Durant Stéphanie, Pederzoli Magali, Lepelletier Yves, Canteloup Sandrine, Nusbaum Patrick, Lesavre Philippe, Witko-Sarsat Véronique
Inserm U507, Hôpital Necker, 161, rue de Sèvres, 75015 Paris, France.
J Leukoc Biol. 2004 Jan;75(1):87-98. doi: 10.1189/jlb.0203079. Epub 2003 Oct 2.
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.
蛋白酶3（PR3）和人中性粒细胞弹性蛋白酶（HNE）是储存于中性粒细胞嗜天青颗粒中的丝氨酸蛋白酶。与HNE不同，PR3是韦格纳肉芽肿中抗中性粒细胞胞浆抗体（ANCA）的靶抗原。导致PR3和HNE膜表达的机制仍不清楚，而这似乎对于理解ANCA的病理生理作用至关重要。利用稳定转染了PR3或HNE的大鼠嗜碱性细胞系（RBL）来分析PR3和HNE的分泌机制并对它们加以区分。RBL细胞缺乏内源性PR3和HNE。它们被稳定转染了HNE或PR3或PR3的无活性突变体（PR3S203A）。使用钙离子载体A23187作为促分泌剂，RBL/HNE上清液中分泌的丝氨酸蛋白酶活性高于RBL/PR3。有趣的是，PR3和PR3/S203A也在质膜上表达，因此表明质膜表达不需要丝氨酸蛋白酶活性。相比之下，在RBL/HNE中未检测到质膜HNE的表达。通过DNA片段化、细胞质组蛋白相关DNA片段的存在以及膜联蛋白V标记来评估依托泊苷诱导的细胞凋亡。在RBL/HNE中未检测到膜HNE。相反，在RBL/PR3和RBL/PR3S203A中，PR3和PR3S203A的膜表达随依托泊苷浓度增加，且似乎与膜联蛋白V标记密切相关。我们的数据表明，膜PR3来源于两个不同的池，一个是脱颗粒后动员的颗粒池，另一个是凋亡时动员的质膜池。
