Background and objectives Upon activation, neutrophils (PMN) form neutrophil extracellular traps (NET). Those structures are expelled chromatin fibres composed of DNA and associated proteins. The process, NETosis, is dependent on citrullination, a post-translational modification of proteins. NETosis has been suggested to be pathogenic in certain autoimmune diseases, particularly in rheumatoid arthritis (RA), characterised by the production of anti-citrullinated protein autoantibodies (ACPA) which are specific for the disease. Although RA PMN show enhanced NETosis and RA autoantibodies recognise NET, the pathogenic mechanisms triggered by NET are not elucidated. Therefore, we have analysed the antigenic and immunogenic properties of NET in RA.
Materials and methods PMN and monocytes were freshly isolated by dextran sedimentation or purified by magnetic bead sorting, respectively, from the blood of healthy donors and RA patients. IgG were purified from healthy donors, from ACPA-positive RA patients and from ACPA-negative patients with rheumatic diseases. Monocytes were differentiated into macrophages by culturing in the presence of M-CSF. Cell purity and IgG binding were estimated by flow cytometry. NETosis was induced in vitro on poly-L-lysine-adherent PMN and was analysed by fluorescence microscopy, together with IgG binding. NET were detached from plastic by mild nuclease digestion to produce soluble NET which were enriched, quantified by fluorescence (PicoGreen) and by spectrophotometry (NanoDrop) and characterised by SDS-PAGE and agarose gel. Macrophages and PMN were cultured with soluble NET in the presence/absence of LPS or C1q. Cell activation was estimated by measuring cytokine secretion by ELISA.
Results Only low binding of IgG was observed by flow cytometry on untreated freshly isolated PMN. Upon NETosis, a strong binding of ACPA+ IgG was observed, specifically on the NET structures, as demonstrated by the co-staining of DNA by fluorescence microscopy. Soluble NET activated both macrophages and PMN, leading to IL-8 secretion, independently of ACPA+ IgG. The stimulatory activity of soluble NET was increased in the presence of C1q. Soluble NET from both healthy donors and RA patients induced cell activation. Likewise, both PMN and macrophages from healthy donors and RA patients responded to soluble NET. On the contrary, soluble NET specifically inhibited LPS-induced IL-6 secretion by macrophages, and to a lower extent TNF secretion, but not IL-8 secretion.
Conclusions ACPA+ IgG from RA patients strongly and specifically recognise NET and not untreated PMN. NET are immunogenic and possess both pro- and anti-inflammatory properties depending on the cell type, the activation status and the presence of co-factors like C1q.