Mechanisms of a novel chemotactic cofactor for C5a

描述(由申请人提供)：补体(C)的激活与C5衍生的多肽C5a和C5a des Arg的产生是感染或损伤部位的主要趋化刺激。C5a介导的白细胞过度聚集到各种组织中被认为是许多炎症性疾病发病机制中的关键步骤。C5衍生的多肽对几乎整个免疫系统的细胞都是非常有效的趋化剂，因此是治疗干预的一个有吸引力的靶点。但在生理环境中对C5a趋化活性的调节知之甚少。此前，几个研究小组已经证明，维生素D结合蛋白(DBP)，也被称为GC-球蛋白，可以显著增强C5衍生多肽的趋化活性，即作为趋化辅助因子发挥作用。DBP的这种活性是C5衍生的多肽所特有的。然而，它在体内的作用尚未确定，趋化增强的机制也不清楚。我们的假设是，DBP是C5a趋化活性的重要生理调节因子，这种活性是通过在细胞表面形成一个新的多方面的DBP结合/信号复合体来启动的。这项提议的目标有两个。首先，将利用DBP-/-和DBP+/+小鼠在C依赖炎症模型中确定DBP作为C5a趋化辅助因子的体内功能。本研究旨在探讨DBP在免疫复合物介导的(I)腹膜炎、(II)肺泡炎、(III)C激活血清和纯化的C5a诱导的肺泡炎中的作用，以及(IV)用小鼠白细胞和纯化的蛋白进行体外趋化试验。第二个具体目标将研究DBP如何在体外利用人中性粒细胞和U937细胞增强对C5a的趋化作用。这一目标将采用反向遗传方法来剖析这一过程，方法是利用(I)选择性删除共趋化和细胞结合序列产生的突变DBP，以及(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、钙信号、趋化和结合/信号复合体脱落的影响。这一研究结果将有助于更好地了解C5a在生理环境中如何招募白细胞，也可能揭示DBP可能成为降低C激活部位白细胞负担的治疗干预的靶点。 与公共健康相关：补体激活肽C5a是诱导组织中白细胞聚集(趋化)的最有效和最重要的生理分子之一。在许多炎症性疾病中，C5a的过度或异常产生与组织损伤密切相关。维生素D结合蛋白(DBP)是一种血液蛋白，已被证明能显著增强C5a的趋化活性。本研究旨在探讨DBP在生理环境下调节C5a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.