Mechanisms of a novel chemotactic cofactor for C5a

描述（由申请方提供）：补体（C）的活化以及随后产生的C5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。已知过度的C5 a介导的白细胞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。C5衍生肽是免疫系统几乎整个细胞库的非常有效的化学引诱物，因此是治疗干预的有吸引力的靶标。但在生理环境中C5 a趋化活性的调节知之甚少。以前，几个研究小组已经表明，维生素D结合蛋白（DBP），也称为GC-球蛋白，可以显著增强C5衍生肽的趋化活性，即，作为趋化辅因子发挥作用。DBP的这种活性对C5衍生肽具有特异性。然而，其在体内的作用尚未确定，其趋化性增强的机制尚不清楚。我们的假设是，DBP作为C5 a趋化活性的生理学重要调节剂发挥作用，并且这种活性是通过在细胞表面形成新型多面DBP结合/信号传导复合物而启动的。这项建议有两个目的。首先，将使用DBP-/-和DBP+/+小鼠在C依赖性炎症的鼠模型中确定DBP作为C5 a趋化辅因子的体内功能。本研究旨在研究DBP在免疫复合物介导的（I）腹膜炎、（II）肺泡炎、（III）由C-活化血清和纯化C5 a诱导的肺泡炎以及（IV）在体外使用小鼠白细胞和纯化蛋白进行的趋化性测定中的作用。第二个具体的目标将调查DBP如何增强趋化性C5 a使用人中性粒细胞和U937细胞在体外。该目的将采用反向遗传方法来剖析该过程，其通过利用（I）通过选择性缺失共趋化和细胞结合序列产生的突变DBP和（II）针对DBP结合/信号传导复合物的单个成员的RNAi来沉默它们在U937细胞中的表达。两者都将用于确定对DBP-细胞结合、钙信号传导、结合/信号传导复合物的趋化性和脱落的影响。这项研究的结果将提供一个更好的了解如何C5 a招募白细胞在生理环境中，也可能揭示DBP可能是一个目标的治疗干预，以减少白细胞负荷的网站C激活。 公共卫生相关性：补体激活肽C5 a是诱导白色血细胞在组织中积聚（趋化性）的最有效和生理上重要的分子之一。C5 a的过度或异常产生与许多炎性疾病中的组织损伤密切相关。维生素D结合蛋白（DBP）是一种血液蛋白，已显示可显著增强C5 a的趋化活性。本研究将探讨DBP在生理条件下调节C5 a趋化活性的机制。

DESCRIPTION (provided by applicant): Activation of complement (C) with consequent generation of the C5-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 (DBP), 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. The objective of this proposal is two- fold. First, the in vivo function of DBP as a C5a chemotactic cofactor will be determined 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, (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, calcium 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. PUBLIC HEALTH RELEVANCE: Complement activation peptide C5a is one of the most potent and physiologically important molecules that induce accumulation of white blood cells in tissues (chemotaxis). Excessive or aberrant production of C5a is strongly associated with tissue injury in numerous inflammatory diseases. The vitamin D binding protein (DBP) is a blood protein that has been shown to significantly enhance the chemotactic activity of C5a. This proposal will investigate the mechanisms by DBP regulates the chemotactic activity of C5a in a physiological setting.