versus high serum IgM, hypo- versus hypersensitivity to BCR cross-linking) suggest that these kinases may also have opposing roles in BCR signaling. To clarify this issue, we examined B cell development in mice lacking both Btk and Lyn. If Btk and Lyn oppose each other, Btk deficiency might be expected to rescue the lyn–/– phenotype, analogous to the rescue of the me B cell phenotype by CD45 deficiency (35). If Lyn is the sole upstream activator of Btk, then effects on B cell development should be no more severe in Btk–/–lyn–/– mice than in lyn–/– mice alone. Increased severity of phenotype would indicate that Btk and Lyn are partially redundant components of one signaling pathway or participants in independent pathways. A combination of these possibilities was observed, indicating that Lyn both opposes Btk-mediated signals and plays a positive signaling role independent of or partially redundant with Btk. 
(for review see reference 2). B cell development is generally blocked at the proB to preB transition in the absence of preB receptor or BCR subunits (3–6). syk–/– mice have a similar phenotype (7, 8), but B lymphopoiesis is less severely affected in mice lacking other molecules downstream of the BCR such as Bruton's tyrosine kinase (Btk; references 9–11), Lyn (12–14), Fyn (15, 16), PKCβ (17), and Vav (18, 19). This suggests that, although Syk plays a unique role early in B cell development, there may be a significant degree of redundancy among some components of BCR signaling pathways. , Syk, shc, and several other substrates in B cells from lyn–/– mice (13, 14). The residual phosphorylation is probably catalyzed by other Src family kinases present in these cells. Despite delayed signal initiation, lyn–/– murine B cells are hypersensitive to anti-IgM stimulation (14, 20). This results from impaired downregulation of BCR signaling via both Fc
RIIb-dependent and -independent mechanisms (14).
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Mice
Btk–/– (10) and lyn–/– mice (14), each on a mixed C57B/6 x 129/Sv genetic background, were crossed to generate Btk+/–lyn+/– F1 progeny. These F1 animals were mated resulting in wild-type (wt), Btk-deficient, Lyn-deficient, and Btk/Lyn-deficient progeny. Genotypes were determined by Southern blot (10) or PCR (14) analysis of tail biopsy DNA as described. For the analysis of limiting Btk dosage in Fig. 5, crosses were performed as above starting with Btk–/– mice carrying an Ig heavy chain enhancer/ promoter–driven Btk transgene expressing 25% of endogenous Btk levels in B cells (34). The resulting progeny were on a mixed C57B/6 x 129/Sv x Balb/c background. The presence of the Btk transgene was determined by Southern blot as previously described (34).
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Analysis of In Vivo Bromodeoxyuridine Incorporation.
Mice were fed 0.25 mg/ml bromodeoxyuridine (BrdU) and 2.5% glucose in their drinking water continuously for up to 15 d. Single cell suspensions of spleens and peripheral blood were depleted of red blood cells, stained with anti-BrdU FITC (Becton Dickinson) and anti-B220 (RA3-6B2) PE (PharMingen) as previously described (36), and analyzed as above.
Proliferation Assays
BrdU Labeling.
Total splenocytes were depleted of red blood cells and plated in RPMI with 10% heat-inactivated FCS at 106/ml. Where indicated, goat anti–mouse IgM F(ab')2 fragments (Jackson ImmunoResearch Labs., West Grove, PA) were added at either 2 or 20 µg/ml. At 24 h, BrdU (Sigma Chemical Co., St. Louis, MO) was added to a final concentration of 10 µM. Cells were harvested at 48 h and FACS® analysis was performed as above.
[3H]Thymidine Labeling.
B220+ spleen cells were isolated using the Minimacs magnetic bead system (Miltenyi Biotec, Inc., Auburn, CA) according to the manufacturer's instructions. Single cell suspensions were depleted of red blood cells before incubation with magnetic beads. B cell–enriched populations were >90% B220+ by FACS® analysis. B220+ splenic B cells were seeded into 96-well plates at 5 x 105/ml in RPMI with 10% heat-inactivated FCS. Where indicated, cells were incubated for 60 h with 2 or 20 µg/ml goat anti–mouse IgM F(ab')2 fragments (Jackson ImmunoResearch Labs.). 1 µCi [3H]thymidine (NEN Life Science Products, Boston, MA) was added per well for the final 12-18 h. Cells were harvested and counted on a scintillation counter.
ELISA
Serum Ig.
