Figure 5

Anti-neutrophil cytoplasmic antibody-induced neutrophil extracellular traps release auto-antigens and LL37. Neutrophils were incubated with anti-hLAMP2-IgG (H4B4, 20 μg/ml) for 180 minutes with buffer from a healthy donor. Auto-antigens for (A) LAMP-2, (B) myeloperoxidase (MPO), (C) proteinase-3 (PR3) and (D) LL37 were examined (green) in the neutrophil extracellular traps (NETs). Histones were stained with an H3 rabbit polyclonal antibody (red), and DNA was stained with 4′,6-diamidino-2-phenylindole (blue). Neutrophil features were observed under scanning electron microscopy for (E) healthy controls (HCs) and (F) anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) patients after incubation for 180 minutes in vitro. (G) Quantification of the supernatant levels of LL37 by enzyme-linked immunosorbent assay: supernatant of neutrophils from healthy control incubated with anti-hLAMP2-IgG (HC+H4B4), neutrophils from AAV patients (AAV) and neutrophils from AAV patients incubated with anti-hLAMP2-IgG (AAV+H4B4)groups showed higher levels of LL37 compared with HC group (***P <0.001), respectively. Supernatant of neutrophils from AAV +H4B4 group showed higher levels of LL37 compared with the HC + H4B4 group (^###P <0.001). ISO; PMA, phorbol 12-myristate 13-acetate.