TNFR2 Regulation of Leukocyte Recruitment 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有效的调节白细胞转运的细胞因子，在动物模型肾小球肾炎的发生发展中起着至关重要的作用。肿瘤坏死因子在体内的许多促炎反应可归因于其对血管内皮细胞和白细胞内流的影响。肿瘤坏死因子通过两种不同的受体TNFR1和TNFR2传递其生物学活性，其对血管内皮细胞的作用主要归因于TNFR1。TNFR2的作用可能被低估了，因为它对膜结合的细胞因子比可溶性形式的细胞因子有更高的亲和力，而且它并不是在所有的内皮细胞培养中都有结构性表达。我们最近发表的数据表明，在培养的内皮细胞中，肿瘤坏死因子受体2的参与触发了干扰素调节因子-1(IRF-1)诱导干扰素的合成，并随后通过干扰素α/β受体的自分泌信号产生单核细胞趋化物质。在体内，静脉注射重组肿瘤坏死因子引起的急性肾炎导致中性粒细胞和单核细胞在肾脏聚集，这需要TNFR1，而TNFR2-IRF-1-干扰素自分泌环只对单核/巨噬细胞聚集是必不可少的。在慢性抗肾小球基底膜肾炎模型中，肾脏的TNFR2和IRF-1而不是TNFR1对巨噬细胞的持续聚集是必不可少的。因此，我们的数据确定了在内皮细胞中存在一条先前未被识别的促进单核细胞募集的受体信号通路，该信号通路促进单核细胞募集。这些发现可能与人类相关，因为在正常人肾脏中缺失的TNFR2在抗GBM肾炎患者的肾小球内皮细胞上被强烈诱导。我们的结果提出了两个重要的问题：TNFR2如何诱导IRF-1，以及TNFR2及其相关的干扰素自分泌环路调控肾内白细胞募集的哪些步骤？这些问题将通过三个具体的目标来解决：i)描绘将TNFR2与IRF-1和干扰素的产生联系起来的TNFR2近端细胞内信号；ii)在完整的肾脏中进行活体显微镜观察，以阐明由肿瘤坏死因子引发的肾脏白细胞募集的途径；以及iii)阐明单核细胞募集动力学中对肾脏TNFR2的分子要求和自分泌干扰素环路的组件。对于AIMS II和AIMS III，我们开发了一种多光子活体显微镜方法，用于对小鼠肾小球和肾小管周围毛细血管中的白细胞行为进行时间推移成像。确定肿瘤坏死因子诱导的血管内皮细胞局部干扰素的表达和信号的放大如何导致单核细胞在肾脏中积聚，可能为肾小球肾炎的发病机制带来重要的新见解，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.