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.     

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.     

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.     

A primed state exists in vivo following histological regression of amyloidosis.

Using a sensitive, quantitative, and non-invasive in vivo method, based on the specific binding of serum amyloid P component to amyloid fibrils, we have directly documented the spontaneous resolution of AA amyloid deposits in mice, and the prolonged existence thereafter of a primed state of enhanced susceptibility to further amyloid deposition. These results may have important implications for understanding and management of amyloidosis in humans.     

Transformation from SAA2-fibrils to AA-fibrils in amyloid fibrillogenesis: In vivo observations in murine spleen using anti-SAA and anti-AA antibodies

Early amyloid fibrillogenesis from serum amyloid A protein (SAA) has been observed in the murine spleen after an injection of casein-Freund's complete adjuvant in the presence of amyloid enhancing factor, using anti-SAA C-terminal (anti-SAA) and anti-amyloid A (AA) antibodies. In Western immunoblotting of sera, both SAA1 and SAA2 reached a maximum after 24 h and began to decrease after 48 h. In spleen extracts, SAA2, but not SAA1 or AA, was found from 48 h, when amyloid was first deposited in the marginal zone. Electron microscopic immunohistochemistry of this stage showed reaction products from SAA in the marginal zone as fine granules along the cell membrane of mononuclear cells and focal intercellular aggregates, which contained fine fibrils originating from the cell membrane. Amyloid nodules, surrounded by mononuclear cells, developed from this stage. In the nodules, fibrils were positive for anti-SAA only in the vicinity of the cell membrane, while anti-AA stained fibrils throughout. Our hypothesis for fibrillogenesis is thus as follows: Serum SAA2 is specifically deposited on mononuclear cells in the marginal zone and polymerized extracellularly into fibrils, retaining its antigenicity (SAA2 amyloid fibrils); these fibrils are then processed to AA amyloid fibrils in situ by cleavage of the C-terminal portion of SAA2.     

Histomorphological variations in systemic AA amyloidosis: clues of AA protein polymorphism

The histological location of amyloid within various organs in 25 cases of systemic AA amyloidosis was studied with a view to determine whether different morphological patterns exist in this category of amyloidosis. Although morphological variations due to progressive severity of disease were observed, there were appreciable variations in the patterns of amyloid deposition in the kidney and spleen that could not be simply explained on those grounds. Eleven (61%) of 18 kidneys examined showed severe glomerular involvement with mild degrees of vascular deposition while the remaining seven showed predominantly vascular involvement. The glomerular pattern appeared to be more ominous, being significantly associated with severe proteinuria or chronic renal failure. In nine (69%) of 13 spleens examined, amyloid was confined to the walls of small and medium-sized arteries while in the remaining four, vascular involvement was less severe and amyloid was deposited mainly along the reticulin of the white pulp. Possible explanations for these different patterns included resorption and redistribution of amyloid within the body during the course of the disease, and variation in tissue deposition as a manifestation of polymorphism of amyloid proteins. The latter appeared more feasible in view of the recent demonstration of SAA polymorphism and AA heterogeneity in man.     

An ELISA assay for murine amyloid a and serum amyloid a utilizing monoclonal antibodies

An enzyme-linked immunoassay has been developed to quantitate the amyloid A (AA) and serum amyloid A (SAA) proteins of mice. The assay utilizes monoclonal rat anti-mouse AA as the antibody. The principle advantage of this method is that it avoids the need to denature the mouse sera before its SAA content can be measured.     

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.     

Amyloid A amyloidosis and systemic lupus erythematosus

Amyloid A (AA) amyloidosis occurs secondary to long-standing inflammation and causes nephropathy and various internal manifestations, which leads to mortality. It is very rare in some rheumatic diseases, such as systemic lupus erythematosus (SLE). Therefore, there are few articles that report AA amyloidosis in SLE. This article focuses on the previously reported cases of 24 patients with SLE that are complicated by AA amyloidosis, and on the underlying mechanisms.     

