Cell surface expression of mouse macrosialin and human CD68 and their role as macrophage receptors for oxidized low density lipoprotein
Cell surface expression of mouse macrosialin and human CD68 and their role as macrophage receptors for oxidized low density lipoprotein
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DOI：
10.1073/pnas.93.25.14833
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发表时间：
1996-12-10
影响因子：
11.1
通讯作者：
Steinberg, D
中科院分区：
文献类型：
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作者：
Ramprasad, MP;Terpstra, V;Steinberg, D
我们先前在小鼠巨噬细胞中鉴定出一种94 - 97kDa的氧化低密度脂蛋白（LDL）结合蛋白为大唾液酸蛋白（MS），它是溶酶体相关膜蛋白（lamp）家族的成员。早期的免疫染色研究表明，MS及其人类同源物CD68主要是细胞内蛋白。然而，利用诸如流式细胞术（FACS）和细胞表面特异性生物素化等灵敏技术，我们现在发现这些蛋白有显著的表面表达。使用单克隆抗体FA/11对完整细胞进行FACS分析表明，在未受刺激的小鼠腹腔巨噬细胞中MS有少量但明确的表面表达，但在巯基乙酸盐诱导后这种表达大大增强。对完整细胞进行生物素化以及对洗涤剂溶解的细胞制剂进行免疫沉淀，结果显示在诱导的巨噬细胞中，质膜上的MS含量占总MS含量的10 - 15%。对未处理的RAW 264.7细胞也得到了类似结果。对完整的THP - 1单核细胞进行FACS分析表明，未活化细胞上CD68的表面表达极少（占细胞总含量的4%）。用佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯刺激后，表面和总CD68表达都显著增加。此外，活化的THP - 1细胞在4℃的特异性结合以及在37℃对¹²⁵I标记的氧化LDL的摄取被CD68单克隆抗体KP - 1和EBM - 11抑制了30 - 50%。因此，尽管在稳态下MS/CD68的表面表达仅占其细胞总含量的一小部分，但这些蛋白在体外活化巨噬细胞摄取氧化LDL中可发挥重要作用，并可能有助于动脉粥样硬化病变中泡沫细胞的形成。
We have previously identified a 94- to 97-kDa oxidized low density lipoprotein (LDL)-binding protein in mouse macrophages as macrosialin (MS), a member of the lamp family. Earlier immunostaining studies have shown that MS and its human homolog, CD68, are predominantly intracellular proteins, However, using sensitive techniques such as flow cytometry (FAGS) and cell-surface-specific biotinylation, we now show that there is significant surface expression of these proteins, FAGS analysis of intact cells using mAb FA/11 showed small but definite surface expression of MS in resident mouse peritoneal macrophages but this,vas greatly enhanced with thioglycollate elicitation, Biotinylation of intact cells and detergent-solubilized cell preparations followed by immunoprecipitation revealed 10-15% of the total MS content of elicited macrophages on the plasma membrane, Similar results were obtained with untreated RAW 264.7 cells, FAGS analysis of intact THP-1 monocytic cells showed minimal surface expression of CD68 on unactivated cells (4% of total cell content). Stimulation with phorbol 12-myristate W-acetate increased both surface and total CD68 expression considerably, Furthermore, the specific binding at 4 degrees C and uptake at 37 degrees C of I-125-labeled oxidized LDL by activated THP-1 cells was inhibited by 30-50% by CD68 mAbs KP-1 and EBM-11. Thus, although the sill-face expression of MS/CD68 at steady-state represents only a small percentage of their total cellular content, these proteins can play a significant role in oxidized LDL uptake by activated macrophages in vitro and could contribute to foam cell formation in atherosclerotic lesions.