Abstract
Neutrophil extracellular traps (NETs) are chromatin filaments decorated with enzymes from neutrophil cytoplasmic granules. Anti-neutrophil cytoplasmic antibodies (ANCAs) bind to enzymes from neutrophil cytoplasmic granules and are biomarkers for the diagnosis of systemic vasculitides. ANCA diagnostics are based on indirect immunofluorescence (IIF) of ethanol-fixed neutrophils. IIF shows a cytoplasmic staining pattern (C-ANCA) due to autoantibodies against proteinase 3 (PR3) or a perinuclear staining pattern (P-ANCA) due to autoantibodies against myeloperoxidase (MPO). The distinct ANCA-staining patterns are an artifact of ethanol fixation. Here, we tested NETs as a substrate for the detection of ANCAs in human sera. We observed that P-ANCAs specifically stained NETs, while C-ANCAs targeted the cell bodies of netting neutrophils. The distinct ANCA-staining patterns were caused by the presence of MPO, but not PR3, in NETs. Using NETs as a substrate for IIF, we characterized ANCAs in sera of patients with ANCA-associated vasculitis (AAV). Furthermore, we inhibited serine proteases by diisopropylfluorophosphate to prevent chromatin unfolding and the release of NETs and thus generated neutrophils with MPO-positive nuclei and PR3-positive cytoplasm, which resembled the appearance of ethanol-fixed neutrophils. In conclusion, our data suggest that NETs are selectively loaded with antigens recognized by P-ANCAs, and netting neutrophils provide a physiological substrate for ANCA detection in patients with AAV.
Highlights
Neutrophil extracellular traps (NETs) are lattices of intact DNA filaments containing histones and neutrophil enzymes [1]
To test whether Anti-neutrophil cytoplasmic antibodies (ANCAs) bind to NETs, we induced NETosis by activating human neutrophils for 4 h with PMA, a potent inducer of NETs [1, 3]
C-ANCApositive sera did not bind to NETs but targeted the cell bodies of netting neutrophils (Figure 1B)
Summary
Neutrophil extracellular traps (NETs) are lattices of intact DNA filaments containing histones and neutrophil enzymes [1]. The loss of internal membranes enables the intracellular loading of NETs Bind P-ANCAs chromatin filaments with enzymes from neutrophil cytoplasmic granules [3, 4]. Anti-neutrophil cytoplasmic antibodies (ANCAs) bind to enzymes from neutrophil cytoplasmic granules [5,6,7]. ANCAs are associated with vasculitis, glomerulonephritis, and several autoimmune diseases of the gastrointestinal tract [8,9,10]. The detection of ANCAs is based on the screening of patient sera by indirect immunofluorescence (IIF) using ethanol-fixed unstimulated neutrophils as a substrate [7]. NETs have been reported to contain enzymes from all types of neutrophil granules including MPO and PR3 [12, 13]. We hypothesized that NETs are a substrate for the detection of ANCAs in patient sera
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