Intraperitoneal amyloid formation by amyloid enhancing factor--rich macrophages in ascitic fluid.

Although resident peritoneal cells from amyloidotic mice (amyloidotic peritoneal cells) are capable of processing the precursor protein of secondary amyloidosis, serum amyloid A (SAA) to amyloid fibrils, the peritoneum is a rare site for amyloid deposition. This is considered to be due to a deficiency of SAA in the peritoneum. To increase the supply of SAA to the peritoneum, ascitic fluid containing about the same protein constituents as in the serum was induced in mice. Amyloidotic peritoneal cells were packed in a microchamber which was shielded with filter membranes, and cultured in ascitic fluid supplemented with additional inflammatory factors. On the 7th day, Congo red-positive structures which showed green birefringence under polarized light were found inside and occasionally outside the chamber. By anti-AA or -SAA immunostaining, amyloid deposits and the cell surfaces of macrophages were positive. Immunologic depletion of T- and B-lymphocytes from the amyloidotic peritoneal cells did not adversely effect the amyloid formation in microchambers. These results suggest that either ascitic fluid containing sufficient amounts of SAA, or peritoneal macrophages with a high amyloid enhancing factor (AEF) activity are indispensable for AA amyloid fibrillogenesis in the peritoneum.     

Organ-specific (localized) synthesis of Ig light chain amyloid

Ig amyloidosis is usually a systemic disease with multisystem involvement. However, in a significant number of cases amyloid deposition is limited to one specific organ. It has not been determined if the Ig light chain (LC) amyloid precursor protein in localized amyloidosis is synthesized by circulating plasma cells with targeting of the amyloid fibril-forming process to one specific organ, or whether the synthesis of Ig LC and fibril formation occurs entirely as a localized process. In the present study local synthesis of an amyloid fibril precursor LC was investigated. Amyloid fibrils were isolated from a ureter that was obstructed by extensive infiltration of the wall with amyloid. Amino acid sequence analysis of the isolated fibril subunit protein proved it to be derived from a lambdaII Ig LC. Plasma cells within the lesion stained positively with labeled anti-lambda Ab and by in situ hybridization using an oligonucleotide probe specific for lambda-LC mRNA. RT-PCR of mRNA extracted from the tumor and direct DNA sequencing gave the nucleotide sequence coding specifically for the lambdaII amyloid subunit protein, thus confirming local synthesis of the LC protein.     

Depletion of Spleen Macrophages Delays AA Amyloid Development: A Study Performed in the Rapid Mouse Model of AA Amyloidosis

AA amyloidosis is a systemic disease that develops secondary to chronic inflammatory diseases Macrophages are often found in the vicinity of amyloid deposits and considered to play a role in both formation and degradation of amyloid fibrils. In spleen reside at least three types of macrophages, red pulp macrophages (RPM), marginal zone macrophages (MZM), metallophilic marginal zone macrophages (MMZM). MMZM and MZM are located in the marginal zone and express a unique collection of scavenger receptors that are involved in the uptake of blood-born particles. The murine AA amyloid model that resembles the human form of the disease has been used to study amyloid effects on different macrophage populations. Amyloid was induced by intravenous injection of amyloid enhancing factor and subcutaneous injections of silver nitrate and macrophages were identified with specific antibodies. We show that MZMs are highly sensitive to amyloid and decrease in number progressively with increasing amyloid load. Total area of MMZMs is unaffected by amyloid but cells are activated and migrate into the white pulp. In a group of mice spleen macrophages were depleted by an intravenous injection of clodronate filled liposomes. Subsequent injections of AEF and silver nitrate showed a sustained amyloid development. RPMs that constitute the majority of macrophages in spleen, appear insensitive to amyloid and do not participate in amyloid formation.     

