Bruton's tyrosine kinase mediates NF-kappa B activation and B cell survival by B cell-activating factor receptor of the TNF-R family

Loss of Bruton's tyrosine kinase (Btk) function results in mouse Xid disease characterized by a reduction in mature B cells and impaired humoral immune responses. These defects have been mainly attributed to impaired BCR signaling including reduced activation of the classical NF-kappaB pathway. In this study we show that Btk also couples the receptor for B cell-activating factor (BAFF) of the TNF family (BAFF-R) to the NF-kappaB pathway. Loss of Btk results in defective BAFF-mediated activation of both classical and alternative NF-kappaB pathways. Btk appears to regulate directly the classical pathway in response to BAFF such that Btk-deficient B cells exhibit reduced kinase activity of IkappaB kinase gamma-containing complexes and defective IkappaBalpha degradation. In addition, Btk-deficient B cells produce reduced levels of NF-kappaB2 (p100) basally and in response to stimulation via the BCR or BAFF-R, resulting in impaired activation of the alternative NF-kappaB pathway by BAFF. These results suggest that Btk regulates B cell survival by directly regulating the classical NF-kappaB pathway under both BCR and BAFF-R, as well as by inducing the expression of the components of alternative pathway for sustained NF-kappaB activation in response BAFF. Thus, impaired BCR- and BAFF-induced signaling to NF-kappaB may contribute to the observed defects in B cell survival and humoral immune responses in Btk-deficient mice.     

B cell receptor (BCR) cross-talk: CD40 engagement creates an alternate pathway for BCR signaling that activates I kappa B kinase/I kappa B alpha/NF-kappa B without the need for PI3K and phospholipase C gamma

BCR signaling is propagated by a series of intermediaries and eventuates in NF-kappaB activation, among other outcomes. Interruption of several mediators that constitute the signalosome, such as PI3K and phospholipase Cgamma2, completely blocks BCR signaling for NF-kappaB. We show here that this accepted, conventional paradigm is, in fact, limited to naive B cells. CD40L treatment reprograms normal B cells such that a novel, alternate pathway for BCR signaling is created. Through this alternate pathway BCR triggering induces nuclear NF-kappaB without the need for PI3K or for phospholipase Cgamma2. Induction of NF-kappaB via the alternate pathway is accompanied by IkappaB kinase beta (IKKbeta) phosphorylation, IkappaBalpha phosphorylation, and IkappaBalpha degradation, and inhibition of IKKbeta blocked IkappaBalpha degradation. Several key events in the conventional pathway, including early protein tyrosine phosphorylation, were unimpeded by generation of the alternate pathway which appears to operate in parallel, rather than in competition, with classical BCR signaling. These results demonstrate cross-talk between CD40 and BCR, such that the requirements for BCR signaling are altered by prior B cell exposure to CD40L. The alternate BCR signaling pathway bypasses multiple signalosome elements and terminates in IKKbeta activation.     

Cutting edge: T cell migration regulated by CXCR4 chemokine receptor signaling to ZAP-70 tyrosine kinase

Chemokines regulate the homeostatic trafficking of lymphocytes and lymphocyte influx into sites of injury and inflammation. The signaling pathways by which chemokine receptors regulate lymphocyte migration remain incompletely characterized. We demonstrate that Jurkat T cells lacking the ZAP-70 tyrosine kinase exhibit reduced migration in response to the CXCR4 ligand CXCL12 when compared with wild-type Jurkat T cells. Expression of wild-type, but not kinase-inactive, ZAP-70 resulted in enhanced migration of ZAP-70-deficient Jurkat T cells. The tyrosine residue at position 292 in the interdomain B region of ZAP-70 exerts a negative regulatory effect on ZAP-70-dependent migration. Stimulation of Jurkat T cells with CXCL12 also resulted in ZAP-70-dependent tyrosine phosphorylation of the Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) adapter protein. Although CXCL12-dependent migration of SLP-76-deficient Jurkat T cells was impaired, re-expression of SLP-76 did not enhance migration. These results suggest a novel function for ZAP-70, but not SLP-76, in CXCR4 chemokine receptor signaling in human T cells.     

Cutting edge: constitutive B cell receptor signaling is critical for basal growth of B lymphoma

B lymphomas account for the majority of the lymphoma cases. BCR expression appears to be important for B lymphoma because most oncogenes are translocated to nonrearranged Ig loci and because all of the variants that arise in anti-idiotypic Ab-treated lymphoma patients remain BCR positive. Based on this and the fact that BCR is required for mature B cell survival, we tested the requirement for continued expression of BCR for the growth and survival of B lymphoma cells. Using Igalpha or Igbeta-specific small interfering RNA (siRNA) to inhibit BCR expression, we demonstrate for the first time that constitutive signaling by BCR is critical for survival and proliferation of both murine and human B lymphoma cells. The BCR signals in lymphoma appear to be mediated by Syk, as it is constitutively active in a variety of B lymphoma cells. Blocking Syk activity by selective inhibitors suppresses growth of several murine and human B lymphomas.     

