Laboratory Diagnosis Center, Beijing Tiantan Hospital, Capital Medical University, Beijing, China.
Department of Rheumatology and Immunology, The Affiliated Hospital of Inner Mongolia Medical University, Hohhot, China.
Inflammation. 2020 Dec;43(6):2245-2255. doi: 10.1007/s10753-020-01292-z.
Tetramerized single-chain variable fragment (ScFv) of anti-cyclic citrullinated peptide (TeAb-CCP) is a constructed tetramerized ScFv of anti-cyclic citrullinated peptide (CCP) antibodies with p53 tetrameric domain, aim to investigate its effect on fibroblast-like synoviocytes (FLSs) proliferation, migration, invasion, and production of inflammatory mediators in the in vitro co-culture system of peripheral mononuclear cells (PBMCs) and FLSs. TeAb-CCP was constructed by modifying a monovalent ScFv antibody to CCP with p53 tetrameric domain to improve its affinity. FLSs were isolated and cultured from rheumatoid arthritis (RA) patients and control subjects. A co-culture system of peripheral mononuclear cells (PBMCs) and FLSs was used. FLSs proliferation, migration, and invasion were measured by MTT, scratch test, and Transwell chamber. Supernatants were measured for cytokines, chemokines, metalloproteinases, and anti-CCP antibodies by Luminex liquid phase protein chip and ELISA. TeAb-CCP significantly inhibited FLSs proliferation in a dose-dependent mode, with maximal action at concentration of 100 μg/ml on the 7th day in the co-culture system with PBMCs and FLSs, but not the same with only FLSs. TeAb-CCP significantly suppressed FLSs migration and invasive ability compared with the controls. Significantly lower levels of interleukin (IL)-6, IL-8, RANKL, protein arginine deiminase (PAD)-2, PAD4, metalloproteinase (MMP)-1 and MMP-3 and anti-CCP antibodies were found in co-culture supernatant of TeAb-CCP group. In contrast, transforming growth factor-β (TGF-β) and tissue inhibitor of metalloproteinases-2 (TIMP-2) was significantly increased in the TeAb-CCP group. No significant difference of IL-1a, IL-10, IL-17, TNFα, VEGF, and FGF was found between two groups. As a blocking antibody, TeAb-CCP can significantly inhibit PBMCs of RA to produce pro-inflammatory mediators, and furthermore, inhibit the proliferation, activation, migration, and invasion of FLSs in vitro. In turn, it is suggested that citrullinated modified self-epitopes may be a new target for RA therapy.
抗环瓜氨酸肽（TeAb-CCP）的四聚化单链可变片段（ScFv）是一种构建的抗环瓜氨酸肽（CCP）抗体的四聚化 ScFv，具有 p53 四聚体结构域，旨在研究其在体外共培养体系中对成纤维样滑膜细胞（FLSs）增殖、迁移、侵袭和炎症介质产生的影响外周血单个核细胞（PBMCs）和 FLSs。TeAb-CCP 通过修饰具有 p53 四聚体结构域的单价 CCP ScFv 来提高其亲和力。从类风湿关节炎（RA）患者和对照者中分离和培养 FLSs。使用外周血单个核细胞（PBMCs）和 FLSs 的共培养系统。通过 MTT、划痕试验和 Transwell 室测量 FLSs 的增殖、迁移和侵袭。通过 Luminex 液相蛋白芯片和 ELISA 测量上清液中的细胞因子、趋化因子、金属蛋白酶和抗 CCP 抗体。TeAb-CCP 以剂量依赖性方式显著抑制 FLSs 的增殖，在与 PBMCs 和 FLSs 共培养的第 7 天，浓度为 100μg/ml 时达到最大作用，但在仅 FLSs 时则没有相同的作用。TeAb-CCP 显著抑制 FLSs 的迁移和侵袭能力，与对照组相比。TeAb-CCP 组共培养上清液中白细胞介素（IL）-6、IL-8、RANKL、蛋白精氨酸脱亚氨酶（PAD）-2、PAD4、金属蛋白酶（MMP）-1 和 MMP-3 及抗 CCP 抗体水平明显降低，而转化生长因子-β（TGF-β）和金属蛋白酶组织抑制剂-2（TIMP-2）则明显升高。TeAb-CCP 组两组间白细胞介素（IL）-1a、IL-10、IL-17、TNFα、VEGF 和 FGF 无明显差异。作为阻断抗体，TeAb-CCP 可显著抑制 RA 的 PBMCs 产生促炎介质，并进一步抑制 FLSs 的体外增殖、激活、迁移和侵袭。反过来，这表明瓜氨酸化修饰的自身表位可能是 RA 治疗的新靶点。
