Brzezinska Agnieszka A, Johnson Jennifer L, Munafo Daniela B, Crozat Karine, Beutler Bruce, Kiosses William B, Ellis Beverly A, Catz Sergio D
Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Traffic. 2008 Dec;9(12):2151-64. doi: 10.1111/j.1600-0854.2008.00838.x. Epub 2008 Oct 30.
Neutrophil granules contain secretory molecules that contribute to the implementation of all neutrophil functions. The molecular components that regulate the exocytosis of neutrophil granules have not been characterized. In this study, using small interfering RNA gene-targeting approaches and granulocytes from genetically modified mice, we characterized the Rab27a effectors JFC1/Slp1 and Munc13-4 as components of the exocytic machinery of granulocytes. Using total internal reflection fluorescence microscopy analysis, we show that Rab27a and JFC1 colocalize in predocked and docked vesicles in granulocytes. Next, we demonstrate that JFC1-downregulated granulocytes have impaired myeloperoxidase secretion. Using immunological interference, we confirm that JFC1 plays an important role in azurophilic granule exocytosis in human neutrophils. Interference with Rab27a but not with JFC1 impaired gelatinase B secretion in neutrophils, suggesting that a different Rab27a effector modulates this process. In similar studies, we confirmed that Munc13-4 regulates gelatinase secretion. Immunofluorescence analysis indicates that Munc13-4 localizes at secretory organelles in neutrophils. Using neutrophils from a Munc13-4-deficient mouse model (Jinx), we demonstrate that Munc13-4 plays a central role in the regulation of exocytosis of various sets of secretory organelles. However, mobilization of CD11b was not affected in Munc13-4-deficient neutrophils, indicating that secretory defects in these cells are limited to a selective group of exocytosable organelles.
中性粒细胞颗粒含有有助于实现所有中性粒细胞功能的分泌分子。调节中性粒细胞颗粒胞吐作用的分子成分尚未得到明确鉴定。在本研究中，我们使用小干扰RNA基因靶向方法和来自转基因小鼠的粒细胞，将Rab27a效应蛋白JFC1/Slp1和Munc13-4鉴定为粒细胞胞吐机制的组成成分。通过全内反射荧光显微镜分析，我们发现Rab27a和JFC1在粒细胞中预对接和对接的囊泡中共定位。接下来，我们证明JFC1下调的粒细胞髓过氧化物酶分泌受损。通过免疫干扰，我们证实JFC1在人类中性粒细胞嗜天青颗粒胞吐中起重要作用。干扰Rab27a而非JFC1会损害中性粒细胞中明胶酶B的分泌，这表明不同的Rab27a效应蛋白调节这一过程。在类似研究中，我们证实Munc13-4调节明胶酶分泌。免疫荧光分析表明Munc13-4定位于中性粒细胞的分泌细胞器。使用来自Munc13-4缺陷小鼠模型（Jinx）的中性粒细胞，我们证明Munc13-4在调节各种分泌细胞器的胞吐作用中起核心作用。然而，Munc13-4缺陷的中性粒细胞中CD11b的动员不受影响，这表明这些细胞中的分泌缺陷仅限于一组可胞吐的选择性细胞器。