Complementary roles for CD19 and Bruton's tyrosine kinase in B lymphocyte signal transduction

CD19 and Bruton's tyrosine kinase (Btk) may function along common signaling pathways in regulating intrinsic and B cell Ag receptor (BCR)-induced signals. To identify physical and functional interactions between CD19 and Btk, a CD19-negative variant of the A20 B cell line was isolated, and CD19-deficient (CD19(-/-)) and CD19-overexpressing mice with the X-linked immunodeficient (Xid; Btk) mutation were generated. In A20 cells, Btk physically associated with CD19 following BCR engagement. CD19 and Btk interactions were not required for initial Btk phosphorylation, but CD19 expression maintained Btk in an activated state following BCR engagement. In primary B cells, CD19 signaling also required downstream Btk function since CD19-induced intracellular Ca(2+) ([Ca(2+)](i)) responses were modest in Xid B cells. In addition, CD19 overexpression did not normalize the Xid phenotype and most phenotypic and functional hallmarks of CD19 overexpression were not evident in these mice. However, CD19 and Btk also regulate independent signaling pathways since their combined loss had additive inhibitory effects on BCR-induced [Ca(2+)](i) responses and CD19 deficiency induced a severe immunodeficiency in Xid mice. Thus, CD19 expression amplifies or prolongs Btk-mediated signaling, rather than serving as a required agent for Btk activation. Consistent with this, phosphatidylinositol 3-monophosphate kinase and Akt activation were normal in CD19(-/-) B cells following IgM engagement, although their kinetics of activation was altered. Thus, these biochemical and compound gene dosage studies indicate that Btk activation and [Ca(2+)](i) responses following BCR engagement are regulated through multiple pathways, including a CD19/Src family kinase-dependent pathway that promotes the longevity of Btk signaling.     

Cutting edge: B cell antigen receptor signaling occurs outside lipid rafts in immature B cells

B cell Ag receptor (BCR) signaling changes dramatically during B cell development, resulting in activation in mature B cells and apoptosis, receptor editing, or anergy in immature B cells. BCR signaling in mature B cells was shown to be initiated by the translocation of the BCR into cholesterol- and sphingolipid-enriched membrane microdomains that include the Src family kinase Lyn and exclude the phosphatase CD45. Subsequently the BCR is rapidly internalized into the cell. Here we show that the BCR in the immature B cell line, WEHI-231, does not translocate into lipid rafts following cross-linking nor is the BCR rapidly internalized. The immature BCR initiates signaling from outside lipid rafts as evidenced by the immediate induction of an array of phosphoproteins and subsequent apoptosis. The failure of the BCR in immature B cells to enter lipid rafts may contribute to the dramatic difference in the outcome of signaling in mature and immature B cells.     

B cell antigen receptor and CD40 differentially regulate CD22 tyrosine phosphorylation

Cell surface molecules on lymphocytes positively or negatively modulate the Ag receptor signaling, and thus regulate the fate of the cell. CD22 is a B cell-specific cell surface protein that contains multiple ITIMs in the cytoplasmic tail, and critically regulates B cell activation and survival. CD22 regulation on B cell signaling is complex because CD22 can have both positive and negative roles in various contexts. We generated phosphospecific polyclonal Abs reacting four major CD22 tyrosine motifs (Y762, Y807, Y822, and Y842) and analyzed the pattern and intensity of phosphorylation of these tyrosine residues. The tyrosine motifs, Y762, Y822, and Y842, are considered as ITIM, whereas the other, Y807, is suggested to be important for Grb2 recruitment. Approximately 10% of the four tyrosine residues were constitutively phosphorylated. Upon anti-IgM ligation, CD22 Y762 underwent most rapid phosphorylation, whereas all four tyrosine residues were eventually phosphorylated equally at approximately 35% of all CD22 molecules in the cell. By contrast, anti-CD40 stimulation specifically up-regulated anti-IgM-induced phosphorylation of tyrosines within two ITIM motifs, Y762 and Y842, which was consistent with in vivo finding of the negative role of CD22 in CD40 signaling. Thus, CD22 phosphorylation is not only quantitatively but also qualitatively regulated by different stimulations, which may determine the outcome of B cell signaling.     

