The role of fibronectin in the development of experimental amyloidosis. Evidence of immunohistochemical codistribution and binding property with serum amyloid protein A.

Azocasein-induced amyloid A (AA) amyloidosis in CBA/K1Jms mice was investigated to elucidate a preference of serum amyloid A (SAA) deposition in the spleen. By indirect immunofluorescence using anti-SAA/AA antibodies the initial deposition of SAA/AA was recognized in the marginal zone of spleen at 20 days after azocasein injection. Indirect immunofluorescence using anti-fibronectin antibodies also showed meshwork positivity in the corresponding area more intensely than that in controls. Immunoelectron microscopy using anti-SAA/AA revealed the presence of positively stained flocculent materials on cell surfaces of macrophages in the marginal area in addition to amyloid fibril. The tissue fibronectin rapidly increased in the spleen and maintained 10 times more than that of controls until the 20th day. Binding assay of SAA on frozen sections revealed the presence of SAA-binding substances in the perifollicular area. Affinity chromatographic assay showed fibronectin have a binding capacity to SAA1 and SAA2. By binding assay on the microtiter plate, SAA had more affinity to fibronectin than those of heparan sulfate, collagen type I, or serum amyloid P component. These results indicate that fibronectin plays an important role in the development of amyloidosis by working as a linking protein between SAA and the cell surface of macrophages.     

Impaired degradation of serum amyloid A (SAA) protein by cytokine-stimulated monocytes

Secondary amyloidosis (AA amyloidosis) is a systemic disease characterized by the extracellular tissue deposition of insoluble amyloid A (AA) protein. Aberrant metabolism of serum amyloid A (SAA) by macrophages is only one of many putative mechanisms which may be important in AA amyloidogenesis. In this study, we investigated the effects of cytokines on human monocyte-mediated SAA proteolysis. Human peripheral blood mononuclear cells (PBMC) or CD14(+) monocytes were cultured with SAA, and the culture supernatants were then subjected to anti-SAA immunoblot. CD14(+) monocytes degraded SAA completely. Whereas, when CD14(+) monocytes were pretreated with IL-1 beta or IFN-gamma, increasing amounts of SAA-related derivatives were detected in culture supernatants. These findings suggest that activation of monocytes by IL-1 beta or IFN-gamma hampers the proteolysis of a precursor protein and leads to a partial degradation of SAA. This down-regulated proteolysis of SAA protein by cytokine-stimulated monocytes may play a role in the mechanism of AA amyloid formation as well as its removal.     

Immunolocalization of serum amyloid A and AA amyloid in lysosomes in murine monocytoid cells: Confocal and immunogold electron microscopic studies

Murine AA amyloid (AA) protein represents the amino-terminal two-third portion of SAA₂, one of the isoforms of serum amyloid A. Whether plasma membrane-bound or lysosomal enzymes in activated murine monocytoid cells degrade SAA₂ to generate amyloidogenic AA-like peptides is not clearly understood, although AA has been localized in the lysosomes. Here we show, using confocal and immunogold microscopy (IEM), that both SAA and AA localize in lysosomes of activated monocytoid cells from amyloidotic mice. Rabbit anti-mouse AA IgG (RAA) and two monoclonal antibodies against murine lysosome-associated membrane proteins (LAMP-1 and LAMP-2) were used to immunolocalize SAA/AA and lysosomes, respectively. Confocal analysis co-localized both anti-RAA and anti-LAMP-1/LAMP-2 reactivities in the perikaryal organelles which by IEM proved to be electron-dense lysosomes. LAMP-1/LAMP-2-specific gold particles were also localized on lysosomal and perikaryal AA. The results suggest sequestration of SAA into the lysosomes. Since monocytoid cells are not known to phagocytose native amyloid fibrils, our results implicate lysosomes in AA formation.     

