WIP regulates signaling via the high affinity receptor for immunoglobulin E in mast cells

Wiskott-Aldrich syndrome protein-interacting protein (WIP) stabilizes actin filaments and is important for immunoreceptor-mediated signal transduction leading to actin cytoskeleton rearrangement in T and B cells. Here we report a role for WIP in signaling pathways downstream of the high affinity receptor for immunoglobulin (Ig)E (FcepsilonRI) in mast cells. WIP-deficient bone marrow-derived mast cells (BMMCs) were impaired in their capacity to degranulate and secrete interleukin 6 after FcepsilonRI ligation. Calcium mobilization, phosphorylation of Syk, phospholipase C-g2, and c-Jun NH2-terminal kinase were markedly decreased in WIP-deficient BMMCs. WIP was found to associate with Syk after FcepsilonRI ligation and to inhibit Syk degradation as evidenced by markedly diminished Syk levels in WIP-deficient BMMCs. WIP-deficient BMMCs exhibited no apparent defect in their subcortical actin network and were normal in their ability to form protrusions when exposed to an IgE-coated surface. However, the kinetics of actin changes and the cell shape changes that follow FcepsilonRI signaling were altered in WIP-deficient BMMCs. These results suggest that WIP regulates FcepsilonRI-mediated mast cell activation by regulating Syk levels and actin cytoskeleton rearrangement.     

SLP-76 deficiency impairs signaling via the high-affinity IgE receptor in mast cells

SLP-76 is an adapter protein expressed in T cells and myeloid cells that is a substrate for ZAP-70 and Syk. SLP-76-deficient mice exhibit a profound block in T-cell development. We found that although SLP-76 is expressed in mouse mast cells, SLP-76(-/-) mice have normal numbers of mast cells in their skin and bronchi. SLP-76(-/-) mice are resistant to IgE-mediated passive anaphylaxis. SLP-76(-/-) mice sensitized with IgE anti-dinitrophenyl (DNP) and then challenged with DNP-HSA developed only mild and transient tachycardia, failed to increase their plasma histamine level, and all survived the antigen challenge. Bone marrow-derived mast cells (BMMCs) from SLP76(-/-) mice failed to release beta-hexosaminidase and to secrete IL-6 after FcepsilonRI cross-linking. Tyrosine phosphorylation of phospholipase C-gamma1 (but not of Syk) and calcium mobilization in response to IgE cross-linking were reduced in SLP-76-deficient BMMCs. These results suggest that SLP-76 plays an important role in FcepsilonRI-mediated signaling in mast cells.     

Hapten-specific tolerance. Preferential depression of the high affinity antibody response

The induction of tolerance in guinea pigs with a 2,4-dinitrophenyl (DNP) derivative of a copolymer of copolymer of D-glutamic acid and D-lysine (D-GL) leads to a preferential depression of the capacity to produce high affinity anti-DNP antibody in response to immunization with DNP-guinea pig albumin. Thus, immunization 2 wk after tolerance induction with 3 mg of DNP-D-GL results in an immune response in which individual plaque-forming cells (PFC) secreting high affinity anti-DNP antibody are absent and in which the affinity of circulating anti-DNP antibody is reduced. A similar, but less marked, suppression is seen when 0.3 mg of DNP-D-GL is used for tolerance induction. If immunization is delayed until 2 months after tolerance induction, then suppression is restricted to the highest avidity PFC group. Our data is consistent with a state of tolerance in the pool of precursors of anti-DNP antibody-secreting cells induced as a result of their interaction with DNP-D-GL in the absence of specific "helper" cells, which appear to be lacking for DNP-D-GL. In such a situation, the affinity of receptors on precursor cells for tolerogen and the concentration of tolerogen appear to be crucial determinants of whether an individual cell will become tolerant.     