Plates were coated with 2 µg/ml goat anti–mouse Ig (Southern Biotechnology Associates, Huntington, AL) and blocked with 1% BSA in borate-buffered saline (BBS). Serum or Ig standards (mouse IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA; Sigma Chemical Co.) were diluted serially into BBS and added to wells in duplicate. Plates were washed, incubated with secondary antibody (goat anti–mouse IgM, IgG1, IgG2a, IgG2b, IgG3, or IgA-alkaline phosphatase [ALPH], Southern Biotechnology Associates) diluted 1:500 in BBS/0.05% Tween 20/1% BSA and developed with an ALPH substrate kit (Bio-Rad, Hercules, CA). OD405 was read on a Vmax kinetic microplate reader (Molecular Devices Corp., Sunnyvale, CA).
Keyhole Limpet Hemocyanin.
Mice were immunized intraperitoneally with 100 µg of keyhole limpet hemocyanine (KLH) (Sigma Chemical Co.) in incomplete Freund's adjuvant (GIBCO BRL, Gaithersburg, MD), boosted on day 21 with 50 µg of KLH in PBS, and bled on day 28. ELISAs were performed as above with the following modifications: plates were coated with 8 µg/ ml of KLH, and secondary antibodies were goat anti–mouse IgM-ALPH and goat anti–mouse IgG1-ALPH.
2,4,6-Trinitrophenyl–Ficoll.
Mice were immunized intraperitoneally with 10 µg of 2,4,6-trinitrophenyl (TNP)-Ficoll (a gift of Dr. John Inman, National Institutes of Health, Bethesda, MD) and bled 6 d later. ELISA was performed as above with the following modifications: plates were coated with 25 µg/ml of TNP-BSA in PBS, and serum and secondary antibody (goat anti–mouse IgM-ALPH) were diluted into PBS/0.1% BSA, and 0.05% Tween 20.
dsDNA.
Unimmunized mice were bled at 16–20 wk of age. Serum antibodies to dsDNA were measured in triplicate by ELISA as previously described (14).
Immunofluorescence
Unimmunized mice were bled at 16–20 wk of age. Serum antibodies to nuclear antigens were measured by immunofluorescence as previously described (14).
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Reduced Numbers of Peripheral B Cells In Btk–/–lyn–/– Mice.
To understand whether Lyn and Btk are components of a common signaling pathway or function independently in B cells, we examined B cell development in the absence of Btk alone, Lyn alone, or both Btk and Lyn. Both the frequency and number of splenic B cells in 8-wk-old Btk–/–lyn–/– mice was reduced two- to fourfold relative to either Btk–/– or lyn–/– animals and four- to sixfold compared with wild-type controls (Fig. 1 A and Table 1). The remaining B cells had an immature IgMhiIgDlo phenotype similar to Btk–/– B cells (Fig. 1 B). This decrease was specific to the B lineage. No significant difference in myeloid cell numbers was observed in the spleens of young mice, and T cell numbers were reduced less than twofold compared with wild-type controls (Table 1). The frequency of conventional (B220+CD5–) B cells in the peritoneum was also diminished in Btk–/–lyn–/– mice compared with single knockouts alone (Fig. 1 D, Table 1).
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The turnover rate of the peripheral B cell population was assessed with in vivo BrdU labeling since an early developmental block did not account for the reduced number of splenic B cells in Btk–/–lyn–/– mice. Short-lived B cells in the periphery are replaced by newly generated cells that have incorporated BrdU, whereas long-lived resting cells remain unlabeled. Both splenic and peripheral blood B cells from Btk–/–lyn–/– mice turned over faster than wild-type, Btk–/–, or lyn–/– B cells (Fig. 2). Greater than 70% of Btk–/– lyn–/– B220+ cells were labeled with BrdU after 8 d, compared with 35% in wild-type mice and 50% in mice lacking either Btk or Lyn alone (Fig. 2). These observations indicate that the reduced number of B cells in mice lacking both Btk and Lyn is due to poor survival in the periphery rather than decreased production of B cells in the bone marrow.
