217. PROTEINASE 3 PROMOTES MULTINUCLEATE GIANT CELL FORMATION IN GRANULOMATOSIS WITH POLYANGIITIS AND CAN BE MODELLED IN VITRO AND IN VIVO
217. PROTEINASE 3 PROMOTES MULTINUCLEATE GIANT CELL FORMATION IN GRANULOMATOSIS WITH POLYANGIITIS AND CAN BE MODELLED IN VITRO AND IN VIVO
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217. 蛋白酶 3 促进肉芽肿性多血管炎中的多核巨细胞形成，并且可以在体外和体内建模
DOI：
10.1093/rheumatology/kez061.032
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发表时间：
2019
期刊：
影响因子：
5.5
通讯作者：
Henderson S
中科院分区：
文献类型：
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作者：
Henderson S
背景资料：肉芽肿伴多血管炎（GPA）患者的特征为ANCA对蛋白酶3（PR 3）的反应性，在肉芽肿病变中有PR 3沉积，并显示中性粒细胞膜PR 3表达增加，这抑制了巨噬细胞的凋亡细胞吞噬作用。肉芽肿的中心是多核巨细胞（MNGC）。我们测试是否持续和过度的酶活性或失活的PR 3表达GPA，可能是巨细胞和肉芽肿formation.Methods：在体外模型，通过分离PBMC和单核细胞从健康对照组（n= 15），GPA（n= 15）和显微镜下多血管炎（MPA）（n= 10）患者。用酶活性（aPR 3）或失活PR 3（iPR 3）或对照自身抗原髓过氧化物酶（MPO）刺激细胞。使用光、落射荧光和共聚焦显微镜，使用双细胞和膜标记在不同时间点确认细胞融合。还使用了扫描电子显微镜。在斑马鱼中生成体内模型。在受精后24小时，向转基因（Mpeg cherry：GFP）斑马鱼注射酶促活性或失活的PR 3或白蛋白。6天后通过光片显微镜对鱼成像。结果：与MPA患者和健康对照组相比，GPA患者在aPR 3和iPR 3刺激后发生单核细胞聚集和巨细胞形成，融合指数（p< 0.001）。在GPA患者中使用具有更大融合指数（p< 0.001）的PBMC制备物观察到典型肉芽肿。aPR 3和iPR 3之间没有显著差异。未观察到MPO的影响。上清液分析涉及促炎细胞因子，特别是IL-6的作用。在斑马鱼（n= 9/组）中，当与注射白蛋白的对照相比时，aPR 3和iPR 3两者都与聚集体体积的显著增加相关（p< 0.001）（图1）。重要的是，IL-6抑制与聚集体体积的显著减少相关结论：这些模型支持抗原性PR 3在体外和体内促进单核细胞和巨噬细胞融合以及肉芽肿形成中的作用，并提供了在潜在的高通量模型系统中测试特异性治疗剂的机会。英国国家健康研究所血管炎英国摘要217图1摘要217图1
Background: Patients with Granulomatosis with polyangiitis (GPA) are characterized by ANCA reactivity towards proteinase 3 (PR3), have PR3 deposition in granulomatous lesions and show increased neutrophil membrane PR3 expression, which inhibits apoptotic cell phagocytosis by macrophages. At the heart of the granuloma are multinucleate giant cells (MNGC). We tested whether persistent and excessive enzymatically active or inactive PR3 expression in GPA, may underlie giant cell and granuloma formation.Methods: In vitro model was created by isolating PBMCs and monocytes from healthy controls (n= 15), GPA (n= 15) and microscopic polyangiitis (MPA)(n= 10) patients. Cells were stimulated with either enzymatically active (aPR3) or inactive PR3 (iPR3) or control auto-antigen myeloperoxidase (MPO). Light, epifluorescence and confocal microscopy was used to confirm cell fusion at different time points using dual cell and membrane labelling. Scanning electron microscopy was also used. In vivo model was generated in Danio rerio. Transgenic (Mpeg cherry: GFP) zebrafish at 24 hours post-fertilization were injected with enzymatically active or inactive PR3 or albumin. Fish were imaged by lightsheet microscopy 6 days later. Co-localisation of T cells was also tested by immunostaining.Results: Monocyte aggregation and giant cell formation occurred following stimulation with both aPR3 and iPR3 with a fusion index (p< 0.001) in GPA patients compared to MPA patients and healthy controls. Typical granuloma were observed using PBMC preparations with a greater fusion index (p< 0.001) in GPA patients. There was no significant difference between aPR3 and iPR3. No effect was seen with MPO. Supernatant profiling implicated the role of pro-inflammatory cytokines, particularly IL-6. In zebrafish (n= 9/group), both aPR3 and iPR3 were associated with a significant increase in aggregate volume (p< 0.001) when compared to albumin injected controls (figure 1). Importantly, IL-6 inhibition was associated with a significant reduction in aggregate volume (p< 0.01).Conclusion: These models support a role for antigenic PR3 in promoting monocyte and macrophage fusion and granuloma formation in vitro and in vivo, and provide an opportunity to test specific therapeutics in potentially high throughout model systems.Disclosures: National Institute of Health Research, UKVasculitis UK.Abstract 217 Figure 1Abstract 217 Figure 1