Cutting edge: molecular mechanisms of synergy between CD40 and the B cell antigen receptor: role for TNF receptor-associated factor 2 in receptor interaction

Optimal Ag-specific B lymphocyte activation requires both recognition of Ag by the B cell Ag receptor (BCR) and contact-mediated interactions with Ag-specific Th lymphocytes. One of these interactions involves ligation of B cell CD40 by T cell-expressed CD154. CD40 signaling is crucial for Ab production, isotype switching, up-regulation of surface molecules, development of germinal centers, and the humoral memory response. The signaling pathways emanating from the BCR and CD40 are able to cooperate, but the molecular mechanisms responsible for this interaction are incompletely understood. The present study explored the roles of signaling motifs in the CD40 cytoplasmic tail in this synergy. We find that threonine in the PXQXT motif in the TNFR-associated factor-2 binding site is critical for synergistic effects of CD40 and BCR signals, independent of its phosphorylation. Furthermore, data suggest an indirect role for TNFR-associated factor-2 in the cooperative signaling.     

CD40 signaling in B cells regulates the expression of the Pim-1 kinase via the NF-kappa B pathway.

The ability of CD40 signaling to regulate B cell growth, survival, differentiation, and Ig class switching involves many changes in gene expression. Using cDNA expression arrays and Northern blotting, we found that CD40 signaling increased the mRNA levels for pim-1, a protooncogene that encodes a serine/threonine protein kinase. Subsequent experiments showed that CD40 engagement also increased both Pim-1 protein levels and Pim-1 kinase activity in B cells. We then investigated the signaling pathways by which CD40 regulates Pim-1 expression and found that CD40 up-regulates Pim-1 primarily via the activation of NF-kappaB. Inhibiting the activation of NF-kappaB, either by treating cells with a chemical inhibitor, BAY11-7082, or by inducibly expressing a superrepressor form of IkappaBalpha, significantly impaired the ability of CD40 to increase Pim-1 protein levels. Because Pim-1 expression is associated with cell proliferation and survival, we asked whether this correlated with the ability of CD40 signaling to prevent anti-IgM-induced growth arrest in the WEHI-231 murine B cell line, a model for Ag-induced clonal deletion. We found that the anti-IgM-induced growth arrest in WEHI-231 cells correlated with a substantial decrease in Pim-1 levels. In contrast, culturing WEHI-231 cells with either anti-CD40 Abs or with the B cell mitogen LPS, both of which prevent the anti-IgM-induced growth arrest, also prevented the rapid decline in Pim-1 levels. This suggests that Pim-1 could regulate the survival and proliferation of B cells.     

BCR engagement induces Fas resistance in primary B cells in the absence of functional Bruton's tyrosine kinase

B cell susceptibility to Fas-mediated apoptosis is regulated in a receptor-specific fashion. CD40 engagement produces marked sensitivity to Fas killing, whereas surface Ig (sIg) engagement blocks Fas signaling for cell death in otherwise sensitive, CD40-stimulated B cell targets, and thus, induces a state of Fas resistance. The signaling mediator, Bruton's tyrosine kinase (Btk), is required for certain sIg-triggered responses, and Btk is reported to directly bind Fas and block Fas-mediated apoptosis. For these reasons, the role of Btk as a mediator of sIg-induced Fas resistance was examined. Dysfunction of Btk through mutation, and absence of Btk through deletion did not interfere with induction of Fas resistance by anti-Ig. This may be due, at least in part, to induction of Btk-dependent Bcl-2 family members by anti-Ig after CD40 ligand treatment. However, the susceptibility to Fas-mediated apoptosis of B cell targets stimulated by CD40 ligand alone was increased in the absence of Btk. These results indicate that Fas resistance produced by sIg triggering does not require Btk, but suggests that in certain situations Btk modulates B cell susceptibility to Fas killing.     

Cutting edge: CD4 T cell-mast cell interactions alter IgE receptor expression and signaling

Mast cell activation is associated with atopic and inflammatory diseases, but the natural controls of mast cell homeostasis are poorly understood. We hypothesized that CD4(+)CD25(+) regulatory T cells (Treg) could function in mast cell homeostasis. In this study, we demonstrate that mast cells can recruit both Treg and conventional CD4(+) T cells (Tconv). Furthermore, Treg, but not Tconv, suppress mast cell FcepsilonRI expression. Despite the known inhibitory functions of IL-10 and TGFbeta1, FcepsilonRI suppression was independent of IL-10 and TGF-beta1 and required cell contact. Surprisingly, coculture with either Treg or Tconv cells suppressed IgE-mediated leukotriene C(4) production but enhanced cytokine production by mast cells. This was accompanied by a selective increase in FcepsilonRI-mediated Stat5 phosphorylation, which is a critical mediator of IgE-mediated cytokine secretion. These data are the first direct demonstration that mast cells can recruit Treg and illustrate that T cell interactions can alter the mast cell response.     