40例急性胰腺炎患者血清淀粉样蛋白A及炎症因子的变化

目的 :观察血清SAA、CRP、IL - 6、IL - 8及SIL - 2R在急性胰腺炎病程中的变化 ,探讨SAA在急性胰腺炎发生发展中的辅助诊断及疾病严重度评价的实用价值。方法 :SAA、CRP定量检测 :采用乳胶增强速率散射比浊法。IL - 6、IL - 8、SIL - 2R水平采用ABC -ELISA法检测。结果 :急性重症胰腺炎患者五项指标显著高于急性轻症胰腺炎及正常对照组 (p <0 0 1 )。同时血清SAA水平与CRP、IL - 6、IL - 8水平呈显著相关 (p <0 0 1 )。急性轻症胰腺炎组SAA、CRP、IL - 6水平高于正常对照组 (p <0 0 5 )。 结论 :联合检测血清SAA、CRP、IL - 6、IL - 8水平对判断急性重症胰腺炎的严重程度及提示急性胰腺炎坏死性形成具有重要参考价值。在急性胰腺炎的病程中SAA的检测价值优于CRP。     

Idiopathic Amyloidosis Due to AA Protein: A Report of 2 Cases

Abstract

Two cases of idiopathic amyloidosis are described. AA protein was found to be the major constituent of tissue amyloid in both, based on sensitivity of Congo red birefringence to pretreatment of slides with potassium permanganate and immunoperoxidase staining with a monospecific antiserum. However, an underlying inflammatory, infectious or neoplastic disorder was not identified. The occurrence of AA amyloidosis without clearcut underlying disease is rare, having been described in only thirteen previous instances. Recent clinical series suggest that such cases may have a different course and response to therapy from “primary” amyloidosis due to systemic light chain deposition. Such cases may also underscore the importance of genetic factors in the pathogenesis of AA amyloid and the importance of characterizing the amyloidoses biochemically, as well as clinically. (The J Histotechnol 12:137, 1989.)     

Studies in vivo and in vitro of serum amyloid P component in normals and in a patient with AA amyloidosis.

Pure serum amyloid P component (SAP) was isolated from a normal donor pool, from individuals with the different genotypes of an MspI restriction fragment length polymorphism (RFLP) linked to the SAP gene, and from a patient with AA amyloidosis. The SAP preparations were all identical and all behaved as a single homogeneous species in polyacrylamide gel electrophoresis, isoelectric focussing, reverse-phase chromatography, binding in vitro to phosphoethanolamine-Sepharose (binding constant 2.4 x 10(7) l/mol) and AL amyloid fibrils (1.6 x 10(8) l/mol), and binding to amyloid deposits in vivo in mice with casein-induced amyloidosis. The in vivo metabolism of 125I-SAP from a single donor was normal and identical in three healthy individuals representing the three different MspI RFLP genotypes. There is thus no frequent polymorphism of SAP in normal subjects, and SAP altered with respect to the characteristics studied here is not a necessary condition for pathogenesis of systemic AA amyloidosis.     

Acute-phase protein synthesis in human hepatoma cells: differential regulation of serum amyloid A (SAA) and haptoglobin by interleukin-1 and interleukin-6.

Interleukin-6 (IL-6, BSF-2 or IFN-beta 2) is thought to be the major regulator of the acute-phase protein response that follows tissue injury and inflammation, with interleukin-1 (IL-1), tumour necrosis factor and more recently, LIF or HSF III, slightly stimulatory on only certain acute phase proteins. The synthesis of the major acute-phase protein SAA, originally described as being synthesized in response to IL-1, has been claimed recently to be mainly under IL-6 regulation. Our results show that in the human hepatoma cell line HuH-7, IL-1 is the major stimulating cytokine increasing SAA synthesis by a factor in excess of 100-fold. We also show that under most conditions interleukin-6 and tumour necrosis factor stimulate additively in combination with IL-1. Isoelectric focusing has demonstrated that SAA1 and SAA2 alpha are expressed but not SAA2 beta. The HuH-7 cell line is IL-6 responsive since haptoglobin is stimulated mainly by IL-6.     

Production of serum amyloid A and C-reactive protein by HepG2 cells stimulated with combinations of cytokines or monocyte conditioned media: the effects of prednisolone.

