Characterisation of the regulation and functional consequences of p21^ras activation in neutrophils by antineutrophil cytoplasm antibodies

Antineutrophil cytoplasm antibodies (ANCA) are implicated in the pathogenesis of systemic vasculitis. ANCA are directed against antigens expressed on the surface of cytokine-primed neutrophils. It was shown previously that whole IgG ANCA and its fraction antigen binding [F(ab')(2)] fragment can activate the GTPase p21(ras). This study shows a functional involvement of this molecule in the ANCA activation of neutrophils by inhibiting the production of superoxide with farnesylthiosalicylic acid. Using the ras activation assay, farnesylthiosalicylic acid inhibits p21(ras). binding to its substrate at comparable concentrations to those seen for superoxide inhibition. It is also shown that activation of p21(ras) by ANCA is transient, peaking at 3 to 10 min and returning to baseline by 30 min. The use of ras isoform-specific antibodies in Western blots established, for the first time, that Harvey-ras is not present in human neutrophils, but both Kirsten-ras (K-ras) and Neuronal-ras are. Stimulation with ANCA is able to differentially activate K-ras without effects on neuronal-ras. The activation of p21(ras) by ANCA and its F(ab')(2) is prevented by inhibition of both Src kinases and phosphatidylinositol-3-kinase, indicating a cooperative role for both molecules in the G protein pathway activated by ANCA F(ab')(2) upstream of p21(ras). It is concluded that ANCA selectively activates K-ras during induction of a respiratory burst via pathways involving multiple upstream kinases.