Endothelia mechanisms of leukocyte accumulation in glomerulonephritis

描述(申请人提供)：白细胞聚集是炎症性肾脏疾病的标志。肿瘤坏死因子是一种强大的调节白细胞转运的细胞因子，在动物模型肾小球肾炎(GN)的发生发展中起着至关重要的作用。肿瘤坏死因子的生物学活性是由两个功能不同的TNFR--TNFR1和TNFR2介导的。我们已经证明了TNFR2对肾小球肾炎至关重要。TNFR2基因缺陷小鼠完全免受抗体介导的肾毒性肾炎的影响，这种肾炎与肾白细胞聚集和肾小球补体沉积减少有关。此外，肾小球肾炎患者外周血白细胞上的TNFR2不是必需的，而是固有实质细胞上的TNFR2。值得注意的是，TNFR2在肾炎肾脏的肾小球内皮细胞上被诱导。相反，TNFR1缺陷导致免疫反应增强，肾脏T细胞聚集导致肾小球肾炎。因此，TNFR在肾小球肾炎中扮演着不同的角色。这项建议的主要目的是确定导致TNFR2依赖的白细胞聚集和肾小球损伤的内在细胞类型和机制。目的：我将阐述一种假设，即TNFR2与内皮细胞的结合诱导了白细胞募集和随后的肾小球损伤所需的转录程序。白细胞迁移所需的内皮细胞黏附受体依赖的信号机制仍然是个谜。我们的初步数据表明，肿瘤坏死因子诱导的白细胞迁移依赖于DOCK4，DOCK4是最近发现的小GTP酶鸟嘌呤交换因子COM家族的成员，我们发现DOCK4在培养的内皮细胞和肾小球内皮细胞上高表达。目的II将验证内皮细胞黏附受体信号转导DOCK4调节白细胞迁移和肾小球损伤的假说。这些目标将开发：a)已建立的抗体诱导的肾小球肾炎小鼠模型，以及抗体和肿瘤坏死因子诱导的白细胞募集模型，这些模型适用于活体显微镜检查；b)肾病肾脏的转录图谱和生理流动条件下的体外白细胞迁移分析；c)仅在内皮细胞可诱导表达TNFR2的转基因小鼠，以及在内皮细胞系中特异性缺失DOCK4的小鼠。这些目标的完成将增加我们对炎症介导的肾小球损伤的内皮依赖机制的理解，这可能有助于开发治疗GN的新的治疗策略。

DESCRIPTION (provided by applicant): Leukocyte accumulation is the hallmark of inflammatory renal diseases. TNF, a potent cytokine that regulates leukocyte trafficking, is essential for the development of glomerulonephritis (GN) in animal models. The biological activities of TNF are mediated by two functionally distinct TNFRs, TNFR1 and TNFR2. We have shown that TNFR2 is critical for GN. TNFR2 deficient mice were completely protected from antibody- mediated nephrotoxic nephritis that was associated with decreased renal leukocyte accumulation and glomerular complement deposition. Furthermore, TNFR2 on intrinsic parenchymal cells but not circulating leukocytes was essential for GN. Notably, TNFR2 was induced on glomerular endothelium of nephritic kidneys. In contrast, a deficiency in TNFR1 resulted in an enhanced immune response and renal T cell accumulation resulting in GN. Thus TNFRs play differential roles in GN. The major objective of this proposal is to identify the intrinsic cell type and mechanisms responsible for TNFR2 dependent leukocyte accumulation and glomerular damage. Aim I will address the hypothesis that TNFR2 engagement on endothelial cells induces a transcriptional program required for leukocyte recruitment and subsequent glomerular injury. The endothelial adhesion receptor dependent signaling mechanisms required for leukocyte transmigration remain enigmatic. Our preliminary data demonstrates that TNF-induced leukocyte transmigration is dependent on DOCK4, a recently identified member of the COM family of guanine exchange factors for small GTPases that we show is highly expressed on cultured endothelial cells and glomerular endothelium. Aim II will test the hypothesis that endothelial adhesion receptor signaling to DOCK4 modulates leukocyte transmigration and glomerular damage. These aims will exploit a) established mouse models of antibody induced GN, and antibody and TNF-induced leukocyte recruitment that are amenable to intravital microscopy, b) transcriptional profiling of nephritic kidneys and in vitro leukocyte transmigration assays under physiological flow conditions, c) transgenic mice with inducible expression of TNFR2 only in the endothelium, and mice lacking DOCK4 specifically in the endothelial lineage. Completion of the aims will increase our understanding of endothelial dependent mechanisms of inflammation mediated glomerular injury that could aid in the development of new therapeutic strategies for the treatment of GN.