Identification of Critical Signaling Pathways Modulating Mast Cell Activation

肥大细胞在变态反应性炎症的发病机制中起着关键作用。这些反应通常是由细胞表面表达的高亲和力IgE受体(Fc-epsilon-RI)的抗原依赖性聚集和随后的促炎介质的释放启动的。其他受体的配体，如KIT和各种GPCRs，可能起到启动肥大细胞的作用，或作为抗原介导的肥大细胞激活的共同激活剂。将Fc-epsilon-RI聚集与人类肥大细胞激活和功能联系起来的信号通路，以及其他受体如何修改这些Fc介导的信号事件，目前还不清楚。因此，本研究的重点是阐明与通过Fc-epsilon-RI激活肥大细胞相关的信号机制，特别是其他受体启动的信号通路如何与Fc-epsilon-RI启动的信号通路整合，以协同激活和/或抑制肥大细胞。 肥大细胞在体内影响疾病状态的能力还取决于它们从祖细胞的生长和分化，肥大细胞向其常驻组织的迁移，以及它们在这些部位的存活。因此，调节这些过程的整合受体介导的信号事件也在研究中。最近在IL-33对人类肥大细胞功能、细胞骨架重排和包含MS4A2的基因座的影响方面已经进行了关键的观察。 这些研究在一定程度上也导致了一种新的小鼠肥大细胞系的鉴定，该细胞系现在已经被报道，并允许研究正常和突变的KIT结构。这些细胞起源于骨髓来源的小鼠肥大细胞培养，作为快速分裂的肥大细胞亚群。随着时间的推移，这些细胞失去了KIT的表达，而继续表达功能性的高亲和力IgE受体。逆转录病毒转导的细胞与人KIT构建导致了人KIT的表面表达，它对人干细胞因子(SCF；KIT配体)有反应。 在检测IL-33对肥大细胞功能的影响时，我们发现人和小鼠的肥大细胞长期暴露于IL-33会导致肥大细胞对抗原的激活显著减少。这似乎是由于依赖于MyD88的肥大细胞激活所必需的信号过程的减弱所致，包括抗原介导的钙动员和细胞骨架重组。这些变化与PLCG1和HCK的表达下调有关。 连锁分析表明，包含MS4A2的基因座(编码Fc-epsilon-RI)是过敏易感性的候选基因。我们已经在人类中发现了Fc-epsilon-RI(t-Fc-epsilon-RI)的一个截短，它包含一个可能的钙调蛋白结合域。因此，我们试图确定该变体在肥大细胞功能中的作用。我们确定了t-Fc-epsilon-RI与Fyn激酶、Gab2、P85PI3K和-微管蛋白形成了一个复合体。钙离子存在时，钙调蛋白与t-Fc-epsilon-RI结合，启动磷酸化，这对t-Fc-epsilon-RI的功能至关重要。共聚焦显微镜显示，在IgE依赖和非IgE依赖的激活后，t-Fc-epsilon-RI复合体定位于中心体周围的高尔基体。T-Fc-epsilon-RI基因敲除可抑制钙信号下游微管的形成、脱颗粒和IL-8的产生。这些观察结果与t-Fc-epsilon-RI介导钙依赖的微管形成，从而促进脱颗粒和细胞因子释放的结论一致。 已知肥大细胞向炎症部位的迁移受到SCF等趋化因子的调控。尽管诱导了与抗原类似的早期信号事件，但趋化因子(包括SCF)在没有其他刺激的情况下产生的脱颗粒最少。因此，我们调查了调控肥大细胞趋化的过程是否对肥大细胞介质的释放进行了速率限制。在这些实验中，我们扰乱了肌动蛋白聚合，这是肥大细胞趋化所必需的。然后，我们检测了抗原或干细胞因子诱导的人肥大细胞的趋化和介质释放。我们发现并报道了肌动蛋白聚合的中断对早期信号通路的影响最小，但减弱了干细胞因子诱导的人肥大细胞的趋化作用。出乎意料的是，在没有其他刺激的情况下，SCF在肌动蛋白分解后以浓度依赖的方式诱导了实质性的脱颗粒。我们将这一数据解释为与调控细胞迁移的过程限制了肥大细胞脱颗粒是细胞骨架重组的结果一致的结论。

Mast cells play a pivotal role in the pathogenesis of allergic inflammation. These reactions are generally initiated by antigen-dependent aggregation of the high affinity IgE receptor (Fc-epsilon-RI) expressed on the cell surface and subsequent release of pro-inflammatory mediators. Ligands for other receptors such as KIT and various GPCRs may serve to prime mast cells for, or act as co-activators of, antigen-mediated mast cell activation. The signaling pathways linking Fc-epsilon-RI aggregation to human mast cell activation and function and how other receptors modify these Fc-mediated signaling events are not well understood. Thus the primary focus of the research is the elucidation of signaling mechanisms associated with the activation of mast cells via the Fc-epsilon-RI and especially how the signaling pathways initiated by other receptors may integrate with those initiated by the Fc-epsilon-RI for synergistic mast cell activation and/or inhibition. The ability of mast cells to impact disease states in vivo also depends on their growth and differentiation from their progenitor cells, migration of the mast cells to their resident tissues, and their survival at these sites. Therefore, the integrated receptor-mediated signaling events regulating these processes are also being examined. Recent key observations have been made relating to the effect of IL-33 on human mast cell function, cytoskeletal rearrangement, and the MS4A2-containing gene locus. These studies in part also resulted in identification of a