山田 俊幸

Serum amyloid A (SAA), an acute-phase protein and a precursor of fibrous components in reactive amyloid deposits, is synthesized mainly in the liver under the stimulation of inflammation-related cytokines. In addition, the SAA gene is expressed in monocytes/macrophages, which are believed to play a central role in amyloid fibrillogenesis. Consequently, the pathogenic implication of SAA produced from these cells has been of major concern. Because SAA synthesis at the protein level in such cells has never been analyzed quantitatively, in this study an enzyme-linked immunosorbent assay was generated with a detection level sufficiently high to measure SAA concentrations in the culture supernatants of the human monocytic leukaemia cell line THP-1. SAA secretion by THP-1 with interleukin (IL)-1 beta required the presence of dexamethasone as proposed previously. We also found that unidentified components in fetal calf serum (FCS) could induce SAA production by THP-1 in the presence of dexamethasone. These findings are in contrast to the results obtained from hepatoma cell line HepG2, in which IL-1 beta alone could induce SAA secretion, while dexamethasone-supplemented FCS could not. The method was able to quantify SAA secreted from cultured human peripheral monocytes. The findings suggest that monocytes produce SAA in almost the same manner as THP-1. Thus, THP-1 cells can be utilized to investigate a distinctive manner of SAA production from monocytes.

Nucleotide variation in the triplet codon coding for amino acid position 72 of human serum amyloid A1 (SAA1), which was suggested by amino acid sequence analysis, was analyzed here in order to identify the genomic sequences coding SAA1.2 and to characterize further the SAA1 allele frequency in a Japanese population. The SAA1 exon 4 was amplified by PCR and treated with Nco I. Sequencing of PCR products from genomic DNA of individuals who were heterozygous for the Nco I site revealed GGT(Gly) and GAT (Asp) at the position 72. The allele having (72)Asp showed the exon 3 polymorphism coding (52)Ala and (57)Val. This allele should thus be identified as SAA 1.2. Alleles with (72)Gly were either (52)Val and (57)Ala(SAA1.1) or (52)Ala and (57)Ala (SAA1.3) or (52)Ala and (57)Val (SAA1.5). The frequency of SAA1 alleles in the 321 Japanese subjects was 0.310, 0.012, 0.347 and 0.330 for each SAA allele of 1.1, 1.2, 1.3 and 1.5, respectively. The presence of the SAA.4 allele was not evaluated.

Serum amyloid A protein (SAA), an acute-phase reactant in reactive amyloidosis, has high affinity for high-density Lipoprotein (HDL). When SAA is added to HDL, SAA displaces apolipoprotein A-I (apoA-I) and phospholipid from the HDL particles. These dissociated components may form pre beta 1-HDL because free apoA-I can associate with phospholipid to become a lipoprotein having pre beta mobility. To determine whether SAA generates pre beta 1-HDL from alpha-migrating HDL, we investigated the effects of recombinant SAA on HDL subfraction concentration using nondenaturing two-dimensional gradient gel electrophoresis. When we added SAA (0.5 mg/mL) to plasma, the pre beta 1-HDL concentration increased by 164% (from 4.7% +/- 1.3% to 12.4% +/- 3.2% of apoA-I, p < 0.005), The increase in pre beta 1-HDL was proportional to the dose of SAA, When we added SAA to a column of Sepharose beads coupled to the isolated HDL (alpha-migrating HDL), pre beta 1-HDL was dissociated from the column together with the SAA-associated HDL. In summary, we demonstrate that SAA generates pre beta 1-HDL from alpha-migrating HDL. We speculate that SAA-mediated HDL remodeling may take place in inflammation.

