Stem cell factor (SCF) acts in synergy with antigen to enhance the calcium signal, degranulation, activation of transcription factors, and cytokine production in human mast cells. However, the underlying mechanisms for this synergy remain unclear. Here we show, utilizing bone marrow-derived mast cells (BMMCs) from Btk and Lyn knock-out mice, that activation of Btk via Lyn plays a key role in promoting synergy. As in human mast cells, SCF enhanced degranulation and cytokine production in BMMCs. In Btk ؊/؊ BMMCs, in which there was a partial reduction in the capacity to degranulate in response to antigen, SCF was unable to enhance the residual antigen-mediated degranulation. Furthermore, as with antigen, the ability of SCF to promote cytokine production was abrogated in the Btk ؊/؊ BMMCs. Mast cell activation leads to the release of both preformed and de novo synthesized inflammatory mediators. The intracellular signaling cascade regulating these responses is initiated by aggregation of high affinity receptors for IgE (Fc⑀RI) 4 following antigen binding to receptorbound IgE (1). However, antigen-induced triggering of mast cells in vivo is likely to occur with a background of stem cell factor (SCF)-mediated Kit activation, as SCF is essential for the growth, differentiation, homing, and survival of mast cells (2). By mimicking this situation in vitro, we have demonstrated that SCF dramatically augments both antigen-mediated degranulation and cytokine generation in these cells (3, 4). Kitmediated signals are thus required for optimal mast cell degranulation and cytokine production induced by Fc⑀RI aggregation.Antigen-mediated degranulation and cytokine production are thought to be initiated by the activation of the Src family tyrosine kinase, Lyn (5). The resulting tyrosine phosphorylation of the ␤ and ␥ chains of Fc⑀RI promotes the binding of the tyrosine kinase Syk to Fc⑀RI (6). This permits the trans/auto-phosphorylation and activation of Syk (7, 8), which in turn phosphorylates the transmembrane adaptor molecules LAT (9) and NTAL (3, 10). These adaptor molecules orchestrate the recruitment of downstream signaling molecules to the receptor-signaling molecular complex by providing docking sites for cytosolic adaptor molecules, including SLP-76, Vav, Gads, Grb2, Gab1, and Gab2 (11) and signaling enzymes such as phospholipase (PL)C␥ 1 , PLC␥ 2 , and phosphoinositide (PI) 3-kinase (12, 13). The subsequent elevation of intracellular calcium levels and activation of protein kinase C (PKC) leads to degranulation (14), whereas activation of the Ras-Raf-MAPK pathway induces arachidonic acid metabolite release (15) and downstream phosphorylation and activation of specific cytokine gene-related transcription factors (16). A parallel pathway controlled by the Src kinase, Fyn, also appears to help regulate Fc⑀RI-dependent mast cell activation (17).Many of these same signaling events are initiated upon binding of SCF to Kit (18) but are insufficient on their own to induce degranulation (4). Our previous studies have suggest...

The development of autoimmunity is correlated with heightened sensitivity of B cells to B cell Ag receptor (BCR) cross-linking. BCR signals are down-regulated by Lyn, which phosphorylates inhibitory receptors. lyn−/− mice have reduced BCR signaling thresholds and develop autoantibodies, glomerulonephritis, splenomegaly due to myeloid hyperplasia, and increased B-1 cell numbers. Bruton’s tyrosine kinase (Btk), a critical component of BCR signaling pathways, is required for autoantibody production in lyn−/− mice. It is unclear whether Btk mediates autoimmunity at the level of BCR signal transduction or B cell development, given that lyn−/−Btk−/− mice have a severe reduction in conventional B and B-1 cell numbers. To address this issue, we crossed a transgene expressing a low dosage of Btk (Btklow) in B cells to lyn−/−Btk−/− mice. Conventional B cell populations were restored to levels similar to those in lyn−/− mice. These cells were as hypersensitive to BCR cross-linking as lyn−/− B cells as measured by proliferation, Ca2+ flux, and activation of extracellular signal-regulated kinase and Akt. However, lyn−/−Btklow mice did not produce anti-ssDNA, anti-dsDNA, anti-histone, or anti-histone/DNA IgM or IgG. They also lacked B-1 cells and did not exhibit splenomegaly. Thus, B cell hyperresponsiveness is insufficient for autoimmunity in lyn−/− mice. These studies implicate B-1 and/or myeloid cells as key contributors to the lyn−/− autoimmune phenotype.

