A pulse-chase study tracking the conversion of macrophage-endocytosed serum amyloid A into extracellular amyloid

Objective. To determine whether serum amyloid A (SAA) is internalized by and processed in macrophages en route to deposition as extracellular amyloid. Methods. 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 (I-125-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. I-125-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. Results. 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 I-125-SAA showed that while 90-95% of internalized I-125-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 I-125-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. Conclusion. The results of this study provide direct evidence that SAA internalized by and processed in macrophages forms extracellular amyloid. Based on the presence of I-125-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.