Mechanisms of a Novel Chemotactic Cofactor for C5a

C5a 新型趋化辅助因子的机制

• 批准号: 7417724
• 负责人: RICHARD R KEW
• 金额: $ 31万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2008-09-24
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7417724
• 关键词: ANXA2 gene; Address; Alveolitis; Amino Acid Sequence; Antigen-Antibody Complex; Binding; Binding Proteins; Biological; Biological Assay; Blood Platelets; Bronchoalveolar Lavage Fluid; C5a anaphylatoxin receptor; CD44 gene; Cell membrane; Cell surface; Cells; Chemotactic Factors; Chemotaxis; Complement; Complement 5a; Complement Activation; Complex; Disease; Generations; Human; Immune; Immune system; In Vitro; Individual; Infection; Inflammation; Inflammatory; Injury; Knowledge; Leukocytes; Ligands; Liquid substance; Mediating; Modeling; Mus; Pathogenesis; Pathology; Patients; Peptides; Peritonitis; Physiological; Plasma Proteins; Play; Process; Protein Binding; Proteins; RNA Interference; Recruitment Activity; Regulation; Reporting; Research; Role; Serum; Signal Transduction; Signaling Molecule; Site; Stimulus; Synovial Fluid; Therapeutic Intervention; Thrombospondin 1; Tissues; U937 Cells; Vitamin D; Vitamin D-Binding Protein; adapter protein; cell type; cofactor; in vivo; macromolecule; member; mutant; neutrophil; novel; positional cloning; protein function; prototype; receptor; response

Activation of complement (C) with consequent generation of the CS-derived peptides C5a and C5a des Arg is a primary chemotactic stimulus at sites of infection or injury. Excessive C5a-mediated leukocyte recruitment into various tissues is known to be a critical step in the pathogenesis of many inflammatory disorders. C5-derived peptides are very potent chemoattractants for almost the entire cellular repertoire of the immune system, and consequently are an attractive target for therapeutic intervention. But the regulation of C5a chemotactic activity in a physiological setting is poorly understood. Previously, several groups have shown that the vitamin D binding protein (DBF), also known as Gc-globulin, can significantly enhance the chemotactic activity of C5-derived peptides, i.e., function as a chemotactic cofactor. This activity of DBP is specific for the C5-derived peptides. However, its role in vivo has not been determined and the mechanism of chemotaxis enhancement is not known. It is our HYPOTHESIS that DBP functions as a physiologically important regulator of the chemotactic activity for C5a, and that this activity is initiated by formation of a novel multi-faceted DBP binding/signaling complex on the cell surface. Thus, the objective of this proposal is two-fold. The first specific aim will determine if DBP functions as a C5a chemotactic cofactor in murine models of C-dependent inflammation using DBP-/- and DBP+/+ mice. This aim will investigate the role of DBP in immune complex mediated (I) peritonitis and (II) alveolitis, (III) alveolitis induced by C-activated serum and purified C5a, and (IV) in vitro using murine leukocytes and purified proteins in a chemotaxis assay. The second specific aim will investigate how DBP enhances chemotaxis to C5a using human neutrophils and U937 cells in vitro. This aim will employ a reverse genetic approach to dissect the process by utilizing (I) mutant DBPs generated by selective deletion of the co-chemotactic and cell binding sequences and (II) RNAi directed at individual members of the DBP binding/signaling complex to silence their expression in U937 cells. Both will be used to determine the effect on DBP-cell binding, Ca2+ signaling, chemotaxis and shedding of the binding/signaling complex. Results of this study will provide a better understanding of how C5a recruits leukocytes in a physiological setting, and also may reveal that DBP could be a target for therapeutic intervention to reduce the leukocyte burden at sites of C activation.

激活补体（C），随后产生CS衍生肽C5 a和C5 a des Arg 是感染或损伤部位的主要趋化刺激物。C5 a介导的白细胞过多 已知募集到各种组织中是许多炎性疾病发病机制中的关键步骤， 紊乱C5-衍生肽是非常有效的化学引诱物，几乎用于所有的细胞库， 免疫系统，因此是治疗干预的有吸引力的靶点。但是， 人们对生理环境中C5 a趋化活性的了解甚少。此前，多个团体 显示维生素D结合蛋白（DBF），也称为GC-球蛋白，可以显着增强 C5衍生肽的趋化活性，即，作为趋化辅因子发挥作用。DBP的活性是 对C5衍生肽具有特异性。然而，其在体内的作用尚未确定， 对趋化性增强的作用尚不清楚。我们的假设是DBP的功能是生理上的 C5 a趋化活性的重要调节剂，并且该活性是通过形成 在细胞表面上的新型多面DBP结合/信号传导复合物。因此，本建议的目的是 双重的第一个具体目标是确定DBP是否在小鼠中作为C5 a趋化辅因子起作用。 使用DBP-/-和DBP+/+小鼠的C依赖性炎症模型。这一目标将调查的作用， DBP在免疫复合物介导的（I）腹膜炎和（II）肺泡炎中的作用，（III）C-激活的 血清和纯化的C5 a，以及（IV）在体外使用小鼠白细胞和纯化的蛋白质进行趋化 比色法第二个具体的目的将研究DBP如何增强对C5 a的趋化性，使用人 中性粒细胞和U937细胞。这一目标将采用逆向遗传学方法来剖析这一过程 通过利用（I）通过选择性缺失共趋化性和细胞结合性而产生的突变体DBP， 序列和（II）针对DBP结合/信号传导复合物的各个成员的RNAi，以沉默 在U937细胞中的表达。两者都将用于确定对DBP-细胞结合、Ca 2 + 信号传导、趋化性和结合/信号传导复合物的脱落。这项研究的结果将提供一个 更好地了解C5 a如何在生理环境中招募白细胞，也可能揭示， DBP可能是治疗干预的目标，以减少C激活部位的白细胞负荷。