Background Synovial fibroblasts (SFs) contribute to rheumatoid arthritis (RA) pathogenesis by growing into the synovial space and by producing pro-angiogenic and tissue remodelling factors, chemokines and inflammatory cytokines that recruit and stimulate various immune cells. We have recently demonstrated that anti-citrullinated proteins antibodies (ACPAs) promote osteoclast development. In the present work we investigated whether ACPAs can also modulate SFs.
Methods SFs were isolated from synovial tissue of RA patients by enzymatic digestion. Polyclonal ACPA and others non-ACPA IgGs were separated from plasma of RA patients by affinity purification on cyclic citrullinated peptide (CCP)-2 column. Monoclonal ACPAs were generated from synovial B-cells. Antibodies were tested in scratch-assays for SF migration and the results were evaluated by NIH ImageJ software. SF adhesion was analyzed by xCELLigence System Real-Time Cell Analyzer (ACEA bioscience). ACPA-induced signalling pathways were examined using inhibitors of phosphoinositide 3-kinase (PI3K), phosphatase and tensin homolog (PTEN), G-protein coupled receptors, focal adhesion kinase (FAK) and peptidylarginine deiminases (PAD) in the migration assays. Protein phosphorylations were monitored by western blot.
Results Polyclonal ACPAs but not others IgGs induced both SFs migration (a fold increase of 2.6±0.5, mean±SD, p<0,05) and SFs adhesion (a fold increase of 1.3±0.1 at 6 hours, p<0.05). Similar effects were observed with two (D10 and B02) out of three tested monoclonal ACPAs, with a fold increase of the migration index of 2.4±0.4 for D10 antibody and 2.2±0.4 for B02 antibody (p<0.05 for both). IL-8, a chemokine that is upregulated in developing ostecolasts by ACPAs, synergistically increased the ACPA-induced SF migration. Inactivation of the PAD enzymes completely abolished the ACPA-induced SFs migration. GPCR and PI3K blocking completely abolished ACPAs effects, indicating an important role for GPCR and PI3K but not for FAK in the ACPA effects.
Conclusions ACPAs promote SFs migration and adhesion acting synergistically with IL-8 through a PAD-dependent pathway. Our findings suggest that SFs may have a potential role in the ACPA-dependent disease propagation from the bone marrow to synovial tissue.
Disclosure of Interest None declared
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