SUMMARYTGF-¯is a multifunctional cytokine modulating the onset and course of autoimmune diseases as shown in experimental models. The aim of this study was to investigate TGF-¯expression in ANCA-associated vasculitis (AAV), and the possible interactions of this cytokine with lysosomal enzymes identified as ANCA autoantigens (e.g. PR3). This included TGF-¯effects on the translocation of the lysosomal enzymes to the cell surface of polymorphonuclear neutrophils (PMN), and the presumed activation of non-bioactive, latent TGF-¯by these enzymes. Patients with various types of systemic vasculitis (SV) were studied, including three different types of AAV (Wegener's granulomatosis (WG), Churg-Strauß syndrome (CSS) and microscopic polyangiitis (MPA)). Regardless of the type of assay applied, the TGF-¯1 isoform was found to be overexpressed in SV, including AAV, and to correlate with disease activity as shown for WG. Mean TGF-¯1 plasma levels in AAV patients ranged from 8 . 9 ng/ml (WG) to 13 . 3 ng/ml (CSS) (control 4 . 2 ng/ml; P < 0 . 01), while TGF-¯2 levels were not elevated. Flow cytometry analysis showed TGF-¯1 to be a potent translocation factor for PR3 comparable to other neutrophilactivating factors such as IL-8. PR3 membrane expression on primed PMN increased by up to 51% after incubation with TGF-¯1. PR3 itself was revealed as a potent activator of latent TGF-¯, thus mediating bioeffects of this cytokine. These findings, together with other features of TGF-¯such as induction of angiogenesis and its strong chemotactic capacity, indicate that TGF-¯might serve as a proinflammatory factor in SV, especially in AAV.

Autoantibodies directed against cytoplasmic antigens of neutrophils (ANCA), especially proteinase 3 (PR-3), have proved to be a useful clinical tool confirming the diagnosis or monitoring disease activity of Wegener's granulomatosis (WG). Although several concepts concerning the pathophysiologic potentials of ANCA have been discussed, only sparse data about ANCA-endothelium interactions have been available. In this study, we have investigated the expression of PR-3 in cytokine- treated human endothelial cells using purified anti-PR-3 antibodies of patients with WG, murine and human monoclonal anti-PR-3 antibodies as probes. We were able to show that tumor necrosis factor-alpha, interleukin-1 alpha/beta, and interferon-gamma led to an increased PR-3 expression in the cytoplasm of endothelial cells by performing polymerase chain reaction analysis, Western blot, cyto-enzyme-linked immunosorbent assays, and confocal laser scanning microscopy. Moreover, PR-3 was also translocated into the cell membrane, becoming accessible to ANCA. Our data suggest a possible direct pathogenic effect of anti- PR-3 antibodies in WG and other vasculitides. Anti-PR-3 antibodies represent an important missing link in ANCA-endothelial interactions.

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