Figure 4

Influence of RA serum and synovial fluid on normal PMNs. (A) Incubation of healthy donor PMNs with serum (Se) from healthy donors or from RA patients, synovial fluid (SF) from patients with noninflammatory osteoarthritis (OA) or synovial fluid from RA patients. Immunohistochemical analysis of four main components of NETs (NE, MPO, PAD4, and citH3) revealed that RA-derived serum and SF enhanced NETosis in normal PMN compared to healthy control serum or noninflammatory OA SF. PAD4 (white arrowheads) and citH3 (empty arrowheads) colocalize with unmodified histones on NETs. Magnification, 20×; scale bars, 50 nm. (B) Release of cell-free nucleosomes during in vitro culture by PMNs from healthy controls incubated with control serum, RA serum, OA SF or RA SF, or PMA. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01. (C) Increased ROS generation during in vitro culture by PMNs from healthy controls incubated with control serum, RA serum, OA SF or RA SF, or PMA. Data are presented as mean ± SEM. (D) Release of cell-free nucleosomes during in vitro culture by PMNs from healthy controls, ACPA-positive and ACPA-negative RA patients incubated with serum (Se +), IgG-depleted serum (Se -), or serum reconstituted with their respective eluted IgG, or with PMA. Data are presented as mean ± SEM. *P < 0.05; ***P < 0.001; ****P < 0.0001; n.s., statistically not significant. All data are representative of at least four independent experiments in Figures  4A–C, and 3 in 4D.