Independent and Opposing Roles For Btk and Lyn in B and Myeloid Signaling Pathways

Transphosphorylation by Src family kinases is required for the activation of Bruton''s tyrosine kinase (Btk). Differences in the phenotypes of Btk^−/− and lyn^−/− mice suggest that these kinases may also have independent or opposing functions. B cell development and function were examined in Btk^−/−lyn^−/− mice to better understand the functional interaction of Btk and Lyn in vivo. The antigen-independent phase of B lymphopoiesis was normal in Btk^−/−lyn^−/− mice. However, Btk^−/−lyn^−/− animals had a more severe immunodeficiency than Btk^−/− mice. B cell numbers and response to T cell–dependent antigens were reduced. Btk and Lyn therefore play independent or partially redundant roles in the maintenance and function of peripheral B cells. Autoimmunity, hypersensitivity to B cell receptor (BCR) cross-linking, and splenomegaly caused by myeloerythroid hyperplasia were alleviated by Btk deficiency in lyn^−/− mice. A transgene expressing Btk at ∼25% of endogenous levels (Btk^lo) 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 Btk^lo mice are a useful sensitized system to identify regulatory components of Btk signaling pathways.     

Role of Tyrosine Phosphorylation of HS1 in B Cell Antigen Receptor-mediated Apoptosis

The 75-kD HS1 protein is highly tyrosine-phosphorylated during B cell antigen receptor (BCR)-mediated signaling. Owing to low expression of HS1, WEHI-231-derived M1 cells, unlike the parental cells, are insensitive to BCR-mediated apoptosis. Here, we show that BCR-associated tyrosine kinases Lyn and Syk synergistically phosphorylate HS1, and that Tyr378 and Tyr-397 of HS1 are the critical residues for its BCR-induced phosphorylation. In addition, unlike wild-type HS1, a mutant HS1 carrying the mutations Phe-378 and Phe-397 was unable to render M1 cells sensitive to apoptosis. Wild-type HS1, but not the mutant, localized to the nucleus under the synergy of Lyn and Syk. Thus, tyrosine phosphorylation of HS1 is required for BCR-induced apoptosis and nuclear translocation of HS1 may be a prerequisite for B cell apoptosis.Stimulation of the antigen receptor on B lymphocytes (BCR) induces intracellular biochemical events that include rapid tyrosine phosphorylation of cellular proteins. Accumulating data reveal that cytoplasmic kinases such as the Syk kinase and Src-like kinases are associated with the BCR (1, 2) and play important roles in the signal transduction cascade through the BCR (3–5). We previously demonstrated that tyrosine phosphorylation of various cellular proteins was greatly enhanced in COS7 fibroblasts transfected with both Lyn and Syk expression plasmids as compared with those transfected with either the Lyn or Syk plasmid alone (6). Thus, these kinases may cooperate in phosphorylating substrates crucial for BCR-mediated B cell activation.The 75-kD HS1 protein is highly tyrosine phosphorylated upon BCR cross-linking (7). Studies with HS1 ^−/− mice (8) and with a mutant WEHI-231 cell line that expresses very low level of HS1 (9) suggest that HS1 plays roles in not only B cell proliferation but also apoptosis upon BCR cross-linking. In this study, we addressed molecular mechanisms of HS1 phosphorylation and significance of HS1 phosphorylation in BCR-mediated apoptosis.     

The Motheaten Mutation Rescues B Cell Signaling and Development in CD45-deficient Mice

The cytosolic SHP-1 and transmembrane CD45 protein tyrosine phosphatases (PTP) play critical roles in regulating signal transduction via the B cell antigen receptor (BCR). These PTPs differ, however, in their effects on BCR function. For example, BCR-mediated mitogenesis is essentially ablated in mice lacking CD45 (CD45⁻), but is enhanced in SHP-1–deficient motheaten (me) and viable motheaten (me^v) mice. To determine whether these PTPs act independently or coordinately in modulating the physiologic outcome of BCR engagement, we assessed B cell development and signaling in CD45-deficient me^v (CD45⁻/SHP-1⁻) mice. Here we report that the CD45⁻/SHP-1⁻ cells undergo appropriate induction of protein kinase activity, mitogen-activated protein kinase activation, and proliferative responses after BCR aggregation. However, BCR-elicited increases in the tyrosine phosphorylation of several SHP-1–associated phosphoproteins, including CD19, were substantially enhanced in CD45⁻/SHP-1⁻, compared to wild-type and CD45⁻ cells. In addition, we observed that the patterns of cell surface expression of μ, δ, and CD5, which distinguish the PTP-deficient from normal mice, are largely restored to normal levels in the double mutant animals. These findings indicate a critical role for the balance of SHP-1 and CD45 activities in determining the outcome of BCR stimulation and suggest that these PTPs act in a coordinate fashion to couple antigen receptor engagement to B cell activation and maturation.     

