Objectives Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) might contribute to bone loss and arthralgia before the onset of joint inflammation. We aimed to dissect additional mechanisms by which ACPAs might contribute to development of joint pathology.
Methods Fibroblast-like synoviocytes (FLS) were isolated from the synovial membrane of patients with RA. The FLS cultures were stimulated with polyclonal ACPAs (anti-CCP-2 antibodies) purified from the peripheral blood of patients with RA or with monoclonal ACPAs derived from single synovial fluid B cells. We analysed how ACPAs modulate FLS by measuring cell adhesion and mobility as well as cytokine production. Expression of protein arginine deiminase (PAD) enzymes and protein citrullination were analysed by immunofluorescence, and signal transduction was studied using immunoblotting.
Results Challenge of FLS by starvation-induced stress or by exposure to the chemokine interleukin-8 was essential to sensitise the cells to ACPAs. These challenges led to an increased PAD expression and protein citrullination and an ACPA-mediated induction of FLS migration through a mechanism involving phosphoinositide 3-kinase activation. Inhibition of the PAD enzymes or competition with soluble citrullinated proteins or peptides completely abolished the ACPA-induced FLS migration. Different monoclonal ACPAs triggered distinct cellular effects in either fibroblasts or osteoclasts, suggesting unique roles for individual ACPA clones in disease pathogenesis.
Conclusion We propose that transient synovial insults in the presence of a certain pre-existing ACPA repertoire might result in an ACPA-mediated increase of FLS migration.
- Rheumatoid Arthritis
- Autoimmune Diseases
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Handling editor Josef S Smolen
Contributors MS, BR and AIC designed the experiments, analysed the data and wrote the manuscript along with input from VM, HW, CS, AHH, SBC and LK. MS designed and performed all fibroblast experiments with help from BR. MS and CO designed and performed adhesion assay. VJ, YL and HW measured chemokines and cytokines in supernatants. MS and ME performed all immunohistochemistry experiments with help from AC. EO perform and analysed mass spectrometry data. AK conducted osteoclast assays. AHH recruited patients and characterised all clinical data. HW purified polyclonal ACPAs. JS, CG and VM produced and validated monoclonal ACPAs. AIC, MS, BR, AK, VJ, HW, AC, VM and LK discussed and developed the concept. All authors critically reviewed and approved the final form of the manuscript.
Funding This project has received funding from FOREUM, Foundation for Research in Rheumatology, from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement CoG 2017—7722209_PREVENT RA and grant agreement 777357_RTCure), from the Swedish Research Council and Konung Gustaf V:s och Drottning Victorias Frimurarestiftelse.
Competing interests None declared.
Patient consent for publication Not required.
Ethics approval This study involves human participants with ethical permit listed in blow: (1) Kartläggning av prediktiva biomarkörer vid kronisk artrit ID: 2009-358-31-3; (2) Kartläggning av inflammatoriska mediatorers betydelse för sjukdomsförlopp vid kroniskaledsjukdomar ID:2009-1262-31-3.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement Data are available on reasonable request.
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