Bruton's tyrosine kinase is essential for hydrogen peroxide-induced calcium signaling.

Using Btk-deficient DT40 cells and the transfectants expressing wild-type Btk or Btk mutants in either kinase (Arg(525) to Gln), Src homology 2 (SH2, Arg(307) to Ala), or pleckstrin homology (PH, Arg(28) to Cys) domains, we investigated the roles and structure-function relationships of Btk in hydrogen peroxide-induced calcium mobilization. Our genetic evidence showed that Btk deficiency resulted in a significant reduction in hydrogen peroxide-induced calcium response. This impaired calcium signaling is correlated with the complete elimination of IP3 production and the significantly reduced tyrosine phosphorylation of PLCgamma2 in Btk-deficient DT40 cells. All of these defects were fully restored by the expression of wild-type Btk in Btk-deficient DT40 cells. The data from the point mutation study revealed that a defect at any one of the three functional domains would prevent a full recovery of Btk-mediated hydrogen peroxide-induced intracellular calcium mobilization. However, mutation at either the SH2 or PH domain did not affect the hydrogen peroxide-induced activation of Btk. Mutation at the SH2 domain abrogates both IP3 generation and calcium release, while the mutant with the nonfunctional PH domain can partially activate PLCgamma2 and catalyze IP3 production but fails to produce significant calcium mobilization. Thus, these observations suggest that Btk-dependent tyrosine phosphorylation of PLCgamma2 is required but not sufficient for hydrogen peroxide-induced calcium mobilization. Furthermore, hydrogen peroxide stimulates a Syk-, but not Btk-, dependent tyrosine phosphorylation of B cell linker protein BLNK. The overall results, together with those reported earlier [Qin et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 7118], are consistent with the notion that functional SH2 and PH domains are required for Btk to form a complex with PLCgamma2 through BLNK in order to position the Btk, PLCgamma2, and phosphatidylinositol 4,5-bisphosphate in close proximity for efficient activation of PLCgamma2 and to maximize its catalytic efficiency for IP3 production.     

Cutting edge: repurification of lipopolysaccharide eliminates signaling through both human and murine toll-like receptor 2

Toll-like receptor (TLR) 2 has recently been associated with cellular responses to numerous microbial products, including LPS and bacterial lipoproteins. However, many preparations of LPS contain low concentrations of highly bioactive contaminants described previously as "endotoxin protein," suggesting that these contaminants could be responsible for the TLR2-mediated signaling observed upon LPS stimulation. To test this hypothesis, commercial preparations of LPS were subjected to a modified phenol re-extraction protocol to eliminate endotoxin protein. While it did not influence the ability to stimulate cells from wild-type mice, repurification eliminated the ability of LPS to activate cells from C3H/HeJ (Lpsd) mice. Additionally, only cell lines transfected with human TLR4, but not human or murine TLR2, acquired responsiveness to both re-extracted LPS and to a protein-free, synthetic preparation of lipid A. These results suggest that neither human nor murine TLR2 plays a role in LPS signaling in the absence of contaminating endotoxin protein.     

B cell receptor (BCR) cross-talk: CD40 engagement enhances BCR-induced ERK activation

Bystander B cells may be initially stimulated through CD40, which enhances susceptibility to Fas-mediated apoptosis, before encountering Ag, which produces Fas resistance. A key issue in this process is to what extent CD40 cross-talk might affect subsequent BCR signaling. It has previously been shown that CD40 engagement bypasses or mitigates the need for Bruton's tyrosine kinase in subsequent BCR signaling for NF-kappaB activation. However, the full extent of the effects of CD40 on BCR signaling has not been delineated. In the present study we evaluated the possibility that CD40-mediated cross-talk also affects another principal outcome of BCR signaling: MAPK activation. We found that prior stimulation of primary murine B cells with CD40L markedly enhanced the level of ERK and JNK (but not p38 MAPK) phosphorylation produced by subsequently added anti-Ig Ab, and much, but not all, of this enhancement was independent of PI3K and phospholipase C. CD40L treatment similarly enhanced BCR-induced MAPK kinase (MEK) phosphorylation, and MEK was required for enhancement of ERK. Although BCR-induced c-Raf phosphorylation was also enhanced by prior CD40L treatment, c-Raf was not required for MEK/ERK phosphorylation. These results identify a novel system of receptor cross-talk between CD40 and BCR and indicate that the effects of CD40 engagement on subsequent BCR stimulation spread beyond NF-kappaB to involve the MAPK pathway.