Humoral proinflammatory cytokine and SAA generation profiles and spatio-temporal relationship between SAA and lysosomal cathepsin B and D in murine splenic monocytoid cells during AA amyloidosis.

Evidence shows that tissue macrophages (MPhis), in mice undergoing AA amyloidosis, endocytose acute-phase humoral serum amyloid A (SAA) and traffic it to lysosomes where it is degraded. Incomplete degradation of SAA leads to intracellular nascent AA fibril formation. In vitro, cathepsin (Cat) B is known to generate amyloidogenic SAA derivatives, whereas Cat D generates non-amyloidogenic SAA derivatives, and interferon (IFN-gamma)-treated MPhis show selective increase in Cat B concentration, a factor conducive to AA amyloidogenesis. To understand the cumulative effect of these factors in AA amyloidosis, humoral levels of SAA, IFN-gamma, tumour necrosis factor (TNF-alpha) and granulocyte-macrophage colony-stimulating factor were determined in azocasein (AZC)-treated CD-1 mice. We correlated these responses with the spatio-temporal distribution of SAA, Cat B- and Cat D-immunoreactive splenic reticuloendothelial (RE) cells. AZC-treated CD-1 mice similar to that of A/J mice showed partial amyloid resistance; their peak humoral IFN-gamma and SAA responses overlapped during the pre-amyloid phase. Unexpectedly, Cat D immunoreactivity (IR), instead of Cat B IR, was predominant in the splenic RE cells, indicating an apparent lack of causal relationship between IFN-gamma-mediated increase in Cat B expression. Partial amyloid resistance in CD-1 mice, probably a genetic trait, may be linked to high levels of Cat D expression, causing a delay in nascent AA fibril formation.

N-terminal sequence analysis of SAA-derivatives purified from murine inflammatory macrophages.

The pathogenesis of secondary amyloidosis in vivo is not well-understood. Experimental studies suggest that incomplete degradation of acute phase serum amyloid A (SAA), presumably endocytosed by activated monocytoid cells, may lead to intralysosomal formation of amyloid A (AA). To establish a possible link between these two events, we have carried out partial N-terminal sequence analysis of affinity purified SAA derivatives from peritoneal macrophages isolated at 4 weeks post-infection from alveolar hydatid cyst infected C57BL/6 mice. The macrophage lysates yielded five N-terminally intact SAA derivatives of approximately 5 to approximately 12 kDa which reacted with anti-mouse AA IgG, and contained a mixture of SAA1 and SAA2 isoforms. The SAA2:SAA1 ratio, evaluated from their proportion present in each M(r) SAA derivative, showed a decrease with the decreasing apparent mass of the N-terminally infected SAA material. These results not only confirm that both SAA1 and SAA2 are processed by activated monocytoid cells but, more importantly, establish a plausible link between N-terminally intact SAA derivatives and formation of AA within activated monocytoid cells.

Fourier transform infrared spectroscopic investigation of temperature- and pressure-induced disaggregation of amyloid A.

The conformation-sensitive amide I band in the Fourier transform infrared (FTIR) spectra of amyloid A suspensions in D2O was examined as a function of temperature (25-95 degrees C) and applied hydrostatic pressure (1-12 kbar) to assess the stability of the peptide. The principal changes observed upon heating were a significant loss of intermolecular beta-sheet structure, and an increase in the broad band centred at 1644 cm(-1) assigned to unordered structure and alpha-helices of the dissociated species. Application of hydrostatic pressure at ambient temperature resulted in a limited degree of aggregate dissociation. These structural changes were partially reversible with cooling or release of the applied pressure. Dissolving the aggregated peptide in alkaline solution (pH 12) also resulted in disaggregation. Dissociation of organ-deposited amyloid substance bears clinical relevance. The present data indicate that residual amounts of undissociated amyloid in the milieu at physiological and acidic pH may act as nucleating foci rendering dissociated amyloid to reaggregate into organized amyloid.

Ubiquitin (Ub) interacts non-covalently with Alzheimer amyloid precursor protein (betaPP): isolation of Ub-betaPP conjugates from brain extracts.

