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.

Proteinase 3, the Autoantigen in Granulomatosis with Polyangiitis, Associates with Calreticulin on Apoptotic Neutrophils, Impairs Macrophage Phagocytosis, and Promotes Inflammation

“…In contrast with neutrophils, neither PR3 nor CRT was expressed at the cell surface of resting RBL/PR3 cells (18). However, after gliotoxin-induced apoptosis, the majority of the cells externalized PS and expressed both PR3 and CRT at their surface.…”

“…The coimmunoprecipitation of CRT with PR3 was confirmed in RBL/PR3 cells (Fig. 1B), which we have used previously to study membrane PR3 (5,6,18). PR3 binding on different domains of immobilized CRT was then analyzed using SPR spectroscopy (Fig.…”

“…To induce physiological apoptosis, we resuspended the neutrophils at 2 3 10 6 /ml in RPMI-1640 (Life Technologies) with 10% FCS and incubated them for 15 h at 37˚C. PS externalization was quantified by PE-Annexin V labeling after necrotic 7-amino-actinomycin D (7-AAD)-labeled cells had been excluded from the analysis using a FACScan flow cytometer (Cell Quest software and Becton Dickinson Immunocytometry Systems), as described previously (18).…”

“…Apoptosis was induced by 16-h incubation at 37˚C with 2 mg/ml gliotoxin (Sigma), as described previously (6). Degranulation was induced with 2 mM A23187 calcium ionophore and was evaluated by measuring b-hexosaminidase activity in the degranulation supernatant, using the chromogenic substrate p-nitrophenyl-N-acetyl-b-D-glucosaminide (Sigma), as described previously (18). To study protein traffic, we treated RBL cells (10 6 /ml) for 1 h with 10 mg/ml brefeldin A (BFA) to block the endoplasmic reticulum to Golgi complex transport.…”

“…Rat basophilic cell line (RBL) cells were cultured as described previously (18,19). Recombinant human PR3 or the membrane-anchoragedeficient PR3 mutant, PR34H4A, was expressed in stably transfected RBL cells as described previously (6,19).…”