Sequential class switching is required for the generation of high affinity IgE antibodies

IgE antibodies with high affinity for their antigens can be stably cross-linked at low concentrations by trace amounts of antigen, whereas IgE antibodies with low affinity bind their antigens weakly. In this study, we find that there are two distinct pathways to generate high and low affinity IgE. High affinity IgE is generated through sequential class switching (μ→γ→ε) in which an intermediary IgG phase is necessary for the affinity maturation of the IgE response, where the IgE inherits somatic hypermutations and high affinity from the IgG1 phase. In contrast, low affinity IgE is generated through direct class switching (μ→ε) and is much less mutated. Mice deficient in IgG1 production cannot produce high affinity IgE, even after repeated immunizations. We demonstrate that a small amount of high affinity IgE can cause anaphylaxis and is pathogenic. Low affinity IgE competes with high affinity IgE for binding to Fcε receptors and prevents anaphylaxis and is thus beneficial.     

Identification of the low affinity receptor for immunoglobulin E on mouse mast cells and macrophages as Fc gamma RII and Fc gamma RIII

In addition to their well characterized high affinity immunoglobulin E (IgE) receptors (Fc epsilon RI) mast cells have long been suspected to express undefined Fc receptors capable of binding IgE with low affinity. In this paper, we show that Fc gamma RII and Fc gamma RIII, but not Mac-2, on mouse mast cells and macrophages bind IgE-immune complexes. This binding is efficiently competed by 2.4G2, a monoclonal antibody against the extracellular homologous region of both Fc gamma RII and Fc gamma RIII. Furthermore, IgE-immune complexes bind specifically to Fc gamma RII or Fc gamma RIII transfected into COS-7 cells. The association constants of IgE binding estimated from competition experiments are about 3.1 x 10(5) M-1 for Fc gamma RII, and 4.8 x 10(5) M-1 for Fc gamma RIII. Engagement of Fc gamma RII and Fc gamma RIII with IgE-immune complexes (after blocking access to Fc epsilon RI) or with IgG-immune complexes triggers C57.1 mouse mast cells to release serotonin. This release is inhibited by 2.4G2, and at maximum, reaches 30-40% of the intracellular content, about half of the maximal release (60-80%) obtained after Fc epsilon RI engagement. These data demonstrate that mouse Fc gamma RII and Fc gamma RIII are not isotype specific, and that the binding of IgE-immune complexes to these receptors induces cell activation.     

Mast cells lacking the high affinity immunoglobulin E receptor are deficient in Fc epsilon RI gamma messenger RNA

A population of cells that express mast cell markers, including the membrane protein p161, but that lack expression of the high affinity IgE receptor, Fc epsilon RI, can be routinely grown from bone marrow. Ionomycin, but not IgE immune complexes, causes these cells to release serotonin and to express IL-3 and IL-13 mRNA, consistent with their being FC epsilon RI-deficient mast cells. These p161+/Fc epsilon RI- mast cells expressed normal amounts of Fc epsilon RI alpha and beta chain mRNA, but extremely low levels of Fc epsilon RI gamma chain mRNA. In addition, this novel mast cell population expressed CD3 zeta chain mRNA, which p161+/Fc epsilon RI+ mast cells did not. CD3 zeta stable transfectants of Abelson-murine leukemia virus-transformed p161+/Fc epsilon RI+ mast cells continued to express Fc epsilon RI. This strongly suggests that the failure of p161+/Fc epsilon RI- mast cells to express IgE receptors was not caused by the presence of CD3 zeta chain. Transfection of human Fc epsilon RI gamma cDNA into p161+/Fc epsilon RI- mast cells rescued IgE binding. These stable transfectants released serotonin in response to cross-linkage of Fc epsilon RI, demonstrating that the molecular defect of p161+/Fc epsilon RI- mast cells is indeed the loss of Fc epsilon RI gamma expression.     

