Neutrophi l Act ivat ion by Ant i - Prote inase 3 Ant ibod ies in

Among the anti-neutrophil cytoplasmic antibodies (ANCA), those targeting proteinase 3 (PR3) have a high specificity for Wegener's granulomatosis (WG). It is known that a preceding priming of neutrophils with cytokines is a prerequisite for membrane surface expression of PR3, which is then accessible to autoantibody binding. Employing a monoclonal antibody directed against human PR3 and ANCA-positive serum from WG patients with specificity for PR3, we now investigated the role of free arachidonic acid (AA) in autoantibody-related human neutrophil activation. Priming of neutrophils with tumor necrosis factor (TNF-or for 15 rain or exposure to anti-PR3 antibodies or incubation with free AA (10 IxM) as sole events did not provoke superoxide generation, elastase secretion or generation of 5-1ipoxygenase products of AA. Similarly, the combination of TNF-oL-priming and AA incubation was ineffective. When TNF-cl-primed neutrophils were stimulated by anti-PR3 antibodies, superoxide and elastase secretion was provoked in the absence of lipid mediator generation. However, when free AA was additionally provided, a strong activation of the 5-1ipoxygenase pathway was demasked, with the appearance of excessive quantities ofleukotriene (LT)B4, LTA4, and 5-hydroxyeicosatetraenoic acid. Moreover, superoxide and elastase secretion were markedly amplified, and studies with 5-1ipoxygenase inhibitors and a LTB4-antagonist demonstrated this was due to an LTB4-related autocrine loop of celt activation. In contrast, the increased synthesis ofplatelet-activating factor in response to TNF-~x-priming and anti-PR3 stimulation did not contribute to the amplification loop of neutrophil activation under the given conditions. We conclude that anti-PR3 antibodies are potent inductors of the 5-1ipoxygenase pathway in primed human neutrophils, and extracellular free AA, as provided at an inflammatory focus, synergizes with the autoantibodies to evoke full-blown lipid mediator generation, granule secretion and respiratory burst. Such events may be enrolled in the pathogenesis of focal necrotizing vascular injury in Wegener's granulomatosis.