Ubiquitin profile in inflammatory leukocytes and binding of ubiquitin to murine AA amyloid: Immunocytochemical and immunogold electron microscopic studies

Lysosomes in activated murine monocytoid cells have been implicated in AA amyloid formation. The pathophysiology of this process is not well understood. Previous studies into the nature of the relationship between ubiquitin (UB), possessing intrinsic amyloid enhancing factor (AEF) activity; serum amyloid A (SAA), the precursor protein of AA amyloid; and activated monocytoid cells have indicated a temporal and spatial relationship between these proteins and tissue AA amyloid deposits. To extend these findings, we have examined murine peritoneal leukocytes and splenic tissues during the early amyloid deposition phase by immunocytochemical and immunogold electron microscopic methods using monospecific anti-ubiquitin and anti-mouse AA amyloid antibodies. We show here enrichment of endosome–lysosome-like (EL) vesicles in the activated monocytoid cells with UB and SAA, and the presence of UB-bound AA amyloid fibrils in the EL vesicles, perikarya, and interstitial spaces. The importance of these findings is emphasized by the fact that activated monocytoid cells, containing UB in the EL vesicles, sequester and eventually localize SAA in their EL vesicles, and that UB binds to the EL-contained AA amyloid fibrils. These findings may also have functional consequences for studies on the role of EL and UB in amyloidogenesis.     

Multinodular Deposition of AA-type Amyloid Localized in the Adrenal Glands of an Old Man

Multinodular amyloid deposits localized in non neoplastic adrenal glands were found incidentally at autopsy in an 83-year-old Japanese man. Clinically, the patient lacked evident deficiency of adrenal hormones. The nodules of the stromal amyloid deposits were scattered in the adrenal cortex, where the parenchymal cells were compressed and atrophic. The deposits were confirmed to be amyloid by Congo red staining and polarization microscopy. Amyloid fibrils were also demonstrated in the deposits by electron microscopy. The amyloid deposits were permanganate-sensitive and showed immunohistochemical staining for serum amyloid P component and serum amyloid A protein (SAA), implying that they were AA amyloid. There have been no reports describing localized amyloid deposits of the AA type in non neoplastic adrenal glands. The patho-genesis and clinical significance of the amyloid deposition in the present case remain only speculative. Acta Pathol Jpn 42: 893–896, 1992.     

Identification in Human Placentae of Antigenic Activity Related to the Amyloid Serum Protein SAA

Antigenic activity related to the amyloid serum protein SAA was observed in indirect immunofluorescence studies on human placental tissue. Positive staining with anti-SAA antisera was localized to the cytoplasm of cells scattered within the mesenchymal stroma, thought to be fibroblasts, and to foetal stem vessel endothelium and some individual fibrillar structures in villous stroma and perivascular tissue. This immunofluorescent staining was specifically inhibited by protein SAA. In contrast, no immunofluorescent staining was achieved using antisera to the amyloid protein AA. Absorption and immunodiffusion studies have further suggested that anti-SAA antisera may recognize in human placentae only a very limited number of the antigenic determinants present in protein SAA but not in the smaller protein AA. The results support previous observations that protein SAA-like antigenic material can be found in normal human tissue.     

AA-amyloidosis. Tissue component-specific association of various protein AA subspecies and evidence of a fourth SAA gene product.

Protein AA, the major repetitive protein subunit present in fibrils deposited in AA-amyloidosis, is an N-terminal cleavage product of a 104-amino acid precursor, serum amyloid A (SAA). Protein AA subspecies varying between 45 and 94 amino acids in length have been described. In this study it is shown that the different protein AA subspecies are not evenly distributed in amyloid deposits and that in single patients, certain subspecies of protein AA are deposited in specific tissue component sites. Thus larger protein AA subspecies occur in lower concentration in amyloid in the glomeruli compared to other sites and are especially found in amyloid in vessel walls. Three different SAA forms have been predicted from genomic and complementary DNA studies. The existence of a fourth type has been suspected from amino acid sequence studies of purified SAA. Protein AA derived from this fourth type of SAA is now shown to be present in amyloid fibrils in one of the patients studied in this paper.     