Role of the high affinity immunoglobulin E receptor in bacterial translocation and intestinal inflammation

A role for immunoglobulin E and its high affinity receptor (Fc epsilon RI) in the control of bacterial pathogenicity and intestinal inflammation has been suggested, but relevant animal models are lacking. Here we compare transgenic mice expressing a humanized Fc epsilon RI (hFc epsilon RI), with a cell distribution similar to that in humans, to Fc epsilon RI-deficient animals. In hFc epsilon RI transgenic mice, levels of colonic interleukin 4 were higher, the composition of fecal flora was greatly modified, and bacterial translocation towards mesenteric lymph nodes was increased. In hFc epsilon RI transgenic mice, 2,4,6-tri-nitrobenzenesulfonic acid (TNBS)-induced colitis was also more pronounced, whereas Fc epsilon RI-deficient animals were protected from colitis, demonstrating that Fc epsilon RI can affect the onset of intestinal inflammation.     

Histamine H4 receptor mediates chemotaxis and calcium mobilization of mast cells

The diverse physiological functions of histamine are mediated through distinct histamine receptors. Mast cells are major producers of histamine, yet effects of histamine on mast cells are currently unclear. The present study shows that histamine induces chemotaxis of mouse mast cells, without affecting mast cell degranulation. Mast cell chemotaxis toward histamine could be blocked by the dual H3/H4 receptor antagonist thioperamide, but not by H1 or H2 receptor antagonists. This chemotactic response is mediated by the H4 receptor, because chemotaxis toward histamine was absent in mast cells derived from H4 receptor-deficient mice but was detected in H3 receptor-deficient mast cells. In addition, Northern blot analysis showed the expression of H4 but not H3 receptors on mast cells. Activation of H4 receptors by histamine resulted in calcium mobilization from intracellular calcium stores. Both G alpha i/o proteins and phospholipase C (PLC) are involved in histamine-induced calcium mobilization and chemotaxis in mast cells, because these responses were completely inhibited by pertussis toxin and PLC inhibitor 1-[6-[[17 beta-3-methoxyestra-1,3,5 (10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122). In summary, histamine was shown to mediate signaling and chemotaxis of mast cells via the H4 receptor. This mechanism might be responsible for mast cell accumulation in allergic tissues.     

甲异靛对K562细胞bcr-abl信号通路的影响以及诱导细胞凋亡机制研究

目的 探讨甲异靛对K562细胞bcr-abl信号通路的影响以及甲异靛诱导K562细胞凋亡的机制.方法 应用流式细胞术检测甲异靛诱导K562细胞凋亡的情况以及线粒体膜电位稳定性.应用Western blot技术检测bcr-abl信号通路相关蛋白、caspase以及Bcl-2家族成员在甲异靛作用前后的表达情况.RT-PCR技术检测甲异靛作用前后bcr-abl mRNA表达水平,用电泳移动迁移速度分析检测STAT3、STATS的DNA结合能力.结果 20 μmol/L甲异靛町降低ber-abl融合蛋白的表达及其磷酸化水平,但并不改变其mRNA表达水平,甲异靛可降低STATS以及CRKL的磷酸化水平,抑制STAT3、STATS的DNA结合能力.5-20μmoL/L甲异靛可诱导K562细胞凋亡并且具有浓度依赖性.甲异靛诱导K562细胞凋亡中伴随caspase 3、8、9活化以及细胞线粒体膜电位降低,caspase 3、8、9特异抑制剂z-DEVD-fmk、z-IETD-cho和z-LETD-fmk可阻断甲异靛诱导的K562细胞凋亡.甲异靛诱导K562细胞凋亡前后不伴有Bcl-2、Bax、Bid蛋白表达水平的改变.结论 甲异靛通过降低bcr-abl融合蛋白的表达并抑制bcr-abl信号通路中相关蛋白活性从而抑制K562细胞的增殖.甲异靛诱导K562细胞凋亡依赖于caspase活化以及线粒体途径,但Bcl-2家族成员不参与凋亡的调控.     

