The overall goal of this project is to elucidate the pathogenic processes underlying reactive (secondary) amyloidosis. This disease develops as a complication of chronic inflammation and is characterized by extracellular deposits of amyloid A (AA) protein, a peptide proteolytically derived from the acute phase protein, serum amyloid A (SAA). Although the pathogenesis of reactive amyloidosis remains largely undefined, macrophages have long been implicated to play a role possibly through altered metabolic processing of SAA. Macrophages cultured in the presence of recombinant SAA2 exhibit amyloid deposition and thus represent a manipulatable system for studying pathogenesis at the cellular level. This system will be employed to test the hypothesis that the amyloidogenic pathway of SAA involves trafficking through macrophages along a course similar to that followed by transferrin, i.e., concentration in coated vesicles, delivery to acidic sorting endosomes, and subsequent trafficking through recycling compartments with translocation to the cell surface, the site of amyloid fibril deposition. Experiments will be carried out to: (a) characterize the binding, uptake, accumulation in/on cells, release into the medium, and degradation of highly amyloidogenic SAA2 under optimal amyloid-forming conditions, (b) simultaneously track SAA2 and transferrin through macrophages using fluorescent immunocytochemistry, and (c) localize SAA2 at the ultrastructural level using immunogold electron microscopy. The same studies will then be performed using non-amyloidogenic CE/J SAA proteins. By comparing the fates of these proteins, it will be possible to identify metabolic events which distinguish the amyloidogenic pathway from non-amyloidogenic (degradative) pathway. The metabolic step at which amyloid-enhancing factor (AEF) exerts its influence will also be determined. An understanding of the cellular pathway leading to amyloid formation should greatly facilitate the development of strategies to prevent or arrest amyloid fibril formation.
