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-alpha 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 betaII 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.

Fig. 1

Effects of priming on O₂⁻ release from neutrophils. O₂⁻ production was measured in response to 100 μg/ml of either MPO-ANCA, or normal IgG and to 1 μm fMLP in the absence of priming (a) or in the presence 2 ng/ml TNF-α, alone (b, open bars) or in combination with 5 μg/ml cytochalasin B (b, solid bars). Results are expressed as nmol/1 × 10⁵ cells/60 min. Data shown are mean ± SEM for at least two experiments performed in triplicate.

Fig. 2

Time course of neutrophil O₂⁻ production (1 × 10⁵ cells) in response to fMLP (♦), 100 ng/ml PMA (▪), 100 μg/ml ANCA IgG (□) or 100 μg/ml normal IgG (X). The results show mean ± SEM of a single experiment representative of at least three experiments performed in triplicate.

Fig. 3

Effect of increasing IgG dose on neutrophil O₂⁻ production. Each value represents mean ± SEM of three experiments performed in triplicate

Fig. 4

O₂⁻ production from neutrophils stimulated with 100 μg/ml MPO-ANCA IgG (♦) or 150 μg/ml of a corresponding F(ab′)₂ fragment (○) or 100 μg/ml normal IgG (▪). Each point represents mean ± SEM of a single experiment performed in triplicate and is representative of at least three individual experiments.

Fig. 5

Effects of increasing concentrations of chelerythrine chloride (IC₅₀ = 0.66 μm) on neutrophil O₂⁻ production stimulated by either, 200 μg/ml PR3-ANCA, 100 μg/ml MPO-ANCA or 1 μm fMLP. Results are expressed as percentage inhibition of the maximal effect seen with each stimulus. Data are shown as mean ± SEM of at least three individual experiments performed in triplicate.

Fig. 6

Effects of increasing concentrations of bisindolylmaleimide I (IC₅₀ = 0.02 μm) on neutrophil O₂⁻ production stimulated by either, 200 μg/ml PR3-ANCA, 100 μg/ml MPO-ANCA or 1 μm fMLP. Results are expressed as percentage inhibition of the maximal effect seen with each stimulus. Data are shown as mean ± SEM of at least three individual experiments performed in triplicate.

Fig. 7

Effects of increasing concentrations of Gö6976 (IC₅₀ = 5 nm) on neutrophil O₂⁻ production stimulated by either, 200 μg/ml PR3-ANCA, 100 μg/ml MPO-ANCA or 1 μm fMLP. Results are expressed as percentage inhibition of the maximal effect seen with each stimulus. Data are shown as mean ± SEM of at least three individual experiments performed in triplicate.

Fig. 8

Effects of 25 μm (solid bar) and 150 μm (hatched bar) genistein on neutrophil O₂⁻ production stimulated by either, 200 μg/ml PR3-ANCA, 100 μg/ml MPO-ANCA, 1 μm fMLP or 100 ng/ml PMA. Results are expressed as percentage inhibition of the maximal effect seen with each stimulus. Data are shown as mean ± SEM of at least three individual experiments performed in triplicate.

Fig. 9

Translocation of PKC isoforms in response to ANCA or normal IgG. Cytosolic and membrane proteins were isolated from cytochalasin B and TNF-α primed neutrophils stimulated with either 500 μg/ml of ANCA IgG or 500 μg/ml normal IgG, with the reaction terminated at the time points indicated. The results were obtained using 20 μg of cytosolic protein or 40 μg/ml membrane protein for each lane.

Fig. 10

Time course of tyrosine phosphorylation in neutrophils stimulated with ANCA or normal IgG. Primed neutrophils (5 × 10⁶/ml) were stimulated in the presence or absence of 500 μg/ml ANCA or normal IgG, or 500 μg/ml ANCA following pretreatment of the neutrophils with 150 μm genistein for 30 min. The reactions were terminated at the time points indicated. Results shown are representative of at least two other experiments.