Fig. 1

Cyclic AMP (cAMP) suppresses NETosis in response to APS IgG. Neutrophils were isolated from healthy volunteers and then treated with either control IgG or APS IgG (pooled from four patients with primary APS) for 3 h. Some samples were additionally treated with 8-Br-cAMP at various concentrations. In panel a, total extracellular DNA was measured as relative fluorescence units upon the addition of Sytox Green. In panel b, an independent set of experiments was performed, with NETosis quantified by measuring the enzymatic activity of nuclease-liberated myeloperoxidase (MPO). Mean and standard deviation are presented for n = 3 independent experiments; *p < 0.05 as compared with the 0 µM group by one-way ANOVA corrected with Dunnett’s test. c NETosis was assessed qualitatively by immunofluorescence microscopy. 8-Br-cAMP = 1 µM, blue = DNA, green = extracellular neutrophil elastase, and scale bar = 100 microns. d Neutrophils were treated with either control IgG or APS IgG. Forskolin and PDE4 inhibitor were additionally added to some samples. NETosis was quantified by the enzymatic activity of nuclease-liberated MPO; *p < 0.05 and **p < 0.01 by one-way ANOVA corrected with Sidak’s test