The Inositol Polyphosphate 5-Phosphatase Ship Is a Crucial Negative Regulator of B Cell Antigen Receptor Signaling

Ship is an Src homology 2 domain containing inositol polyphosphate 5-phosphatase which has been implicated as an important signaling molecule in hematopoietic cells. In B cells, Ship becomes associated with Fcγ receptor IIB (FcγRIIB), a low affinity receptor for the Fc portion of immunoglobulin (Ig)G, and is rapidly tyrosine phosphorylated upon B cell antigen receptor (BCR)–FcγRIIB coligation. The function of Ship in lymphocytes was investigated in Ship^−/− recombination-activating gene (Rag)^−/− chimeric mice generated from gene-targeted Ship^−/− embryonic stem cells. Ship^−/−Rag^−/− chimeras showed reduced numbers of B cells and an overall increase in basal serum Ig. Ship^−/− splenic B cells displayed prolonged Ca²⁺ influx, increased proliferation in vitro, and enhanced mitogen-activated protein kinase (MAPK) activation in response to BCR–FcγRIIB coligation. These results demonstrate that Ship plays an essential role in FcγRIIB-mediated inhibition of BCR signaling, and that Ship is a crucial negative regulator of Ca²⁺ flux and MAPK activation.     

Tyrosine Phosphorylation of Pyk2 Is Selectively Regulated by Fyn During TCR Signaling

The Src family protein tyrosine kinases (PTKs), Lck and Fyn, are coexpressed in T cells and perform crucial functions involved in the initiation of T cell antigen receptor (TCR) signal transduction. However, the mechanisms by which Lck and Fyn regulate TCR signaling are still not completely understood. One important question is whether Lck and Fyn have specific targets or only provide functional redundancy during TCR signaling. We have previously shown that Lck plays a major role in the tyrosine phosphorylation of the TCR-ζ chain and the ZAP-70 PTK. In an effort to identify the targets that are specifically regulated by Fyn, we have studied the tyrosine phosphorylation of Pyk2, a recently discovered new member of the focal adhesion kinase family PTK. We demonstrated that Pyk2 was rapidly tyrosine phosphorylated following TCR stimulation. TCR-induced tyrosine phosphorylation of Pyk2 was selectively dependent on Fyn but not Lck. Moreover, in heterologous COS-7 cells, coexpression of Pyk2 with Fyn but not Lck resulted in substantial increases in Pyk2 tyrosine phosphorylation. The selective regulation of Pyk2 tyrosine phosphorylation by Fyn in vivo correlated with the preferential phosphorylation of Pyk2 by Fyn in vitro. Our results demonstrate that Pyk2 is a specific target regulated by Fyn during TCR signaling.Engagement of the TCR evokes a series of signal transduction events critical for the functional activation of T cells (reviewed in reference 1). Signal transduction through the TCR is also important for T cell development (1). The earliest detectable signaling event after TCR stimulation is the activation of protein tyrosine kinases (PTKs)¹, resulting in the tyrosine phosphorylation of cellular proteins (1). Lck and Fyn, two cytoplasmic PTKs of the Src family, have been implicated as the initiating PTKs for TCR signaling. Lck is critical for TCR signaling. Mutant T cell lines lacking functional Lck or T cells from lck ^−/− mice respond to TCR stimulation with very limited tyrosine phosphorylation of cellular proteins, greatly decreased calcium mobilization, and reduced proliferation (2–4). Lck also plays a critical role in T cell development, as lck ^−/− mice have a pronounced reduction in thymocyte numbers and a block in thymocyte development at the early CD4⁺CD8⁺ stage (2). The residual progression of thymocytes from CD4⁻CD8⁻ to CD4⁺CD8⁺ stage in lck ^−/− mice depends upon the redundant function of Fyn. Combined disruption of both Lck and Fyn (lck ^−/−/fyn ^−/−) completely arrests thymocyte development at the CD4⁻CD8⁻ stage (5, 6). Fyn is also important for TCR signaling. Mature CD4⁺ and CD8⁺ thymocytes from fyn ^−/− mice are severely impaired in TCR signaling as measured by calcium mobilization, protein tyrosine phosphorylation, IL-2 production, and proliferation (7, 8). Peripheral T cells from fyn ^−/− mice are also impaired in TCR signaling, albeit to a lesser degree (7, 8).The mechanisms by which Lck or Fyn regulates proximal TCR signaling are still not completely understood. One important issue is whether Lck and Fyn have specific targets or only provide functional redundancy during TCR signaling. We have studied the TCR signaling pathway in T cells from lck ^−/− or fyn ^−/− mice in an attempt to identify targets for Lck and Fyn. We have shown previously that Lck is the primary PTK that regulates the tyrosine phosphorylation of the TCR subunits and of ZAP-70 (9), a Syk family PTK critical for TCR signaling (reviewed in reference 10). The identity of the downstream target(s) for Fyn has not been identified. In the present study, we have focused our efforts in identifying target(s) whose phosphorylation is specifically regulated by Fyn. Previous studies have shown that Fyn interacts with a number of proteins in T cells (11–16), including several proteins migrating from 110 to 130 kD. These proteins are potential targets for Fyn, though their identities have not been fully elucidated. A recently discovered cytoplasmic PTK Pyk2 has a molecular mass of 112 kD (17–19). Pyk2 is a member of the focal adhesion kinase (FAK) family and has been shown to play important roles in signal transduction of neuronal cells (17, 20, 21). Because Pyk2 is also expressed in T cells (18, 19), we examined whether it might be a target for Fyn during TCR signaling. Our data demonstrate that Pyk2 is a novel Fyndependent tyrosine-phosphorylated substrate during TCR signaling.     

