Heat Shock Protein, Citrullinated Antigen and Dendritic Cell Interactions

热休克蛋白、瓜氨酸抗原和树突状细胞相互作用

• 批准号: 7617038
• 负责人: Frances Ivette Santiago-Schwarz
• 金额: $ 6.79万
• 依托单位: FARMINGDALE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-23 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7617038
• 关键词: Affinity; Antigen-Presenting Cells; Antigens; Autoantigens; Autoimmune Process; Cell Communication; Co-Immunoprecipitations; Common Epitope; Complex; Confocal Microscopy; Dendritic Cells; Disease; Epitopes; Event; Family; Fibrin; Fibrinogen; Goals; HLA-DR1 Antigen; HLA-DR4 Antigen; Heat Stress Disorders; Heat shock proteins; Heat-Shock Proteins 70; Immune response; Immune system; In Vitro; Inflammatory; Joints; Link; Lymphocyte; Patients; Peptides; Process; Proteins; Research; Rheumatoid Arthritis; Serum; Staging; Surface Plasmon Resonance; Synovial Fluid; Techniques; Therapeutic; Time; biological adaptation to stress; effective therapy; immunopathology; response; trafficking; uptake

DESCRIPTION (provided by applicant): The long-term objective of this research is to develop highly directed and long-lasting therapy for rheumatoid arthritis (RA). The specific aims are to, for the first time: 1) Study physical associations between heat shock protein (HSP70) and citrullinated-fibrinogen 2) Demonstrate mechanism of delivery of cit-fibrinogen into immature dendritic cells (DC). The recognition that cit-proteins (especially cit-fibrinogen) are key auto-antigens (Ags) initiating and perpetuating immunopathology in RA may represent a huge step toward achieving more effective therapy. To date, however, many of the events related to the handling of cit-Ags are unknown, including mechanisms of cit-autoAg uptake/processing by antigen presenting cells such as DC. The hypothesis is that in RA, citrullinated Ags such as cit-fibrinogen are transported via HSP into immature DC to activate autoimmune lymphocyte responses. In RA, HSP are highly abundant and likely to facilitate the transport of auto-Ags into immature DC. Because citrullination of proteins results in unfolding and partial denaturing, citproteins are prime candidates for association with HSP, which partner with proteins in non-native states. Emphasis will be placed on studying inducible HSP70 and cit-fibrin, which are specifically elevated in RA synovial fluid (SF) vs non-RA SF and considered key self-cit-Ags in RA, resp. In Aim 1, HSP70-cit-fibrinogen interactions will be studied using surface plasmon resonance (SPR) and co-immunoprecipitation (IP) techniques. For SPR, purified proteins will be used. HSP70-cit-fibinogen complexes present in RA SF, RA serum, non-RA SF and normal serum will be revealed by IP. In Aim 2, immature DC will be primed in vitro to undergo an inflammatory/heat stress response under precise conditions present in the RA joint. After the addition of cit-fibrinogen, uptake/intracellular trafficking of cit-fibinogen will be assessed by IP and confocal microscopy. Due to the increased affinity of cit-Ags to HLA-DR4, studies of cit-fibrinogen-HLA-DR interactions and cit-fibrinogen uptake in DC prepared from RA patients expressing the common epitope will be included. These studies will employ synthetic cit-fibrinogen 10mer peptides (containing cit-epitopes) and native fib peptides and purified HLA-DR4 and HLA-DR1 (non RA linked). Understanding how key self-antigens in RA are handled by the immune system at the earliest stages of the autoimmune process may reveal a universal disease mechanism that can be specifically interrupted, before the onset of severe disease activity.

描述（由申请人提供）： 这项研究的长期目标是开发针对类风湿关节炎（RA）高度定向和长期疗法。具体目的是，第一次：1）研究热休克蛋白（HSP70）和瓜氨基氨基纤维蛋白原之间的物理关联2）证明了将柠檬纤维蛋白原传递到未成熟的树突状细胞（DC）中的机制。认识到，柠檬蛋白（尤其是柠檬纤维蛋白原）是关键的自动抗原（AGS）在RA中引发和永久性免疫病理学可能是朝着实现更有效治疗的巨大一步。然而，迄今为止，与CIT-AGS处理有关的许多事件尚不清楚，包括通过抗原呈递细胞（例如DC）对CIT-AutoAg摄取/加工的机制。假设是，在RA中，将Citruplinated AG（例如Cit-纤维蛋白原）通过HSP运输到未成熟的DC中，以激活自身免疫性淋巴细胞反应。在RA中，HSP高度丰富，并且很可能促进自动速度转运到未成熟的DC中。由于蛋白质的柠檬化会导致展开和部分变性，因此柠檬蛋白是与HSP相关的主要候选者，HSP与非本地状态的蛋白质合作。重点将放在研究诱导HSP70和CIT-纤维蛋白上，它们在RA滑液（SF）与非RA SF中的特异性升高，并被认为是RA中的关键自动cit-ags，resp。在AIM 1中，将使用表面等离子体共振（SPR）和共免疫沉淀（IP）技术研究HSP70-CIT-纤维蛋白原相互作用。对于SPR，将使用纯化的蛋白质。 RA SF，RA血清，非RA SF和正常血清中存在的HSP70-CIT纤维纤维组合物将由IP揭示。在AIM 2中，未成熟的DC将在体外启动，以在RA关节中存在的精确条件下进行炎症/热应激反应。添加了柠檬纤维蛋白原后，将通过IP和共聚焦显微镜评估CIT纤维蛋白原的摄取/细胞内运输。由于CIT-AGS与HLA-DR4的亲和力增加，将包括对表达公共表位的RA患者制备的DC中CIT-FIRBILIN HALA-DR相互作用和CIT-纤维蛋白原吸收的研究。这些研究将采用合成的CIT-纤维蛋白原10mer肽（含有柠檬含量）和天然FIB肽以及纯化的HLA-DR4和HLA-DR1（非RA链接）。在自身免疫过程的最早阶段，了解RA中的关键自我抗原是如何处理的，可能会揭示出一种普遍的疾病机制，该机制可能会在严重疾病活动开始之前特异性中断。