Mechanisms of a Novel Chemotactic Cofactor for C5a

补体(C)的激活与CS衍生多肽C5a和C5a des Arg的产生 是感染或损伤部位的主要趋化刺激。过量C5a介导的白细胞 已知，在许多炎症性疾病的发病机制中，重新聚集到各种组织中是一个关键步骤。 精神错乱。C5衍生的多肽对几乎整个细胞库都是非常有效的化学诱导剂。 免疫系统，因此是一个有吸引力的治疗干预的目标。但这项规定 C5a在生理环境中的趋化活性还知之甚少。此前，有几个组织 研究表明，维生素D结合蛋白(DBF)，也称为GC-球蛋白，可以显著增强 C5衍生的多肽的趋化活性，即作为趋化辅助因子发挥作用。DBP的这一活动是 专用于C5衍生的多肽。然而，其在体内的作用和机制尚未确定。 趋化增强的作用尚不清楚。我们的假设是，DBP在生理上起着 C5a趋化活性的重要调节因子，这种活性是通过形成一种 细胞表面新的多面DBP结合/信号复合体。因此，这项提案的目标是 两倍。第一个特定目标将确定DBP是否在小鼠体内发挥C5a趋化辅助因子的功能 使用DBP-/-和DBP+/+小鼠建立C依赖炎症模型。这一目标将调查 DBP在免疫复合物介导的(I)腹膜炎和(II)肺泡炎，(III)C活化的肺泡炎中的作用 血清和纯化的C5a，以及(IV)用小鼠白细胞和纯化的蛋白体外趋化 化验。第二个具体目标将研究DBP如何利用人类增强对C5a的趋化作用 中性粒细胞和U937细胞的体外培养。这一目标将采用反向遗传方法来剖析这一过程 通过利用(I)选择性删除共趋化和细胞结合而产生的突变型DBPs 序列和(Ii)针对DBP结合/信号复合体的单个成员的RNAi以沉默 它们在U937细胞中的表达。两者都将被用来确定对DBP-细胞结合，钙离子的影响 结合/信号复合体的信号、趋化和脱落。这项研究的结果将提供 更好地理解C5a如何在生理环境中招募白细胞，也可能揭示 DBP可作为治疗干预的靶点，以降低C活化部位的白细胞负荷。

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.