SLP-65: A New Signaling Component in B Lymphocytes which Requires Expression of the Antigen Receptor for Phosphorylation

The B cell antigen receptor (BCR) consists of the membrane-bound immunoglobulin (Ig) molecule as antigen-binding subunit and the Ig-α/Ig-β heterodimer as signaling subunit. BCR signal transduction involves activation of protein tyrosine kinases (PTKs) and phosphorylation of several proteins, only some of which have been identified. The phosphorylation of these proteins can be induced by exposure of B cells either to antigen or to the tyrosine phosphatase inhibitor pervanadate/H₂O₂. One of the earliest substrates in B cells is a 65-kD protein, which we identify here as a B cell adaptor protein. This protein, named SLP-65, is part of a signaling complex involving Grb-2 and Vav and shows homology to SLP-76, a signaling element of the T cell receptor. In pervanadate/H₂O₂-stimulated cells, SLP-65 becomes phosphorylated only upon expression of the BCR. These data suggest that SLP-65 is part of a BCR transducer complex.     

Identification of the tyrosine phosphatase PTP1C as a B cell antigen receptor-associated protein involved in the regulation of B cell signaling

Recent data implicating loss of PTP1C tyrosine phosphatase activity in the genesis of the multiple hemopoietic cell defects found in systemic autoimmune/immunodeficient motheaten (me) and viable motheaten (mev) mice suggest that PTP1C plays an important role in modulating intracellular signaling events regulating cell activation and differentiation. To begin elucidating the role for this cytosolic phosphatase in lymphoid cell signal transduction, we have examined early signaling events and mitogenic responses induced by B cell antigen receptor (BCR) ligation in me and mev splenic B cells and in CD5+ CH12 lymphoma cells, which represent the lymphoid population amplified in motheaten mice. Despite their lack of functional PTP1C, me and mev B cells proliferated normally in response to LPS. However, compared with wild-type B cells, cells from the mutant mice were hyperresponsive to normally submitogenic concentrations of F(ab')2 anti- Ig antibody, and they exhibited reduced susceptibility to the inhibitory effects of Fc gamma IIRB cross-linking on BCR-induced proliferation. Additional studies of unstimulated CH12 and wild-type splenic B cells revealed the constitutive association of PTP1C with the resting BCR complex, as evidenced by coprecipitation of PTP1C protein and phosphatase activity with BCR components and the depletion of BCR- associated tyrosine phosphatase activity by anti-PTP1C antibodies. These results suggest a role for PTP1C in regulating the tyrosine phosphorylation state of the resting BCR complex components, a hypothesis supported by the observation that PTP1C specifically induces dephosphorylation of a 35-kD BCR-associated protein likely representing Ig-alpha. In contrast, whereas membrane Ig cross-linking was associated with an increase in the tyrosine phosphorylation of PTP1C and an approximately 140-kD coprecipitated protein, PTP1C was no longer detected in the BCR complex after receptor engagement, suggesting that PTP1C dissociates from the activated receptor complex. Together these results suggest a critical role for PTP1C in modulating BCR signaling capacity, and they indicate that the PTP1C influence on B cell signaling is likely to be realized in both resting and activated cells.     

