TNFR2 Regulation of Leukocyte Recruitment in Glomerulonephritis

描述（由申请方提供）：白细胞蓄积是炎症性肾病的标志。TNF是一种调节白细胞运输的强效细胞因子，在动物模型中对肾小球肾炎的发展至关重要。TNF在体内的许多促炎反应可归因于其对血管内皮和白细胞内流的作用。TNF通过两种不同的受体TNFR 1和TNFR 2传递其生物活性，其中TNF对内皮的大部分作用归因于TNFR 1。TNFR 2的贡献可能被低估，因为它对膜结合的细胞因子的亲和力高于可溶性形式的细胞因子，并且它不是在所有内皮细胞培养物中组成型表达的。我们最近发表的数据表明，TNFR 2在培养的内皮细胞中的参与触发干扰素调节因子-1（IRF-1）诱导的IFN α合成，以及随后通过IFNα/IFN α受体的自分泌信号传导，以产生单核细胞化学引诱物。在体内，由静脉内施用的重组TNF诱导的急性肾脏炎症导致需要TNFR 1的肾脏中的中性粒细胞和单核细胞募集，而TNFR 2-IRF-1-IFN β自分泌环仅对于单核细胞/巨噬细胞积累是必需的。在抗肾小球基底膜肾炎的慢性模型中，肾TNFR 2和IRF-1而不是TNFR 1对于持续的巨噬细胞积聚是必需的。因此，我们的数据确定了一个以前未被识别的TNFR 2-IRF-1-IFN α-IFNα/IFN α受体信号通路，在内皮细胞中促进单核细胞募集。这些发现在人类中可能具有相关性，因为在正常人肾脏中不存在的TNFR 2在患有抗GBM肾炎的患者的肾小球内皮上被强烈诱导。我们的研究结果提出了两个重要的问题：TNFR 2如何诱导IRF-1，以及TNFR 2及其相关的IFN-γ自分泌回路调节肾脏中白细胞募集的步骤。这些问题将在三个具体目标中得到解决：I）描绘将TNFR 2与IRF-1和IFN β产生联系起来的TNFR 2近端细胞内信号; II）在完整肾脏中进行活体显微镜检查，以阐明TNF触发的肾脏白细胞募集途径;和III）阐明单核细胞募集动力学中肾脏TNFR 2和IFN β自分泌环组分的分子要求。对于目标II和III，我们已经开发了一种多光子活体显微镜方法，用于小鼠肾小球和管周毛细血管中白细胞行为的延时成像。确定TNF-诱导的IFN-γ表达的扩增和IFN-γ信号传导如何局部地在内皮中导致单核细胞在肾脏中的积累，可能导致对肾小球肾炎发病机制的重要新见解，并导致新的治疗靶点。

DESCRIPTION (provided by applicant): Leukocyte accumulation is a hallmark of inflammatory renal diseases. TNF, a potent cytokine that regulates leukocyte trafficking is essential for the development of glomerulonephritis in animal models. Many of the pro- inflammatory responses of TNF in vivo can be attributed to its effects on the vascular endothelium and leukocyte influx. TNF relays its biological activities by two distinct receptors, TNFR1 and TNFR2 with most of TNF's effects on the endothelium being attributed to TNFR1. The contribution of TNFR2 is likely underestimated as it has a higher affinity for the membrane bound versus the soluble form of the cytokine and it is not constitutively expressed in all endothelial cell cultures. Our recently published data demonstrate that engagement of TNFR2 in cultured endothelial cells triggers Interferon regulatory factor-1 (IRF-1) induced IFNß synthesis, and subsequent autocrine signaling via the IFNα/ß receptor, to generate mononuclear cell chemoattractants. In vivo, acute renal inflammation induced by intravenously administered recombinant TNF results in neutrophil and monocyte recruitment in the kidney that requires TNFR1, while the TNFR2-IRF-1- IFNß autocrine loop is essential only for monocyte/macrophage accumulation. In a chronic model of anti- glomerular basement membrane nephritis, renal TNFR2 and IRF-1 but not TNFR1 are essential for sustained macrophage accumulation. Thus, our data identify a previously unrecognized TNFR2-IRF-1-IFNß-IFNα/ß receptor-signaling pathway in endothelial cells that promotes monocyte recruitment. These findings may have relevance in humans as TNFR2, absent in normal human kidney, is robustly induced on the glomerular endothelium of patients with anti-GBM nephritis. Our results raise two important questions: How does TNFR2 induce IRF-1, and what steps of leukocyte recruitment in the kidney are regulated by TNFR2 and its associated IFNß autocrine loop? These questions will be addressed in three specific aims: I) Delineate TNFR2 proximal intracellular signals that link TNFR2 to IRF-1 and IFNß production; II) Conduct intravital microscopy in the intact kidney to elucidate pathways of renal leukocyte recruitment triggered by TNF; and III) Elucidate the molecular requirements for renal TNFR2 and components of the IFNß autocrine loop in the dynamics of monocyte recruitment. For Aims II and III, we have developed a multiphoton intravital microscopy approach for time-lapse imaging of leukocyte behavior in the glomerular and peritubular capillaries of mice. Determining how TNF-induced amplification of IFNß expression and IFNß-signaling locally in the endothelium leads to monocyte accumulation in the kidney could result in important new insights into the pathogenesis of glomerulonephritis and lead to new therapeutic targets.