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A Btk transgene expressing 25% of endogenous Btk levels in splenic B cells (34) was crossed onto both Btk–/– (Btklo) and Btk–/–lyn–/– (Btklo lyn–/–) backgrounds. The frequency and absolute number of B220+ and IgMloIgDhi cells were increased two- to threefold in Btklo spleens relative to Btk–/– spleens in both the presence and absence of Lyn (reference 34 and data not shown), indicating that the transgene restored Btk-dependent signals for maintenance of B cell numbers. Btklo B cells were less sensitive to BCR engagement than were wild-type B cells (reference 34 and Fig. 5 A). In contrast, the BCR-induced proliferative response of Btklo lyn–/– B cells was indistinguishable from that of lyn–/– B cells even at low doses of anti-IgM (Fig. 5 A). Btk–/–lyn–/– B cells failed to proliferate upon anti-IgM stimulation (Fig. 5 A). This was not due to altered T or myeloid cell function, as the same result was obtained with purified B cells (Fig. 5 B) and total splenocytes (Fig. 5 A). Lyn deficiency therefore enhances Btk-dependent signaling by the BCR but cannot bypass a requirement for Btk. These observations suggest that Lyn has a net inhibitory effect on Btk-dependent BCR signaling pathways.
Autoimmunity in lyn–/– Mice Is Dependent on Btk.
The development of autoimmunity in aged lyn–/– mice is another feature that distinguishes them from Btk–/– and xid mice (12–14). The xid mutation has been shown to prevent autoantibody production in both NZBxNZW mice (39, 40) and me mice (41). Although six out of six lyn–/– mice older than 16 wk developed IgM and IgG antibodies against both dsDNA and nuclear antigens, no Btk–/–lyn–/– animals of similar age displayed signs of autoimmunity (Table 2). Peritoneal B1 cells are believed to be a major source of anti-self antibodies in autoimmune strains of mice (42, 43). Btk–/–lyn–/– mice, like Btk–/– mice, have a reduced frequency of B220+CD5+ cells in the peritoneum relative to wild-type or lyn–/– animals (Fig. 1 D, Table 1).
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RI-mediated cytokine induction has been reported in xid and Btk–/– mast cells (44). Consistent with these observations, the increased frequency of myeloid and erythroid cells characteristic of lyn–/– spleens was also observed in old Btk–/–lyn–/– mice (Fig. 6 B). Surprisingly, splenomegaly did not occur in Btk–/–lyn–/– mice. Spleens of 10–11-mo-old Btk–/–lyn–/– mice were four- to fivefold smaller by both weight (0.208 ± 0.042 g vs. 1.1 ± 0.4 g, n = 2) and cell count (3.74 x 107 ± 2.5 x 107, n = 5, vs. 1.6 x 108 ± 0.72 x 108, n = 6, nucleated cells) than those of age-matched lyn–/– mice (Fig. 6 A). These results suggest that although extramedullary hematopoiesis does not require Btk, the subsequent expansion of myeloid and erythroid elements in lyn–/– mice is Btk dependent.
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Although Lyn may contribute to the activation of Btk in concert with other Src kinases, it also plays a Btk-independent role in the maintenance of the peripheral B cell population. lyn–/– mice have fewer B cells than did wild-type littermates (12–14). This has been suggested to result from increased negative selection of B cells that are hypersensitive to BCR cross-linking (14). Diminished B cell numbers in Btk–/–lyn–/– mice could be explained by the additive effect of reduced positive selection in the absence of Btk and increased negative selection in the absence of Lyn. However, this is unlikely since lyn–/– B cells do not respond to BCR engagement when they also lack Btk. Lyn is therefore likely to transmit a positive signal for B cell survival that is independent of Btk.
Reduced Life-span of B Cells in the Periphery of Btk–/–lyn–/– Mice.
B cells from both Btk–/– and lyn–/– mice have an increased turnover rate relative to wild-type B cells. The number of long-lived B cells is even further reduced in the absence of both Btk and Lyn. This could be secondary to a block in maturation as the long-lived B cell pool consists predominantly of mature B cells (45). However, this possibility is unlikely as Btk–/– and Btk–/–lyn–/– mice have a similar developmental block at the IgMhiIgDhi to IgMloIgDhi transition. The reduced half-life of Btk–/–lyn–/– B cells could also be a result of impaired positive BCR signaling due to the combined effects of Lyn deficiency on signal initiation and Btk deficiency on signal transmission. The BCR is required for survival of B cells in the periphery (46), and deletion of the cytoplasmic tail of Ig in mice results in a B cell phenotype (4) similar to that of Btk–/–lyn–/– mice. Independent roles for Btk and Lyn in BCR signaling are also supported by the recent demonstration that the Btk/Tec family kinase Itk and the Src family kinase Fyn have independent functions in TCR signaling (47). Alternatively, Btk and Lyn may be redundant for CD40 signaling. Btk–/– lyn–/– mice resemble mice deficient in both Btk and CD40 (48, 49). The impaired response to T cell–dependent antigens would also be explained by failure to transmit CD40 signals (50–52). Finally, defects in homing of Btk–/– lyn–/– B cells to the proper compartments in secondary lymphoid organs could contribute to their poor survival (53).
