Anti-citrullinated protein antibodies are associated with neutrophil extracellular trap formation in rheumatoid arthritis

Objectives We evaluated the associations of anti-citrullinated protein antibodies (ACPAs) and serological and cytological levels of neutrophil extracellular trap (NET) formation in rheumatoid arthritis (RA) patients. Methods Serum levels of myeloperoxidase-DNA and elastase-DNA complexes (NET remnants) were examined in 51 patients with RA and 40 healthy controls using a modified enzyme-linked immunosorbent assay. Neutrophils were isolated by density gradient centrifugation. IgG antibodies were purified by affinity chromatography. NET formation in RA and control neutrophils was assessed by microscopy in vitro. NETs were purified and co-incubated with fibroblast-like synoviocyte (FLS) cells. Interleukin (IL)-6 and IL-8 mRNA expression and protein levels in FLS cells were determined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Results In RA patients, NET remnants in the peripheral circulation were higher in extremely high ACPA titers when compared to in moderate ACPA titers. And IgG antibodies containing ACPA can stimulate neutrophils to form NETs in a concentration-dependent manner. Furthermore, significantly higher expression of the pro-inflammatory cytokines IL-6 and IL-8 is detected after FLS cells interacted with NETs which derived from neutrophils stimulated with ACPA-containing IgG antibodies. Conclusions Anti-citrullinated protein antibodies may enhance NET formation and contribute to inflammation development in RA by stimulating NET formation, such as by subsequent activation of FLS cells by NETs.