Background/Purpose: Activated neutrophils (PMN) form neutrophil extracellular traps (NET). Those structures are expelled chromatin fibers composed of DNA and associated proteins. The process, NETosis, is dependent on citrullination, and has been suggested to be pathogenic in rheumatoid arthritis (RA). RA is characterized by the production of anti-citrullinated protein autoantibodies (ACPA) which are specific for the disease. Although RA PMN show enhanced NETosis and RA autoantibodies recognize NET, the potential pathogenic mechanisms triggered by NET are not elucidated. Therefore, we have analyzed the antigenic and inflammatory properties of NET in RA.

Methods: PMN and monocytes were purified from the blood. We have used primary cells freshly isolated from 115 independent 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 by PMA on adherent PMN and was analyzed by fluorescence microscopy, together with IgG binding. To produce soluble NET, NET were detached from glass by mild nuclease digestion. They were enriched, quantified by fluorescence and spectrophotometry, and characterized by SDS-PAGE and agarose gel. Macrophages/PMN were cultured with soluble NET in the presence/absence of LPS, IgG, NH₄Cl or C1q. C1q receptor expression and cell activation were estimated by flow cytometry and 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 recognition by ACPA+ IgG was observed, specifically on the NET structures, as demonstrated by fluorescence microscopy. The NET staining with ACPA from RA patients was stronger than with ACPA-negative IgG in 15 out of 19 experiments (79 %; p < 0.05) and normal NET were as antigenic as RA NET. Soluble NET activated both steady-state macrophages and PMN, leading to IL-8 secretion and CD11b up-regulation, independently of ACPA+ IgG. The stimulatory activity of soluble NET on macrophages was increased in the presence of C1q, whereas PMN activation was not influenced by inhibition of endosomal acidification. Soluble NET from both healthy donors and RA patients induced cell activation and have similar immuno-modulatory properties. 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.

Conclusion: ACPA+ IgG from RA patients strongly and specifically recognize NET and not untreated PMN. NET are antigenic and possess both pro- and anti-inflammatory properties depending on the cell type, the activation level and the presence of co-factors like C1q or high expression of its receptors but independently of endolysosomal activity or ACPA. Therefore, an excess of NETosis rather than altered NETosis may be pathogenic in RA and trigger ACPA. This is probably the largest analysis comparing RA/normal NET on RA/normal cells reported so far.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/neutrophil-extracellular-traps-are-not-only-targets-for-acpa-positive-igg-from-rheumatoid-arthritis-patients-but-also-directly-trigger-pro-and-anti-inflammatory-effects-partly-mediated-by-the-c/