High-mobility group box 1 potentiates antineutrophil cytoplasmic antibody-inducing neutrophil extracellular traps formation

Recent studies found that the circulating high-mobility group box 1 (HMGB1) levels could reflect the disease activity of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). HMGB1 could prime neutrophils by increasing ANCA antigens translocation for ANCA-mediated respiratory burst and degranulation. The current study aimed to investigate whether HMGB1 participates in ANCA-induced neutrophil extracellular traps (NETs) formation, which is one of the most important pathogenic aspects in the development of AAV. NETs were induced by treating neutrophils with HMGB1 and ANCA-positive IgG in vitro. NETs formation was assessed using immunofluorescence microscopy and fluorescence probe. Antagonist for relevant receptors Toll-like receptor (TLR)2, TLR4 and the receptor for advanced glycation end products (RAGE), as well as NADPH oxidase molecules were employed. The percentage of NETs formation was significantly higher in neutrophils stimulated with HMGB1 plus ANCA-positive IgG than that in neutrophils incubated with HMGB1 or ANCA-positive IgG alone. Consistently, compared with the nonstimulated neutrophils, the cell-free DNA (cfDNA) concentration of NETs was significantly increased from 334.09 ± 46.89 ng/ml to 563.32 ± 122.07 ng/ml in the neutrophils incubated with HMGB1 plus MPO-ANCA-positive IgG (P