Myeloid cells,BAFF, and IFN-γ establish an inflammatory loop that exacerbates autoimmunity in Lyn-deficient mice

Autoimmunity is traditionally attributed to altered lymphoid cell selection and/or tolerance, whereas the contribution of innate immune cells is less well understood. Autoimmunity is also associated with increased levels of B cell–activating factor of the TNF family (BAFF; also known as B lymphocyte stimulator), a cytokine that promotes survival of self-reactive B cell clones. We describe an important role for myeloid cells in autoimmune disease progression. Using Lyn-deficient mice, we show that overproduction of BAFF by hyperactive myeloid cells contributes to inflammation and autoimmunity in part by acting directly on T cells to induce the release of IFN-γ. Genetic deletion of IFN-γ or reduction of BAFF activity, achieved by either reducing myeloid cell hyperproduction or by treating with an anti-BAFF monoclonal antibody, reduced disease development in lyn^−/− mice. The increased production of IFN-γ in lyn^−/− mice feeds back on the myeloid cells to further stimulate BAFF release. Expression of BAFF receptor on T cells was required for their full activation and IFN-γ release. Overall, our data suggest that the reciprocal production of BAFF and IFN-γ establishes an inflammatory loop between myeloid cells and T cells that exacerbates autoimmunity in this model. Our findings uncover an important pathological role of BAFF in autoimmune disorders.Systemic lupus erythematosus is a prototypic autoimmune disease with complex and unclear etiology (Rahman and Isenberg, 2008). Most studies of this disease have focused on the defects of B and T cell tolerance as an underlying cause of the disorder. Recently, however, greater attention has been given to the pathological roles of myeloid cells in autoimmunity (Cohen et al., 2002; Hanada et al., 2003; Zhu et al., 2005; Stranges et al., 2007).Mice lacking Lyn, an Src family kinase mainly expressed in B and myeloid cells, are a well-established model of lupus-like autoimmunity (Xu et al., 2005). lyn^−/− mice develop progressive autoimmunity characterized by autoantibody production, lymphocyte activation, immune complex deposition, and nephritis (Hibbs et al., 1995; Nishizumi et al., 1995; Chan et al., 1997; Yu et al., 2001). The development of autoimmunity in lyn^−/− mice has been mainly attributed to alterations in B cell signaling thresholds, leading to abnormal B cell selection and/or tolerance resulting in production of self-reactive antibodies (Chan et al., 1998; Xu et al., 2005). The Lyn mutation directly affects B cell development, as lyn^−/− mice have an ∼30–50% reduction in mature B cell numbers because of the reduction of specific B cell subtypes such as marginal zone and follicular B cells (Xu et al., 2005; Gross et al., 2009).Lyn is also expressed in innate immune cells, where it regulates cell signaling thresholds to several CSFs, such as G-CSF, GM-CSF, and M-CSF (Harder et al., 2001, 2004; Scapini et al., 2009). lyn^−/− myeloid cells are hyperresponsive to engagement of surface integrins, leading to hyperadhesion, enhanced respiratory burst, and increased secondary granule release (Pereira and Lowell, 2003). Despite this experimental evidence in vitro, the contribution of myeloid cells to the development of autoimmunity in lyn^−/− mice has not been investigated.Autoimmunity is often associated, both in mice and humans, with excess production of B cell–activating factor of the TNF family (BAFF), a member of the TNF superfamily of cytokines also known as B lymphocyte stimulator (Mackay et al., 2007; Stadanlick and Cancro, 2008; Mackay and Schneider, 2009). Both autoimmune-prone mice (such as MRL^lpr/lpr and NZB×W F1) and human patients suffering from autoimmune disorders such as systemic lupus erythematosus or rheumatoid arthritis have elevated serum levels of BAFF (Kalled, 2005; Mackay and Schneider, 2009). This cytokine is thought to exert its pathogenic role, under conditions of excess production, through its ability to support survival and proliferation of autoreactive B cells, which have a higher BAFF dependence (Lesley et al., 2004). However, in addition to its effect on B cells, recent work has suggested that BAFF can also promote T cell activation (Ye et al., 2004; Sutherland et al., 2005; Mackay and Leung, 2006; Lai Kwan Lam et al., 2008). Despite this evidence, it remains unclear if BAFF exerts a direct pathogenic role on T cells in vivo during autoimmunity. Furthermore, the mechanisms responsible for deregulated BAFF production in autoimmune diseases have been poorly investigated. Studies in mice have shown that there are two distinct pools of BAFF: a constitutive pool produced by stromal cells, which is thought to regulate the size and maturation stage of the peripheral B cell compartment, and an accessory pool, produced mainly by myeloid cells during inflammatory or immune responses (Schneider, 2005). Which of these two pools contributes to autoimmune pathologies is unknown. Different attempts to neutralize BAFF activity in autoimmune disorders have been performed in both mice and humans, but despite a general agreement on the efficacy of the treatments, the mechanisms of this protection are still not fully understood (Ding, 2008; Ramanujam and Davidson, 2008; Moisini and Davidson, 2009).Another important cytokine that has been shown to be involved in lupus pathogenesis is IFN-γ. Several studies in MRL^lpr/lpr and NZB×W F1 autoimmune-prone mice observed significant reduction of histological and serological disease characteristics, and extended survival in these strains after IFN-γ genetic deletion or after anti–IFN-γ mAb treatment (Theofilopoulos et al., 2001).We found that the levels of BAFF were dramatically higher in the sera of lyn^−/− mice compared with WT animals, and that the deregulated production of BAFF by lyn^−/− myeloid cells can contribute to autoimmunity in these animals by affecting not only B cell activation but, more interestingly, by directly promoting T cell activation and IFN-γ production by the latter cells. These findings shed new insight on the pathological mechanisms of interplay between innate and adaptive immunity, as well as the consequences of BAFF and IFN-γ overproduction, in autoimmune disorders.     

