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. 2019 Jun 12;10:1334. doi: 10.3389/fimmu.2019.01334

Figure 2.

Figure 2

BPI-ANCAs induce NETs with hyper citrullinated histones. (A) Representative NETs staining of healthy or BPI-AAV patient IgGs-stimulated neutrophils using CitH3 (red) and DAPI (BLUE). (B) Representative Sytox-positive neutrophils live image. From the left, healthy IgGs (n = 3), MPO-AAV patient IgGs (n = 5), BPI-AAV patient IgGs. Right figure shows Sytox-positive areas quantified by Image J. (C) Representative CitH3-positive NETs and Sytox-positive neutrophils treated with TNFα, TNFα+mBPI-Ab, TNFα+mBPI-Ab+rBPI. Right figures show CitH3-positive area (upper) and Sytox-positive area (lower) under each situation. Data represent the mean±SEM of three independent experiments and were analyzed using unpaired t-test (PRISM software, GraphPad). *p < 0.05. (D) BPI expression with or without TNFα stimulation. Green; BPI, BLUE; DAPI. (E) Sytox positivity of neutrophils treated with 3% serum, or 3% IgG depleted serum of patients with BPI-AAV, MPO-AAV (n = 3) and healthy control (n = 3). Scale bar in all figures, 50 mm. CitH3, citrullinated histone3; DAPI, 49,6-diamidin-2-phenylindol; BPI, bactericidal/permeability increasing protein; ANCA, anti neutrophil cytoplasmic antibody; AAV, ANCA associated vasculitis; IgG, immunoglobulin G; TNFα, tumor necrosis factor alpha; mBPI-Ab, monoclonal BPI antibody; rBPI, recombinant BPI.