AA-amyloidosis can be transferred by peripheral blood monocytes

Spongiform encephalopathies have been reported to be transmitted by blood transfusion even prior to the clinical onset. Experimental AA-amyloidosis shows similarities with prion disease and amyloid-containing organ-extracts can prime a recipient for the disease. In this systemic form of amyloidosis N-terminal fragments of the acute-phase reactant apolipoprotein serum amyloid A are the main amyloid protein. Initial amyloid deposits appear in the perifollicular region of the spleen, followed by deposits in the liver. We used the established murine model and induced AA-amyloidosis in NMRI mice by intravenous injections of purified amyloid fibrils ('amyloid enhancing factor') combined with inflammatory challenge (silver nitrate subcutaneously). Blood plasma and peripheral blood monocytes were isolated, sonicated and re-injected into new recipients followed by an inflammatory challenge during a three week period. When the animals were sacrificed presence of amyloid was analyzed in spleen sections after Congo red staining. Our result shows that some of the peripheral blood monocytes, isolated from animals with detectable amyloid, contained amyloid-seed that primed for AA-amyloid. The seeding material seems to have been phagocytosed by the cells since the AA-precursor (SAA1) was found not be expressed by the monocytes. Plasma recovered from mice with AA amyloidosis lacked seeding capacity. Amyloid enhancing activity can reside in monocytes recovered from mice with AA-amyloidosis and in a prion-like way trigger amyloid formation in conjunction with an inflammatory disorder. Human AA-amyloidosis resembles the murine form and every individual is expected to be exposed to conditions that initiate production of the acute-phase reactant. The monocyte-transfer mechanism should be eligible for the human disease and we point out blood transfusion as a putative route for transfer of amyloidosis.     

A fish oil diet retards experimental amyloidosis, modulates lymphocyte function, and decreases macrophage arachidonate metabolism in mice

Several recent reports have shown that diets in which the only source of fat was fish oil can modify the course of diseases with an inflammatory or immune component. In these experiments we examined the effect of a fish oil diet on experimental amyloidosis in mice. In most azocasein-treated mice, amyloid deposits were found in the spleen, varying from a trace to wide and contiguous perifollicular bands. The spleens of mice receiving fish oil had significantly less amyloid than did spleens of mice fed corn oil. A marked increase in spontaneous blastogenesis that occurred in azocasein-treated mice on corn oil was suppressed in azocasein-treated mice on fish oil. Substitution of the unsaturated fatty acids of corn oil with those of fish oil was also found to modify the prostaglandin profile of macrophages. This altered profile may change cellular immune function and/or enhance the processing of serum amyloid A to retard the induction of secondary amyloidosis in mice.     

Regulation of immunoglobulin production in human peripheral bood leukocytes: cellular interactions.

The intercellular influences regulating immunoglobulin (Ig) synthesis by normal human peripheral blood leukocytes (PBL) were investigated in cells stimulated by pokeweed mitogen (PWM). This system was shown to be totally T lymphocyte dependent as purified B lymphocytes (less than or equal to 1% T lymphocytes) failed to make significant amounts of Ig. No evidence was obtained for an Ig class switch as all classes of Ig (IgM, IgG, IgA) were shown to be produced in increasing amounts over a 6-day time period. T lymphocytes demonstrated maximum helper effect when mixed with equal numbers of B cells. This helper effect was mediated through the dual mechanisms of increasing the number of B lymphocytes containing cytoplasmic Ig and by increasing the maturity of these B lymphocytes as demonstrated by an increasing Ig production per B lymphocyte. When present in higher numbers, T lymphocytes were also capable of suppressing Ig production. This T-mediated suppression was first evident as a decrease in the Ig produced per B lymphocyte (decreased maturity). With maximum T suppression Ig-containing B lymphocyte numbers were also diminished. T lymphocyte help was relatively independent of macrophages (phagocytic cells) and did not require DNA synthesis for expression. Both T help and suppression were shown to cross allogeneic barriers. Immature T lymphocytes (thymocytes) were incapable of mediating either activity. Normal human PBL contain T lymphocytes campable of mediating both T help and suppression and the Ig produced by PBL was shown to be the balance of these activities. This balance probably represent the participation of distinct T lymphocyte subpopulations analogous to the T helper (Ly 1+) and T suppressor (Ly 2+, 3+) populations in the mouse.     

