Figure 1

RA-derived PMNs exhibited increased spontaneous NETosis and elevated levels of NET-component release. (A) Schematic representation of the time-course design for studying in vitro spontaneous NET release. (B) Detection of in vitro NETosis by immunohistochemistry for neutrophil elastase (NE) (green) and DAPI (blue). Magnification: 20×; scale bar, 50 nm. (C) Concentration of cell-free nucleosomes in PMN culture supernatant by ELISA. (D) Quantification of NET-associated MPO/DNA complexes. These assays indicate that more-rapid and extensive progression of NET formation is observed in RA versus control PMNs. (E) Changes in PMN nuclear morphology during NETosis detected with immunohistochemistry for NE and DAPI. (F) Steady-state (T1) RA-derived PMNs exhibited a greater proportion of delobulated/diffused cells, and progressed rapidly to a NETotic spread phenotype during in vitro culture. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. All data are representative of at least six independent experiments.