Based on the recent findings that patients with systemic amyloidosis have reduced plasma apolipoprotein An (apoAII) concentrations, this study assessed the behaviour of apolipoprotein AI (apoAI) and apoAII in a murine model of reactive amyloidosis. ICR mice were subjected to a single inflammatory stimulus and then sacrificed at the end of week 1 (group I), week 2 (group II) or week 8 (group III). Amyloid deposits were found in approximately one-half of the animals. Plasma apoAI and apoAII concentrations were reduced in all stimulated animals compared with control animals. In groups II and III, apoAII and apoAII/apoAI ratios were lower in the amyloidotic animals than in nonamyloidotic animals, similar to findings in the human amyloidosis patients. Both apoAI and apoAII mRNA in the liver were reduced in group I, but not in group II or III. Both apoAI and apoAII were detected immunohistochemically in the amyloid deposits in groups I and II, but not in group III. Thus, the reduction of plasma apoAs in the amyloid-forming phase may be due, in part, to the involvement of high-density lipoproteins in the deposits. However, that in the chronic phase may be affected by other mechanisms.

Serum amyloid A1 (SAA1), the predominant isotype of acute phase SAA in plasma and the predominant precursor of fibrillar deposits in reactive amyloidosis, is encoded by a gene, for which six allelic variants have been described. Recent studies proposed that the allele SAA1.3 was positively correlated with the development of reactive amyloidosis in Japanese. This study examined whether the plasma concentration of total SAA is influenced by specific SAA1 alleles. Two hundred and eighty healthy Japanese subjects were examined to determine the allelic distribution of SAA1 and SAA2 genes by the PCR-RFLP method, and to measure the total plasma SAA concentrations. SAA concentrations were significantly higher (p<0.001) in subjects with the allele SAA1.5 than those without it, suggesting that SAA1.5 may have a distinctive feature in the process of synthesis or catabolism. Subjects with the allele SAA1.3 had lower SAA concentrations, though not statistically significant, than those with SAA1.1. There was not significant correlation of SAA2 alleles with SAA concentrations. These results are discussed in terms of amyloidogenicity.

The incidence of serum amyloid A2 alleles (SAA(2 alpha) and SAA(2 beta)) in the Japanese population was analyzed by PCR-RFLP analysis. The SAA(2 alpha) allele was predominant (approximately 90%) in both healthy controls and adult patients with rheumatoid arthritis. There was no significant association of allele patterns with AA amyloidosis in the patient group. These results are discussed in comparison with those of previous reports from other areas.

Objective-To investigate if serum apolipoprotein A-I and A-II (apoAI and apoAII) concentrations change in subjects with systemic amyloidosis secondary to underlying disorders. Methods-Serum concentrations of apoAI and apoAII were measured in 21 multiple myeloma patients, including eight with amyloidosis; 95 rheumatoid arthritis patients, including 45 with amyloidosis; and 73 haemodialysis patients, including 32 with amyloidosis. Results-ApoAII values tended to be reduced in subjects with amyloidosis in each group, but could not effectively distinguish amyloidosis. However, apoAII/AI ratios were significantly lower in subjects with amyloidosis in all groups. The ratio of 0.2 had diagnostic sensitivity and specificity for amyloidosis; 50% and 100%, respectively, in multiple myeloma; 80% and 78%, respectively, in rheumatoid arthritis; and 46% and 90%, respectively, in patients requiring long term haemodialysis. Conclusion-The apoAII/AI ratio can be a useful biochemical marker of suspect amyloidosis in patients with underlying diseases, especially those with rheumatoid arthritis.

Monoclonal antibodies against human serum amyloid A (SAA) were generated in rat (which seems not to have mature SAA proteins) by immunizing intact human SAA. Thirteen clones selected by initial screening were analysed based on reactivity with synthetic peptides of SAA and with carboxyl-terminal truncated recombinant SAA. Antibodies were divided into four types, i.e. those recognizing the area around residue 18, 30, 90, and 100, respectively, of SAA. The antibody to the carboxyl terminus (around residue 100) of SAA, when subjected to immunohistochemistry for amyloid deposits in specimens from patients with reactive amyloidosis, always yielded negative reactivity, supporting the general concept that the carboxyl terminus of SAA is absent from human AA deposits.