A Critical Role for Syk in Signal Transduction and Phagocytosis Mediated by Fcγ Receptors on Macrophages

Receptors on macrophages for the Fc region of IgG (FcγR) mediate a number of responses important for host immunity. Signaling events necessary for these responses are likely initiated by the activation of Src-family and Syk-family tyrosine kinases after FcγR cross-linking. Macrophages derived from Syk-deficient (Syk⁻) mice were defective in phagocytosis of particles bound by FcγRs, as well as in many FcγR-induced signaling events, including tyrosine phosphorylation of a number of cellular substrates and activation of MAP kinases. In contrast, Syk⁻ macrophages exhibited normal responses to another potent macrophage stimulus, lipopolysaccharide. Phagocytosis of latex beads and Escherichia coli bacteria was also not affected. Syk⁻ macrophages exhibited formation of polymerized actin structures opposing particles bound to the cells by FcγRs (actin cups), but failed to proceed to internalization. Interestingly, inhibitors of phosphatidylinositol 3-kinase also blocked FcγR-mediated phagocytosis at this stage. Thus, PI 3-kinase may participate in a Syk-dependent signaling pathway critical for FcγR-mediated phagocytosis. Macrophages derived from mice deficient for the three members of the Src-family of kinases expressed in these cells, Hck, Fgr, and Lyn, exhibited poor Syk activation upon FcγR engagement, accompanied by a delay in FcγR-mediated phagocytosis. These observations demonstrate that Syk is critical for FcγR-mediated phagocytosis, as well as for signal transduction in macrophages. Additionally, our findings provide evidence to support a model of sequential tyrosine kinase activation by FcγR's analogous to models of signaling by the B and T cell antigen receptors.     

