Effect of anti–HNA-3a antibody on NET formation by human neutrophils. (A) Quantification of NETs after anti–HNA-3a antibody treatment by fluorescence microscopy analysis. HNA-3a-positive neutrophils primed with TNF-α were incubated for 180 minutes with PBS (TNF-α + PBS), control IgG purified from healthy volunteer plasma (TNF-α + Ctrl Ab), anti–HNA-3a antibody purified from 2 donors whose plasma induced TRALI (TNF-α + Ab1 and TNF-α + Ab2), or 25nM PMA as a positive control (TNF-α + PMA). DNA release was visualized after DNA staining with Hoechst 33342. The experiment was independently performed 9 times. Bars represent mean ± SEM. **P < .01. (B) Examples of the fluorescence images quantified in panel A. White arrowheads indicate selected cells forming NETs; and white arrows, examples of delobulated (large) neutrophil nuclei. Their presence indicates nuclear decondensation, an early step in NETosis. Scale bar represents 50 μm. (C) Quantification of NETs after anti–HNA-3a F(ab′)2 fragment treatment by fluorescence microscopy analysis. HNA-3a–positive neutrophils were incubated and analyzed in the same conditions as in panel A but were also treated with anti–HNA-3a F(ab′)2 fragments (TNF-α + F(ab′)2 anti–HNA-3a). The experiment was independently performed 3 times. Bars represent means ± SEM. ***P < .005. NS indicates not significant.
