Figure 5. MPO-ANCA generates oxidized phospholipids.

(A) The most abundant oxidized phospholipids extracted from peripheral blood monocytes incubated for 18 hours with LPS and anti-myeloperoxidase antibodies (MPO-ANCA) (n = 3) or control IgG (n = 3). The data are presented as a percentage of the intensity of the native saturated phospholipid dipalmitoylphosphatidylcholine at m/z 734 in each sample. *P < 0.05 by 2-tailed Student’s t test). (B) Extracted ion chromatograms (XICs) for the control IgG–treated (n = 3) and MPO-ANCA–treated (n = 3) samples, showing the intensity of the long-chain oxidized phospholipid species at m/z 828.8 eluting at ~29.5 minutes, ahead of nonoxidized species of the same m/z ratio. The labels above the peaks indicate their respective area as calculated by Peakview software. Based on these characteristics, the early eluting species at m/z 828.8 was identified (arrow) as 1-palmitoyl-2-(epoxyisoprostane E2)-sn-glycero-3-phosphocholine (PEIPC). The XICs were prepared with a window of 0.7 Da and without smoothing. (C) Surface E06 staining on peripheral blood monocytes incubated for 18 hours with LPS and MPO-ANCA (n = 3) or control IgG (n = 3). (D) Median fluorescence intensity (MFI) data from the experiment shown in C and 2 other monocyte donors to give 3 monocyte donors in total. The effect of adding the MPO inhibitor AZD5904 (4 μM) to MPO-ANCA–treated monocytes is also shown. Control IgG was compared with MPO-ANCA using a 2-tailed Student’s t test. MPO-ANCA was compared with MPO-ANCA + AZD5904 using a 2-tailed paired Student’s t test (with lines indicating paired samples). *P < 0.05. Error bars represent mean ± SEM. For a given monocyte donor, n is the number of IgG preparations from different individuals. They are not technical replicates or repeated measures of the same IgG samples.