The activation of the neutrophil respiratory burst by anti-neutrophil cytoplasm autoantibody (ANCA) from patients with systemic vasculitis requires tyrosine kinases and protein kinase C activation

Abstract

The ability of antineutrophil cytoplasm autoantibodies (ANCA) from patients with systemic vasculitis to stimulate protein kinase C (PKC) and tyrosine kinases was examined in human neutrophils. Using the superoxide dismutase-inhibitable reduction of ferricytochrome C, the kinetics of ANCA-induced superoxide (O₂⁻) production were characterized and subsequently manipulated by specific inhibitors of PKC and tyrosine kinases. With this approach, ANCA IgG, but not normal IgG or ANCA F(ab′)₂ fragments caused a time and dose dependent release of O₂⁻ from TNF-α primed neutrophils. The kinetics of ANCA-induced O₂⁻ production showed an initial 10–15 min lag phase compared to the N-formyl- l-methionyl- l-leucyl- l-phenylalanine response, suggesting differences in the signalling pathways recruited by these two stimuli. Inhibitor studies revealed that ANCA-activation involved members of both the Ca²⁺-dependent and -independent PKC isoforms and also tyrosine kinases. ANCA IgG resulted in the translocation of the β_II isoform of PKC at a time corresponding to the end of the lag phase of O₂⁻ production, suggesting that PKC activity may be instrumental in processes regulating the activity of the NADPH oxidase in response to ANCA. Tyrosine phosphorylation of numerous proteins also peaked 10–15 min after stimulation with ANCA but not normal IgG. These data suggest that PKC and tyrosine kinases regulate O₂⁻ production from neutrophils stimulated with autoantibodies from patients with systemic vasculitis.