Identification of Critical Signaling Pathways Modulating Mast Cell Activation

肥大细胞在过敏性炎症的发病机制中起着关键作用。这些反应通常由细胞表面上表达的高亲和力IgE受体（Fc-IgE-RI）的抗原依赖性聚集和随后促炎介质的释放引发。其他受体如KIT和各种GPCR的配体可用于引发肥大细胞进行抗原介导的肥大细胞活化或充当抗原介导的肥大细胞活化的共活化剂。将Fc-γ-RI聚集与人肥大细胞活化和功能联系起来的信号传导途径以及其他受体如何修饰这些Fc介导的信号传导事件尚不清楚。因此，研究的主要焦点是阐明与通过Fc-γ-RI活化肥大细胞相关的信号传导机制，特别是由其他受体引发的信号传导途径如何与Fc-γ-RI引发的信号传导途径整合，以协同肥大细胞活化和/或抑制。 肥大细胞在体内影响疾病状态的能力还取决于它们从它们的祖细胞的生长和分化、肥大细胞向它们的驻留组织的迁移以及它们在这些部位的存活。因此，整合的受体介导的信号事件调节这些过程也正在研究。 最近的关键观察结果与IL-33对人肥大细胞功能、细胞骨架重排和含MS 4A 2基因位点的影响有关。 这些研究部分也导致了一种新的小鼠肥大细胞系的鉴定，该细胞系现已报道，并允许研究正常和突变的KIT构建体。这些细胞来源于骨髓来源的小鼠肥大细胞培养物，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表达，同时继续表达IgE的功能性高亲和力受体。用人KIT构建体逆转录病毒转导细胞导致人KIT的表面表达，其响应于人干细胞因子（SCF; KIT配体）。 在检查IL-33对肥大细胞功能的影响时，我们发现人和小鼠肥大细胞长期暴露于IL-33导致肥大细胞响应抗原的活化显著降低。 这似乎是肥大细胞活化所必需的信号传导过程的MyD 88依赖性衰减的结果，包括抗原介导的钙动员和细胞骨架重组。这些变化与PLCg 1和Hck表达下调有关。 连锁分析表明，含有MS 4A 2的基因座（编码Fc-γ-RI）是过敏易感性的候选基因。我们已经确定了一个截断的Fc-ε-RI（t-Fc-ε-RI）在人体内含有一个假定的钙调素结合结构域。 因此，我们试图确定肥大细胞功能的这种变体的作用。我们确定t-Fc-ε-RI与Fyn激酶、Gab 2、p85 PI 3 K和β-微管蛋白形成复合物。钙调素在Ca 2+存在下与t-Fc-epsilon-RI结合，启动磷酸化，这对t-Fc-epsilon-RI功能至关重要。共聚焦显微镜显示，在IgE依赖性和IgE非依赖性激活后，t-Fc-ε-RI复合物定位于中心体周围的高尔基体。t-Fc-epsilon-RI的敲低减弱了微管形成、脱粒和Ca 2+信号下游的IL-8产生。这些观察结果与t-Fc-epsilon-RI介导Ca 2+依赖性微管形成的结论一致，该微管形成促进脱粒和细胞因子释放。 已知肥大细胞向炎症部位的迁移受趋化因子如SCF调节。尽管诱导类似的早期信号事件抗原，趋化因子（包括SCF）产生最小的脱粒在没有其他刺激。因此，我们研究是否调节肥大细胞趋化性的过程是肥大细胞介质释放的速率限制。在这些实验中，我们破坏了肌动蛋白聚合，这是肥大细胞趋化性的一个必要条件。然后，我们研究了抗原或SCF诱导的人肥大细胞的趋化性和介质释放。我们发现并报道了肌动蛋白聚合的破坏对早期信号通路的影响最小，但减弱了SCF诱导的人肥大细胞趋化性。出乎意料的是，在没有其他刺激的情况下，SCF诱导大量的脱粒在浓度依赖性的方式后肌动蛋白解体。我们认为这些数据与调节细胞迁移的过程限制肥大细胞脱颗粒是细胞骨架重组的结果这一结论一致。

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.