Levi-Schaffer F, Lacy P, Severs N J, Newman T M, North J, Gomperts B, Kay A B, Moqbel R
Department of Allergy and Clinical Immunology, Royal Brompton Hospital, London, UK.
Blood. 1995 May 1;85(9):2579-86.
We have previously shown that normal-density human peripheral blood eosinophils transcribe and translate mRNA for granulocyte-macrophage colony-stimulating factor (GM-CSF) and that the intracellular distribution was granular as assessed by light microscopy immunocytochemistry. The present study was conducted to confirm this apparent association between GM-CSF and the crystalloid granule using a subcellular fractionation method for human eosinophils and immunogold electron microscopy (EM). Highly purified (> 99%, by negative selection using anti-CD16 immunomagnetic microbeads) human peripheral blood eosinophils were obtained from four asthmatic subjects (not taking systemic medication), homogenized and density fractionated (5 x 10(7) cells/subject) on linear Nycodenz gradients. Twenty-four fractions were collected from each cell preparation and analyzed for marker enzyme activities as well as total protein. Dot blot analysis with specific monoclonal antibodies (MoAbs) was used to detect the eosinophil granule proteins major basic protein (MBP) and eosinophil cationic protein (ECP). An anti-CD9 MoAb was used as an eosinophil plasma membrane marker. Lactate dehydrogenase (LDH) was used as a cytosolic marker. Immunoreactivity for GM-CSF was detected by a specific enzyme-linked immunosorbent assay using a polyclonal antihuman GM-CSF antibody and confirmed by dot blot. GM-CSF coeluted with the cellular fractions containing granule markers (MBP, ECP, eosinophil peroxidase, hexosaminidase, and arylsulphatase), but not those containing cytoplasm (LDH+) or membrane (CD9+) markers. EM examination of pooled fractions associated with the peak of GM-CSF immunoreactivity confirmed that they contained crystalloid and small granules, but not plasma membrane. In addition, quantification, using immunogold labeling with an anti/GM-CSF MoAb, indicated preferential localization of gold particles over the eosinophil granule cores of intact cells. Thus, our results indicate that GM-CSF resides as a granule-associated, stored mediator in unstimulated human eosinophils.
我们之前已经表明，正常密度的人外周血嗜酸性粒细胞能够转录和翻译粒细胞-巨噬细胞集落刺激因子（GM-CSF）的mRNA，并且通过光学显微镜免疫细胞化学评估，其细胞内分布呈颗粒状。本研究采用人嗜酸性粒细胞亚细胞分级分离方法和免疫金电子显微镜（EM）来证实GM-CSF与晶体颗粒之间的这种明显关联。从四名哮喘患者（未服用全身药物）中获取高度纯化（通过使用抗CD16免疫磁珠阴性选择，纯度> 99%）的人外周血嗜酸性粒细胞，匀浆后在线性Nycodenz梯度上进行密度分级分离（每位受试者5×10⁷个细胞）。从每个细胞制剂中收集24个级分，并分析标记酶活性以及总蛋白。使用特异性单克隆抗体（MoAbs）进行斑点印迹分析，以检测嗜酸性粒细胞颗粒蛋白主要碱性蛋白（MBP）和嗜酸性粒细胞阳离子蛋白（ECP）。使用抗CD9 MoAb作为嗜酸性粒细胞质膜标记物。乳酸脱氢酶（LDH）用作胞质标记物。使用多克隆抗人GM-CSF抗体通过特异性酶联免疫吸附测定法检测GM-CSF的免疫反应性，并通过斑点印迹法进行确认。GM-CSF与含有颗粒标记物（MBP、ECP、嗜酸性粒细胞过氧化物酶、己糖胺酶和芳基硫酸酯酶）的细胞级分共洗脱，但不与含有细胞质（LDH⁺）或膜（CD9⁺）标记物的级分共洗脱。对与GM-CSF免疫反应性峰值相关的合并级分进行EM检查证实，它们含有晶体和小颗粒，但不含质膜。此外，使用抗/GM-CSF MoAb进行免疫金标记的定量分析表明，完整细胞的嗜酸性粒细胞颗粒核心上金颗粒优先定位。因此，我们的结果表明，GM-CSF作为一种颗粒相关的储存介质存在于未受刺激的人嗜酸性粒细胞中。
