Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ, United Kingdom; and
Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ, United Kingdom; and.
J Immunol. 2018 Sep 1;201(5):1373-1381. doi: 10.4049/jimmunol.1800346. Epub 2018 Jul 25.
Rheumatoid arthritis (RA) is characterized by formation of synovial ectopic lymphoid structures (ELS) supporting B cell autoreactivity toward locally generated citrullinated (cit) antigens, including those contained in neutrophil extracellular traps (NETs). However, only a minority of RA-rmAbs from B cells isolated from ELS RA tissues react against NETs. Thus, alternative cellular sources of other potential autoantigens targeted by locally differentiated B cells remain undefined. RA fibroblast-like synoviocytes (FLS) have been implicated in the release of RA-associated autoantigens. In this study, we aimed to define stromal-derived autoantigens from RA-FLS targeted by RA-rmAbs. Seventy-one RA-rmAbs were screened toward RA-FLS by living-cell immunofluorescence (IF). Western blotting was used to identify potential autoantigens from RA-FLS protein extracts. Putative candidates were validated using colocalization immunofluorescence confocal microscopy, ELISA, immunoprecipitation assay, and surface plasmon resonance on unmodified/cit proteins. Serum immunoreactivity was tested in anti-citrullinated peptide/protein Abs (ACPA) versus ACPA RA patients. Ten out of 71 RA-rmAbs showed clear reactivity toward RA-FLS in immunofluorescence with no binding to NETs. One stromal-reactive RA-rmAb (RA057/11.89.1) decorated a ∼58-kDa band that mass spectrometry and Western blotting with a commercial Ab identified as calreticulin (CRT). Confocal microscopy demonstrated significant cellular colocalization between anti-CRT RA057/11.89.1 in RA-FLS. RA057/11.89.1 was able to immunoprecipitate rCRT. Deimination of CRT to cit-CRT moderately increased RA057/11.89.1 immunoreactivity. cit-CRT displayed increased blocking capacity compared with unmodified CRT in competitive binding assays. Finally, anti-cit-CRT Abs were preferentially detected in ACPA versus ACPA RA sera. We identified a synovial B cell-derived RA-rmAb locally differentiated within the ELS RA synovium reacting toward CRT, a putative novel autoantigen recently described in RA patients, suggesting that FLS-derived CRT may contribute to fuel the local autoimmune response.
类风湿关节炎（RA）的特征是形成滑膜异位淋巴样结构（ELS），支持针对局部产生的瓜氨酸化（cit）抗原的 B 细胞自身反应性，包括包含在中性粒细胞细胞外陷阱（NETs）中的抗原。然而，从 ELS RA 组织中分离的 RA-rmAbs 中只有少数针对 NETs 反应。因此，针对局部分化的 B 细胞的其他潜在自身抗原的替代细胞来源仍未确定。RA 成纤维样滑膜细胞（FLS）已被牵连到 RA 相关自身抗原的释放中。在这项研究中，我们旨在确定由 RA-rmAbs 靶向的 RA-FLS 衍生的基质衍生自身抗原。通过活细胞免疫荧光（IF）筛选 71 种 RA-rmAbs 针对 RA-FLS。Western blot 用于鉴定 RA-FLS 蛋白提取物中的潜在自身抗原。使用共聚焦免疫荧光显微镜、ELISA、免疫沉淀测定和未修饰/cit 蛋白的表面等离子体共振对推定候选物进行验证。在抗瓜氨酸肽/蛋白 Abs（ACPA）与 ACPA RA 患者中测试血清免疫反应性。在免疫荧光中，71 种 RA-rmAbs 中有 10 种对 RA-FLS 显示出明显的反应性，而与 NETs 无结合。一种基质反应性 RA-rmAb（RA057/11.89.1）修饰了一个约 58 kDa 的带，通过质谱和与商业 Ab 的 Western blot 鉴定为钙网蛋白（CRT）。共聚焦显微镜显示 RA-FLS 中抗 CRT RA057/11.89.1 之间存在明显的细胞共定位。RA057/11.89.1 能够免疫沉淀 rCRT。CRT 的脱氨化为 cit-CRT 适度增加了 RA057/11.89.1 的免疫反应性。在竞争性结合测定中，与未修饰的 CRT 相比，cit-CRT 显示出增加的阻断能力。最后，在 ACPA 与 ACPA RA 血清中优先检测到抗 cit-CRT Abs。我们鉴定了一种滑膜 B 细胞衍生的 RA-rmAb，该 Ab 在 ELS RA 滑膜内局部分化，针对 CRT 反应，CRT 是最近在 RA 患者中描述的一种新的自身抗原，这表明 FLS 衍生的 CRT 可能有助于引发局部自身免疫反应。
