Figure 1.

Independent pathways induce chromatin decondensation in neutrophils. (A) Isolated neutrophils were stimulated in HBSS media with phorbol myristyl acetate (PMA, 50 nM) or ionomycin (5 μM). Imaging was performed 3 h after stimulation. The reproducibly observed response of peripheral blood neutrophils derived from healthy donors and a patient suffering from chronic granulomatous disease (CGD) is depicted. DNA of neutrophils is stained by SYTOX Green (in green), once the plasma membrane is disrupted and/or DNA has been expulsed. Decondensation of chromatin results in increased area and decreased intensity of SYTOX staining (scale bar = 20 μm). (B) SYTOX Green fluorescence intensity of microscopic images from equal numbers of cells as presented in (A) was quantified and represented as relative signal intensity (^***p < 0.001 Student's t-test of technical replicates, n > 4 healthy donors, n = 2 independent experiments with CGD neutrophils). (C,D) Stimulation of healthy donor-derived human neutrophils in the presence or absence of Diphenyliodonium (DPI) and the previously mentioned stimuli and (C) whole-well DNA^SYTOX quantification as well as (D) whole well detection of reactive oxygen species (H2-DCFDA) was performed after 180 min in a Tecan M200 microplate reader. Results representative of 4 independent healthy donors. (E) Neutrophil granulocytes were stimulated with the indicated stimuli for 120 min before subsequent protein isolations. Citrullinated Histone H3, total histone H3 and citrulline-containing proteins were assessed subsequently. (F) Recombinant Histone H3 and Histone H1 as well as nuclear protein fractions of A549 cells were treated with recombinant PAD4. Protein citrullination was assessed by antibody based detection of modified citrulline residues after reaction with diacetyl monoxime/antipyrine. Unless otherwise indicated, results are representative of at least 3 independent experiments using independent healthy donors.