Investigation into the role of macrophages in the formation and degradation of beta2-microglobulin amyloid fibrils

Dialysis related amyloidosis is a serious complication of long-term hemodialysis in which beta(2)-microglobulin (beta(2)m) forms amyloid fibrils that deposit predominantly in cartilaginous tissues. How these fibrils form in vivo, however, is poorly understood. Here we perform a systematic investigation into the role of macrophages in the formation and degradation of beta(2)m amyloid fibrils, building on observations that macrophages are found in association with beta(2)m amyloid deposits in vivo and that these cells contain intra-lysosomal beta(2)m amyloid. In live cell imaging experiments we demonstrate that macrophages internalize monomeric beta(2)m, whereupon it is sorted to lysosomes. At lysosomal pH beta(2)m self-associates in vitro to form amyloid-like fibrils with an array of morphologies as visualized by atomic force microscopy. Cleavage of the monomeric protein by both macrophages and lysosomal proteases isolated from these cells results in the rapid degradation of the monomeric protein, preventing amyloid formation. Incubation of macrophages with preformed fibrils revealed that macrophages internalize amyloid-like fibrils formed extracellularly, but in marked contrast with the monomeric protein, the fibrils were not degraded within macrophage lysosomes. Correspondingly beta(2)m fibrils were highly resistant to degradation by high concentrations of lysosomal proteases isolated from macrophages. Despite their enormous degradative capacity, therefore, macrophage lysosomes cannot ameliorate dialysis-related amyloidosis by degrading pre-existing amyloid fibrils, but lysosomal proteases may play a protective role by eliminating amyloid precursors before beta(2)m fibrils can accumulate in what may represent an otherwise fibrillogenic environment.     

The antisense approach in amyloid light chain amyloidosis: identification of monoclonal Ig and inhibition of its production by antisense oligonucleotides in in vitro and in vivo models

Primary amyloid L chain (AL) amyloidosis is a plasma cell disorder in which depositions of AL cause progressive organ failure. The lack of effective therapies for this fatal disease prompts exploration of newer treatment avenues. We have investigated the application of antisense oligonucleotides (AS) for the inhibition of monoclonal Ig production. The monoclonal L chain was identified by using primers designed for amplifying the human lambda Ig V (Vlambda) region. We demonstrated that AS against L chain complementarity-determining regions inhibited the production of L chain in vitro. RPMI 8226 myeloma cells injected in SCID mice developed s.c. tumors. RT-PCR analysis showed Vlambda mRNA expression in the tumors. In addition, the presence of human Ig in the sera of mice given injection of RPMI 8226 cells was confirmed by ELISA. Administration of AS inhibited the expression of Vlambda mRNA in the s.c. tumors and decreased the concentration of L chain in serum. Therefore, we have shown that it is possible to determine the sequence of Vlambda mRNA and design specific complementary oligonucleotides, suggesting that treatment with Vlambda antisense could represent a rational novel approach to improve treatment outcome in AL amyloidosis.     

Differential expression of the amyloid SAA 3 gene in liver and peritoneal macrophages of mice undergoing dissimilar inflammatory episodes

The three active serum amyloid A (SAA) genes of mice, SAA 1, SAA 2, and SAA 3, are coordinately expressed in liver during acute and chronic inflammatory stimulation and experimental amyloidosis. The genes, primarily SAA 3, are also expressed extrahepatically. The apoprotein SAA 2 is the precursor of the amyloid A (AA) fibril protein that is deposited as insoluble fibrils extracellularly in spleen and other organs when amyloidosis occurs secondarily to inflammation. The exact cause of AA fibril formation is unknown. Amyloid enhancing factor is a high m.w. glycoprotein extracted from amyloidotic organs. Administration of amyloid enhancing factor alters experimental inflammation to bring about accelerated deposition of amyloid A fibrils first in spleen and later in other organs. In this study, hepatic and extrahepatic expression of the SAA genes were compared during accelerated amyloidosis relative to inflammation uncomplicated by amyloidosis. Differences in kinetics and pattern of SAA gene expression by resident peritoneal macrophages and liver were detected during four dissimilar inflammatory episodes. Macrophages expressed the SAA 3 gene solely, and to a greater extent in chronic than in acute inflammation. In accelerated amyloid induction, macrophage SAA 3 expression increased as SAA 1 and SAA 2 expression in liver decreased. However, alpha-1-acid glycoprotein expression remained elevated throughout the course of amyloid induction. The greatly increased expression of the SAA 3 gene by macrophages and decreased expression of the SAA 1 and SAA 2 genes in liver during amyloidosis, suggests that altered SAA gene expression may play a pathogenetic role in experimental amyloid deposition.     