Accumulation of plasma cells and autoantibodies against nuclear antigens characterize both human and murine lupus. Understanding how these processes are controlled may reveal novel therapeutic targets for this disease. Mice deficient in Lyn, a negative regulator of B and myeloid cell activity, develop lupus-like autoimmune disease. Here, we show that lyn À/À mice exhibit increased splenic plasmablasts and plasma cells and produce IgM against a wide range of self-antigens. Both events require Btk, a target of Lyn-dependent inhibitory pathways. A Btk-dependent increase in the expression of the plasma cell survival factor IL-6 by lyn À/À splenic myeloid cells was also observed. Surprisingly, IL-6 was not required for plasma cell accumulation or polyclonal IgM autoreactivity in lyn À/À mice. IL-6 was, however, necessary for the production of IgG autoantibodies, which we show are focused towards a limited set of nucleic acid-containing and glomerular autoantigens in lyn À/À mice. A similar uncoupling of plasma cell accumulation from IgG autoantibodies was seen in lyn 1/À mice. Plasma cell accumulation and polyclonal IgM autoreactivity are therefore controlled separately from, and are insufficient for, the production of IgG against lupus-associated autoantigens. Regulators of either of these two checkpoints may be attractive therapeutic targets for lupus.Key words: Autoimmunity . Btk . IL-6 . Lyn . Plasma cell Supporting Information available online IntroductionThe autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of tolerance to nuclear antigens resulting in autoantibody production, immune complex deposition, inflammation, and end organ damage. Aberrant accumulation of antibody-secreting cells, which include among them autoreactive specificities, is a common feature of human SLE [1] and several genetically distinct models of murine lupus, including NZB Â NZW and Lyn-deficient mice [2][3][4][5][6][7][8][9]. This has been attributed to an unusually favorable splenic plasma cell survival niche and impaired plasma cell homing to the bone marrow in the NZB Â NZW model [5,6] Lyn inhibits responses to BCR-crosslinking by phosphorylating the ITIM of several inhibitory receptors [12]. B cells from lyn À/À mice are thus hyperresponsive to BCR stimulation [4,12]. lyn À/À mice also demonstrate numerous defects in myeloid cells, such as increased myelopoiesis [13], increased integrin signaling [14], and increased production of cytokines by activated macrophages, mast cells, and, in some circumstances, dendritic cells [15][16][17]. These cytokines include IL-6, a plasma cell survival factor [18][19][20], the expression of which has been shown to be elevated in both murine and human lupus [21][22][23]. It plays a role in the production of anti-DNA antibodies in murine lupus models both in vitro and in vivo [23,24].A major target of Lyn-dependent inhibitory pathways in B cells is the BCR signaling component Btk [25][26][27]. Btk and Lyn also have opposing effects in macrophages [15,28], dendritic cells [...

The autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of tolerance to nuclear antigens such as chromatin, DNA, and RNA. This focused autoreactivity is thought to arise from the ability of DNA or RNA specific B cells to receive dual signals from the BCR and TLR9 or TLR7, respectively. The Tec kinase Btk is necessary for the production of anti-DNA antibodies in several murine models of SLE. To assess the role of Btk in the fate of DNA reactive B cells, we generated Btk−/− mice carrying the 56R anti-DNA Ig transgene on the C57BL/6 background. dsDNA specific B cells were present in 56R.Btk−/− mice, although they were not preferentially localized to the marginal zone. These cells were able to proliferate in response to large CpG DNA containing fragments that require BCR-induced internalization to access TLR9. However, anti-DNA antibodies were not observed in the serum of 56R.Btk−/− mice. A transgene expressing a low level of Btk in B cells (Btk lo ) restored anti-DNA IgM in these mice. This correlated with partial rescue of proliferative response to BCR engagement and TLR9-induced IL-10 secretion in Btk lo B cells. anti-DNA IgG was not observed in 56R.Btk lo mice, however. This was likely due, at least in part, to a role for Btk in controlling the expression of T-bet and AID in cells stimulated with CpG DNA. Thus, Btk is required for the initial loss of tolerance to DNA and the subsequent production of pathogenic autoantibodies once tolerance is breached.

