Background and objectives Activated neutrophils (PMN) form neutrophil extracellular traps (NET), expelled chromatin fibres. NETosis is dependent on citrullination and may be pathogenic in rheumatoid arthritis (RA), which is characterised by the production of anti-citrullinated protein autoantibodies (ACPA). However, the potential pathogenic mechanisms triggered by NET are not elucidated. Therefore, we have analysed the antigenic and inflammatory properties of NET in RA.
Materials and methods Primary blood PMN and monocytes were freshly purified from 115 independent healthy donors (HD) and RA patients. IgG were purified from HD, ACPA-positive RA patients or ACPA-negative patients. Monocytes were differentiated into macrophages. NETosis was induced in vitro by PMA on adherent PMN and analysed by fluorescence microscopy, together with IgG binding. To produce soluble NET (sNET), NET were detached from glass by mild nuclease digestion. They were enriched, quantified by spectrophotometry, and characterised by SDS-PAGE and agarose gel. Macrophages/PMN were cultured with sNET in the presence/absence of LPS, IgG, NH4Cl, LL-37, ATP or C1q. Cell purity, IgG binding and cell activation were estimated by flow cytometry. Cytokine secretion was measured by ELISA.
Results Low binding of IgG was observed on untreated PMN. Upon activation, a strong recognition by ACPA+ IgG was observed on NET. The NET staining with ACPA from RA patients was stronger than with ACPA-negative IgG. HD NET were as antigenic as RA NET. NETosis was higher with RA PMN. Soluble NET activated steady-state macrophages and PMN, independently of ACPA+ IgG or soluble LL-37. The stimulatory activity of sNET on macrophages was increased in the presence of C1q but was not influenced by inhibition of endosomal acidification/TLR in PMN. Both HD NET and RA NET induced cell activation, although RA NET were more potent. Likewise, both HD and RA PMN and macrophages responded to sNET but IL-1β secretion was not induced. On the contrary, sNET specifically inhibited LPS-induced IL-6 secretion by macrophages.
Conclusions ACPA+ IgG from RA patients recognise NET and not untreated PMN. NET are antigenic and possess both pro- and anti-inflammatory properties. Therefore, an excess of NETosis together with higher NET activity may be pathogenic in RA.