Fibrillar islet amyloid polypeptide (amylin) is internalised by macrophages but resists proteolytic degradation

Pancreatic islet amyloid, formed from islet amyloid polypeptide, is found in 96% of Type II (non-insulin-dependent) diabetic patients. Islet amyloidosis is progressive and apparently irreversible. Fibrils immunoreactive for islet amyloid polypeptide are found in macrophages associated with amyloid, suggesting that deposits can be phagocytosed. To determine the mechanism for the recognition and internalisation of fibrils, mouse peritoneal macrophages were cultured with fibrillar synthetic human islet amyloid polypeptide. Fibrils did not exert a cytotoxic effect over 72 h of culture. The uptake and degradation of fibrils was analysed by quantitative light-and electron-microscopic immunocytochemistry and immunoreactivity was detectable in 86±3% cells within 6 h of culture. Neither polyinosinic acid (200 µg/ml) nor nocodazole (10 µg/ml) inhibited fibril uptake, suggesting that internalisation is not blocked by poly-ions and is independent of microtubule assembly. Inhibition of pseudopodia formation by cytochalasin B blocked fibriI uptake. Fibril aggregates became condensed in lysosomes to form protofilaments and were resistant to intracellular proteolysis. Fibrils can be phagocytosed by macrophages in vitro but amyloid-associated factors may block the recognition of fibrils in vivo preventing the removal of islet amyloid in diabetes.     

Oligodeoxynucleotides containing CpG motifs induce low levels of TNF-alpha in human B lymphocytes: possible adjuvants for Th1 responses

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) represent potential adjuvants for specific immunotherapy of type I allergies because they foster Th1-like immune responses. However, previous work has shown that CpG-ODN induce systemically active levels of TNF-alpha in murine macrophages. The goal of the present study was to evaluate the release of TNF-alpha in human cells by a CpG-ODN proven to induce Th1 immune responses in cells from atopic individuals and in mice. CpG-ODN induced TNF-alpha in cells from atopic and healthy individuals. However, the amounts were low, as determined by comparison with commonly used Ags. Intracellular cytokine staining of PBMC revealed that CpG-ODN-induced TNF-alpha derived exclusively from B lymphocytes. TNF-alpha contributed to the CpG-ODN-augmented proliferation and Ig synthesis in PBMC, but was not involved in IFN-gamma synthesis. In conclusion, our findings indicate that certain CpG-ODN induce low amounts of TNF-alpha in human B lymphocytes and may therefore be used to modulate Th2-biased immune responses in allergic patients.     

Colocalization of apolipoprotein AI in various kinds of systemic amyloidosis.

Apolipoprotein AI (apoAI), a major component of high-density lipoproteins, is one of the major amyloid fibril proteins and a minor constituent of the senile plaques observed in Alzheimer's disease. We examined colocalization of apoAI in various kinds of systemic amyloidosis in this study. Forty-three of 48 formalin-fixed paraffin-embedded heart specimens with various forms of systemic amyloidosis reacted immunohistochemically with anti-human apoAI antibody. ApoAI was also detected in water-extracted amyloid material by immunoblotting. In addition, we observed colocalization of apoAI and murine amyloid A (AA) amyloidosis in human apoAI transgenic mice. This is the first report of colocalization of apoAI with amyloid deposits in various forms of human systemic amyloidosis and murine AA amyloidosis in human apoAI transgenic mice. ApoAI may not always be a major component of amyloid fibrils, even when it is present in systemic amyloid deposits.     

