G-Protein-Coupled Signal Transduction in Allergy and Anaphylaxis

过敏和过敏反应中的 G 蛋白偶联信号转导

• 批准号: 7964553
• 负责人: Kirk m Druey
• 金额: $ 7.12万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7964553
• 关键词: 1-Phosphatidylinositol 3-Kinase; Adenosine; Allergens; Allergic; Anaphylaxis; Angioneurotic Edema; Ascaridil; Basophils; Binding; CXCL12 gene; Calcium; Cell Degranulation; Cell Line; Cells; Cellular biology; Classification; Codeine; Collaborations; Complement 5a; Conflict (Psychology); Cutaneous; DNA; Data; Disease; G-Protein-Coupled Receptors; GTP-Binding Protein Regulators; GTP-Binding Proteins; Generations; Genes; Genetic Polymorphism; Goals; Guanosine Diphosphate; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Histamine; Human; Hypersensitivity; Hypotension; Idiopathic anaphylaxis; IgE; IgE Receptors; In Vitro; Inflammation Mediators; Ligands; Mediating; Morphine; Mus; Mutation; Patients; Phenotype; Phosphatidylinositols; Phosphorylation; Phosphotransferases; Production; Protein Tyrosine Kinase; RNA; Recurrence; Rodent Model; Series; Serum; Signal Pathway; Signal Transduction; Signaling Molecule; Skin; Stridor; Systemic Mastocytosis; Tachycardia; Testing; Tryptase; United States National Institutes of Health; Urticaria; Wheezing; chemokine; crosslink; cytokine; inorganic phosphate; insight; interest; mast cell; receptor; release of sequestered calcium ion into cytoplasm; response; small hairpin RNA; sphingosine 1-phosphate; src-Family Kinases; sudden cardiac death

Idiopathic anaphylaxis (IA) was first described in 1978 in a series of patients with recurrent anaphylactic episodes where no specific trigger could be identified. IA usually manifests as urticaria, angioedema, wheezing, stridor, and, most importantly, may include hypotension, tachycardia, and sudden cardiac death. Although no offending allergens can be determined in patients with IA, mast cell degranulation and subsequent release of inflammatory mediators is thought to cause the disease. This hypothesis is corroborated by the elevated levels of mast cell-derived tryptase found in the serum of IA patients after an episode. The evidence for enhanced mast cell reactivity in IA is conflicting. An early study at NIH (Keffer et al. J Allergy Clin Immunol 1989) found no difference in the cutaneous response to either morphine or histamine in patients with IA compared to normal controls or patients with systemic mastocytosis. In contrast, a more recent study (2004) found that IA patients demonstrated higher skin responses to codeine than atopic controls. In mast cells and basophils, IgE receptor crosslinking leads to receptor phosphorylation by lyn, a src family kinase, which phosphorylates a second tyrosine kinase, syk. Phosphorylation and activation of syk leads to recruitment of numerous downstream signaling molecules such as phosphoinositide-3-kinase (PI3K), which catalyzes the formation of phosphatidylinositol-3, 4-5 phosphate (PIP3). PIP3 generation is critical to the release of calcium from intracellular stores leading to degranulation. Agents acting on G-protein coupled receptors (GPCRs), such as histamine and morphine, induce mast cell and basophil degranulation independently of IgE. GPCRs activate heterotrimeric G proteins, which bind guanosine triphosphate (GTP) in exchange for guanosine diphosphate (GDP). The GTP-bound form of the G protein induces downstream signaling cascades, including intracellular calcium flux responsible for mast cell degranulation. In recent years, several compounds acting on GPCRs, such as chemokines or the serum factors sphingosine 1-phosphate and adenosine, have been shown to either activate mast cells themselves or to be required for optimal IgE-mediated degranulation. We have identified a regulator of G protein signaling (RGS13) expressed in mast cells, which appears to regulate both GPCR and IgE-mediated mast cell degranulation by distinct mechanisms. Mice deficient in RGS13 had markedly increased anaphlyactic responses due to more IgE-mediated mast cell degranulation. This phenotype was due to inhibition of IgE-Ag induced PI3 kinase activation by RGS13. In 2009, we extended these findings by showing that RGS13 also regulates GPCR-induced degranulation and cytokine production by human mast cells. HMC-1 and LAD-2 mast cell lines depleted of RGS13 by shRNA showed increased calcium influx in response to several endogenous ligands such as C5a, sphingosine-1-phosphate, and the chemokine CXCL12. LAD-2 cells with reduced RGS13 expression degranulated more to sphingosine-1-phosphate than control cells. Although evidence from rodent models and the aforementioned patient data suggest that some signaling molecules profoundly influence mast cell reactivity, a systematic analysis of signaling components from patients with IA has not been performed. The goals of this project are 1) to examine the IgE- and GPCR induced degranulation of mast cells grown in vitro from IA patients in comparison to allergic patients and normal controls (in collaboration with the Mast Cell Biology Section of LAD) and 2) to analyze the occurrence and functional significance of specific polymorphisms in the RGS13 gene (in germline DNA and RNA derived from cultured mast cells). These studies may provide insight into how signaling pathways leading to mast cell degranulation differ in IA from healthy controls.