The hepatic production of the acute phase proteins in response to inflammatory cytokines, and the interaction of corticosteroids within this response, has been the subject of considerable recent research. In this study we have examined the effects of the corticosteroid prednisolone on the production of IL-1 alpha and IL-1 beta by lipopolysaccharide (LPS)-stimulated monocytes, and the ability of the monocyte conditioned media (MOCM) obtained under these conditions to induce human hepatoma HepG2 cells to produce serum amyloid A (SAA) and C-reactive protein (CRP). We also examined the production of SAA and CRP by HepG2 cells exposed to different combinations and concentrations of recombinant human (rh) IL-1 alpha, rhIL-1 beta, rhIL-6, recombinant human tumour necrosis factor-alpha (rhTNF-alpha) and prednisolone. The findings indicate: (i) prednisolone substantially inhibits the production of both IL-1 alpha and IL-1 beta by LPS-stimulated monocytes. The MOCM from prednisolone-treated monocytes induced less SAA and CRP production by HepG2 cells; (ii) IL-1 alpha and IL-1 beta both induced CRP and SAA synthesis by HepG2 cells, but only in the presence of IL-6. IL-1 beta was the more potent inducer for SAA production, but for CRP production IL-1 alpha and IL-1 beta were equivalent; (iii) prednisolone enhances the production of SAA by HepG2 cells, but does not enhance the production of CRP; (iv) TNF-alpha in the presence or absence of IL-6 and/or prednisolone did not induce the production of SAA or CRP by HepG2 cells. These findings offer a tenable solution to a disparate production of SAA compared with CRP in corticosteroid-treated cystic fibrosis (CF) patients.     

Ubiquitin biology in neurodegenerative disorders: From impairment to therapeutic strategies

The abnormal accumulation of neurotoxic proteins is the typical hallmark of various age-related neurodegenerative disorders (NDDs), including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Amyotrophic lateral sclerosis and Multiple sclerosis. The anomalous proteins, such as Aβ, Tau in Alzheimer’s disease and α-synuclein in Parkinson’s disease, perturb the neuronal physiology and cellular homeostasis in the brain thereby affecting the millions of human lives across the globe. Here, ubiquitin proteasome system (UPS) plays a decisive role in clearing the toxic metabolites in cells, where any aberrancy is widely reported to exaggerate the neurodegenerative pathologies. In spite of well-advancement in the ubiquitination research, their molecular markers and mechanisms for target-specific protein ubiquitination and clearance remained elusive. Therefore, this review substantiates the role of UPS in the brain signaling and neuronal physiology with their mechanistic role in the NDD’s specific pathogenic protein clearance. Moreover, current and future promising therapies are discussed to target UPS-mediated neurodegeneration for better public health.     

The significance of cerebrovascular amyloid in the aetiology of superficial (lobar) cerebral haemorrhage and its incidence in the elderly population.

Cerebrovascular amyloid deposition (CVAD), caused by deposition of the beta/A4 protein, has been previously identified as a cause of cerebral haemorrhage, yet its prevalence is uncertain. The presence of vascular amyloid was studied in brains of 169 patients by immunohistochemical and Congo red staining. Fifty patients had cerebral haemorrhage (CH), 56 had cerebral infarction (CI), and 63 had neither haemorrhage nor infarction (control group). CVAD was found in 38 per cent of the CH group, 25 per cent of the CI group, and 32 per cent of the control group. The incidence of CVAD increased with age in each group. Immunohistochemical staining with an antibody to beta/A4 protein was more sensitive than Congo red staining the demonstrating the extent of vascular amyloid. Within the CH group, CVAD was present in the vessels at the site of haemorrhage in 6/8 (75 per cent) of pure superficial (lobar) cerebral haemorrhages. While amyloid was detected in vessels in the brain of 10/37 (27 per cent) of pure deep cerebral haemorrhages, none was present in vessels at the site of haemorrhage. CVAD is a common pathological finding in the elderly and has a significant association with pure superficial (lobar) cerebral haemorrhages.     

