PHAGOCYTOSIS IN PULMONARY ALVEOLAR MACROPHAGES

肺泡巨噬细胞的吞噬作用

• 批准号: 2872917
• 负责人: STEVEN M GREENBERG
• 金额: $ 24.5万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-02-01 至 2001-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2872917
• 关键词: SDS polyacrylamide gel electrophoresis; actins; affinity chromatography; alveolar macrophages; antibody receptor; biological signal transduction; chimeric proteins; cytoskeletal proteins; enzyme substrate; guanine nucleotide binding protein; laboratory mouse; laboratory rat; leukocyte activation /transformation; lymphocyte; phagocytosis; phosphorylation; polymerization; protein tyrosine kinase; tissue /cell culture; transfection; western blottings; SDS polyacrylamide gel electrophoresis; actins; affinity chromatography; alveolar macrophages; antibody receptor; biological signal transduction; chimeric proteins; cytoskeletal proteins; enzyme substrate; guanine nucleotide binding protein; laboratory mouse; laboratory rat; leukocyte activation /transformation; lymphocyte; phagocytosis; phosphorylation; polymerization; protein tyrosine kinase; tissue /cell culture; transfection; western blottings

Phagocytosis is an important arm of the lung's host defense against a variety of pathogenic organisms, including Streptococcal pneumonia, Mycobacterium tuberculosis, and Pneumocystis carinii. Defects of phagocytosis have been documented in patients with diabetes mellitus, cirrhosis, uremia, various autoimmune diseases, and AIDS. In addition to its medical significance, phagocytosis serves as a convenient model system to study cytoskeletal dynamics that occur at the leading edge of all eukaryotic cells. During Fc receptor.mediated phagocytosis, pseudopods are elaborated which are enriched in F-actin and numerous tyrosine- phosphorylated proteins. Fc receptor-mediated phagocytosis is blocked by cytochalasins and tyrosine kinase inhibitors; thus, it is dependent on F- actin assembly and one or more tyrosine kinases. The identity of specific tyrosine kinases that are capable of mediating these cytoskeletal changes is unknown. We have developed several model systems to probe specific steps in the formation of pseudopods and engulfment of IgG-coated particles. These employ cell lines transfected with chimeric constructs containing Fc receptor subunits, and chimeric fusion proteins containing the entire coding region of several cytosolic tyrosine kinases. These and other studies have enabled us to identify the protein tyrosine kinase syk as a likely candidate to play a central role in mediating cytoskeletal rearrangements that culminate in phagocytosis. The overall goal of this proposal is to determine how syk becomes activated to signal cytoskeletal alterations and pseudopod formation. We will address the following related hypotheses: (1) Do Src family tyrosine kinases participate directly in phagocytic signaling by activating syk tyrosine kinase? (2) Is syk tyrosine kinase critical for Fc receptor-mediated phagocytosis? (3) Does syk activate downstream pathways that mediate phagocytosis? We will address the role of the Src family tyrosi~e kinases in Specific Aim 1, in which we employ a model system for pseudopod formation: a lymphocyte cell line that expresses a chimerib receptor whose cytosolic domain consists of the y subunit of Fc receptors. We will use lyn-deficient lymphocytes to address the requirement for this tyrosine kinase in pseudopod formation. The spatial relationship between lyn and hck kinases and Fc receptors will be explored. Are lyn and/or hck required for the activation of syk kinase? In Specific Aim 2, we will address the role of syk tyrosine kinase in pseudopod formation using sykdeficient lymphocytes. We will characterize a model system for phagocytosis in COS cells expressing transmembrane fusion proteins bearing syk, and various mutated forms of syk. Finally, in Specific Aim 3 we will explore the mechanisms by which syk signals phagocytosis, and identify possible downstream targets of syk, such as phosphatidylinositol 3-kinase, the small molecular weight GTP-binding protein ras and potentially novel substrates of syk.

吞噬作用是肺部宿主防御的重要部门 多种致病生物，包括链球菌肺炎， 结核分枝杆菌和肺炎囊肿。缺陷 糖尿病患者已记录了吞噬作用， 肝硬化，尿毒症，各种自身免疫性疾病和艾滋病。此外 吞噬作用是其医学意义，是一种方便的模型系统 研究在所有前缘发生的细胞骨架动力学 真核细胞。在FC受体介导的吞噬作用期间，伪足 详细阐述了富含F-肌动蛋白和许多酪氨酸 - 磷酸化蛋白。 FC受体介导的吞噬作用被阻止 细胞切拉蛋白和酪氨酸激酶抑制剂；因此，它取决于F- 肌动蛋白组装和一个或多个酪氨酸激酶。特定的身份 能够介导这些细胞骨架变化的酪氨酸激酶 是未知的。我们已经开发了多个模型系统来探测特定的 形成伪足的步骤和IgG涂层的吞噬 颗粒。这些采用了用嵌合构建体转染的细胞系 包含FC受体亚基和嵌合融合蛋白 几种胞质酪氨酸激酶的整个编码区域。这些和 其他研究使我们能够鉴定蛋白质酪氨酸激酶SYK 作为候选人在介导细胞骨架中发挥核心作用 重排，最终导致吞噬作用。总体目标 建议是确定SYK如何激活以信号细胞骨架 变化和伪足形成。我们将解决以下相关的 假设：（1）DO SRC家族酪氨酸激酶直接参与 通过激活Syk酪氨酸激酶来传导吞噬信号？ （2）是Syk FC受体介导的吞噬作用至关重要的酪氨酸激酶？ （3）做 SYK激活介导吞噬作用的下游途径？我们将 探讨了Src家族酪氨酸酶在特定目的1中的作用，在 我们采用伪足形成的模型系统：淋巴细胞细胞 表达Chimerib受体的线，其胞质结构域由 FC受体的Y亚基。我们将使用Lyn缺陷淋巴细胞 解决了伪足形成中这种酪氨酸激酶的需求。 Lyn和HCK激酶与FC受体之间的空间关系将 被探索。 Lyn和/或HCK是否需要SYK激酶的激活？ 在特定目标2中，我们将解决Syk酪氨酸激酶在 使用Sykdeigient淋巴细胞形成假脚架。我们将表征 表达跨膜的COS细胞中吞噬作用的模型系统 带有SYK的融合蛋白和各种突变形式的SYK。最后，在 特定目标3我们将探索SYK信号的机制 吞噬作用，并确定SYK的可能下游靶标，例如 磷脂酰肌醇3-激酶，小分子量GTP结合 蛋白RAS和SYK的潜在新型底物。