NEUTROPHIL ACTIVATION BY ANTIPROTEINASE-3 ANTIBODIES IN WEGENERS-GRANULOMATOSIS - ROLE OF EXOGENOUS ARACHIDONIC-ACID AND LEUKOTRIENE B-4 GENERATION

Among the anti-neutrophil cytoplasmic antibodies (ANCA), those targeti ng proteinase 3 (PR3) have a high specificity for Wegener's granulomat osis (WG). It is known that a preceding priming of neutrophils with cy tokines is a prerequisite for membrane surface expression of PR3, whic h is then accessible to autoantibody binding. Employing a monoclonal a ntibody directed against human PR3 and ANCA-positive serum from WG pat ients with specificity for PR3, we now investigated the role of free a rachidonic acid (AA) in autoantibody-related human neutrophil activati on. Priming of neutrophils with tumor necrosis factor (TNF-alpha) for 15 min or exposure to anti-PR3 antibodies or incubation with free AA ( 10 mu M) as sole events did not provoke superoxide generation, elastas e secretion or generation of 5-lipoxygenase products of AA. Similarly, the combination of TNF-alpha-priming and AA incubation was ineffectiv e. When TNF-alpha-primed neutrophils were stimulated by anti-PR3 antib odies, superoxide and elastase secretion was provoked in the absence o f lipid mediator generation. However, when free AA was additionally pr ovided, a strong activation of the 5-lipoxygenase pathway was demasked , with the appearance of excessive quantities of leukotriene (LT)B-4, LTA(4), and 5-hydroxyeicosatetraenoic acid. Moreover, superoxide and e lastase secretion were markedly amplified, and studies with 5-lipoxyge nase inhibitors and a LTB(4)-antagonist demonstrated this was due to a n LTB(4)-related autocrine loop of cell activation. In contrast, the i ncreased synthesis of platelet-activating factor in response to TNF-al pha-priming and anti-PRS stimulation did not contribute to the amplifi cation loop of neutrophil activation under the given conditions. We co nclude that anti-PR3 antibodies are potent inductors of the 5-lipoxyge nase 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.