Sustained activation of Lyn tyrosine kinase in vivo leads to autoimmunity

Genetic ablation of the Lyn tyrosine kinase has revealed unique inhibitory roles in B lymphocyte signaling. We now report the consequences of sustained activation of Lyn in vivo using a targeted gain-of-function mutation (Lyn(up/up) mice). Lyn(up/up) mice have reduced numbers of conventional B lymphocytes, down-regulated surface immunoglobulin M and costimulatory molecules, and elevated numbers of B1a B cells. Lyn(up/up) B cells are characterized by the constitutive phosphorylation of negative regulators of B cell antigen receptor (BCR) signaling including CD22, SHP-1, and SHIP-1, and display attributes of lymphocytes rendered tolerant by constitutive engagement of the antigen receptor. However, exaggerated positive signaling is also apparent as evidenced by the constitutive phosphorylation of Syk and phospholipase Cgamma2 in resting Lyn(up/up) B cells. Similarly, Lyn(up/up) B cells show a heightened calcium flux in response to BCR stimulation. Surprisingly, Lyn(up/up) mice develop circulating autoreactive antibodies and lethal autoimmune glomerulonephritis, suggesting that enhanced positive signaling eventually overrides constitutive negative signaling. These studies highlight the difficulty in maintaining tolerance in the face of chronic stimulation and emphasize the pivotal role of Lyn in B cell signaling.     

Ion channel TRPV1-dependent activation of PTP1B suppresses EGFR-associated intestinal tumorigenesis

The intestinal epithelium has a high rate of turnover, and dysregulation of pathways that regulate regeneration can lead to tumor development; however, the negative regulators of oncogenic events in the intestinal epithelium are not fully understood. Here we identified a feedback loop between the epidermal growth factor receptor (EGFR), a known mediator of proliferation, and the transient receptor potential cation channel, subfamily V, member 1 (TRPV1), in intestinal epithelial cells (IECs). We found that TRPV1 was expressed by IECs and was intrinsically activated upon EGFR stimulation. Subsequently, TRPV1 activation inhibited EGFR-induced epithelial cell proliferation via activation of Ca²⁺/calpain and resulting activation of protein tyrosine phosphatase 1B (PTP1B). In a murine model of multiple intestinal neoplasia (Apc^Min/+ mice), TRPV1 deficiency increased adenoma formation, and treatment of these animals with an EGFR kinase inhibitor reversed protumorigenic phenotypes, supporting a functional association between TRPV1 and EGFR signaling in IECs. Administration of a TRPV1 agonist suppressed intestinal tumorigenesis in Apc^Min/+ mice, similar to — as well as in conjunction with — a cyclooxygenase-2 (COX-2) inhibitor, which suggests that targeting both TRPV1 and COX-2 has potential as a therapeutic approach for tumor prevention. Our findings implicate TRPV1 as a regulator of growth factor signaling in the intestinal epithelium through activation of PTP1B and subsequent suppression of intestinal tumorigenesis.     

Inhibition of the B Cell by CD22: A Requirement for Lyn

Mice in which the Lyn, Cd22, or Shp-1 gene has been disrupted have hyperactive B cells and autoantibodies. We find that in the absence of Lyn, the ability of CD22 to become tyrosine phosphorylated after ligation of mIg, to recruit SHP-1, and to suppress mIg-induced elevation of intracellular [Ca²⁺] is lost. Therefore, Lyn is required for the SHP-1–mediated B cell suppressive function of CD22, accounting for similarities in the phenotypes of these mice.     

Altered antigen receptor signaling and impaired Fas-mediated apoptosis of B cells in Lyn-deficient mice

Mice deficient in the src related protein tyrosine kinase, Lyn, exhibit splenomegaly and accumulate lymphoblast-like and plasma cells in spleen as they age, resulting in elevated levels of serum IgM (10-20-fold of control) and glomerulonephritis due to the presence of immune complexes containing auto-reactive antibodies. It remains unclear, however, how antibody-producing cells are accumulated in the lymphoid tissues of Lyn- /- mice. To elucidate the role of Lyn in B cell function, we have studied the proliferative responses to various stimuli and Fas-mediated apoptosis in B cells from young Lyn-/- mice which do not yet show apparent abnormality such as splenomegaly. Compared with control B cells, Lyn-/- B cells were hyper responsive to anti-IgM-induced proliferation and defective in Fc gamma RIIB-mediated suppression of B cell antigen receptor (BCR) signaling, indicating that Lyn is involved in the negative regulation of BCR signaling. In addition, the BCR- mediated signal in Lyn-/- B cells, unlike that in control B cells, failed to act in synergy with either CD40- or IL-4 receptor-triggered signal in inducing a strong proliferative response, suggesting that the BCR signaling pathway in Lyn-/- B cells is altered from that in control B cells. Furthermore, Lyn-/- B cells were found to be impaired in the induction of Fas expression after CD40 ligation and exhibited a reduced susceptibility to Fas-mediated apoptosis. Moreover, BCR cross-linking in Lyn-/- B cells suppressed Fas expression induced by costimulation with CD40 ligand and IL-4. Collectively, these results suggest that the accumulation of lymphoblast-like and plasma cells in Lyn-/- mice may be caused in part, by the accelerated activation of B cells in the absence of Lyn, as well as the impaired Fas-mediated apoptosis after the activation.     

