Figure 2

Increased expression of NET-associated signaling elements and nuclear localization of PAD4, citrullination of histone H3 in RA-derived PMN, and possible extrusion of PAD4 on NETs. Baseline ROS levels, measured with flow cytometry, were higher in RA-derived PMNs than in control PMNs (A). Quantitative real-time PCR analysis of NE mRNA (B) and MPO mRNA expression (C), as well as Western blot analysis of MPO protein levels (D), indicated that the levels of these two components required for NETosis were elevated in RA-derived PMNs. Total PAD4 protein (E) or PAD4 mRNA expression levels (F) did not indicate any significant difference between control and RA-derived PMNs. (G) Quantification of PAD4 protein levels in cytoplasmic and nuclear fractions of PMNs from healthy controls and RA patients. Nuclear levels of PAD4 were significantly increased in RA patients, whereas the cytoplasmic levels were lower compared with the healthy control PMNs. (H) Elevated citrullinated histone H3 levels in RA PMN extracts detected with Western blot. (I) Co-localization of citrullinated histone H3 (green) with histone components detected with a pan-histone antibody (red), spread over the entire NET surface (blue). Magnification: upper panel 20×, scale bar, 50 nm; lower panel 63× scale bar, 10 nm. (J) Extracellular localization of PAD4 (red) on extruded NETs by multicolor fluorescent immunohistochemistry. NET DNA is stained blue (DAPI), and histones (panH) are stained green. Magnification, 20×; scale bar, 50 nm (K). Detection of PAD4/cell-free DNA complexes in the culture supernatants of isolated PMN undergoing spontaneous NETosis. Higher levels of these complexes were detected in RA-derived PMN cultures. Time points correspond to those of Figure  1A. Data are represented as mean ± SEM. **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., statistically not significant. All data are representative of at least six independent experiments.