title: Apoptosis-induced proteinase 3 membrane expression is independent from degranulation.
abstract: Proteinase 3 (PR3) and human neutrophil elastase (HNE) are serine proteinases stored in the azurophilic granules of neutrophils. In contrast to HNE, PR3 is the target of antineutrophil cytoplasm antibodies (ANCA) in Wegener's granulomatosis. The mechanisms leading to the membrane expression of PR3 and HNE are still unclear and appear to be critical to understand the pathophysiological role of ANCA. Stably transfected rat basophilic cell lines (RBL) with PR3 or HNE were used to analyze the PR3 and HNE secretion mechanisms and differentiate between them. RBL cells were lacking endogenous PR3 and HNE. They were stably transfected with HNE or PR3 or an inactive mutant of PR3 (PR3S203A). Using the calcium ionophore A23187 as a secretagogue, higher serine proteinase activity was secreted in the supernatant of RBL/HNE than in RBL/PR3. It is interesting that PR3 and PR3/S203A were also expressed at the plasma membrane, thus demonstrating that serine protease activity was not required for plasma membrane expression. In contrast, no expression of plasma membrane HNE could be detected in RBL/HNE. Apoptosis induced by etoposide was evaluated by DNA fragmentation, the presence of cytoplasmic histone-associated DNA fragments, and annexin V labeling. No membrane HNE was detected in RBL/HNE. In contrast, in RBL/PR3 and in RBL/PR3S203A, the membrane expression of PR3 and PR3S203A increased with etoposide concentrations and appeared closely related to annexin V labeling. Our data suggest that membrane PR3 originates from two distinct pools, the granular pool mobilized following degranulation or a plasma membrane pool mobilized upon apoptosis.
authors.affiliation: Inserm U507, Hôpital Necker, 161, rue de Sèvres, 75015 Paris, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Durant
authors.forename: Magali
authors.initials: M
authors.lastname: Pederzoli
authors.forename: Yves
authors.initials: Y
authors.lastname: Lepelletier
authors.forename: Sandrine
authors.initials: S
authors.lastname: Canteloup
authors.forename: Patrick
authors.initials: P
authors.lastname: Nusbaum
authors.forename: Philippe
authors.initials: P
authors.lastname: Lesavre
authors.forename: Véronique
authors.initials: V
authors.lastname: Witko-Sarsat
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000032:analysis; D019268:Antibodies, Antineutrophil Cytoplasmic / Q000097:blood; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology; D002462:Cell Membrane / Q000201:enzymology; D003594:Cytoplasmic Granules / Q000201:enzymology*; D053938:DNA Fragmentation; D005047:Etoposide / Q000494:pharmacology; D005434:Flow Cytometry; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D015471:Leukemia, Basophilic, Acute; D019272:Leukocyte Elastase / Q000235:genetics; D053146:Myeloblastin; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D017304:Annexin A5; D019268:Antibodies, Antineutrophil Cytoplasmic; D011994:Recombinant Proteins; D000001:Calcimycin; D005047:Etoposide; D012697:Serine Endopeptidases; D019272:Leukocyte Elastase; D053146:Myeloblastin; D002118:Calcium