IgG from myeloperoxidase-antineutrophil cytoplasmic antibody-positive patients stimulates greater activation of primed neutrophils than IgG from proteinase 3-antineutrophil cytoplasmic antibody-positive patients

Objective. Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA) have been reported to be pathologically and clinically different. The aim of this study was to assess whether these differences could be explained by differing abilities of proteinase 3-antineutrophil cytoplasmic antibody (P R3-ANCA)positive IgG preparations or myeloperoxidase-ANCA (MPO-ANCA)-positi ve IgG preparations to activate neutrophils (polymorphonuclear cells [PMN]) in vitro. Methods. Using Percoll density gradients, PMN were isolated (concentration 2 X 10(6)/ml) and primed with cytochalasin B (1 ng/ml) and tumor necrosis f actor alpha (TNF alpha; 2 ng/ml). The PMN were activated with 200 mug/ml of normal IgG or ANCA. Activation was determined by 1) superoxide anion gener ation as determined by the superoxide dismutase-inhibitable reduction of fe rricytochrome c, 2) monitoring fluxes in Ca2+ concentration using Fura 2-AM -loaded PMN, and 3) degranulation using an MPO assay. Surface expression of PR3 and MPO was determined by fluorescence-activated cell sorter analysis. ANCA isotypes were investigated by enzyme-linked immunosorbent assay. Results. Activation of PMN by MPO-ANCA-positive IgG preparations compared w ith PR3-ANCA-positive IgG preparations resulted in greater generation of su peroxide anions (MPO-ANCA-positive IgG preparations 9.13 +/- 0.39 nmoles [m ean +/- SEM], PR3-ANCA-positive IgG preparations 6.32 +/- 0.35 nmoles; P < 0.001), Ca2+ fluxes (MPO-ANCA-positive IgG preparations 0.735 +/- 0.10, PR3 -ANCA-positive IgG preparations 0.33 +/- 0.098; P < 0.01), and MPO degranul ation (MPO-ANCA-positive IgG preparations 251.98 +/- 26.7 ng, PR3-ANCA-posi tive IgG preparations 145.19 +/- 19.4 ng; P < 0.001). The increased activat ion seen with MPO-ANCA-positive IgG preparations was not due to increased e xpression of MPO on the cell surface, because following TNF alpha priming P R3 was expressed on significantly more cells than was MPO (PR3 expression 5 4.2 +/- 5.18%, MPO 31.6 +/- 3.55%; P < 0.001). IgG1 and IgG4 were the predo minant isotypes in both MPO-ANCA-positive IgG preparations and PR3-ANCA. MP O-ANCA contained significantly more IgG1 than did PR3-ANCA, and PR3-ANCA-po sitive IgG preparations contained significantly more IgG3. Conclusion. In vitro MPO-ANCA-positive IgG preparations are more activating than PR3-ANCA-positive IgG preparations. The increased activation cannot b e explained by increased MPO expression on the cell surface or greater IgG3 present in MPO-ANCA-positive IgG preparations. Differences in activation o f PMN by these antibodies may determine some differences between WG and MPA .