The Role of Dendritic Cells in Renal Immune Responses

描述（由申请人提供）：包括淋巴细胞在内的炎症细胞浸润肾实质是肾脏疾病的共同特征，即使在明显缺乏外源性免疫刺激的情况下。肾脏疾病中t淋巴细胞激活的机制尚不清楚，健康肾脏中阻止免疫激活的机制也是如此。本实验方案将检查树突状细胞（dc）的动态行为、反应和功能，树突状细胞（dc）是一种具有抗原摄取和递呈特殊功能的迁移细胞群，在健康期间在肾脏及其引流淋巴组织内，并探索它们在不同形式的肾损伤中的作用。尽管密度、分布和周转率各不相同，但包括肾脏在内的所有器官都有dc常住人口。DC“成熟”是由疾病或损伤的炎症产物诱导的。成熟的树突状细胞从器官转移到引流淋巴结，在那里它们激活t细胞启动细胞免疫反应。在缺乏成熟刺激的情况下，树突状细胞也可能将抗原转移到淋巴组织，以激活防止自身免疫的调节机制。dc - t细胞相互作用的调节被认为是预防/治疗免疫介导性疾病的重要治疗靶点。该提议的主要假设是：(a)蛋白质抗原从肾脏转运到引流淋巴结发生在健康期间，并积极维持对肾组织的免疫耐受；(b)肾DC表型和转换的改变是由多种形式的肾损伤引起的，并显著导致炎症性肾实质损伤。实验策略将着重于使用体内技术，直接检测dc介导的抗原在小鼠肾脏和肾淋巴结中的转运和呈递。第一个特异性目标是确定健康期间肾脏DC的生物学，将采用体内brdu标记来确定DC的转换，同源骨髓移植来检查肾脏DC的前体来源，将荧光颗粒或蛋白质单侧接种到肾脏中以跟踪肾脏DC介导的抗原运输，产生表达肾脏限制性新抗原的转基因小鼠，以及抗原特异性TCR转基因t细胞的过继转移。第二个特定目标是确定肾dc在不同形式肾损伤病理生理中的作用，将这些相同的体内实验策略应用于遭受以下四种肾损伤之一的动物：(a)缺血，(b)尿路梗阻，(c)急性肾小球肾炎，(d)基于基因的胶原缺乏（Alport肾炎模型）。在这些实验中，疾病相关的肾脏DC转换、表面表型、抗原转运、引流淋巴结内抗原特异性t细胞活化以及活化t细胞浸润肾实质的改变将被表征。对于这两个特定目的，观察性研究将随后进行机制研究，包括阻断特定分子途径，消耗

DESCRIPTION (provided by applicant): Infiltration of the renal parenchyma by inflammatory cells, including lymphocytes, is a common feature of kidney diseases even in the apparent absence of exogenous immune stimuli. The mechanisms underlying T-lymphocyte activation in kidney disease are poorly understood, as are the mechanisms that prevent immune activation in the healthy kidney. This experimental protocol will examine the dynamic behavior, responses, and functions of Dendritic cells (DCs) -a migratory population of cells with specialized function in antigen uptake and presentation - within the kidney and its draining lymphoid tissue during health and explore their role in different forms of renal injury. All organs, including the kidney, have a resident population of DCs although the density, distribution and turnover vary. DC "maturation" is induced by the inflammatory products of disease or injury. Mature DCs migrate from an organ to its draining lymph node where they activate T-cells to initiate cellular immune responses. In the absence of maturing stimuli, DCs may also transfer antigens to lymphoid tissue in order to activate regulatory mechanisms that prevent autoimmunity. Modulation of DC-T-cell interactions is recognized as an important therapeutic target for the prevention/treatment of immune-mediated disease. The primary hypotheses of the proposal are that: (a) trafficking of protein antigens from the kidney to the draining Iymph nodes occurs during health and actively maintains immune tolerance to renal tissue, and (b) alterations in renal DC phenotype and turnover result from diverse forms of kidney injury and contribute significantly to inflammatory renal parenchvmal damage. The experimental strategy will focus strongly on the use of in vivo techniques that directly examine DC-mediated antigen trafficking and presentation in the kidney and renal lymph nodes of mice. The first Specific Aim, to determine the biology of renal DCs during health, will employ in vivo BrdU-labeling to determine DC turnover, congenic bone marrow transfer to examine the precursor origins of renal DCs, unilateral inoculation of fluorescent particles or proteins into the kidney to track renal DC-mediated antigen trafficking, generation of transgenic mice expressing a kidney-restricted neo-antigen, and adoptive transfer of antigen-specific TCR transgenic T-cells. The second Specific Aim, to determine the role of renal DCs in the pathophysiology of diverse form of renal injury, will apply these same in vivo experimental strategies to animals subjected to one of the following four types of renal injury: (a) ischemia, (b) urinary obstruction, (c) acute glomerulonephritis, (d) genetically-based collagen deficiency (a model of Alport's nephritis). In these experiments, the disease-associated alterations to renal DC turnover, surface phenotype, and antigen trafficking as well as antigen-specific T-cell activation within the draining lymph nodes, and infiltration of the renal parenchyma by activated T-cells will be characterized. For both Specific Aims, observational studies will be followed by mechanistic studies involving blockade of specific molecular pathways, depletion of regulatory cell populations, and cross-breeding to genetically modified strains. Analytic tools will include flow cytometry, immunohistochemistry, immunofluorescence microscopy, morphometric analysis, cytokine/chemokine ELISA.