Impaired in vitro activation of LDNs, while in vitro activation of NDNs correlated with autoantigen gene expression. Intracellular reactive oxygen species (ROS) were determined by fluorescence of rhodamine. Histograms (a) show fluorescence signal in LDNs from HC (left) and Patient (right) following no stimulation (light blue), activation with HC IgG (orange), activation with MPO-ANCA IgG (purple), or PMA (green). Red histogram illustrates background autofluorescence of LDNs. (b) Geometric mean fluorescence intensity (MFI) indicates the baseline intracellular ROS in LDNs with no stimulation for HC (circles, n = 6) and ANCA vasculitis patients (triangles, n = 9). (c and d) Intracellular ROS were determined by fluorescence of rhodamine in NDNs. Geometric MFI values are shown for NDNs activated with HC IgG (circles) or (c) MPO-ANCA IgG (triangles) or (d) anti-PR3 monoclonal antibody (triangles). (c) HC, n = 13; patients with active disease, n = 11; patients in remission, n = 15. (d) HC, n = 11; patients with active disease, n = 3; patients in remission, n = 10. Significant p-values shown were adjusted for multiple comparisons. P-values for NDNs from HC, Active patients, or Remission patients activated with HC IgG compared to the same samples activated with MPO-ANCA IgG were all <0.0003 (not shown). (Comparison of oxidative burst with monoclonal anti-PR3 antibodies and PR3-ANCA IgG are shown in Supplementary Table 5.) (e and f) Linear regression was used to compare MFI in NDNs activated with (e) MPO-ANCA IgG to MPO expression in NDNs from HC (circles, n = 11) and ANCA vasculitis patients (triangles, n = 29), and (f) in NDNs activated with anti-PR3 monoclonal antibody to PRTN3 expression in NDNs from HC (circles, n = 11) and ANCA vasculitis patients (triangles, n = 12). Gray shading illustrates the 95% confidence limit. (g) Intracellular ROS were determined by fluorescence of rhodamine in neutrophils from mouse strain C57BL/6 (circles, n = 4) and 129-S6 (triangles, n = 4). Plotted values are the ratio of the median fluorescence intensity (FI) with anti-mouse MPO IgG to median fluorescence intensity without anti-mouse MPO IgG.