Ubiquitin (Ub)-immunocytochemistry on Alzheimer's disease (AD) brain sections shows diverse Ub-associated deposits in the neuropil and senile plaques, elevated levels of Ub reactivity in hippocampal neurons and glia, and co-localization of Ub and beta-amyloid precursor protein (betaPP) epitope reactivity in dystrophic axons. These observations may suggest a role for Ub and stress-related mechanisms in AD pathogenesis. Here we show for the first time that Ub interacts avidly but non-covalently with betaPP and such complexes, apparently formed in vivo, can be isolated from AD brain extracts by Ub-gel matrix affinity chromatography. Polyclonal antibodies specific to Ub and to different regions of betaPP were employed to characterize these proteins. The implication of Ub-betaPP complex formation is discussed in the context of betaPP processing.

Ubiquitin and Alzheimer's amyloid beta precursor protein colocalize to endosomes-lysosomes in cultured human cells.

Chloroquine (CHQ)-sensitive cellular compartments, identified as endosomes-lysosomes (ELs), have been implicated in the proteolysis of amyloid beta precursor protein (A beta PP) in Alzheimer's disease. Here we show using immunocytochemistry and immunogold electron microscopy that not only A beta PP but also ubiquitin (Ub) co-localize to ELs in CHQ-treated human neuroblastoma (SK-N-SH) and glioblastoma (U-373). Immunoblotting analysis of cell lysates indicated a significant degree of CHQ-mediated interference in A beta PP metabolism in a time- and concentration-dependent manner. The implication is that abnormal intracellular accumulation of A beta PP and its C-terminal fragments beyond a certain threshold may trigger the Ub response. We hypothesize that Ub may play a role in A beta PP processing and/or trafficking to ELs, particularly in stress-related conditions.

Animal model for the pathogenesis of reactive amyloidosis.

The pathogenesis of amyloidosis is not well understood. Here, Zafer Ali-Khan, Weihua Li and Sic L. Chan present a metazoan parasite mouse model of reactive amyloidosis, review the relationship between chronic inflammation and multiorgan AA amyloidosis and postulate how ubiquitin might function in the processing of serum amyloid A and in AA amyloid formation in the endosomes-lysosomes of activated murine reticuloendothetial cells.

Alveolar hydatid cyst (AHC): inflammation-induced reactive gastrointestinal (GL) amyloidosis in AHC-infected mice and chemical characterization of the GL amyloid.

A high incidence of GI amyloidosis has been described in patients with various forms of systemic amyloidosis but its evolution and progression in different subregions of the GI tract are not well documented. These aspects including the chemical nature of GI amyloid were examined in the AHC mouse model of inflammation-associated reactive amyloidosis. C57BL/6 mice were infected intraperitoneally with 250 AHC. Paraffin sections from the stomach and the small and large intestines of AHC mice were stained at different time intervals with Congo red or immunocytochemically with monospecific RAA. The submucosal blood vessels at 1 week postinfection were found to be the first target of amyloid deposition. With time the amyloid deposits extended to the mucosa and the Peyer's patches and immunoreacted with RAA; ileum was the most severely affected region. Amyloid was extracted from the GI tract and purified by size exclusion chromatography using 5 M guanidine-formic acid, pH 3. The purified amyloid was identified by Western blotting using RAA and by partial N-terminal microsequencing up to 10 cycles. The GI amyloid showed homology with murine SAA2, although SAA2 mRNA is not expressed in murine GI tract. These results shows that (a) the GI amyloid is derived, similar to that of splenic/hepatic amyloid, from circulating SAA2 and (b) the GI tract submucosal blood vessels are the first target of AA deposition. The data also suggest that AA-mediated damage to the submucosal blood capillaries may lead to SAA leakage followed by cascading of AA deposition in other layers of the GI tract.

Both murine SAA1 and SAA2 yield AA amyloid in alveolar hydatid cyst-infected mice.

