Proteinase 3 (PR3), a serine proteinase contained in neutrophil azurophilic granules, is considered a risk factor for vasculitides and rheumatoid arthritis when expressed on the outer leaflet of neutrophil plasma membrane and is the preferred target of antineutrophil cytoplasm autoantibodies (ANCA) in Wegener granulomatosis. ANCA binding to PR3 expressed at the surface of neutrophils activates them. Evidence is provided that neutrophil apoptosis induced significantly more membrane PR3 expression without degranulation (but no enhanced membrane CD35, IntroductionProteinase 3 (PR3), also called myeloblastin, 1 belongs to the family of neutrophil microbicidal serine proteinases that are stored within azurophilic granules. 2 They are considered proinflammatory proteinases because they mediate deleterious effects on host tissues during inflammation. 3,4 Intriguingly, among the numerous proteins contained within azurophilic granules, only PR3 and myeloperoxidase (MPO) are the main targets of antineutrophil cytoplasm antibodies (ANCA) associated with systemic vasculitides. 5,6 More than 80% of Wegener granulomatosis patients have anti-PR3 ANCA, whereas only 10% have anti-MPO ANCA. By contrast, microscopic polyangiitis, Churg-Strauss syndrome, and pauci-immune crescentic glomerulosclerosis are generally associated with anti-MPO ANCA. 7 Unlike MPO, whose subcellular localization is restricted to azurophilic granules, PR3 has been detected on secretory vesicle membranes and the outer leaflet of plasma membranes. 8,9 ANCA have a pathophysiologic role because they activate neutrophils when ANCA antigens are expressed at the neutrophil membrane. 7 We and others demonstrated that high percentages of membrane PR3-positive neutrophils could favor the development or progression of chronic inflammatory diseases, namely vasculitides and rheumatoid arthritis. [10][11][12] Moreover, enhanced membrane PR3 expression was observed on neutrophils from patients with sepsis. 13 Taken together, these data strongly suggest that membrane PR3 expression constitutes a pathogenic factor in ANCA-associated vasculitis and other inflammatory diseases involving neutrophils.Because of the unexpected complex subcellular localization of PR3 in neutrophils, we previously investigated whether it could have specific molecular substrates and, consequently, unanticipated functions. To explore this possibility, we used the rat mast cell lines (RBL-2H3) stably transfected with human neutrophil elastase (HNE), PR3, or its inactive mutant PR3S203A and demonstrated that PR3, unlike its homolog HNE, was able to access cytosolic (eg, the cyclin-dependent kinase inhibitor p21/waf1) 14 or membrane substrates (eg, procaspase-3). 15 We also observed that PR3 could be expressed at the cell surface during apoptosis, independently of degranulation, and was strongly associated with phosphatidylserine (PS) externalization, 16 which is an "eat-me" signal recognized by macrophages. 17,18 Thus, it became evident, at least in our model of stable transfectants, that PR3 was lo...

Engagement of the IgE receptor (Fc⑀RI) on mast cells leads to the release of preformed and newly formed mediators as well as of cytokines. The signaling pathways responsible for these responses involve tyrosine phosphorylation of multiple proteins. We previously reported the phosphorylation on tyrosine of phospholipid scramblase 1 (PLSCR1) after Fc⑀RI aggregation. Here, PLSCR1 expression was knocked down in the RBL-2H3 mast cell line using short hairpin RNA. Knocking down PLSCR1 expression resulted in significantly impaired degranulation responses after Fc⑀RI aggregation and release of vascular endothelial growth factor, whereas release of MCP-1 was minimally affected. The release of neither leukotriene C4 nor prostaglandin D2 was altered by knocking down of PLSCR1. Analysis of Fc⑀RI-dependent signaling pathways revealed that whereas tyrosine phosphorylation of ERK and Akt was unaffected, tyrosine phosphorylation of LAT was significantly reduced in PLSCR1 knocked down cells. Tyrosine phosphorylation of phospholipase C␥1 and consequently the mobilization of calcium were also significantly reduced in these cells. In nonactivated mast cells, PLSCR1 was found in part in lipid rafts where it was further recruited after cell activation and was constitutively associated with Lyn and Syk but not with LAT or Fyn. Altogether, these data identify PLSCR1 as a novel amplifier of Fc⑀RI signaling that acts selectively on the Lyn-initiated LAT/phospholipase C␥1/calcium axis, resulting in potentiation of a selected set of mast cell responses.Mast cells actively participate in the defense against bacterial and parasitic infections (1, 2) and are key players in the allergic reaction. They express receptors that bind IgE with a high affinity (Fc⑀RI) and that are aggregated by the cognate allergen when they are occupied by allergen-specific IgE (3). This results in a number of cellular responses, such as immediate release of granule contents (e.g. histamine and ␤-hexosaminidase), synthesis of the lipid mediators of inflammation leukotrienes (LT) 6 and prostaglandins (PG), and delayed production and release of cytokines and chemokines (4).The pathways leading from Fc⑀RI aggregation to cellular responses have been extensively studied and depend on tyrosine phosphorylations (5, 6). The two main pathways involve tyrosine kinases belonging to the Src family, Lyn and Fyn (4). Lyn is constitutively associated with Fc⑀RI ␤ chain (7). Upon aggregation of the receptor it phosphorylates both the ␤ and the ␥ chains of Fc⑀RI on tyrosine residues within the immunoreceptor tyrosine-based activation motif that is found in their intracellular domains (8). This results in the recruitment of additional Lyn molecules and of the tyrosine kinase Syk to the phosphorylated receptors and in the activation of Syk (9 -11). Lyn and Syk then phosphorylate a number of substrates including the scaffold proteins LAT (12) and LAT2 (13-15). Activation of PLC␥1 and PLC␥2 recruited to phosphorylated LAT leads to the release in the cytosol of inositol 1,4,5-trisphospha...

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