A role for Bruton's tyrosine kinase in B cell antigen receptor-mediated activation of phospholipase C-gamma 2

Defects in the gene encoding Bruton's tyrosine kinase (Btk) result in a disease called X-linked agammaglobulinemia, in which there is a profound decrease of mature B cells due to a block in B cell development. Recent studies have shown that Btk is tyrosine phosphorylated and activated upon B cell antigen receptor (BCR) stimulation. To elucidate the functions of this kinase, we examined BCR signaling of DT40 B cells deficient in Btk. Tyrosine phosphorylation of phospholipase C (PLC)-gamma 2 upon receptor stimulation was significantly reduced in the mutant cells, leading to the loss of both BCR-coupled phosphatidylinositol hydrolysis and calcium mobilization. Pleckstrin homology and Src-homology 2 domains of Btk were required for PLC-gamma 2 activation. Since Syk is also required for the BCR-induced PLC-gamma 2 activation, our findings indicate that PLC-gamma 2 activation is regulated by Btk and Syk through their concerted actions.     

T Cell Receptor–initiated Calcium Release Is Uncoupled from Capacitative Calcium Entry in Itk-deficient T Cells

Itk, a Tec family tyrosine kinase, plays an important but as yet undefined role in T cell receptor (TCR) signaling. Here we show that T cells from Itk-deficient mice have a TCR-proximal signaling defect, resulting in defective interleukin 2 secretion. Upon TCR stimulation, Itk^−/− T cells release normal amounts of calcium from intracellular stores, but fail to open plasma membrane calcium channels. Since thapsigargin-induced store depletion triggers normal calcium entry in Itk^−/− T cells, an impaired biochemical link between store depletion and channel opening is unlikely to be responsible for this defect. Biochemical studies indicate that TCR-induced inositol 1,4,5 tris-phosphate (IP3) generation and phospholipase C γ1 tyrosine phosphorylation are substantially reduced in Itk^−/− T cells. In contrast, TCR-ζ and ZAP-70 are phosphorylated normally, suggesting that Itk functions downstream of, or in parallel to, ZAP-70 to facilitate TCR-induced IP3 production. These findings support a model in which quantitative differences in cytosolic IP3 trigger distinct responses, and in which only high concentrations of IP3 trigger the influx of extracellular calcium.     

The Molecular Mechanism of B Cell Activation by toll-like Receptor Protein RP-105

The B cell–specific transmembrane protein RP-105 belongs to the family of Drosophila toll-like proteins which are likely to trigger innate immune responses in mice and man. Here we demonstrate that the Src-family protein tyrosine kinase Lyn, protein kinase C β I/II (PKCβI/II), and Erk2-specific mitogen-activated protein (MAP) kinase kinase (MEK) are essential and probably functionally connected elements of the RP-105–mediated signaling cascade in B cells. We also find that negative regulation of RP-105–mediated activation of MAP kinases by membrane immunoglobulin may account for the phenomenon of antigen receptor–mediated arrest of RP-105–mediated B cell proliferation.     

Gαq-containing G proteins regulate B cell selection and survival and are required to prevent B cell–dependent autoimmunity

