Figure 3

Increased NETotic response of RA-derived PMNs to PMA. (A) Scanning electron micrographs of NETs induced by PMA (25 nM) indicate the excessive NETotic response of RA-derived PMN. Scale bar, 20 nm. (B) Assessment of NETs induced by PMA treatment by fluorescent immunohistochemistry for MPO (red) and DAPI (blue), indicating the increased response of RA PMNs to PMA (25 nM). Magnification, 20×; scale bar, 50 nm. (C) Analysis of the nuclear phenotype indicated a vast decrease in delobulated/diffused RA PMN nuclei after treatment with PMA and rapid increase in the NETotic-spread phenotype. (D) Release of cell-free nucleosomes after PMA treatment is abrogated by chloramidine, a PAD4 inhibitor. (E) Increased nuclear localization and concomitant decrease in cytoplasmic PAD4 protein levels after PMA treatment, with a clear tendency for an increased responsiveness to the PMA stimulus by RA PMN. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., statistically not significant. All data are representative of at least four independent experiments.