Transfection of Syk protein tyrosine kinase reconstitutes high affinity IgE receptor-mediated degranulation in a Syk-negative variant of rat basophilic leukemia RBL-2H3 cells

Aggregation of the high affinity receptor for immunoglobulin E (Fc epsilon RI) on mast cells results in rapid tyrosine phosphorylation and activation of Syk, a cytoplasmic protein tyrosine kinase. To examine the role of Syk in the Fc epsilon RI signaling pathway, we identified a variant of RBL-2H3 cells that has no detectable Syk by immunoblotting and by in vitro kinase reactions. In these Syk-deficient TB1A2 cells, aggregation of Fc epsilon RI induced no histamine release and no detectable increase in total cellular protein tyrosine phosphorylation. However, stimulation of these cells with the calcium ionophore did induce degranulation. Fc epsilon RI aggregation induced tyrosine phosphorylation of the beta and gamma subunits of the receptor, but no increase in the tyrosine phosphorylation of phospholipase C-gamma 1 and phospholipase C-gamma 2 and no detectable increase in intracellular free Ca2+ concentration. By transfection, cloned lines were established with stable expression of Syk. In these reconstituted cells, Fc epsilon RI aggregation induced tyrosine phosphorylation of phospholipase C- gamma 1 and phospholipase C-gamma 2, an increase in intracellular free Ca2+ and histamine release. These results demonstrate that Syk plays a critical role in the early Fc epsilon RI-mediated signaling events. It further demonstrates that Syk activation occurs downstream of receptor phosphorylation, but upstream of most of the Fc epsilon RI-mediated protein tyrosine phosphorylations.     

CD40 and IgE: synergism between anti-CD40 monoclonal antibody and interleukin 4 in the induction of IgE synthesis by highly purified human B cells

A novel pathway of IgE-B cell differentiation has been identified. Engagement of the B cell antigen CD40 by F(ab')2 fragments of monoclonal antibody (mAb) 626.1 in the presence of recombinant interleukin 4 (rIL-4) induced intense IgE synthesis, but modest IgG synthesis, by highly purified human B cells. Surface IgE- B cells isolated by cell sorting were induced to produce IgE by mAb 626.1 and IL-4. Thus, IgE synthesis is unlikely to result from expansion of a B cell population precommitted to IgE in vivo. A neutralizing anti-IL-6 antibody strongly, but not completely, inhibited the IgE response. This indicates that autocrine production of IL-6 plays an important amplification role in IgE synthesis triggered by anti-CD40 mAb and IL-4. Although the exact role played by CD40 in IgE responses in vivo remains to be established, this T cell-independent system represents a useful model to characterize the biochemical and molecular events leading to IgE synthesis in human B cells.     

Regulation of proximal T cell receptor signaling and tolerance induction by deubiquitinase Usp9X

The T cell hyperproliferation and autoimmune phenotypes that manifest in mice lacking E3 ubiquitin ligases such as Cbl, ITCH, or GRAIL highlight the importance of ubiquitination for the maintenance of peripheral T cell tolerance. Less is known, however, about the deubiquitinating enzymes that regulate T cell proliferation and effector function. Here, we define a cell intrinsic role for the deubiquitinase Usp9X during proximal TCR signaling. Usp9X-deficient T cells were hypoproliferative, yet mice with T cell–specific Usp9x deletion had elevated numbers of antigen-experienced T cells and expanded PD-1 and OX40-expressing populations consistent with immune hyperactivity. Aged Usp9x KO mice developed lupus-like autoimmunity and lymphoproliferative disease, indicating that ubiquitin ligases and deubiquitinases maintain the delicate balance between effective immunity and self-tolerance.Ignorance of self-antigens is one example of a cell intrinsic mechanism for inducing tolerance in peripheral T cell populations and results from either a failure to encounter self-antigens or more pertinently, because the avidity or amount of self-antigen is not sufficient to trigger a response. Naive CD4⁺ T cells function at the apex of the adaptive immune response, where, during an initial encounter with antigen, their principal function is to discriminate between low and high signal strength. If a signal is high, as is typically the case for encounters with foreign antigens, then the antigen-specific T cell will undergo clonal expansion; however, if the signal strength is low, an immune response fails to occur. Insights into the regulation of the earliest signaling events downstream of TCR engagement will inform our understanding of how signal strength is modulated during this critical decision making process.The enzymatic conjugation of the 76-aa protein ubiquitin to lysine residues, either singly or as polymeric chains, impacts cell signaling by modifying the stability, localization, or interactions of a protein (Komander and Rape, 2012). Ubiquitin is attached by the concerted actions of E1, E2, and E3 enzymes and it is removed by ubiquitin hydrolases (also called deubiquitinating enzymes). Therefore, the balance between these activities will determine the magnitude and duration of signaling. The E3 ubiquitin ligases Cbl, ITCH, and GRAIL are established regulators of peripheral T cell tolerance due to their provision of negative signals that shift the balance toward lower TCR signal strength. For example, in activated peripheral T cells, Cbl-b binds and ubiquitinates the TCR-proximal kinase ZAP70, resulting in its proteasomal-mediated degradation (Lupher et al., 1996; Rao et al., 2000). The fact that deubiquitinating enzymes counteract these ligases to shift the balance toward higher signal strength has received little attention.Ubiquitin-specific protease 9X (Usp9X) is a mammalian orthologue of the Drosophila developmental gene fat facets (Wood et al., 1997) that exhibits specificity for diverse ubiquitin linkages, not only removing degradative K48-linked ubiquitin chains (Nagai et al., 2009; Schwickart et al., 2010) but also nondegradative monoubiquitin from SMAD4 (Dupont et al., 2009) and atypical Lys29/33 polyubiquitin chains from NUAK1 or MARK4 (Al-Hakim et al., 2008). Here, we show that Usp9X is a positive regulator of proximal TCR signaling in peripheral T cells and also contributes to T cell tolerance established during intrathymic development.     