Specific pathogen free conditions prevent transthyretin amyloidosis in mouse models

Transthyretin (TTR) associated amyloidosis is an autosomal dominant disorder characterized by peripheral and autonomic neuropathy. Both genetic and environmental factors are thought to be involved in development of TTR associated amyloidosis. Previously, we demonstrated that amyloid deposition was observed in various tissues of transgenic mouse lines carrying a human mutant TTR (Met30) gene. To analyze the influence of environmental factors on TTR amyloidosis, these amyloidogenic transgenic mouse models were kept under conventional (CV) or specific pathogen free (SPF) conditions. Although the serum levels of Met30 for mice housed in the CV and SPF conditions were similar, amyloid deposition was observed in CV conditions, but not in SPF conditions. In addition, the extent of amyloid deposition in transgenic mice was dependent on duration kept under CV conditions. There were significant differences in proportion of amyloid deposition in several tissues between CV and SPF conditions. Maintenance of these mice at 30 degrees C did not induce amyloid deposition in SPF conditions. These results suggest that the SPF conditions can completely prevent amyloid deposition, and that environmental factors can affect the onset and progression even in a single gene disorder.     

The induction of accelerated murine amyloid with human splenic extract. Probable role of amyloid enhancing factor

Amyloid enhancing factor (AEF) is derived from the tissues of pre-amyloidotic and amyloidotic animals and, when transferred, greatly accelerates amyloid induction in the recipient murine models. It has also been reported that similarly accelerated amyloid induction can be achieved in mice by injection of human splenic homogenates from patients with amyloidosis. The present study has attempted to characterize further the mechanism of this "heterologous transfer of amyloid". Treatment of mice with the "tissue homogenate" or the "AEF extract" of AA-, AL- and A prealbumin-laden human spleens followed by daily subcutaneous casein injections induced amyloidosis in an accelerated fashion. The resultant amyloid deposits in mice had strongly positive immunohistochemical reactions with anti-mouse AA, and negative reaction with anti-human AA or anti-human prealbumin. The results lend support to the idea that accelerated amyloid induction in the recipient mice is unlikely to be due to transfer of human amyloid substance, but rather to formation of "native" murine amyloid under the influence of a human AEF factor similar to or identical with AEF described in mouse-to mouse transfer models.     

AL Amyloid Imaging and Therapy with a Monoclonal Antibody to a Cryptic Epitope on Amyloid Fibrils

The monoclonal antibody 2A4 binds an epitope derived from a cleavage site of serum amyloid protein A (sAA) containing a -Glu-Asp- amino acid pairing. In addition to its reactivity with sAA amyloid deposits, the antibody was also found to bind amyloid fibrils composed of immunoglobulin light chains. The antibody binds to synthetic fibrils and human light chain (AL) amyloid extracts with high affinity even in the presence of soluble light chain proteins. Immunohistochemistry with biotinylated 2A4 demonstrated positive reaction with ALκ and ALλ human amyloid deposits in various organs. Surface plasmon resonance analyses using synthetic AL fibrils as a substrate revealed that 2A4 bound with a K_D of ∼10 nM. Binding was inhibited in the presence of the –Glu-Asp- containing immunogen peptide. Radiolabeled 2A4 specifically localized with human AL amyloid extracts implanted in mice (amyloidomas) as evidenced by single photon emission (SPECT) imaging. Furthermore, co-localization of the radiolabeled mAb with amyloid was shown in biodistribution and micro-autoradiography studies. Treatment with 2A4 expedited regression of ALκ amyloidomas in mice, likely mediated by the action of macrophages and neutrophils, relative to animals that received a control antibody. These data indicate that the 2A4 mAb might be of interest for potential imaging and immunotherapy in patients with AL amyloidosis.     

Leishmania (Leishmania) amazonensis: experimental cutaneous leishmaniasis associated with systemic amyloidosis in mice

We infected Swiss and C57BL/6 female mice in the left hind footpad with 10⁴Leishmania (L.) amazonensis promastigotes in stationary phase. The macroscopic examination showed a nodular non-ulcerated lesion at the site of inoculation and hepatic and spleenic enlargement. Histopathologically, the primary lesion showed an extensive liquefactive necrosis and inflammatory infiltrate, mainly consisting of macrophages filled with amastigotes, and rare lymphocytes. The inflammatory reaction in liver, spleen and kidney showed amyloid deposits. Additionally, C57BL/6 had accentuated amyloidosis in both ovarian cortical and medullar region and inflammatory infiltrates in the pancreas and adrenal gland.     

