Determination of proteinase 3—α₁‐antitrypsin complexes in inflammatory fluids

Abstract

Phy~iological inht biters were tested for their in vitro interaction with neutrophil protemase 3 (PR3). The major plasma protemase inhibitor of PR3 is =tAT. We have developed a radioimmunoa~ay (RIA) for quantitative detection of PR3--CtlAT complexes formed in rive in inflammatory exudates such as synovtal fluid and plasma from patients with sepsis. Levels ofPR3-a~AT complexeb correlated significantly with levels of human nemrophtl elastase (HNE)--~tAT complexes. Thus, in vtvo a~AT not only proteet~ against excessive HNE activity, but also against excessive PR3 activity.

Pathogenesis of diseases associated with antineutrophil cytoplasm autoantibodies

“…The concentration of ␣ 1 -AT normally increases during infection or inflammation, allowing a tight control of the activity of serine proteases at the inflammation site. Dolman et al [40] have demonstrated that anti-PR3 antibodies exhibit an inhibitory effect on complexation . Adherence of primed neutrophils and/or monocytes to the endothelium is followed by activation of these cells by ANCA.…”

Relevance of classic anti-neutrophil cytoplasmic autoantibody (C-ANCA)-mediated inhibition of proteinase 3-α1-antitrypsin complexation to disease activity in Wegener's granulomatosis

“…PR3 was purified from polymorphonuelear leucocyte granules aceording to the method described by K,ao et al [21], The preparation appeared to be homogeneous by SDS-PAGEi analysis and PR3 migrated as a 29-kD triplet [12]. Purified HNF.…”

“…PR3-aiAT complexes were detected using a radioimmunoassay (RIA) as recently deseribed [12], Briefly, test samples were incubated with Sepharose-bound MoAb 12.8 (directed against PR3), Bound PR3 i\AJ complexes were detected by subsequent incubation with ''T-labelted MoAb AT 15 (directed against complexed :X]AT). Levels of PR3 y.\/\.T complexes were expressed in nM by reference to a standard dose-response curve of preformed PR3 -X\JKT eoniplexes, that were prepared by ineubating purified, active site titrated PR.3 with an excess of eontrol human plasma fl2].…”

“…Normal values in plasma from healthy donors range from 0'5 to 2 5 nM for both PR3-x,AT and MNE ir.AT eomplexes [12],…”

“…C-ANCA can activate pruned neutrophils to release oxygen radicals and lysosomal enzymes in vitro [9], Another observation of possible pathogenic relevance is that C-ANCA interfere with PR3 enzyme activity by reducing, but not totally abolishing, the proteolytic aetivity oi PR3 [Kl], C-ANCA also interfere with the irreversible complexation of PR3 with ai-antitrypsin (aiAT) [10]. the major inhibitor of PR3 [11,12], This inhibition by C-ANCA of PR3-a,AT complexation was observed with purified IgG. but also after addition of PR3 to C-ANCA-positivc sera [10], The latter experiments more closely rcfleet the in viro situation, and suggest that also in vivo C-ANCA may inhibit complexation of PR3 with a,AT, It is therefore possible that C-ANCA.…”