Mechanisms of Inflammation: Role of CD34 and Podocalyxin in Neutrophil Recruitment

炎症机制：CD34 和 Podocalyxin 在中性粒细胞募集中的作用

• 批准号: RGPIN-2014-04308
• 负责人: Singh, Baljit
• 金额: $ 4.16万
• 依托单位: University of Saskatchewan
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=708000
• 关键词: Mechanisms; Inflammation; Role; CD34; Podocalyxin

Inflammation is a host's innate defense response to biological, chemical or physical insults. Inflammation is induced through infections with bacteria, viruses and parasites. Inflammation underlies many diseases in organs such as the liver (e.g., hepatitis), joints (e.g., arthritis) and lungs (e.g., pneumonia and asthma). Inflammation is characterized by the activation of defense cells such as neutrophils and monocytes while in the blood stream. Activated cells such as neutrophils move from the blood into the organs to fight bacteria. Neutrophils migrate from the blood vessels through interactions with the endothelial cells. Endothelial cells line the inside of the walls of blood vessels. Activated neutrophils kill bacteria through production of chemicals such as free oxygen radicals. Excessive migration of activated neutrophils into the tissues results in significant tissue damage from free oxygen radicals and other chemicals produced by the neutrophils. The tissue damage caused by products of neutrophils, if excessive, contributes to morbidity and mortality. In order to maintain balance and insure that neutrophils do more good than harm, we need to maintain the anti-microbial actions of the neutrophils, and at the same time, fine-tune their migration into the tissues to prevent tissue damage. This can be potentially accomplished through control of migration of the neutrophils. Neutrophil migration occurs through various steps such as slowing, rolling on the endothelium, firm adhesion with the endothelium and finally migration out of the blood vessels. One of the very early events in the migration of neutrophils into the tissues is the interaction between the neutrophils and the endothelium. This interaction is facilitated through the formation of projections (called pseudopods) on the neutrophil membrane through reorganization of cell skeleton proteins such as F-actin. The mechanisms that lead to the cytoskeletal changes in neutrophils are not fully understood. I propose to study the roles of CD34 and podocalyxin (PC) proteins in these processes to eventually discover molecular mechanisms to control neutrophil migration. The studies include evaluation of the mechanisms that influence the expression of CD34 and PC in neutrophils and endothelial cells as well as the study of the role of these proteins in the interactions of neutrophils with the endothelium. The studies will be conducted with an array of in vitro and in vivo methods to investigate the cell morphology, molecules and outcomes: confocal and electron immunocytochemistry; western blots; flow cytometry; endothelial and neutrophil cultures; mouse models of inflammation in cremaster muscle and lungs. Taken together, my trainees and I will conduct studies that range from isolated cultured neutrophil and endothelial cells to two different animal models of inflammation to understand the fundamental nature of the neutrophil migration. The data from these studies are expected to show hitherto unknown roles for CD34 and PC in the formation of pseudopods on neutrophils, which facilitate interactions with endothelial cells lining the inside walls of the blood vessels. Because these initial membrane contacts are critical for the eventual migration of neutrophils, these data will advance our knowledge and contribute towards the development of new molecular management models of inflammatory diseases for animals and humans.

炎症是宿主对生物、化学或物理损伤的先天防御反应。炎症是由细菌、病毒和寄生虫感染引起的。炎症是器官如肝脏（例如，肝炎），关节（例如，关节炎）和肺（例如，肺炎和哮喘）。炎症的特征在于在血流中时激活防御细胞，如中性粒细胞和单核细胞。激活的细胞，如中性粒细胞，从血液进入器官，以对抗细菌。中性粒细胞通过与内皮细胞的相互作用从血管中迁移。内皮细胞排列在血管壁的内侧。活化的中性粒细胞通过产生化学物质如氧自由基来杀死细菌。活化的中性粒细胞过度迁移到组织中导致由中性粒细胞产生的自由氧自由基和其他化学物质引起的显著组织损伤。中性粒细胞产物引起的组织损伤，如果过量，会导致发病率和死亡率。为了保持平衡并确保中性粒细胞利大于弊，我们需要保持中性粒细胞的抗微生物作用，同时，微调它们向组织中的迁移以防止组织损伤。这可以通过控制中性粒细胞的迁移来实现。 神经元迁移通过各种步骤发生，例如减慢、在内皮上滚动、与内皮牢固粘附并最终迁移出血管。中性粒细胞迁移到组织中的非常早期的事件之一是中性粒细胞和内皮之间的相互作用。通过细胞骨架蛋白如F-肌动蛋白的重组，在中性粒细胞膜上形成突起（称为伪足），促进这种相互作用。导致中性粒细胞中细胞骨架变化的机制尚未完全了解。我建议研究CD 34和podocalyxin（PC）蛋白在这些过程中的作用，最终发现控制中性粒细胞迁移的分子机制。这些研究包括评估影响中性粒细胞和内皮细胞中CD 34和PC表达的机制，以及研究这些蛋白在中性粒细胞与内皮细胞相互作用中的作用。 将采用一系列体外和体内方法进行研究，以研究细胞形态、分子和结局：共聚焦和电子免疫细胞化学;蛋白质印迹;流式细胞术;内皮细胞和中性粒细胞培养;提睾肌和肺炎症小鼠模型。总之，我和我的学员将进行研究，从分离培养的中性粒细胞和内皮细胞到两种不同的炎症动物模型，以了解中性粒细胞迁移的基本性质。 这些研究的数据预计将显示CD 34和PC在中性粒细胞伪足形成中迄今未知的作用，伪足促进与血管内壁内皮细胞的相互作用。由于这些最初的膜接触对于中性粒细胞的最终迁移至关重要，这些数据将推进我们的知识，并有助于开发动物和人类炎症性疾病的新分子管理模型。