Inhibition of proteinase 3 by ANCA and its correlation with disease activity in Wegener's granulomatosis

Inhibition of proteinase 3 by ANCA and its correlation with disease activity in Wegener's granulomatosis. Detection of circulating antineutrophil cytoplasmic antibodies (ANCA) to the neutrophil serine proteinase, proteinase 3 (PR3), has proven valuable for the diagnosis of Wegener's granulomatosis (WG). However, the importance of these autoantibodies in the pathogenesis of WG remains unknown. It was recently reported that anti-PR3 autoantibodies (PR3-ANCA) from some patients with WG inhibit the proteolytic activity of PR3 and interfere with the inactivation of PR3 by the physiologic inhibitor, α1-proteinase inhibitor (α1-PI). We have studied the effect of PR3-ANCA on the enzymatic activity of PR3 and its correlation with disease activity in patients with WG. We purified IgG from 21 PR3-ANCA positive sera obtained from 17 patients with WG, and determined its effect on the esterolytic and proteolytic activity of purified human PR3 using Boc-Ala-O-Nitrophenyl ester and fluoresceinated-elastin as enzyme substrates. Controls included seven sera containing anti-MPO autoantibodies (MPO-ANCA) from patients with systemic vasculitis and seven ANCA-negative sera obtained from healthy individuals. We found that PR3-ANCA from 9 of the 17 patients significantly inhibited the activity of PR3. There was no correlation between the titers of PR3-ANCA and their inhibitory activity. For one extensively characterized autoantibody, the inhibition reached 70 to 95% at 20-fold molar excess of IgG to enzyme, with an apparent Kiapp of 56.5 µM. This inhibition was non-competitive in nature, and was additive to that produced by α1-PI. A review of the clinical histories of the patients revealed a strong association between active WG and inhibitory autoantibodies. PR3-ANCA from eight of 10 patients with active disease showed significant inhibition of PR3 activity, whereas only one of seven patients in remission had inhibitory PR3-ANCA. These results suggest that the inhibitory profile of PR3-ANCA may be more reflective of disease activity in patients with WG than the absolute titer, and suggest possible alternative mechanisms for the role of these autoantibodies in the pathogenesis of WG