Fig. 1.

Serum DNase1 degrades NETs. Human neutrophils were activated to form NETs and incubated in the indicated conditions before measurement of the digested and released NET DNA using the fluorescencent DNA dye Picogreen. (A) In the absence of serum (open bars) NETs are stable for at least 90 h in vitro. NET degradation with MNase for 10 min at each time point (black bars) represents the total NETs recovered. (B) Serum-mediated degradation of NETs is concentration and (C) time dependent when exposed to 10% serum, suggesting an enzymatic activity (open bars are medium controls). (D) Activated neutrophils that formed NETs were incubated in media with 10% serum for the indicated time points, fixed, and immunostained for myeloperoxidase (green) and histones (red). DNA (blue) was stained with Draq5. (Scale bar, 10 μm.) (E) Serum NET degradation requires calcium. NETs were incubated with 10% serum for 6 h. EGTA, a calcium chelator, inhibited degradation. Calcium, but not magnesium ions, restored the NET-degrading activity. (F) Inhibition of NET degradation by G-actin, a specific inhibitor of DNase1, is dose dependent. NETs were incubated with 10% serum for 6 h in the presence of the indicated concentrations of G-actin, and NET degradation was measured as described. (G) To control for the specificity of G-actin for DNase1, we incubated NETs with 20 μM of G-actin with either commercially available purified DNase1 or MNase to digest the NETs. G-actin blocked degradation by DNase1 but not MNase. (H) NETs were incubated with serum as described above and with the indicated concentrations of polyclonal anti-DNase1 (black bars) or irrelevant control Abs (open bars). Inhibition of NET degradation by anti-DNase1 Abs was specific and dose dependent. (I) Anti-DNase1 antibodies are specific. We incubated NETs in the presence of 40 μg/mL of anti-DNase1 Abs and incubated with purified DNase1 or MNase. The data shown are representative of experiments performed in triplicate and are presented as mean ± SD.