Ubiquitin: its potential significance in murine AA amyloidogenesis.

Amyloid enhancing factor (AEF), which has recently been shown to have identity with ubiquitin (Ub), is believed to play a causative role in experimentally induced AA amyloidosis in mice. We have examined the profile of Ub in activated leukocytes and splenic reticulo-endothelial (RE) cells and its relationship with serum amyloid A protein (SAA) and AA amyloid deposits in an alveolar hydatid cyst (AHC)-infected mouse model of AA amyloidosis. Two monospecific antibodies, anti-ubiquitin (RABU) and anti-mouse AA amyloid, were used as immunological probes to localize Ub, SAA, and AA amyloid. In response to AHC infection, the dull and diffuse Ub immunoreactivity in normal mouse leukocytes and RE cells promptly changed to a discrete granular pattern suggesting an increase in the intracellular concentration of Ub and the formation of Ub-protein conjugates. This corresponded to an elevation in SAA levels, SAA uptake by RABU-positive phagocytic cells, co-localization of Ub-SAA immunoreactive splenocytes in the perifollicular areas, and deposition of Ub-bound AA amyloid in the splenic and hepatic tissues. These results suggest that Ub-loaded monocytoid cells may play an important role in the physiological processing of the sequestered SAA into AA amyloid. Aspects of AA amyloidogenesis are discussed in relation to other experimental models in which stress-induced Ub-protein conjugate formation and its transport to lysosomal vesicles have been studied.     

Three Assays for the Characterization and Quantitation of Human Serum Amyloid A

Two different radioimmunoassays (RIA) and an enzyme-linked immunosorbent assay (ELISA) were developed for the quantitation and antigenic characterization of amyloid A (AA) and serum amyloid A (SAA) proteins, and the three assays were evaluated and compared with each other. Sensitivity, reproducibility, effect of denaturation and storage of serum and range of determination were considered. All three assays were found useful, but for different purposes. The most suitable method for the determination of SAA in whole serum was a second antibody precipitation RIA with purified SAA as labelled tracer and standard, and polyclonal rabbit anti-SAA as first antibody. This assay provided SAA concentrations in absolute amounts (mg/l) and acceptable reproducibility without need for prior denaturation of serum. Both advantages and disadvantages of ELISA using monoclonal antibodies to SAA and a solid-phase RIA using AA, SAA, anti-AA and anti-SAA were observed. The three assays were found suitable for antigenic studies of AA and SAA.     

Acceleration of murine AA amyloidosis by oral administration of amyloid fibrils extracted from different species

We herein report that experimental murine amyloid A (AA) deposition is accelerated by oral administration of semipurified amyloid fibrils extracted from different species. Three groups of mice were treated with semipurified murine AA amyloid fibrils, semipurified bovine AA amyloid fibrils or semipurified human light chain-derived (A(lambda)) amyloid fibrils for 10 days. After 3 weeks, each mouse was subjected to inflammatory stimulation by subcutaneous injection with a mixture of complete Freund's adjuvant supplemented with Mycobacterium butyricum. The mice were killed on the third day after the inflammatory stimulation, and the spleen, liver, kidney and gastrointestinal tract were examined for amyloid deposits. Amyloid deposits were detected in 14 out of 15 mice treated with murine AA amyloid fibrils, 12 out of 15 mice treated with bovine AA amyloid fibrils and 11 out of 15 mice treated with human A(lambda) amyloid fibrils. No amyloid deposits were detected in control mice receiving the inflammatory stimulant alone or in amyloid fibril-treated mice without inflammatory stimulation. Our results suggest that AA amyloid deposition is accelerated by oral administration of semipurified amyloid fibrils when there is a concurrent inflammatory stimulation.     

Pathogenesis of secondary amyloidosis in an alveolar hydatid cyst-mouse model: histopathology and immuno/enzyme-histochemical analysis of splenic marginal zone cells during amyloidogenesis.

