Ramprasad M P, Terpstra V, Kondratenko N, Quehenberger O, Steinberg D
Department of Medicine, University of California at San Diego, La Jolla 92093-0682, USA.
Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14833-8. doi: 10.1073/pnas.93.25.14833.
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 (FACS) and cell-surface-specific biotinylation, we now show that there is significant surface expression of these proteins. FACS analysis of intact cells using mAb FA/11 showed small but definite surface expression of MS in resident mouse peritoneal macrophages but this was 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. FACS 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 13-acetate increased both surface and total CD68 expression considerably. Furthermore, the specific binding at 4 degrees C and uptake at 37 degrees C of 125I-labeled oxidized LDL by activated THP-1 cells was inhibited by 30-50% by CD68 mAbs KP-1 and EBM-11. Thus, although the surface 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.
我们之前已在小鼠巨噬细胞中鉴定出一种94至97 kDa的氧化型低密度脂蛋白（LDL）结合蛋白，即巨唾液酸蛋白（MS），它是lamp家族的一员。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，现在我们使用诸如流式细胞术（FACS）和细胞表面特异性生物素化等敏感技术表明，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分析显示，在驻留的小鼠腹膜巨噬细胞中，MS有少量但确定的表面表达，但经巯基乙酸诱导后这种表达大大增强。对完整细胞和去污剂溶解的细胞制剂进行生物素化，然后进行免疫沉淀，结果显示，诱导的巨噬细胞中总MS含量的10%至15%存在于质膜上。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分析显示，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 - 乙酸酯刺激可显著增加CD68的表面表达和总表达量。此外，活化的THP-1细胞对125I标记的氧化型LDL在4℃的特异性结合以及在37℃的摄取，可被CD68单克隆抗体KP-1和EBM-11抑制30%至50%。因此，尽管MS/CD68在稳态时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型LDL过程中可发挥重要作用，并可能有助于动脉粥样硬化病变中泡沫细胞的形成。
