The synergistic effects were further demonstrated by combination of different concentrations of ACPAs with a fixed dose of IL-8 (figure 4D) or suboptimal concentration of ACPAs with increasing concentration of IL-8 (figure 4E). 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. strong class=”kwd-title” Keywords: Anti-CCP, Rheumatoid Arthritis, Fibroblasts, Autoantibodies, Autoimmune Diseases Key messages What is already known about this subject? Anticitrullinated protein/peptide antibodies (ACPAs) exist prior to the onset of rheumatoid arthritis (RA), however, it is unclear how autoimmunity in some but not all cases translate into manifest joint inflammation. What does this study add? Cellular stress and pro-inflammatory mediators (interleukin-8) can sensitise synovial fibroblasts to ACPAs by enhancing protein arginine deiminase enzyme expression and cellular citrullination. ACPAs promote synovial fibroblast migration through a phosphoinositide 3-kinase-mediated mechanism. Different monoclonal ACPAs have distinct cellular effects with three clones increasing migration of challenged fibroblasts, with no effect on osteoclasts and Cyclo (-RGDfK) another clone increasing osteoclast differentiation with no effect on Cyclo (-RGDfK) fibroblasts. How might this impact on clinical practice or future developments? Our results suggest that unique ACPAs may be responsible for specific pathological features in ACPA+RA. Inducible protein citrullination could be a key event in the transition of a systemic humoral autoimmunity towards the inflammation of the joints. Introduction Anti-citrullinated protein antibodies (ACPAs) are present in GluN1 a majority of patients with rheumatoid arthritis (RA) and are specific for this disease.1 They consist of a group of antibodies with different Cyclo (-RGDfK) specificities towards citrullinated antigens that might cross-react with other protein modifications but not with the native proteins2C4 and have been suggested to contribute to joint Cyclo (-RGDfK) pain and bone loss already before onset of joint inflammation in RA.5C8 In line with this, we and others have shown that polyclonal ACPAs bind to the surface of developing osteoclasts (OC) and suggested that reactivity to citrullinated targets increase OC differentiation and bone loss.9 10 Furthermore, experiments in mice have shown that polyclonal ACPAs (defined as anti-CCP-2 IgG antibodies) induces pain-related behaviours even though no joint inflammation develops,11 similar to the predisease stage of pain described in ACPA-positive individuals. We originally proposed that this, as well as ACPA-induced bone loss in mice, occurred through an interleukin (IL)-8-dependent and citrulline-specific mechanisms.10 11 However, recent papers and corrections12 13 this year have led to a reconsideration and extension of the concept. As such also other RA-derived monoclonal antibodies than those with citrulline reactivity and immune complexes are able to Cyclo (-RGDfK) cause functional effects similar to those of polyclonal ACPAs, through different mechanisms that are potentially distinct between autoantibody subsets and might include both antigen-driven and Fc receptor activation-driven pathways.14C16 Taken together, these data suggest a new concept where different RA-associated antibodies with different reactivities contribute to bone loss and pain, potentially through different mechanisms, a complex scenario that requires additional investigations. The need for these investigations and the ways of performing them has been highlighted in a recent editorial.17 Previous studies have shown that in the presence of pre-existing joint inflammation in mice, transfer of a monoclonal ACPA may enhance synovial tissue injury,18 suggesting that additional local stimuli might be essential for sensitisation of the synovial compartment to effects.