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The pre-BCR and the BCR regulate B cell development via a signalosome nucleated by the adaptor protein B cell linker protein (BLNK). Formation of this complex facilitates activation of phospholipase C (PLC) c2 by Bruton's tyrosine kinase (Btk). To determine whether Btk and PLCc2 also have separate functions, we generated Btk -/-PLCc2 -/-mice. They demonstrated a block in development at the pre-B stage and increased pre-BCR surface expression. This phenotype was more severe than that of Btk -/-or PLCc2 -/-mice. Although both Btk and PLCc2 were required for proliferation of splenic B cells in response to BCR cross-linking, they contributed differently to anti-IgM-induced phosphorylation of ERK. Btk -/-and PLCc2 -/-mice each had a reduced frequency of Igk-expressing B cells and impaired migration of pre-B cells towards stromal cellderived factor 1. However, the increase in pre-B cell malignancy that occurs in BLNK -/-mice in the absence of Btk was not observed in the absence of PLCc2. Thus, Btk and PLCc2 act both in concert and independently throughout B cell development. IntroductionSignals from the B cell antigen receptor (BCR) regulate multiple stages of B lymphopoiesis [1]. In pre-B cells, the pre-BCR signals for proliferation, allelic exclusion of the IgH locus, down-regulation of VpreB and k5 expression, and light chain rearrangement. The pre-BCR is predominantly intracellular and is believed to signal in a ligand-independent manner. The BCR is first expressed on the surface of immature B cells. Antigen encounter at this stage results in negative selection via deletion, anergy, or receptor editing. A tonic BCR signal is required for differentiation beyond the T2 stage in the periphery and survival of mature B cells. Mature B cells proliferate and differentiate upon stimulation with foreign antigen in the appropriate context. BCR and pre-BCR signaling is mediated by a signalosome composed of Syk, Bruton's tyrosine kinase (Btk), B cell linker protein (BLNK; also known as SLP65 and BASH), and phospholipase C (PLC) c2 [2-4]. Receptor cross-linking activates Syk, which phosphorylates the adaptor protein BLNK. Btk and PLCc2 bind to phosphorylated BLNK via their SH2 domains. Btk then phosphorylates and activates PLCc2, resulting in changes in intracellular Ca 2+ and the activation of protein kinase Cb. Btk can also mediate BCR-stimulated Ca 2+ flux [5] and B cell development [6] independently of its catalytic activity. Btk acts as an adaptor to recruit and activate phosphatidylinositol 4-phosphate 5-kinase (PIP5K) [5], which produces the substrate for PLCc2. Thus, Btk signals through PLCc2 directly, by phosphorylation, and indirectly, by increasing substrate availability.Loss of Btk function causes the human immunodeficiency X-linked agammaglobulinemia (XLA) [7,8], which is characterized by a block in B cell development at the pre-B stage. A similar disease results from

B-1 cells are important players in the first line of defense against pathogens. According to current models for the origin of B-1 cells, they either represent a separate lineage from conventional B-2 cells or differentiate from conventional B-2 cells via an intermediate, B-1(int), in response to positive selection by antigen. Here we show that Btk, a Tec family kinase that mediates B cell antigen receptor (BCR) signaling, is required at multiple stages of B-1 cell development. VH12 anti-phosphatidylcholine (PtC) IgH transgenic mice provide a model for the induced differentiation of B-1 cells. This transgene selects for PtC-reactive cells and induces them to adopt a B-1 phenotype. Both processes have been shown to depend on Btk. To determine whether this is secondary to a requirement for Btk in the development of mature B-2 cells, we crossed VH12 transgenic mice to mice expressing low levels of Btk. B-2 cell development occurs normally in Btk(lo) mice despite reduced responsiveness to BCR crosslinking. Analysis of VH12.Btk(lo) mice reveals that Btk regulates the B-1(int) to B-1 transition and/or the survival of splenic B-1 cells, in part via a mechanism independent of its role in BCR signaling. We also show that Btk mediates the survival of, and expression of IL-10 by, those B-1 cells that do develop and migrate to the peritoneum. Multiple roles for Btk in B-1 cell development and maintenance may explain the particular sensitivity of this population to mutations in components of Btk signaling pathways.

Current models of lymphocyte activation suggest that formation of a signaling complex, or "signalosome", composed of Syk, Bruton's tyrosine kinase (Btk), phospholipase gamma2 and the adaptor protein B cell linker protein (BLNK) is critical for transmission of signals from the BCR. However, impaired B cell development in mice lacking each individual signalosome component has made it difficult to study the functional consequences of the formation of this complex in mature B cells. Sensitized genetic systems, commonly used in Drosophila, define signaling pathways by combining partial loss of function mutations in the components of interest. This allows genetic interactions to be observed in the absence of pleiotropic or lethal effects of complete deficiency of either gene. We used this approach to demonstrate that Btk and BLNK are limiting components of a common signaling pathway that mediates the mitogenic response of mature B cells to antigen. B cells from transgenic mice expressing a limiting dosage of Btk (Btk(lo)) have normal numbers of mature B cells that have reduced, but measurable, responses to BCR cross-linking. Haploinsufficiency of BLNK did not affect the development of Btk(lo) B cells. However, it exacerbated their defects in BCR-induced Ca(2+) flux, IkappaB degradation, and up-regulation of cyclin D2, bcl-x(L) and A1 leading to dramatic impairment of B cell mitogenic responses. In contrast, no effect of reduced Btk and BLNK dosage was observed on extracellular signal-regulated kinase activation. These results suggest that the signals regulating the maintenance and activation of mature B cells are differentially sensitive to the strength of the signal emanating from the signalosome.