Kluve-Beckerman Barbara, Manaloor John J, Liepnieks Juris J
Dpartment of Pathology and laboratoty Medicine, Indiana University School of Medicine, Indianapolis, 46202, USA.
Arthritis Rheum. 2002 Jul;46(7):1905-13. doi: 10.1002/art.10335.
To determine whether serum amyloid A (SAA) is internalized by and processed in macrophages en route to deposition as extracellular amyloid.
SAA was tracked in cultures of peritoneal macrophages, using a pulse-chase protocol. Macrophages were pulsed with either fluorescently (with Texas Red) tagged SAA (TxR-SAA) or iodinated SAA ((125)I-SAA). Cells were then rinsed and shifted to chase medium containing unlabeled SAA and amyloid-enhancing factor (AEF) to induce amyloid formation. At selected times, TxR-SAA in living cells was observed by confocal scanning microscopy. (125)I-SAA was visualized and quantified in cell lysates and medium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and phosphorimaging. The presence of amyloid was confirmed by Congo red staining.
Confocal microscopy immediately after the pulse revealed TxR-SAA in endosomal vesicles, with no extracellular or cell surface accumulation. After 24 hours and 72 hours of chase, virtually all TxR-SAA remained intracellular. By 10 days, extracellular fluorescence was very strong, indicating that SAA had moved out of cells. Congo red staining revealed amyloid colocalized with areas of extracellular fluorescence. Experiments using (125)I-SAA showed that while 90-95% of internalized (125)I-SAA was degraded within 24 hours, 5-10% persisted as intact SAA or SAA peptides. Immediately after the pulse, SAA was full-length, but within 24 hours, discrete (125)I-SAA peptides were seen. Each peptide had an intact SAA amino-terminus, as expected for AA protein. Amyloid was detected in cultures as early as 24 hours after initiation of treatment with SAA and AEF and appeared to be intracellular.
The results of this study provide direct evidence that SAA internalized by and processed in macrophages forms extracellular amyloid. Based on the presence of (125)I-AA protein in macrophage lysates prior to the appearance of extracellular TxR-labeled amyloid, it was concluded that cleavage of SAA to AA occurs intracellularly.
确定血清淀粉样蛋白A（SAA）在巨噬细胞内被摄取并加工处理后是否会沉积为细胞外淀粉样物质。
采用脉冲追踪实验方案，在腹膜巨噬细胞培养物中追踪SAA。用荧光（德克萨斯红）标记的SAA（TxR-SAA）或碘化SAA（¹²⁵I-SAA）对巨噬细胞进行脉冲处理。然后冲洗细胞，并转移至含有未标记SAA和淀粉样增强因子（AEF）的追踪培养基中以诱导淀粉样物质形成。在选定的时间点，通过共聚焦扫描显微镜观察活细胞中的TxR-SAA。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和磷光成像对细胞裂解物和培养基中的¹²⁵I-SAA进行可视化和定量分析。用刚果红染色确认淀粉样物质的存在。
脉冲处理后立即进行共聚焦显微镜观察，发现TxR-SAA存在于内体囊泡中，细胞外或细胞表面无积聚。追踪24小时和72小时后，几乎所有的TxR-SAA仍保留在细胞内。到第10天，细胞外荧光非常强，表明SAA已移出细胞。刚果红染色显示淀粉样物质与细胞外荧光区域共定位。使用¹²⁵I-SAA的实验表明，虽然90 - 95%内化的¹²⁵I-SAA在24小时内被降解，但5 - 10%以完整的SAA或SAA肽的形式持续存在。脉冲处理后立即检测到的SAA是全长的，但在24小时内，可见离散的¹²⁵I-SAA肽。每个肽都有完整的SAA氨基末端，这与AA蛋白预期的情况一致。在用SAA和AEF处理后最早24小时就在培养物中检测到淀粉样物质，且似乎是细胞内的。
本研究结果提供了直接证据，表明在巨噬细胞内被摄取并加工处理的SAA会形成细胞外淀粉样物质。基于在细胞外TxR标记的淀粉样物质出现之前巨噬细胞裂解物中存在¹²⁵I-AA蛋白，得出SAA裂解为AA发生在细胞内的结论。
