Neutrophil priming and apoptosis in anti-neutrophil cytoplasmic autoantibody-associated vasculitis1

Neutrophil priming and apoptosis in anti-neutrophil cytoplasmic autoantibody-associated vasculitis.

Background

Interactions between anti-neutrophil cytoplasmic autoantibody (ANCA) and primed neutrophils (PMNs) may be central to the pathogenesis of primary small vessel vasculitis. PMNs from patients are primed, expressing proteinase 3 (PR3) on the cell surface, which permits interaction with ANCA. In vitro ANCA activates primed PMN to degranulate and generate a respiratory burst. Resultant reactive oxygen species are important in triggering apoptosis, but the fate of PMN in ANCA-associated vasculitis is unknown. Failure to remove apoptotic PMN in a nonphlogistic manner may sustain the inflammatory response.

Methods

PMNs from patients or controls were isolated, and the basal production of superoxide was measured by the superoxide dismutase-inhibitable reduction of ferricytochrome C. ANCA antigen expression on apoptotic PMN was assessed at 0, 12, and 18 hours by flow cytometry using dual staining with FITC-conjugated annexin V and PE-conjugated anti-murine IgG against monoclonal ANCA. Apoptosis was also assessed by morphology. In further studies, apoptotic PMNs were opsonized with monoclonal anti-myeloperoxidase (MPO) or anti-proteinase-3 (PR3) or irrelevant isotype-matched IgG (N IgG) and phagocytosis by macrophages was measured using interaction assays. Cytokines interleukin-8 (IL-8) and interleukin-1 were measured by enzyme-linked immunosorbent assay (ELISA).

Results

Proteinase-3 expression (active 63.04 ± 5.6% of total number of cells, remission 51.47 ± 7.9% of total number of cells, control 17.7 ± 4.7% of total number of cells, P < 0.05) and basal superoxide production (active 6.9 ± 0.8 nmol/L × 10⁶ cells, remission 5.15 ± 0.4 nmol/L/10⁶ cells, control 3.63 ± 0.3 nmol/L/10⁶ cells, P < 0.001) were significantly greater with freshly isolated PMN from patients than controls. PR3 expression and superoxide generation were positively correlated. PMN from patients with active disease became apoptotic at a greater rate than those of controls (at 18 hours, patients 72.3 ± 3.9% apoptosis, controls 53.2 ± 2.7% apoptosis, P < 0.05). PR3 and MPO expression were significantly greater on PMN isolated from patients at 12 and 18 hours. Opsonization of apoptotic PMN with ANCA significantly enhanced recognition and phagocytosis by scavenger macrophages (anti-MPO 88.95 ± 6.27, anti-PR3 93.98 ± 4.90, N IgG 44.89 ± 3.44, P < 0.01) with increased secretion of IL-1 (anti-PR3 34.73 ± 6.8 pg/mL, anti-MPO 42.01 ± 12.3 pg/mL, N IgG 8.04 ± 6.3 pg/mL, P < 0.05) and IL-8 (anti-PR3 8.97 ± 0.93 ng/mL, anti-MPO 8.45 ± 1.46 ng/mL, N IgG 0.96 ± 0.15 ng/mL, P < 0.01).

Conclusion

In vivo circulating PMNs are primed as assessed by PR3 expression and basal superoxide production, thereby enhancing their inflammatory potential. These PMNs undergo apoptosis more readily, at which times they express PR3 and MPO on their surface. These antigens may then provide targets for ANCA. Opsonization of apoptotic PMN will enhance clearance by macrophages but will also trigger the release of pro-inflammatory cytokines that may contribute to chronic inflammation.