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Inhibitory Effect of Tetramerized Single-Chain Variable Fragment of Anti-Cyclic Citrullinated Peptide Antibodies on the Proliferation, Activation, and Secretion of Cytokines of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis In Vitro Co-Culture System

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Abstract

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.

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Funding

This work was funded by grants from the Natural Science Foundation of China (81360460 and 81660276) and Inner Mongolia grassland talents fund.

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Author notes

  1. Hongbin Li and Xixiong Kang contributed equally to this work.

Authors and Affiliations

  1. Laboratory Diagnosis Center, Beijing Tiantan Hospital, Capital Medical University, Beijing, China

    Jing Wang & Xixiong Kang

  2. Department of Rheumatology and Immunology, The Affiliated Hospital of Inner Mongolia Medical University, Hohhot, China

    Jing Wang, Ning Tie & Hongbin Li

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  1. Jing Wang
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Contributions

WJ, XXK, and HBL had full access to all of the data in the study and take responsibility for the integrity of the data and accuracy of the data analysis. Study design: XXK and HBL. Acquisition of data and analysis and interpretation of data: WJ, TN, XXK, and HBL. Manuscript preparation: WJ and HBL. Statistical analysis: WJ. All original data used to support the findings of this study are included within the article. All authors read and approved the final version of the manuscript.

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Correspondence to Hongbin Li or Xixiong Kang.

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Wang, J., Tie, N., Li, H. et al. Inhibitory Effect of Tetramerized Single-Chain Variable Fragment of Anti-Cyclic Citrullinated Peptide Antibodies on the Proliferation, Activation, and Secretion of Cytokines of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis In Vitro Co-Culture System. Inflammation 43, 2245–2255 (2020). https://doi.org/10.1007/s10753-020-01292-z

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