C57BL/6 mice infected with 10, 50 or 250 alveolar hydatid cysts (AHC) were used to study the pathogenesis of secondary amyloidosis. Immuno and enzyme-histochemical analyses on spleen sections were performed to investigate the temporal relationship between AHC antigen, serum amyloid A protein (SAA) and amyloid (AA) deposition and concomitant qualitative and quantitative changes in the concentration of lysosomal acid phosphatase (AP) and nonspecific esterase (NSE) in splenic marginal zone (MZ) and red pulp reticuloendothelial (RE) cells prior to and during amyloidogenesis. AA-induction period was reduced from 5 weeks in the 50 cyst group to 6 or 7 days in the 250-cyst group; the 10-cyst group mice remained negative for splenic AA. Splenic RE cell hyperplasia, deposition of AHC antigen and SAA and peak AP and NSE activities occurred in splenocytes prior to AA deposition. AA-deposition in the MZs coincided with reduced RE cell AP and NSE activities and degenerative changes in the MZs. AA-induction period in the 250-cyst group was further reduced from 7 days to 4 days after intravenous injection of silica which is cytotoxic to RE cells. We suggest that defective clearance of SAA from tissue sites as a result of progressive reduction in lysosomal enzymes coupled with degenerative changes in splenic MZ monocytoid cells trigger amyloidogenesis in the extracellular matrix.     

Morphologic and chemical variation of the kidney lesions in amyloidosis secondary to rheumatoid arthritis

The kidneys of 20 patients who died of secondary systemic amyloidosis due to rheumatoid arthritis were studied histologically, and four of these were shown to have an uncommon pattern of deposition with almost no glomerular involvement but heavy deposits in the outer zone of the medulla. In three of the four patients frozen tissue was available. Immunochemical characterization of amyloid fibrils from these three cases showed that the major subunit amyloid fibril protein was protein AA, typical of secondary amyloidosis. Gel chromatography of fibrils revealed an uncommon elution pattern with two retarded major protein peaks. Both these proteins showed immunologic identity with protein AA and had N-terminal amino acid sequences identical with that protein but differed in size obviously due to a shortening of the C-terminal in one of the proteins. The reason for the correlation between the pattern of deposition of amyloid and alterations in protein AA is unclear but might be due to variations in enzymes responsible for the cleavage of the amyloid fibril subunit precursor protein SAA.     

A serum AA-like protein as a common constituent of secondary amyloid fibrils

Amyloid fibrils, purified from the spleen of four patients with amyloidosis associated with rheumatoid arthritis, had protein AA as a major protein. Besides this protein, all four amyloid fibril preparations contained a protein which in size, amino acid composition and N-terminal amino acid sequence was the same as the postulated serum precursor of protein AA, serum AA (SAA). The SAA-like amyloid fibril protein had a tendency to aggregate in neutral conditions, a phenomenon which is also seen in SAA but not in protein AA.     

Activities of lysosomal enzymes and levels of serum amyloid A (SAA) in blood plasma of hamsters during casein induction of AA-amyloidosis

Casein-induced amyloidosis in hamsters was found to be of the AA-type, as shown by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and amino acid analysis of the major low-molecular weight component of the amyloid fibrils. Levels of serum amyloid A (SAA) and the activities of cathepsin D, beta-N-glucosaminidase, serine esterase, lactate dehydrogenase (LDH) and gamma glutamyl transpeptidase (GGT) were measured in the blood plasma during induction of amyloidosis. During the pre-amyloid phase an increase was observed in all these parameters. During the deposition of amyloid, an increase was observed in the activities of the lysosomal enzymes cathepsin D and beta-N-glucosaminidase, which was significantly correlated with amyloid deposition. Serine esterase activities did not show any relationship to amyloid deposition. LDH and GGT activities were normal in the amyloid phase. SAA levels were lower during amyloid deposition than during the pre-amyloid phase. These findings indicate that a specific release of lysosomal contents from mononuclear phagocytic cells is involved in the pathogenesis of AA-amyloidosis. Amyloid deposition may be the result of: (i) extrusion of intralysosomal protein AA or pre-amyloid, followed by extracellular formation of amyloid fibrils; (ii) secretion of lysosomal enzymes, followed by extracellular cleavage of SAA and subsequent aggregation of protein AA with other components.     

