pepstatin and Amyloidosis

A murine macrophage culture system that is both easy to employ and amenable to manipulation has been developed to study the cellular processes involved in AA amyloid formation. Amyloid deposition, as identified by Congo red-positive, green birefringent material, is achieved by providing cultures with recombinant serum amyloid A2 (rSAA2), a defined, readily produced, and highly amyloidogenic protein. In contrast to fibril formation, which can occur in vitro with very high concentrations of SAA and low pH, amyloid deposition in culture is dependent on metabolically active macrophages maintained in neutral pH medium containing rSAA2 at a concentration typical of that seen in acute phase serum. Although amyloid-enhancing factor is not required, its addition to culture medium results in larger and more numerous amyloid deposits. Amyloid formation in culture is accompanied by C-terminal processing of SAA and the generation of an 8.5-kd fragment analogous to amyloid A protein produced in vivo. Consistent with the possibility that impaired catabolism of SAA plays a role in AA amyloid pathogenesis, treatment of macrophages with pepstatin, an aspartic protease inhibitor, results in increased amyloid deposition. Finally, the amyloidogenicity exhibited by SAA proteins in macrophage cultures parallels that seen in vivo, eg, SAA2 is highly amyloidogenic, whereas CE/J SAA is nonamyloidogenic. The macrophage culture model presented here offers a new approach to the study of AA amyloid pathogenesis.

Topics: Amyloid; Amyloidosis; Animals; Apolipoproteins; Cell Adhesion; Cells, Cultured; Glycoproteins; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Pepstatins; Protease Inhibitors; Protein Isoforms; Recombinant Proteins; Serum Amyloid A Protein