Correlation between NETosis, ANCAs, and autoimmune vasculitis in lupus-prone mice. (A) Spleen PMNs isolated from 8 lpr/lpr mutant mice were tested for spontaneous NETosis. PMNs were seeded onto polylysine-coated glasses without PMA or any type of stimuli, fixed and stained with the SYTOX green DNA dye. PMNs from every single mouse were imaged through confocal microscopy. Spleen purified PMNs normally die of apoptosis when cultured o/n in the absence of any stimuli (as shown in Figure 1), whereas PMNs from the same lpr/lpr mice showed enrichment in NETosis other than dying of apoptosis. Grippingly, PMNs from mice #44, 47, 48, and 50 only showed apoptosis, whereas PMNs from mice #43, 49, 52, and 54 clearly showed signs of NETosis. (B) Quantification of NET formation was performed by plotting SYTOX⁺ nuclear areas against the percentage of SYTOX⁺ cells corresponding to a given nuclear area. Number of nuclei analyzed/experiment = 200. (C) Level of MPO-ANCA, PR3-ANCA, ss-DNA, and ds-DNA Abs in the same lpr/lpr mutant mice analyzed in panel A. (D) Correlation between NETosis and MPO and PR3-ANCA. NETosis correlated with both MPO and PR3-ANCA (r² = .9040, P = .046; and r² = .7857, P = .0279, respectively) but not with the different types of ANA. (E) Histopathologic grading of the overall renal damage in the same lpr/lpr mutant mice described in panel A. (F) Hematoxylin and eosin staining shows intense renal tubulointerstitial lesions, particularly vasculitic lesions, in mice with higher ANCA titers (lpr/lpr 43 and 54), whereas in cases with lower ANCA titers, in which autoantibody patterns were dominated by ds- and ss-DNA ANA antibodies, glomerular nephritic lesions are prevalent (lpr/lpr 48 and 50).