Abstract：
A key molecule for neutrophil degranulation is Rac2 guanosine triphosphatase. Neutrophils from Rac2 knockout mice (Rac2-/-) exhibit impaired primary granule exocytosis in response to cytochalasin B/f-Met-Leu-Phe, while secondary and tertiary granule release is unaffected. Coronin 1A, a protein involved in actin remodeling, is diminished in Rac2-/- neutrophils. However, primary granule exocytosis from Rac2-/- neutrophils has not been determined using more immunologically relevant stimuli. We sought to determine the role of Rac2 in degranulation and actin cytoskeleton rearrangement in response to immobilized immune complexes and relate this to intracellular coronin 1A localization. We used bone marrow neutrophils from wild-type and Rac2-/- mice stimulated with immobilized immune complexes. Secretion of primary (myeloperoxidase), secondary (lactoferrin), and tertiary granule (MMP-2 and MMP-9) products was evaluated. Subcellular colocalization of coronin 1A with actin and the primary granule marker CD63 was determined by deconvolution microscopy. We found major differences in myeloperoxidase, MMP-2, and MMP-9 but not lactoferrin release, along with diminished filopodia formation, CD63 polarization, and colocalization of coronin 1A with CD63 in immune complex-stimulated Rac2-/- bone marrow neutrophils. Rac2 and coronin 1A were found associated with granules in cytochalasin B/f-Met-Leu-Phe-activated human neutrophils. This report confirms a role for Rac2 in immunologically relevant stimulation of neutrophil granule exocytosis. Rac2 appears to attach to neutrophil granules, polarize CD63+ granules to the cell surface in a manner dependent on coronin 1A, and induce filopodia formation. Our studies provide insight into mechanisms of Rac2-mediated regulation of granule exocytosis.
摘要：
中性粒细胞脱颗粒的关键分子是Rac2鸟苷三磷酸酶。来自Rac2敲除小鼠（Rac2-/-）的中性粒细胞对细胞松弛素B/f-Met-Leu-Phe的反应表现出初级颗粒胞吐作用受损，而二级和三级颗粒释放不受影响。Coronin1A,一种参与肌动蛋白重塑的蛋白质，在Rac2-/-中性粒细胞中减少。然而,尚未使用更多的免疫学相关刺激确定Rac2-/-中性粒细胞的初级颗粒胞吐作用。我们试图确定Rac2在响应固定化免疫复合物的脱粒和肌动蛋白细胞骨架重排中的作用，并将其与细胞内冠蛋白1A定位联系起来。我们使用来自用固定化免疫复合物刺激的野生型和Rac2-/-小鼠的骨髓嗜中性粒细胞。原发性（髓过氧化物酶）的分泌，次要（乳铁蛋白），并对三级颗粒(MMP-2和MMP-9)产品进行评价。通过去卷积显微镜确定冠状蛋白1A与肌动蛋白和主要颗粒标记CD63的亚细胞共定位。我们发现髓过氧化物酶的主要差异，MMP-2和MMP-9，但不是乳铁蛋白释放，随着丝状伪足形成的减少，CD63极化，以及免疫复合物刺激的Rac2-/-骨髓中性粒细胞中冠状蛋白1A与CD63的共定位。发现Rac2和coronin1A与细胞松弛素B/f-Met-Leu-Phe激活的人中性粒细胞中的颗粒相关。该报告证实了Rac2在中性粒细胞颗粒胞吐的免疫学相关刺激中的作用。Rac2似乎附着在中性粒细胞颗粒上，将CD63+颗粒以依赖于冠状蛋白1A的方式极化到细胞表面，并诱导丝足形成。我们的研究提供了对Rac2介导的颗粒胞吐调节机制的见解。
