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Abstract: FR-OR40

Alpha1-Antitrypsin Diminishes Neutrophil Activation by PR3-ANCA and Endothelial Injury by Neutrophil Serine Proteases

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation


  • Kettritz, Ralph, Charite Universitatsmedizin Berlin, Berlin, Germany
  • Jerke, Uwe, Experimental and Clinical Research Center, Charité, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany
  • Eulenberg-Gustavus, Claudia, Experimental and Clinical Research Center, Charité, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany
  • Jenne, Dieter E., German Center for Lung Research, Helmholtz Zentrum München, Munich, Germany

Neutrophil serine proteases (NSPs) contribute to ANCA-associated vasculitis (AAV). PR3 is a unique NSP family member because it is both a proteolytic enzyme and an ANCA antigen. Alpha1-antitrypsin (AAT) is the major natural NSP inhibitor. We hypothesized that AAT protects from ANCA-induced neutrophil activation and neutrophil-mediated glomerular endothelial cell injury.


We produced recombinant wild-type (wt) AAT and a mutant (mu) form that does not inhibit proteolytic NSP activity. We used flow cytometry to study membrane PR3 (mPR3) and MPO (mMPO), ferricytochrome C assay for superoxide release, FRET assays for proteolytic NSP activity, human neutrophils and glomerular microvascular endothelial cells (gMVEC), confocal and electron microscopy, and assessed ECs by phalloidin staining and gene expression.


Wt-AAT reduced mPR3 on TNF-primed neutrophils dose-dependently from 0.1 to 10 µM (n=3). Five µM wt-AAT, but not mut-AAT, reduced neutrophil mPR3 in suspension, on fibronectin, and on an EC monolayer to approximately 25% (n=3). Parallel comparisons in neutrophil-EC co-cultures using antibodies to different PR3 epitopes showed that 5 µM AAT reduced mPR3 but not mMPO. Importantly, reduced mPR3 by AAT resulted in significantly less superoxide release by TNFa-primed neutrophils when activated with PR3-, but not with MPO-ANCA IgG from AAV patients (n=4). Next, we studied the NSP transfer from activated neutrophils to gMVEC and found that gMVEC acquired NSPs, exemplified by PR3, from both cell-free supernatants (cf-SN) and under neutrophil-EC co-culture conditions. Importantly, wt-, but not muAAT abrogated the PR3 transfer from cf-SN. In contrast, AAT did not inhibit the PR3 transfer under neutrophil-EC co-culture conditions. Finally, we observed by RT-PCR that cf-SN from activated neutrophils increased two NF-κB dependent genes in gMVEC, namely IkBa and IL-8. This effect was reduced by wt-, but not by mu-AAT suggesting an NSP-dependent activation mechanism.


AAT has protective effects by reducing neutrophil activation in response to PR3-ANCA, and NSP-mediated glomerular microvascular endothelial cell injury. Disturbances of the AAT-NSP balance possibly contribute to neutrophil-mediated vascular injury in AAV, particularly, but not exclusively, in patients with PR3-ANCA.


  • Government Support - Non-U.S.