Fig. 2

Agonism of the adenosine A_2A receptor suppresses NETosis. a, b Neutrophils were isolated from healthy volunteers and then treated with either control IgG or APS IgG for 3 h. Some samples were additionally treated with agonists of the adenosine A_2A receptor (CGS21680), A_2B receptor (BAY60-6583), or A₃ receptor (IB-MECA) as indicated. 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 APS IgG/no-drug group by one-way ANOVA corrected with Sidak’s test. c NETosis was assessed qualitatively by immunofluorescence microscopy. Blue = DNA, green = extracellular neutrophil elastase, and scale bar = 100 microns. d, e Neutrophils were treated with affinity-purified anti-β₂GPI IgG (black bars) in the presence or absence of the A_2A receptor agonist. Extracellular DNA (d) or NETs (e) were quantified as above. Mean and standard deviation are presented for n = 3 independent experiments; **p < 0.01 as compared with the no-drug group by one-way ANOVA corrected with Dunnett’s test. f Neutrophils were treated with APS IgG (black bars) in the presence of a protein kinase A inhibitor (PKAi) or an inhibitor of the exchange protein activated by cyclic-AMP pathway (EPACi) as indicated, and NETs were quantified. Mean and standard deviation are presented for n = 3 independent experiments; *p < 0.05