Cellular events associated with the initial phase of AA amyloidogenesis: insights from a human monocyte model

Abstract

Reactive amyloidosis is a systemic protein deposition disease that develops in association with chronic inflammation. The deposits are composed of extracellular, fibrillar masses of amyloid A (AA) protein, an N-terminal fragment of the acute-phase serum protein serum amyloid A (SAA). The pathogenic conversion of SAA into amyloid has been studied in two human cell culture models, peritoneal cells and peripheral blood monocytes. Human monocyte cultures proved more robust than either mouse or human peritoneal cells at initiating amyloid formation in the absence of a preformed nidus such as amyloid-enhancing factor and particularly well suited for examination of individual cells undergoing amyloid formation. Amyloid-producing monocyte cultures were stained with Congo red and Alcian blue for detection of amyloid and glycosaminglycans, respectively; immunocytochemistry was performed to identify SAA/AA, CD68, CD14, lysosomal protein Lamp-1, and early endosomal protein EEA1. SAA interaction with monocytes was also visualized directly via fluorescence confocal microscopy. Amyloid was initially detected only in intracellular vesicles, but with time was seen extracellularly. Morphologic changes in lysosomes were noted during the early phase of amyloid formation, suggesting that exocytosis of fibrils may occur via lysosome-derived vesicles. Cultures engaged in amyloid formation remained metabolically active; no cytotoxic effects were observed. Mimicking in vivo phenomena, amyloid formation was accompanied by increased glycosaminoglycan content and C-terminal processing of SAA. The ability of human monocytes to endocytose and intracellularly transform SAA into amyloid via a mechanism that requires and maintains, rather than compromises, metabolic activity distinguishes them as a useful model for probing earliest events in the disease process.

Keywords:

• Serum amyloid A
• AA amyloidosis
• monocyte
• human
• cell culture
• acute-phase reactant

Abbreviations
SAA	=	serum amyloid A
AA	=	amyloid A
HDL	=	high-density lipoprotein
AEF	=	amyloid-enhancing factor
GAG	=	glycosaminoglycan
TCA	=	trichloroacetic acid
FBS	=	fetal bovine serum

Abbreviations
SAA	=	serum amyloid A
AA	=	amyloid A
HDL	=	high-density lipoprotein
AEF	=	amyloid-enhancing factor
GAG	=	glycosaminoglycan
TCA	=	trichloroacetic acid
FBS	=	fetal bovine serum