The Antigen-independent Phase of B Cell Development Is Normal in Btk–/–lyn–/– Mice.
Mice lacking the three Src family kinases Lyn, Blk, and Fyn have a block in development at the proB to preB transition (Tarakhovsky, A., personal communication) similar to that observed in Ig heavy chain– (5) or surrogate light chain–deficient (6) mice. These combined results imply that Src family kinases are redundant for the transmission of preB receptor signals. B lymphopoiesis is also blocked at the preB stage in XLA patients (33), suggesting that Btk is an essential substrate of Src family kinases in human preB receptor signaling. In contrast, the antigen-independent phase of B cell development is normal in Btk–/– mice even in the absence of Lyn. The critical target of Src family kinases in murine preB cells is probably Syk rather than Btk since the proB to preB transition is impaired in syk–/– mice (7, 8).
Lyn Negatively Regulates Btk-dependent Signaling Pathways.
Hypersensitivity to BCR cross-linking in lyn–/– B cells indicates that Lyn plays a critical role in the negative regulation of BCR signaling. Both the failure of Btk–/– lyn–/– B cells to proliferate in response to anti-IgM and the observation that Btk is no longer limiting for response to BCR engagement in the absence of Lyn suggest that Lyn downregulates Btk-dependent signaling pathways. The ability of Btk to promote depletion of intracellular calcium stores in response to BCR cross-linking is prevented by FcRIIb signaling (27). This inhibition may be mediated by Lyn since FcRIIb function is partially impaired in lyn–/– B cells (14). The negative regulatory role of Lyn is not limited to Btk-dependent pathways, as BCR-induced activation of the classical mitogen-activated protein kinase (MAPK) pathway does not require Btk (54, 55) but is enhanced in lyn–/– B cells (14).
The transgenic mice expressing low levels of Btk (34) are shown here (Fig. 5 A) to be a useful sensitized system with which to identify negative regulatory components of Btk signaling pathways. Similarly, molecules that contribute positively to Btk signaling could be defined by mutations that further impair BCR signaling in Btklo mice. It will be interesting to determine the effect of mutations in the remaining Src family kinases, BCR signal threshold modulators, and other B cell signaling molecules on the transmission of BCR signals by limiting amounts of Btk.
A Role For Btk in Myeloid Expansion.
A distinguishing characteristic of older lyn–/– mice is the development of splenomegaly due to extramedullary hematopoiesis (12– 14). Surprisingly, although splenomegaly did not occur in old Btk–/–lyn–/– mice, these animals had a similar increase in the frequency of myeloid and erythroid cells as lyn–/– mice. This suggests that the splenomegaly in lyn–/– mice is caused by two separate defects. The first, in which the frequency of splenic myeloid and erythroid cells is increased, is independent of Btk. This phase could result from either a shift in the site of hematopoiesis to the spleen or simply "space filling" (56) secondary to a reduction in the number of lymphoid cells. Myeloid and erythroid elements that are present in lyn–/– spleens then expand in a Btk-dependent manner. Lyn deficiency may render myeloid cells hypersensitive to cytokines, analogous to the reduction of BCR signaling thresholds in B cells. This enhanced response may be attenuated in the absence of Btk. Btk has been implicated as a component of the IL-5 (25, 57, 58), IL-6 (59), and IL-10 (60) cytokine pathways in B cells. However, no alterations in myeloid or erythroid cell development have been reported in xid mice, Btk–/– mice, or XLA patients except for some defects in FcRI signaling in mast cells (44).
Btk may serve in a general capacity to regulate mitogenic responses and cell survival. These functions would normally be observed only in B cells because of redundant signaling pathways in other lineages. Variation in genetic context or the mutation of other signaling molecules on a Btk–/– background may reveal additional roles for Btk in the development and function of hematopoietic cells.
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Acknowledgments |
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Submitted: 6 March 1998
Revised: 22 April 1998
Wasif Khan's current address is Department of Microbiology and Immunology, School of Medicine, Vanderbilt University, Nashville, TN 37232-2363.
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