Figure 3. Anti-β₂GPI IgG stimulates neutrophils to release NETs.

A, Five APS IgG samples were pooled, and then depleted of anti-β₂GPI IgG using purified β₂GPI protein. Control neutrophils were stimulated with IgG (10 ug/ml) as indicated for 3 hours. NET release was scored by immunofluorescence microscopy. B, Control neutrophils were treated with purified monoclonal antiphospholipid Abs (aPL; 10 μg/ml) for 3 hours in the presence or absence of 10% autologous serum. aPL IS4, CL1, and CL24 are known to bind β₂GPI, while IS1 and IS2 do not. P values were determined by comparing data sets to control IgG (−/+ serum as appropriate). C, Control neutrophils were stimulated with control IgG, CL1, or 20 nM PMA. After 2 hours, PicoGreen was added to the culture, and fluorescence intensity (corresponding to extracellular DNA) was measured. Normalization was to cells permeablized with 0.1% Triton. In panels A–C, bars represent the mean and SEM of at least five independent experiments; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. D, Representative live cells stained with PicoGreen as in panel C. In the CL1 sample, expanded cell remnants (dashed lines) are surrounded by a halo of DNA (green). Scale bars=25 microns.