Phospholipid scramblase 1 modulates a selected set of IgE receptor-mediated mast cell responses through LAT-dependent pathway.

Engagement of the IgE receptor (FcepsilonRI) 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 FcepsilonRI 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 FcepsilonRI 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 FcepsilonRI-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 Cgamma1 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 FcepsilonRI signaling that acts selectively on the Lyn-initiated LAT/phospholipase Cgamma1/calcium axis, resulting in potentiation of a selected set of mast cell responses.

Proteinase 3, the Wegener autoantigen, is externalized during neutrophil apoptosis: evidence for a functional association with phospholipid scramblase 1 and interference with macrophage phagocytosis.

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, CD66b, CD63, myeloperoxidase, or elastase expression). This observation was confirmed on cytoplasts, a model of granule-free neutrophils. We hypothesized that PR3 could interact with proteins involved in membrane flip-flop (eg, phospholipid scramblase 1 [PLSCR1]). PR3-PLSCR1 interaction in neutrophils was demonstrated by confocal microscopy and coimmunoprecipitation. In the RBL-2H3 rat mast-cell line stably transfected with PR3 or its inactive mutant (PR3S203A), PR3 externalization depended on PLSCR1, as shown by less PR3 externalization in the presence of rPLSCR1 siRNA, but independently of its serine-proteinase activity. Finally, apoptosis-externalized PR3 decreased the human macrophage-phagocytosis rate of apoptotic PR3 transfectants. Therefore, in addition to ANCA binding in vasculitis, the proinflammatory role of membrane PR3 expression may involve interference with macrophage clearance of apoptotic neutrophils.