Regulation of serum amyloid a gene expression in Syrian hamsters by cytokines

Amyloid A (AA) protein is derived from serum amyloid A (SAA) and deposited as beta-pleated sheet fibrils in reactive amyloidosis, a disease that occurs spontaneously in golden Syrian hamsters. The precursor SAA is an acute-phase reactant in many species including hamsters, and in this report we have defined the in vivo kinetic and dosage responses for SAA mRNA accumulation in hamsters following administration of various cytokines. Elevations in levels of hepatic SAA mRNA were documented when the doses of interleukin-1, interleukin-6, and tumor necrosis factor were increased. The increase in dosages applied ranged from 2 1/2-fold for interleukin-6 to 10-fold for interleukin-1. SAA transcipt levels were highest 8 h following administration of interleukin-6 or tumor necrosis factor, whereas maximal amounts of SAA-specific mRNA were found 24 h after administration of interleukin-1.     

Activities of lysosomal enzymes and levels of serum amyloid A (SAA) in blood plasma of hamsters during casein induction of AA-amyloidosis.

Casein-induced amyloidosis in hamsters was found to be of the AA-type, as shown by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and amino acid analysis of the major low-molecular weight component of the amyloid fibrils. Levels of serum amyloid A (SAA) and the activities of cathepsin D, beta-N-glucosaminidase, serine esterase, lactate dehydrogenase (LDH) and gamma glutamyl transpeptidase (GGT) were measured in the blood plasma during induction of amyloidosis. During the pre-amyloid phase an increase was observed in all these parameters. During the deposition of amyloid, an increase was observed in the activities of the lysosomal enzymes cathepsin D and beta-N-glucosaminidase, which was significantly correlated with amyloid deposition. Serine esterase activities did not show any relationship to amyloid deposition. LDH and GGT activities were normal in the amyloid phase. SAA levels were lower during amyloid deposition than during the pre-amyloid phase. These findings indicate that a specific release of lysosomal contents from mononuclear phagocytic cells is involved in the pathogenesis of AA-amyloidosis. Amyloid deposition may be the result of: (i) extrusion of intralysosomal protein AA or pre-amyloid, followed by extracellular formation of amyloid fibrils; (ii) secretion of lysosomal enzymes, followed by extracellular cleavage of SAA and subsequent aggregation of protein AA with other components.     

Specific Deposition of Serum Amyloid A Protein 2 in the Mouse

The homogenates of amyloid-laden spleens prepared from CBA mice were analysed by SDS-PAGE and immunoblotting employing rat anti-murine monoclonal antibody, MSA 4-26. The results showed that the precursor of amyloid A protein (AA), serum amyloid A protein 2 (SAA2), and SAA intermediates with molecular weights of 10,000, 9000, and 8000 were contained in amyloid-laden tissues. The experiment using sonicated spleen cells and acute phase murine sera showed a delay in the degradation rate of SAA2 on cell fragments and the remains of SAA1 in supernatants. This result can explain disappearance of SAA2 from the murine serum during amyloidogenesis in vivo.     

Expression of mouse apolipoprotein SAA1.1 in CE/J mice: isoform-specific effects on amyloidogenesis

