Figure 4.

Neutrophil extracellular trap (NET) induction by patient IgG before and after absorption of anti-lactoferrin antibodies (aLf). For this purpose, eosinophilic granulomatosis with polyangiitis sera with aLf were divided into the following two groups; Group 1, aLf-positive/myeloperoxidase (MPO)–anti-neutrophil cytoplasmic antibodies (ANCA)-negative (n = 1) and Group 2, aLf-positive/MPO–ANCA-positive (n = 3). Sera from a healthy volunteer and from microscopic polyangiitis patients (Group 3, aLf-negative/MPO–ANCA-positive, n = 3) were used as controls. Peripheral blood neutrophils obtained from healthy volunteers were seeded on chamber slides (1 × 10⁵/mL), incubated for 15 min at 37°C, and then exposed to 0 or 10 nM phorbol myristate acetate (PMA) combined with 250 µg/mL patient IgG (before and after absorption of aLf) or healthy control IgG. After incubation for 3 h at 37°C, the samples were fixed with 4% paraformaldehyde followed by mounting with the solution containing DAPI. For positive control, the neutrophils were exposed to 100 nM PMA for 3 h at 37°C. NET area was represented by DAPI-positive area, which was calculated using Image J software. Data were presented as mean ± SD values of relative NET induction in which the value of the positive control (PMA 100 nM) was set as 1. Experiments were repeated five times. *p < 0.05.