特发性过敏反应 (IA) 于 1978 年首次在一系列反复发生过敏反应的患者中被描述，但无法确定具体的触发因素。 IA 通常表现为荨麻疹、血管性水肿、喘息、喘鸣，最重要的是，可能包括低血压、心动过速和心源性猝死。 尽管无法确定 IA 患者的致病过敏原，但肥大细胞脱颗粒和随后释放的炎症介质被认为是导致该疾病的原因。 IA 患者血清中肥大细胞来源的类胰蛋白酶水平升高证实了这一假设。 IA 中肥大细胞反应性增强的证据是相互矛盾的。 NIH 的一项早期研究（Keffer 等人，J Allergy Clin Immunol 1989）发现，与正常对照或系统性肥大细胞增多症患者相比，IA 患者对吗啡或组胺的皮肤反应没有差异。相比之下，最近的一项研究（2004 年）发现，IA 患者比特应性对照组表现出更高的皮肤对可待因的反应。在肥大细胞和嗜碱性粒细胞中，IgE 受体交联导致受体被 lyn（一种 src 家族激酶）磷酸化，该激酶磷酸化第二种酪氨酸激酶 syk。 syk 的磷酸化和激活导致大量下游信号分子的募集，例如磷酸肌醇 3 激酶 (PI3K)，其催化磷脂酰肌醇 3, 4-5 磷酸 (PIP3) 的形成。 PIP3 的产生对于细胞内储存的钙释放导致脱颗粒至关重要。 作用于 G 蛋白偶联受体 (GPCR) 的药物，例如组胺和吗啡，可独立于 IgE 诱导肥大细胞和嗜碱性粒细胞脱颗粒。 GPCR 激活异三聚体 G 蛋白，该蛋白结合三磷酸鸟苷 (GTP)，以交换二磷酸鸟苷 (GDP)。 G 蛋白的 GTP 结合形式诱导下游信号级联反应，包括负责肥大细胞脱颗粒的细胞内钙流。 近年来，一些作用于 GPCR 的化合物，例如趋化因子或血清因子 1-磷酸鞘氨醇和腺苷，已被证明可以激活肥大细胞本身，或者是最佳 IgE 介导的脱粒所需的。我们已经鉴定出肥大细胞中表达的 G 蛋白信号传导调节剂 (RGS13)，它似乎通过不同的机制调节 GPCR 和 IgE 介导的肥大细胞脱颗粒。 缺乏 RGS13 的小鼠由于 IgE 介导的肥大细胞脱粒作用明显增加，过敏反应明显增加。该表型是由于 RGS13 抑制 IgE-Ag 诱导的 PI3 激酶激活所致。 2009 年，我们扩展了这些发现，表明 RGS13 还调节 GPCR 诱导的人类肥大细胞脱颗粒和细胞因子产生。通过 shRNA 耗尽 RGS13 的 HMC-1 和 LAD-2 肥大细胞系显示出响应多种内源配体（例如 C5a、1-磷酸鞘氨醇和趋化因子 CXCL12）的钙流入增加。 RGS13 表达降低的 LAD-2 细胞比对照细胞更多地脱颗粒为 1-磷酸鞘氨醇。 尽管来自啮齿动物模型的证据和上述患者数据表明一些信号分子深刻影响肥大细胞反应性，但尚未对 IA 患者的信号成分进行系统分析。 该项目的目标是 1) 与过敏患者和正常对照相比，检查 IgE 和 GPCR 诱导的 IA 患者体外生长的肥大细胞脱颗粒（与 LAD 肥大细胞生物学科合作），2)分析 RGS13 基因（来自培养肥大细胞的种系 DNA 和 RNA）中特定多态性的发生和功能意义。这些研究可能有助于了解 IA 中导致肥大细胞脱粒的信号通路与健康对照有何不同。