Amyloid A (AA) amyloid deposition in mice is dependent upon isoform-specific effects of the serum amyloid A (SAA) protein. In type A mice, SAA1.1 and SAA2.1 are the major apolipoprotein-SAA isoforms found on high-density lipoproteins. During inflammation, both isoforms are increased 1000-fold, but only SAA1.1 is selectively deposited into amyloid fibrils. Previous studies showed that the CE/J mouse strain is resistant to amyloid induction. This resistance is not due to a deficiency in SAA synthesis, but is probably related to the unusual SAA isoform present. The CE/J mouse has a single acute-phase SAA protein (SAA2.2), which is a composite of the SAA1.1 and SAA2.1, with an amino terminus similar to the nonamyloidogenic SAA2.1. Recently, genetic experiments suggested that the SAA2.2 isoform might provide protection from amyloid deposition. To determine the amyloidogenic potential of the CE/J mouse, we generated SAA adenoviral vectors to express the various isoforms in vitro and in vivo. Purified recombinant SAA proteins demonstrated that SAA1.1 was fibrillogenic in vitro, whereas SAA2.2 was unable to form fibrils. Incubation of increasing concentrations of the nonamyloidogenic SAA2.2 protein with the amyloidogenic SAA1.1 did not inhibit the fibrillogenic nature of SAA1.1, or alter its ability to form extensive fibrils. Injection of the mouse SAA1.1 or SAA2.2 adenoviral vectors into mice resulted in isoform-specific expression of the SAA proteins. Amyloid induction after viral expression of the SAA1.1 protein resulted in the deposition of amyloid fibrils in the CE/J mouse, whereas SAA2.2 expression had no effect. Similar expression of the SAA2.2 protein in C57BL/6 mice did not alter amyloid deposition. These data demonstrate that the failure of the CE/J mouse to deposit amyloid is due to the structural inability of the SAA2.2 to form amyloid fibrils. This mouse provides a unique system to test the amyloidogenic potential of altered SAA proteins and to determine the important structural features of the protein.     

Multinodular deposition of AA-type amyloid localized in the adrenal glands of an old man.

Multinodular amyloid deposits localized in non-neoplastic adrenal glands were found incidentally at autopsy in an 83-year-old Japanese man. Clinically, the patient lacked evident deficiency of adrenal hormones. The nodules of the stromal amyloid deposits were scattered in the adrenal cortex, where the parenchymal cells were compressed and atrophic. The deposits were confirmed to be amyloid by Congo red staining and polarization microscopy. Amyloid fibrils were also demonstrated in the deposits by electron microscopy. The amyloid deposits were permanganate-sensitive and showed immunohistochemical staining for serum amyloid P component and serum amyloid A protein (SAA), implying that they were AA amyloid. There have been no reports describing localized amyloid deposits of the AA type in non-neoplastic adrenal glands. The pathogenesis and clinical significance of the amyloid deposition in the present case remain only speculative.     

Binding of ubiquitin to experimentally induced murine AA amyloid.

Amyloid enhancing factor (AEF) activity has recently been demonstrated in ubiquitin purified from amyloidotic murine tissues and Alzheimer brain extract. Since AEF is known to bind to amyloid fibrils and 'fibril-AEF' on passive transfer induces accelerated amyloidogenesis in the recipient animals, it was of interest to investigate whether ubiquitin binds to amyloid. Immunohistological studies were carried out on liver sections from amyloidotic mice. Biotin-strepavidin-peroxidase methods using monospecific rabbit anti-mouse AA amyloid IgG (RAAG) and rabbit anti-bovine ubiquitin IgG (RABU) antibodies were employed to immunostain the amyloid and ubiquitin deposits, respectively. RABU-treated liver sections were counterstained with thioflavine S. RAAG reacted strongly with the amyloid, indicating that it is AA type, and RABU-positive immunodeposits were found bound to the thioflavine-S-positive AA deposits. Treatment of the liver sections with 0.1 M sodium acetate containing 0.5 M NaCl, pH 4, for 2-3 h at 37 degrees C nearly completely desorbed the AA amyloid-bound ubiquitin. Since ubiquitin demonstrates AEF activity in vivo and binds non-covalently to AA amyloid, we suggest that ubiquitin may indeed be 'fibril-AEF' and may play a crucial role in the pathogenesis of amyloidosis. To our knowledge, this is the first time that ubiquitin bound to extracellularly deposited amyloid has been demonstrated.