Received December 3, 2006
Accepted on April 27, 2007

BASIC RESEARCH: Basic Research

Increased Membrane Expression of Proteinase 3 during Neutrophil Adhesion in the Presence of Anti-Proteinase 3 Antibodies

Soumeya Brachemi ^*, Agnès Mambole ^*, Fadi Fakhouri , Luc Mouthon , Loïc Guillevin , Philippe Lesavre ^*, and Lise Halbwachs-Mecarelli ^*1

*INSERM U845 and Université Paris V René Descartes, Necker Hospital Nephrology Department, and Internal Medicine Department, Cochin Hospital and Unité Propre de Recherche de l’Enseignement Supérieur (UPRES) EA 4058, Paris, France

¹ To whom correspondence should be addressed. E-mail: mecarelli{at}necker.fr.

	Abstract

We investigated membrane proteinase 3 (mPR3) expression during TNF--induced adhesion of neutrophils in the presence of anti-PR3 antibodies, a situation occurring during anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis. Three increasing levels of mPR3 expression were observed on the mPR3⁺ neutrophil subset after stepwise cell activation. TNF- activation without adhesion, TNF--induced adhesion, and adhesion in the presence of anti-PR3 mAb or human anti-PR3 ANCA resulted, respectively, in a two-, seven-, and 24-fold increase of mPR3 levels. In plasma, anti-PR3 antibodies poorly recognized suspended neutrophils, whereas they bound to mPR3 on adherent cells. mPR3 upregulation was also triggered by IL-8, formyl-methionyl-leucyl-phenylalanine (fMLP), and neutrophil adhesion to activated human umbilical vein endothelial cells. It involved 2 integrins and Fc receptor, because it was prevented by anti-CD18 antibodies and was not observed with anti-PR3 F(ab')₂. Furthermore, it was specific to anti-PR3 mAb, and no mPR3 upregulation was observed with anti-myeloperoxidase or anti-HLA-ABC mAb. Newly expressed mPR3 molecules, after TNF-induced adhesion, were mobilized from secretory vesicles (CD35⁺) and secondary granules (CD11b⁺). The adhesion- and antibody-dependent upregulations of mPR3 expression occurred with little azurophilic granule degranulation, no sign of apoptosis, and no further CD177 upregulation. In conclusion, this study describes an amplifying loop in polymorphonuclear neutrophil activation process, whereby ANCA are involved in the membrane expression of their own antigen during cell adhesion. This could explain the restriction of ANCA-associated vasculitis to small vessels, the main site of neutrophil adhesion.