Amyloid susceptible C57BL/6 and partially amyloid resistant A/J mice, infected intraperitoneally with 250 alveolar hydatid cyst (AHC), the larval stage of a cestode parasite Echinococcus multilocularis, develop multiple organ amyloid deposits at approximately 1 and 4 weeks post infection (p.i.), respectively. Pooled spleens and livers from each mouse strain, at 8 and 10 weeks p.i., were used for the purification of protein AA utilizing a HiLoad Superdex 200 column equilibrated with 5 M guanidine-HCl. Protein AA from each mouse strain was separated on 16% Tris-tricine SDS-PAGE gels and immunoblotted with monospecific rabbit anti-mouse AA IgG; five and six immunoreactive AA subspecies were detected in the C57BL/6 and A/J materials, respectively. N-Terminal amino acid sequence analysis was performed on the bulk column-purified protein AA as well as on the electroblotted AA subspecies from each mouse strain. The results show a mixture of serum amyloid A1 (SAA1) and (SAA2)-derived AA protein from each mouse strain; SAA1-derived AA, although alluded to, has never been demonstrated as tissue deposits in mice. These findings suggest that the intense and persistent inflammatory processes in AHC-infected mice may have induced conversion of weakly amyloidogenic SAA1 to AA. This conversion could be detected by amino acid sequencing of electrophoretically separated AA subspecies.

Direct evidence for SAA deposition in tissues during murine amyloidogenesis.

To study the mechanism of amyloid deposition, the nature of amyloid proteins formed in experimental murine amyloidosis, was examined. Spleen specimens, 15-60 mg, were homogenized and extracted using aqueous acidic acetonitrile, in a recently developed procedure, making it possible to obtain amyloid proteins from minute amounts of tissue. The extracted material, 1.5-4 mg, was analysed by Western blotting and ELISA using antibodies recognizing differentially proteins AA and SAA. Two immunoreactive proteins of 8 and 12 KDa were isolated and subjected to amino acid analysis and N-terminal sequence determination. The results of immunochemical and chemical examination showed that the 8 and 12 KDa proteins represented proteins AA and SAA, respectively. The data obtained provide new direct evidence for SAA in tissues during murine amyloidogenesis.

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.

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.

Ubiquitin profile and amyloid enhancing factor activity in Alzheimer and 'normal' human brain extracts.

Tris-HCl or Laemmli sample buffer extracted frontal lobe and hippocampal samples from normal aged and Alzheimer's disease (AD) subjects were used to determine total ubiquitin (Ub), distribution of monomeric Ub and Ub-protein conjugates and amyloid enhancing factor (AEF) activity using the dot-blot, Western blot and mouse AEF bioassay techniques, respectively. The AD samples, as compared to the normals, demonstrated a 1.7-fold increase in total Ub, elevated levels of Ub-protein conjugates and an appreciably enhanced AEF activity. Many of the hippocampal Ub-protein conjugates were found to be soluble only in the Laemmli sample buffer. The possible roles of elevated Ub levels and of the association of AEF activity with Ub are discussed in regard to pathogenesis of brain amyloidosis.

Amyloid enhancing factor activity is associated with ubiquitin.

Crude amyloid enhancing factor (AEF) drastically reduces the pre-amyloid phase on passive transfer and induces amyloid deposition in the recipient mice in 48-120 h. We attempted to purify AEF from murine amyloidotic liver and spleen extracts by using gel filtration, preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis and ion exchange chromatography and isolated a 5.5 kDa peptide. In the mouse bioassay, this peptide induced accelerated splenic AA deposition in a dose-dependent manner. Based on structural, electrophoretic and immunochemical criteria the peptide was identified as ubiquitin. A polyclonal rabbit anti-bovine ubiquitin IgG antibody (RABU) abolished the in vivo AEF activity of crude murine AEF in a dose-dependent manner. Monomeric ubiquitin and its large molecular weight adducts were isolated from crude AEF using cyanogen bromide-activated sepharose conjugated to RABU and size exclusion chromatography methods. These were assayed and were found to possess AEF activity. Furthermore, increased levels of ubiquitin, a phenomenon similar to that of AEF, were detected by immunocytochemistry in mouse peritoneal leucocytes prior to and during amyloid deposition. Since AEF shares a number of biological and functional properties with ubiquitin, we suggest a possible role of ubiquitin as an AEF, and that serum amyloid protein A and ubiquitin, the two reactants generated during inflammatory stress conditions, may converge to induce AA amyloid deposition.