Antitumoral effects of calcitriol in basal cell carcinomas involve inhibition of hedgehog signaling and induction of vitamin D receptor signaling and differentiation

Activation of the Hedgehog (Hh)-signaling pathway due to deficiency in the Hh receptor Patched1 (Ptch) is the pivotal defect leading to formation of basal cell carcinoma (BCC). Recent reports provided evidence of Ptch-dependent secretion of vitamin D(3)-related compound, which functions as an endogenous inhibitor of Hh signaling by repressing the activity of the signal transduction partner of Ptch, Smoothened (Smo). This suggests that Ptch-deficient tumor cells are devoid of this substance, which in turn results in activation of Hh-signaling. Here, we show that the application of the physiologically active form of vitamin D(3), calcitriol, inhibits proliferation and growth of BCC of Ptch mutant mice in vitro and in vivo. This is accompanied by the activation of the vitamin D receptor (Vdr) and induction of BCC differentiation. In addition, calcitriol inhibits Hh signaling at the level of Smo in a Vdr-independent manner. The concomitant antiproliferative effects on BCC growth are stronger than those of the Hh-specific inhibitor cyclopamine, even though the latter more efficiently inhibits Hh signaling. Taken together, we show that exogenous supply of calcitriol controls the activity of 2 independent pathways, Hh and Vdr signaling, which are relevant to tumorigenesis and tumor treatment. These data suggest that calcitriol could be a therapeutic option in the treatment of BCC, the most common tumor in humans.     

The effect of lymphokines on proliferation and collagen synthesis of cultured human synovial cells

Abstract. Human mononuclear cell supernatants were obtained by incubating 3 times 10⁶ cells per ml of Dulbecco's modified Eagles medium at 37°C for 24 h or 48 h, either in presence or absence of phytohaemagglutinin. After removal of intact cells, the supernatants were dialysed and diluted (1:1, 1:2, 1:5, 1:10) again using the above medium. The diluted supernatants, containing a final concentration of 10% (v/v) human platelet-factor poor serum, were found to stimulate the proliferation of human synovial cells in culture and to increase both the total amount of collagen and the percentage of Type III collagen synthesized by these cells. Incubation of the mononuclear cells in presence of phytohaemagglutinin appeared to further enhance the stimulatory effects of the supernatants upon the synovial cells. Since activated mononuclear leucocytes such as lymphocytes and macrophages are present in rheumatoid synovia, this study suggests that factors released from activated mononuclear leucocytes may play an important role in the proliferation of rheumatoid synovial tissue and development of the pannus.