Transmission of circulating cell-free AA amyloid oligomers in exosomes vectors via a prion-like mechanism

Recent studies clearly demonstrated that several types of pathogenic amyloid proteins acted as agents that could transmit amyloidosis by means of a prion-like mechanism. Systemic AA amyloidosis is one of the most severe complications of chronic inflammatory disorders, particularly rheumatoid arthritis. It is well known that, similar to an infectious prion protein, amyloid-enhancing factor (AEF) acts as a transmissible agent in AA amyloidosis. However, how AEF transmits AA amyloidosis in vivo remained to be fully elucidated. In the present study, we focused on finding cell-free forms of AEF and its carriers in circulation by using the murine transfer model of AA amyloidosis. We first determined that circulating cell-free AEF existed in blood and plasma in mice with systemic AA amyloidosis. Second, we established that plasma exosomes containing AA amyloid oligomers derived from serum amyloid A had AEF activity and could transmit systemic AA amyloidosis via a prion-like mechanism. These novel findings should provide insights into the transmission mechanism of systemic amyloidoses.     

Lymphocyte transformation in casein-induced murine amyloidosis.

Spleen lymphocytes from casein-induced amyloidotic mice demonstrate diminished transformation in vitro to PHA-P, concanavalin A, and pokeweed mitogen but a normal response to lipopolysaccharide. Thymic and peripheral blood lymphocytes respond normally to these mitogens. The amyloidogen casein acted as a mitogen in both normal and casein injected mice. The diminished PHA responsiveness of spleen lymphocytes in vitro could have resulted from an inhibitory effect of amyloid fibrils on lymphocyte proliferation and did not indicate a generalized diminished cellular immunologic responsiveness of amyloidotic mice.     

Establishment of an Inflamed Animal Model of Diabetic Nephropathy

Aims Inflammatory stress plays a crucial role in the progression of diabetic nephropathy (DN). This study aimed to establish a novel inflamed animal model of DN and to evaluate its significance in DN.Methods Nondiabetic db/m mice and diabetic db/db mice were randomly divided into four groups: db/m, db/m+casein, db/db, and db/db+casein for eight weeks. Casein was subcutaneously injected to induce chronic inflammation. Body weight and albumin to creatinine ratio (ACR) in the urine were measured every week. The plasma levels of serum amyloid protein A (SAA) and tumour necrotic factor-α (TNF-α) were determined with the enzyme-linked immunosorbent assay. The morphological changes to the renal pathology and ultra-microstructures were checked by pathological staining and electron microscopy. Immunofluorescent staining and Western blotting were used to determine the protein expression of podocyte-specific molecules and inflammatory cytokines in kidneys.Results ACR, plasma levels of SAA and TNF-α, protein expression of inflammatory cytokines, mesangial expansion, collagen accumulation, and foot process effacement in kidneys of casein-injected db/db mice were significantly increased compared with the db/db mice. Casein injection markedly decreased the protein expression of Wilms'' tumor-1 and nephrin in kidneys of db/db mice, which are specific podocyte biomarkers, suggesting that chronic inflammation accelerates podocyte injuries in db/db mice. Interestingly, no obvious urinary protein, inflammatory cytokine expression, or histological changes in the kidneys of casein-injected db/m mice were found compared with the db/m mice.Conclusion An inflamed animal model of DN was successfully established and may provide a useful tool for investigating the pathogenesis of DN under inflammatory stress.     

Amyloidosis in adrenal glands of hamsters experimentally infected with Leishmania infantum

Thirty 10-week-old hamsters were inoculated intraperitoneally with leishmania infantum amastigotes and were serially killed after 15, 30, 45, 60, 75 and 89-90 days. The adrenal glands of each of them were examined by means of light and electron microscopy. The cortex and medulla of the adrenal gland showed a progressive deposition of amyloid, selectively identified by both optical and ultrastructural techniques. It has been suggested that adrenal amyloidosis during visceral leishmaniasis is directly related to a stimulation of the phagocitary mononuclear system due to the persistence of the antigenic stimulation of the parasite. In addition to these deposits, the presence of inflammatory infiltrates containing lymphocytes, plasmocytes and macrophages with leishmanias confirmed the establishment of leishmaniasis. The deposition of the amyloid fibrils finally leads to the partial destruction of the adrenal parenchyma.