VASCULOPATHY, APOPTOSIS AND AUTOIMMUNITY

这项建议侧重于血管病变导致系统性硬化症的纤维化和自身免疫表现的分子机制。系统性硬化症患者产生的自身抗体唯一地针对核仁蛋白，如DNA拓扑异构酶I(Topo-I)。已有研究表明，Topo-I是细胞凋亡过程中特异性的蛋白酶(S)的底物，导致产生新的切割片段，这些片段可能揭示宿主以前不能耐受的隐蔽表位。最近的研究还表明，在弥漫性硬皮病中靶向的几种自身抗原特别容易被金属催化的氧化反应切割，类似于在适当金属存在下的缺血-再灌注过程中可能发生的情况。这一过程还可能揭示免疫分泌表位，并为为什么系统性硬化症的免疫反应以某些蛋白质为唯一靶点提供分子解释。然而，众所周知，仅有一个隐蔽的表位不足以产生自身反应性，这还需要分子佐剂的参与，以及具有共刺激能力的抗原提呈细胞(APC)摄取潜在的自身抗原。这一建议的中心假设是，系统性硬化症患者的慢性缺血再灌注损伤不仅产生皮肤来源的核仁自身抗原内的免疫分泌表位，而且还产生补体配体，提供打破免疫耐受所需的分子佐剂。这一建议的具体目的是：1)确定携带补体配体的凋亡小泡调节细胞因子表达和抗原提呈细胞共刺激能力的能力；2)表征对含有自身抗原的凋亡小泡的免疫反应；3)检测补体配体C3d在体外诱导T和B细胞对Topo I的自身反应中的作用。尽管系统性硬化症是这一提议的重点，但这些研究产生的数据应该有助于我们对血管病变和自身免疫过程的总体理解。

This proposal is focused upon the molecular mechanism by which vasculopathy leads to fibrotic and autoimmune manifestations of systemic sclerosis. Autoantibodies generated by patients with systemic sclerosis are uniquely targeted to nucleolar proteins such as DNA topoisomerase I (topo-I). It has been demonstrated that topo-I is a substrate for protease(s) specific to the apoptotic process, resulting in novel cleavage fragments that may reveal cryptic epitopes to which the host has not previously been tolerized. It has also been recently demonstrated that several of the autoantigens targeted in diffuse scleroderma are uniquely susceptible to cleavage by metal-catalyzed oxidation reactions similar to what may occur during ischemia-reperfusion in the presence of appropriate metals. This process may also reveal immunocryptic epitopes and provides a molecular explanation for why certain proteins are uniquely targeted by the immune response in systemic sclerosis. However, it is well known that a cryptic epitope alone is not sufficient to generate autoreactivity, which also requires the participation of a molecular adjuvant, and uptake of the potential autoantigen by an antigen presenting cell (APC) with costimulatory capacity. The central hypothesis of this proposal is that chronic ischemia-reperfusion injury in patients with systemic sclerosis not only generates immunocryptic epitopes within nucleolar autoantigens of cutaneous origin, but it also generates complement ligands that provide the molecular adjuvant required to break immune tolerance. The specific aims of this proposal are to: 1) Determine the capacity of apoptotic blebs bearing complement ligands to modulate the cytokine expression and costimulatory capacity of antigen presenting cells, 2) characterize the immune responses to self antigen- containing apoptotic blebs, and 3) examine the role of complement ligand C3d during induction of autoreactive T and B cell responses to topo I in vitro. Although systemic sclerosis is the focus of this proposal, the data generated by these studies should provide insight into our understanding of vasculopathic and autoimmune processes in general.