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.

Alzheimer's disease brain-derived ubiquitin has amyloid-enhancing factor activity: behavior of ubiquitin during accelerated amyloidogenesis.

Amyloid-enhancing factor (AEF) is believed to be a crucial common pathogenetic link in diverse forms of human amyloidosis. Passive transfer of crude AEF is known to trigger accelerated splenic amyloid deposition in mice. We have recently identified AEF activity in ubiquitin isolated from murine amyloidotic tissues. Using similar techniques we have purified ubiquitin, from crude Alzheimer's disease (AD) brain extracts, to apparent homogeneity. Based on the partial amino acid sequence homology, immunochemical and pathophysiological criteria, the approximately 5.5-kDa AD-derived protein was identified as ubiquitin (AD-ubiquitin) with AEF activity. Ten to twenty micrograms of this protein per mouse, with or without CaCl2, in conjunction with four subcutaneous injections of 0.5 ml of 1% aqueous AgNO3, induced accelerated splenic amyloid deposition. By immunohistochemistry, using anti-mouse AA amyloid antibody, the AD-ubiquitin-induced amyloid was identified as AA type. With anti-bovine ubiquitin antibody, using similar spleen sections as above, ubiquitin was found to co-deposit with AA amyloid in the splenic perifollicular areas. These results strongly suggest that ubiquitin may be involved in the pathogenesis of amyloidosis.

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.

Phlogistic and chemotactic activities of alveolar hydatid cyst antigen.

Alveolar hydatid cyst antigen (AHCA) absorbed with Sepharose-coupled anti-mouse Ig or its Sephacryl S-300 fractions was assayed for phlogistic/chemotactic and amyloidogenic properties in C57BL/6J mice. The in vivo and in vitro biological properties of the antigen were assessed by intradermal or intraperitoneal routes in mice or in Boyden chambers, respectively. In both these assays the chemotactic activity of the antigen was found to be dose dependent. A single intradermal injection of the antigen, containing 35 or 70 micrograms protein, showed a peak inflammatory cell response in the dermal layers at 6 hr. Neutrophils were the dominant cellular infiltrates and the number of monocytoid cells, except at 24 hr, remained relatively low. Antigen concentrations ranging from 1 to 200 micrograms protein per Boyden chamber showed peak neutrophilic and monocytoid cell responses with 100 micrograms of the antigen. We therefore conclude that intense inflammatory response and amyloidogenesis in alveolar hydatid cyst-infected murine hosts are directly attributable to the parasite antigen.

Echinococcus multilocularis: characterization of an alveolar hydatid cyst-induced amyloid enhancing factor.

Alveolar hydatid cysts, the larvae of Echinococcus multilocularis, were shown to induce the formation of an amyloid enhancing-like factor (AEF) called alveolar hydatid cyst-AEF (AHC-AEF) in the spleens of infected animals, 5 to 6 weeks post-infection. The adoptive transfer of AHC-AEF results in rapid induction and deposition of amyloid fibrils in the spleens following AgNO3 injection within 24 h. When injected concomitantly with alveolar hydatid cysts, it shortens the induction phase from 5 or 12 weeks to 1 week in C57BL/6J and A/JAX mice respectively. Preliminary characterization of this factor indicates that its bioactivity may be detected in the non-lipid moiety of spleen cell extracts and the sediment of ultracentrifugation. The factor is resistant to trypsin, pepsin, DNA-ase and RNA-ase treatments. It is sensitive to low or high pH (pH 3.0 and 9.5) and boiling. It resists freeze drying, dialysis, and storage at 4 degrees C for 2 weeks or -70 degrees C for more than a year. It may be fractionated using borate buffered saline, pH 7.4, on Sephacryl S-300 gel column. The active moiety eluted mainly in the first peak.