Major co-localization of the extracellular-matrix degradative enzymes heparanase and gelatinase in tertiary granules of human neutrophils
Major co-localization of the extracellular-matrix degradative enzymes heparanase and gelatinase in tertiary granules of human neutrophils
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DOI：
10.1042/bj3270917
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发表时间：
1997-11-01
影响因子：
4.1
通讯作者：
Fabra, A
中科院分区：
文献类型：
--
作者：
Mollinedo, F;Nakajima, M;Fabra, A
细胞表面粘附蛋白的表达和细胞外基质降解酶的释放是中性粒细胞附着于内皮细胞和随后这些细胞通过内皮层外渗的关键过程。我们分析了静止的人类中性粒细胞中肝素酶的亚细胞定位，肝素酶是一种硫酸肝素降解内糖苷酶，可以降解基底膜成分，从而促进炎症反应期间中性粒细胞进入组织。通过对静止的人中性粒细胞核后上清液在连续蔗糖梯度上的亚细胞分离，我们发现肝素酶活性主要位于含有明胶酶的三级颗粒中，使用特异性抗体，96-kDa的肝素酶蛋白进一步位于富含明胶酶的亚细胞部分。在对不同亚细胞组分进行免疫印迹和免疫沉淀分析后，我们还发现在富含明胶酶三级颗粒的组分中，肝素酶和Mol (CD11b/CD18)（一种白细胞整合素，参与中性粒细胞与内皮的附着）共定位。用肿瘤坏死因子或粒细胞/巨噬细胞集落刺激因子处理人中性粒细胞诱导CD11b/CD18细胞表面表达增加，以及明胶酶（基质金属蛋白酶-9）和肝素酶的释放，但其他颗粒标记物不释放，表明明胶酶、肝素酶和CD11b/CD18在同一细胞器中主要共定位。此外，使用针对明胶酶和肝素酶的特异性抗体的共聚焦激光扫描显微镜显示，这两种酶在细胞内细胞质颗粒中主要共定位。肝素酶和CD11b/CD18在含明胶酶的三级颗粒中的主要定位支持了这一细胞器的动员可以调节人类中性粒细胞外渗的观点。
The expression of cell-surface adhesion proteins and the release of extracellular-matrix degradative enzymes constitute crucial processes for the attachment of neutrophils to the endothelium and for the subsequent extravasation of these cells through the endothelial layer. We have analysed in resting human neutrophils the subcellular localization of heparanase, a heparan-sulphate-degrading endoglycosidase that can degrade basement-membrane components, thereby facilitating neutrophil passage into the tissue during an inflammatory reaction. By subcellular fractionation of postnuclear supernatants from resting human neutrophils on continuous sucrose gradients, we have found that heparanase activity was mainly located in gelatinase-containing tertiary granules, Using a specific antibody, the 96-kDa heparanase protein was further located in the gelatinase-rich subcellular fractions. Following immunoblotting and immunoprecipitation analysis in the distinct subcellular fractions, we also found colocalization of heparanase and Mol (CD11b/CD18), a leucocyte integrin involved in the attachment of neutrophils to the endothelium, in the fractions enriched in gelatinase-containing tertiary granules. Treatment of human neutrophils with tumour necrosis factor or granulocyte/macrophage colony-stimulating factor induced an increase in the CD11b/CD18 cell-surface expression, as well as the release of both gelatinase (matrix metalloproteinase-9) and heparanase, but not of other granule markers, indicating a major co-localization of gelatinase, heparanase and CD11b/CD18 in the same organelle. Furthermore, confocal laser scanning microscopy using specific antibodies against gelatinase and heparanase revealed a major co-localization of both enzymes in intracellular cytoplasmic granules. The major localization of heparanase and CD11b/CD18 in the gelatinase-containing tertiary granule supports the notion that mobilization of this organelle can regulate extravasation of human neutrophils.