Quantitative radial immunodiffusion assay for serum amyloid A protein

A radial immunodiffusion assay for serum amyloid A protein (SAA) using a commercially available antiserum is described. Serum is applied untreated to 1% agarose gels prepared in 0.02 M barbitone buffer, pH 8.6, containing 40 g/l polyethylene glycol 6000. Incubation is carried out overnight at 37 degrees C. The assay combines the advantages of simplicity, rapidity, specificity and stability, and avoids the hazards associated with the previously described radioimmunoassays. The method has sufficient sensitivity to measure SAA in the majority (99%) of normal subjects, and confirms the behaviour of SAA as a very sensitive acute phase reactant in inflammatory disease. The method is ideally suited to the rapid processing of a large number of samples.     

Sequential measurement of the murine acute-phase protein serum amyloid P component (SAP) as an indicator of graft-versus-host disease following allogeneic bone marrow transplantation in mice.

Murine models of bone marrow transplantation (BMT) are used commonly for studies of the pathogenesis and treatment of graft-versus-host disease (GVHD). We report here that the sequential measurement of the mouse acute-phase protein SAP can be used to provide a sensitive, quantitative index of the severity of GVHD. Thirty mice underwent allogeneic, and a further 30 syngeneic BMT. GVHD was assessed in vivo by clinical appearances and weight change, and post mortem by histology and calculation of splenic indices. Blood was obtained twice/week for SAP measurement and blood culture. In all mice an initial rise in SAP levels due to irradiation was followed by a return to baseline. Thereafter in syngeneic marrow recipients levels remained low. In contrast, after allogeneic BMT SAP levels rose progressively as mice developed GVHD, reaching a peak of 135 micrograms/ml prior to death, from a nadir at day 20 of 15 micrograms/ml. Mice with high splenic indices and histological evidence of severe GVHD had significantly higher SAP levels than mice with mild GVHD (P = 0.0002). Elevation in SAP levels occurred independently of bacteraemia. We conclude that in murine BMT sequential measurement of SAP provides an objective means of assessing GVHD in vivo.     

Standardisation of the quantitation of serum amyloid A protein (SAA) in human serum

An adequate method for standardising the quantitation of serum amyloid A protein (SAA) in human serum was developed. Acute phase high density lipoprotein3 (HDL3) was used as a standard. The concentration of the SAA in the standard was determined by the use of purified SAA. After protein determination, various concentrations of purified SAA were run on SDS-polyacrylamide gel together with the HDL3 standard containing an unknown amount of SAA amongst the apolipoproteins. From the standard curve obtained by pyridine extraction (Coomassie blue colour yield at A605 nm) the concentration of SAA in the HDL3 standard was determined. An established immunoradiometric assay (IRMA) for SAA was standardised with the HDL3. SAA concentrations in normal and acute phase sera were determined.     

肿瘤坏死因子α mRNA在小鼠病毒性心肌炎中的表达及意义

目的　研究病毒性心肌炎 (VMC)小鼠肿瘤坏死因子α(TNF α)的动态变化及mRNA的表达及其在VMC小鼠发病中的意义。方法　用逆转录 聚合酶链反应 (RT PCR)方法检测小鼠VMCTNF αmRNA的表达 ,同时用酶联免疫吸附试验法 (ELISA)检测VMC小鼠在接种病毒后 3、5、7、9、15、35d血清TNF α的变化 ,并分析了TNF α与VMC发病的可能关系。结果　实验发现VMC小鼠TNF αmRNA表达量明显增加 ,而且接种病毒后 7d的表达量 (1 94 )大于 3d(1 0 9)和对照组 (0 0 7)。VMC小鼠在接种病毒后 3～ 35d各时点的血清TNF α水平 (分别为D3组 15 9 7± 4 0 8;D5组 2 12 7± 4 6 0 ;D7组 2 96 7± 34 3;D9组 2 6 7 3± 4l 4 ;D15组 187 8± 4 9 6 ;D35组 15 5 4± 35 9)均明显高于对照组 (12 7 2± 13 8,P <0 0 5或 0 0 1)。结论　小鼠VMC血清TNF α及其mRNA水平升高 ,TNF α可能参与了VMC的发病