Survival of mature B cells is regulated by B cell receptor and BAFFR-dependent signals. We show that B cells from mice lacking the G_αq subunit of trimeric G proteins (Gnaq^−/− mice) have an intrinsic survival advantage over normal B cells, even in the absence of BAFF. Gnaq^−/− B cells develop normally in the bone marrow but inappropriately survive peripheral tolerance checkpoints, leading to the accumulation of transitional, marginal zone, and follicular B cells, many of which are autoreactive. Gnaq^−/− chimeric mice rapidly develop arthritis as well as other manifestations of systemic autoimmune disease. Importantly, we demonstrate that the development of the autoreactive B cell compartment is the result of an intrinsic defect in Gnaq^−/− B cells, resulting in the aberrant activation of the prosurvival factor Akt. Together, these data show for the first time that signaling through trimeric G proteins is critically important for maintaining control of peripheral B cell tolerance induction and repressing autoimmunity.Autoreactive B cells significantly contribute to the morbidity and mortality associated with many autoimmune diseases (Manjarrez-Orduño et al., 2009). B cell tolerance is normally controlled at several checkpoints in the BM and periphery (Goodnow, 2007). In the periphery, BCR- and BAFFR-dependent signals are required for the differentiation of immature transitional B cells into mature B cells and the continued maintenance of mature B cells (Cancro, 2009). B cells that express BCRs with intermediate affinity for autoantigens are less competitive than nonautoreactive B cells for access to the survival niches in the spleen and are eliminated at the transitional T1 stage of development (Lesley et al., 2004; Thien et al., 2004). However, in the presence of excess BAFF (also known as BLyS), autoreactive B cells can pass the T1 checkpoint and enter the mature B cell pool (Thien et al., 2004). Thus, the appropriate survival and selection of B cells in the periphery appears to be dependent on a dynamic integration of BAFFR and BCR signals.Engagement of either the BCR or BAFFR alone is insufficient to maintain mature B cell survival in the periphery (Cancro, 2009), as BCR signaling is required to sustain NF-κB–dependent BAFFR signaling (Stadanlick et al., 2008). In addition to the NF-κB–dependent cross talk between BAFFR and the BCR, both receptors can also activate PI3K (Fruman and Bismuth, 2009) and its downstream target Akt (Pogue et al., 2000; Patke et al., 2006), a serine threonine kinase which functions as a prosurvival factor in many cell types (Manning and Cantley, 2007). One recent study showed that BCR-dependent survival of mature B cells is highly dependent on PI3K (Srinivasan et al., 2009), and another study showed that activation of the PI3K pathway can rescue normally anergic autoreactive B cells (Browne et al., 2009). The PI3K–Akt signaling pathway is also engaged by activation of seven transmembrane-spanning G protein–coupled receptors (GPCRs; Yanamadala et al., 2009). GPCRs associate with heterotrimeric G proteins in their GDP-bound state (Wettschureck et al., 2004). Upon ligand binding to the GPCR, GDP is exchanged for GTP, which causes G protein release and the disassociation of the GTP-bound α subunit and the βγ dimer. Signal transduction is mediated by both the GTP-bound α subunit and the βγ dimer, but specialization and diversification of the response is often mediated by the GTP-bound α subunits (Wettschureck et al., 2004). There are 16 α subunits that fall into four classes, G_αi, G_αs, G_αq/11, and G_12/13, based on their downstream signaling targets. PI3K can be activated by the βγ dimers released from G_αi-coupled receptors (Wettschureck et al., 2004). In contrast, G_αq, a member of the G_αq/11 family, normally inhibits PI3K activation and prevents activation of Akt (Harris et al., 2006). In cardiomyocytes, Akt activation and cell survival is enhanced when the amount of active GTP-bound G_αq is low (Howes et al., 2006). However, when the amount of active G_αq is increased in cardiomyocytes, Akt activity is inhibited (Ballou et al., 2003) and survival of the cells upon stimulation is reduced (Howes et al., 2003). Examination of cardiomyocytes from transgenic mice expressing G_αq in the cardiomyocytes indicated that the level of cardiomyocyte apoptosis correlated directly with the amount of active G_αq expressed in the cells (Adams et al., 1998). Likewise, increased expression levels of G_αq are associated with changes in cardiomyocyte survival and in the development of cardiac disease in patients (Liggett et al., 2007; Frey et al., 2008). Together, these data suggest that one major function of G_αq is to suppress the PI3K/Akt signaling axis and cell survival. Surprisingly, despite that fact G_αq is expressed ubiquitously in B cells and myeloid cells (Wilkie et al., 1991), nothing is known regarding the requirement for G_αq-containing G proteins in regulating Akt activity or hematopoietic cell survival.In this paper, we show that G_αq regulates peripheral B cell tolerance by suppressing the survival and selection of autoreactive B cells. In the absence of G_αq, B cells constitutively express higher levels of activated Akt and preferentially survive BCR-induced cell death signals and BAFF withdrawal in vitro and in vivo. Most importantly, G_αq-deficient mice rapidly develop an autoreactive B cell repertoire and systemic autoimmunity. Together, these data show that G_αq-containing G proteins, working in concert with the BCR and BAFFR signaling networks, regulate B cell development and peripheral tolerance induction. In addition, the data provide the first example of G protein–dependent suppression of B cell–mediated autoimmunity.