novel mouse mast cell line which now has been reported and allows the study of normal and mutated KIT constructs. These cells originated from a bone marrow-derived mouse mast cell culture as a rapidly dividing mast cell sub-population. Over time, these cells lost KIT expression while continuing to express functional high affinity receptors for IgE. Retroviral transduction of the cells with a human KIT construct resulted in surface expression of human KIT which responded to human stem cell factor (SCF; KIT ligand). In examining IL-33 on mast cell function, we discovered that long-term exposure of human and mouse mast cells to IL-33 results in a substantial reduction of mast cell activation in response to antigen. This appears to be a consequence of MyD88-dependent attenuation of signaling processes necessary for mast cell activation including antigen-mediated calcium mobilization and cytoskeletal reorganization. These changes were related to down-regulation of the expression of PLCg1 and Hck. Linkage analyses have implicated the MS4A2-containing gene locus (encoding for Fc-epsilon-RI) as a candidate for allergy susceptibility. We have identified a truncation of Fc-epsilon-RI (t-Fc-epsilon-RI) in humans which contains a putative calmodulin binding domain. We thus sought to identify the role of this variant in mast cell function. We determined that t-Fc-epsilon-RI forms a complex with Fyn kinase, Gab2, p85 PI3K and -tubulin. Calmodulin bound to t-Fc-epsilon-RI in the presence of Ca2+ initiating phosphorylation, which was critical for t-Fc-epsilon-RI function. Confocal microscopy demonstrated localization of the t-Fc-epsilon-RI complex to the Golgi surrounding the centrosome after IgE-dependent and IgE-independent activation. Knockdown of t-Fc-epsilon-RI attenuated microtubule formation, degranulation and IL-8 production downstream of Ca2+ signals. These observations are consistent with the conclusion that t-Fc-epsilon-RI mediates Ca2+-dependent microtubule formation, which promotes degranulation and cytokine release. Migration of mast cells to sites of inflammation is known to be regulated by chemotactic factors such as SCF. Despite inducing similar early signaling events to antigen, chemotactic factors (including SCF) produce minimal degranulation in the absence of other stimuli. We therefore investigated whether processes regulating mast cell chemotaxis are rate limiting for mast cell mediator release. In these experiments, we disrupted actin polymerization, a requirement for mast cell chemotaxis. We then examined chemotaxis and mediator release in human mast cells induced by antigen or SCF. We found and reported that disruption of actin polymerization minimally affected early signaling pathways, but attenuated SCF-induced human mast cell chemotaxis. Unexpectedly, in the absence of other stimuli, SCF induced substantial degranulation in a concentration-dependent manner following actin disassembly. We interpreted this data as consistent with the conclusion that processes regulating cell migration limit mast cell degranulation as a consequence of cytoskeletal reorganization.