Table 1.
PMN donor characteristics.
Table 2.
Degranulation (% release, median (IQR)).
Table 3.
Degranulation controls (% release, median (IQR)).
Table 4.
Oxidative burst after ANCA stimulation.
Fig 1.
Increased C3bBbP levels in supernatants from AAV PMN.
C3bBbP (stable alternative pathway C3 convertase, a sign of alternative pathway activation) formed in normal serum after incubation with supernatants from AAV PMN (neutrophils from vasculitis patients) or HC PMN (neutrophils from healthy controls), that have been primed with TNF-α and then stimulated with ANCA IgG (immunoglobulin G fraction from PR3-ANCA positive patients). Mean and SEM are shown. N = 50 AAV PMN samples (5 PMN donors, 10 different ANCA IgG stimuli) and 50 HC PMN samples (5 PMN donors, 10 different ANCA IgG stimuli). Statistical analysis: Mann Whitney, p = 0.01.
Fig 2.
Activation with different ANCA IgG leads to varying degree of C3bBbP formation.
C3bBbP (stable alternative pathway C3 convertase) formed in normal serum after incubation with supernatants from five AAV PMN (neutrophils from vasculitis patients) and five HC PMN (neutrophils from healthy controls) primed with TNF-α and then stimulated with 10 different ANCA IgGs (immunoglobulin G fraction from PR3-ANCA positive patients) and one HC IgG (immunoglobulin G fraction from a healthy blood donor). PMA (phorbol 12-myristate 13-acetate) was used as positive control and PBS as negative control. Number of neutrophil supernatants tested = 5 in each bar. Means and SEMs are shown. Statistical analysis: Mann Whitney, p < 0.001.
Fig 3.
Activation with different ANCA IgG leads to varying degree of C5a formation.
C5a measured in normal serum after incubation with supernatants from five AAV PMN (neutrophils from vasculitis patients) and five HC PMN (neutrophils from healthy controls) primed with TNF-α and then stimulated with 10 different ANCA IgGs (immunoglobulin G fraction from PR3-ANCA positive patients) and one HC IgG (immunoglobulin G fraction from a healthy blood donor). PMA (phorbol 12-myristate 13-acetate) was used as positive control and PBS as negative control. Number of neutrophil supernatants tested = 5 in each bar. Means and SEM are shown. Statistical analysis: Mann Whitney, p = 0.01.
Fig 4.
Increased microparticle release from AAV PMN.
Release of CD66+ microparticles from five AAV PMN (neutrophils from PR3-ANCA positive vasculitis patients) and five HC PMN (neutrophils from healthy controls), after ANCA IgG (immunoglobulin G fraction from PR3-ANCA positive patients) stimulation, with 10 different ANCA IgGs. Means and SEM are shown. N = 50 AAV PMN samples and 50 HC PMN samples. Statistical analysis: Mann Whitney, p-value = 0.001.
Fig 5.
C3bBbP formation correlates with microparticle release.
Correlation analysis of the number of CD66+ microparticles in PMN supernatants, and C3bBbP formation in normal serum, after incubation with the same supernatants. PMN supernatants were collected from 10 AAV PMN (neutrophils from 10 different PR3-ANCA positive vasculitis patients), after ANCA IgG stimulation, using two different ANCA IgG (IgG4 and IgG7 in Fig 1B), number of samples = 20, r = 0.72, p < 0,0001. Statistical analysis: Spearman.
Fig 6.
Less C3bBbP formation after microparticle removal.
C3bBbP formation in normal serum after incubation with supernatants from seven AAV PMN (neutrophils from vasculitis patients) after ANCA IgG stimulation, using two different ANCA IgG (IgG4 and IgG7 in Fig 1B), before and after removal of CD66+ microparticles from the supernatants by centrifugation. Means and SEM are shown. Number of samples in each group = 14, p < 0,0001. Statistical analysis: Wilcoxon matched pairs signed rank test.
Fig 7.
Effects of priming on membrane C5aR expression.
Effects of pre-priming with CLB (cytochalasin B) or not, in combination with TNF-α or C5a priming, on membrane C5aR expression on HC PMN (neutrophils from healthy controls). Percentages of cells expressing CD88 and C5L2 are shown. Number of experiments = 2, showing representative values.
Fig 8.
Effects of priming and ANCA IgG stimulation on membrane C5aR expression.
Effects of ANCA IgG stimulation of primed AAV PMN (neutrophils from two different vasculitis patients) on membrane C5aR expression are displayed to the left and HC PMN (neutrophils from two different healthy controls) are displayed to the right. Percentages of cells expressing CD88 and C5L2 are shown. Number of experiments = 2, showing representative values.