Comparative studies of complement responses to ICs

人类和实验动物(小鼠和大鼠)的补体系统在功能上表现出显著的物种差异，特别是在补体激活的经典途径方面。事实上，自20世纪50年代以来，S的研究已经证明，与大鼠和人类相比，小鼠的经典通路功能显著降低。在初步数据中，我们使用抗血小板因子4(PF4)和肝素的单抗(KKO ICs)或DNP的多克隆兔抗DNP抗体(DNP ICs)与锁孔帽状血蓝蛋白(DNP ICs)结合形成的模型免疫复合体(IC)来证实和推广这些发现。具体地说，我们发现：1)KKO和DNP IC在小鼠、大鼠和人类的全血和血浆中激活C3的能力很强；2)KKO IC激活补体的经典途径需要，但小鼠的替代途径需要3)将替代途径蛋白整合到小鼠的KKO IC中，而不是大鼠或人类4)四个品系之间对替代途径的类似需要以及5)通过蛋白质组学方法研究替代途径蛋白质在小鼠中与大鼠和人类的差异表达。基于这些初步数据和已发表的观察结果，我们将检验总体假设，即小鼠中受损的经典途径活动被替代途径C3b反馈周期抵消。为了验证这一假设，我们提出了以下两个目标：1)人、小鼠和大鼠补体系统的比较研究。在这个目标中，我们将检验这一假设，即小鼠经典途径蛋白的遗传变异导致经典途径功能受损。我们将应用分子方法来比较基因序列，并在小鼠、大鼠和人类中执行经典途径蛋白的结构同源性。在其他研究中，我们将研究小鼠、大鼠和人类的血浆蛋白质组，以检查蛋白质/免疫复合物的相互作用，补体和补体相关蛋白的共表达，并将补体蛋白水平与对KKO和DNP IC的功能反应相关联。2)小鼠和人类补体系统的功能研究。在这个目标中，我们将检验这样一个假设，即小鼠利用替代途径的C3b反馈周期作为对低经典途径活动的补偿反应。为了验证这一假设，我们将使用纯化的人和小鼠补体蛋白来证明小鼠C3b反馈周期中受损的经典C3转换酶活性和正常/增强的活性，检查人和小鼠补体蛋白互换的效果，并从小鼠血清中识别潜在的经典途径激活的小鼠抑制剂。总之，这些研究有望描绘出小鼠和人类不同补体途径反应的分子和功能基础。我们希望这些研究能为扩大应用提供初步数据，以阐明小鼠补体反应的分子和结构差异，并以开发翻译相关的动物模型为长期目标。

The complement systems of humans and laboratory animals (mice and rats) show striking species differences in function, particularly with regards to the classical pathway of complement activation. Indeed, studies since the 1950’s have documented markedly reduced classical pathway function in mice as compared to rats and humans. In preliminary data, we confirm and extend these findings using model immune complexes (ICs) formed by monoclonal antibodies to platelet factor 4 (PF4) and heparin (KKO ICs) or polyclonal rabbit anti-dinitrophenol (DNP) antibodies to DNP conjugated with keyhole limpet hemocyanin (DNP ICs). Specifically, we show: 1) robust C3 activation by KKO and DNP ICs in whole blood and plasma from mice, rats and humans 2) classical pathway requirements for complement activation by KKO ICs in rats and human, but alternative pathway requirements in mice 3) incorporation of alternative pathway proteins into KKO ICs in mice, but not rats or humans 4) comparable requirements for the alternative pathway among four murine strains and 5) differential expression of alternative pathway proteins by proteomics in mice as compared to rats and humans. Based on these preliminary data and published observations, we will test the overall hypothesis that impaired classical pathway activity in mice is counterbalanced by the alternative pathway C3b feedback cycle. To test this hypothesis, we propose the following two aims: 1) Comparative studies of human, mouse, and rat complement systems. In this aim, we will test that hypothesis that genetic variation in murine classical pathway proteins contributes to impaired classical pathway function. We will apply molecular approaches to compare genetic sequences and perform structural homologies of classical pathway proteins in mice v rats and humans. In other studies, we will investigate the plasma proteome of mice, rats, and humans to examine protein/immune complex interactions, co-expression of complement and complement-associated proteins and correlate complement protein levels with functional responses to KKO and DNP ICs. 2) Functional studies of murine and human complement systems. In this aim, we will test the hypothesis that mice utilize the C3b feedback cycle of the alternative pathway as a compensatory response to low classical pathway activity. To test this hypothesis, we will use purified human and mouse complement proteins to demonstrate reduced impaired classical C3 convertase activity and normal/heightened activity of the C3b feedback cycle in mice, examine the effects of interchanging human and mouse complement proteins, and identify potential murine inhibitors of classical pathway activation from mouse serum. Together, these studies are expected to delineate the molecular and functional basis of divergent complement pathway responses in mice and humans. We expect these studies to generate preliminary data for an expanded application to elucidate the molecular and structural divergence of murine complement responses with the long term goal of developing translationally relevant animal models.