The polymorphic protein, serum amyloid A (SAA), consists of acute phase isotypes and a constitutive isotype. Both are associated mostly with high density lipoproteins (HDL) in the circulation. In the present study, both SAA isotypes were detected by immunohistochemistry and immunoblotting using monoclonal antibodies in atherosclerotic lesions. As the distribution of SAA was identical with that of apolipoprotein B and SAA is known to be associated also with low density lipoproteins (LDL), SAA may also be delivered to the artery wall by LDL.

The development of amyloidotic diseases is believed to be determined in large part by the structure and metabolism of the amyloid subunit protein. The amino-terminal region of serum amyloid A (SAA), the subunit precursor protein in reactive amyloidosis, appears to confer fibrillogenic potential. Here we present data consistent with the hypothesis that amyloid A fibrillogenesis is favored when proteolysis of the amino-terminal region of SAA is impaired. Murine tissue extracts were found to contain pepstatin-inhibitable protease activity that cleaved mouse SAA2 between Glu8 and Ala9. Tissues obtained from mice that had been treated with pepstatin for 3 days lacked this activity. To investigate a possible relationship between inhibition of aspartic proteases and amyloidogenesis, mice were treated with pepstatin while concurrently undergoing a standard amyloid induction protocol (repeated casein injections). Pepstatin-treated mice showed amyloid deposition significantly sooner than the control group, which had received only casein. During the preamyloidotic phase, pepstatin-treated mice had higher concentrations of SAA in serum and spleen than control mice. In addition, clearance of injected I-125-labeled SAA from plasma was significantly delayed. Based on these findings, it is reasonable to postulate that inhibition of aspartic protease activity can lead to an accumulation of amino-terminally intact SAA molecules and thereby accelerate amyloid fibril formation.

Amyloid A protein (AA), the chief constituent of reactive amyloid deposits, is derived from serum amyloid A (SAA) and most commonly corresponds to the amino-terminal 76 residues (AA76). Digestion of recombinant human SAA1 with a lysosomal thiol protease, cathepsin B, and analysis of the products by SDS-PAGE and amino-terminal sequencing revealed that AA76 was generated as a minor and transient degradation product. Digestion with neutrophil elastase generated intermediates different from AA76. This finding suggests that cathepsin B may play an important role in amyloid fibrilogenesis by converting SAA to AA.

Three isotypes of human serum amyloid A (SAA), SAA1, SAA2 beta, and SAA4 were expressed at high levels in Escherichia coli (E. coli) using a pET vector expression system. Each SAA cDNA was ligated to the vector pET-21a(+) and transformed into E. coli, strain BL21(DE3)pLysS, Expression conditions required high concentrations of antibiotics in order to obtain a high ratio of synthesized SAA to total E. coli proteins. Each recombinant SAA (rSAA) was purified by molecular sieve chromatography followed by chromatofocusing or hydrophobic interaction chromatography. The yield of purified protein was 5-10 mg per 11 of culture. When subjected to in vitro fibril forming conditions, rSAA1 formed amyloid-like fibrils confirmed by Congo red staining and electron microscopy. In contrast, rSAA2 beta and rSAA4 showed negative Congo red staining and curvilinear or flattened fibrillar structures on electron microscopy. This suggests that SAA1 has greater potential for forming amyloid fibrils than either SAA2 beta or SAA4.

A latex agglutination photometric immunoassay method was developed to measure serum amyloid A protein (SAA). Using an automated analyzer, the assay required 10 minutes. Standardization was possible using SAA-enriched high density lipoprotein which was obtained from a patient. The SAA content was determined electrophoretically and the result was used as the assay standard. This rapid and easy method is expected to be applied for the routine uses. Health values of SAA were somewhat higher than those of C-reactive protein (CRP). In healthy subjects and patients with acute myocardial infarction, both proteins changed in parallel. The log SAA vs log CRP ratio showed essentially the same values in patients with rheumatoid arthritis, gastrointestinal cancer, lung canncer, acute myocardial infarction, surgical operation and bacterial pneumonia, while significantly higer values were obtained in patients with rubellosis, cytomegalo virus infection and kidney allograft rejection. The serum levels of SAA and CRP may thus not always be controlled by the same mechanisms, and the measurment of SAA would be of value in certain disorders.