apo-SAA1/apo-SAA2 Isotype Ratios during Casein- and Amyloid-Enhancing-Factor-Induced Secondary Amyloidosis in A/J and C57BL/6J Mice

A/J mice are resistant while C57BL/6J are susceptible to casein-induced secondary amyloidosis. One mechanism responsible for this phenotypic expression of resistance/susceptibility was shown to operate at the level of production of the 'amyloid-enhancing factor' (AEF). AEF and processing of the apo-SAA protein appear almost concomitantly during amyloidogenesis. In order to determine if AEF played a role in the processing of the apo-SAA protein, three major parameters (apo-SAA1/apo-SAA2 ratios, level of AEF, and fibril formation) were determined during casein-induced secondary amyloidosis. Kinetics of AEF production and serum levels of the two major apo-SAA isotypes were compared in A/J and C57BL/6J animals. Both strains showed equal relative amounts of the two isotypes after seven, 15 and 21 casein injections, irrespective of the fact that the A/J strain had no detectable level of AEF and no amyloid deposition; while C57BL/6J mice had a high AEF level and were amyloidotic after 15 and 21 injections. An increased apo-SAA1/apo-SAA2 ratio due to a decrease in apo-SAA2 was noted after 38 days of casein injections when both strains had extensive deposits of amyloid fibrils. Involvement of AEF as an effector molecule was determined by following the ratio of the two major serum apo-SAA isotypes and fibril formation during an accelerated protocol of amyloid induction in C57BL/6J animals. AEF had no direct effect on apo-SAA isotype ratios in the serum.     

Studies of the in vivo synthesis and catabolism of serum amyloid P component (SAP) in the mouse.

The production of mouse serum amyloid P component (SAP), a major acute phase protein of liver origin, was studied immunocytochemically using the peroxidase staining technique. SAP was not detectable in the cytoplasm of hepatocytes from normal, unstimulated mice, nor was it observed before 24 h after an acute phase stimulus. 125I-labelled mouse SAP was cleared from the plasma in vivo with a half-life of 7.0-8.25 h in all animals studied including: normal mice of different strains with different genetically determined plasma SAP concentrations; mice undergoing acute phase responses with greatly elevated plasma SAP levels and mice with casein-induced amyloidosis. The circulating level of SAP is thus independent of its rate of clearance and catabolism and is determined by the rate of synthesis and/or secretion of SAP.     

Amyloid-enhancing factor: production and response in amyloidosis-susceptible and -resistant mouse strains

Genetic variations in the development of casein-induced amyloidosis exist among inbred strains of mice: CBA/J and C57BL/6J mice are susceptible, while A/J strain mice are resistant to this disease. Amyloidosis is usually induced by daily injections of an inflammatory stimulus for 2-3 wk. The deposition of amyloid in experimental animals can be accelerated by injection of a material called amyloid-enhancing factor (AEF); when injected concomitantly with an inflammatory stimulus, AEF provokes appearance of amyloidosis as early as 2 days after injection. AEF is extracted from amyloid laden or from normal organs (although in small amount). Our studies were designed to determine if the resistance to amyloidosis seen in A/J mice was either due to a lack of AEF production or to an inability of these mice to respond to AEF. A standard source of CBA/J-derived AEF facilitated the development of amyloidosis in the organs of both the susceptible (CBA/J, C57BL/6J) and the resistant A/J mice. On the contrary, amyloidosis was only induced in susceptible CBA/J hosts when material derived from susceptible (CBA/J, C57BL/6J) animals was injected. CBA/J mice injected with A/J-derived AEF preparation did not develop amyloidosis. These results thus suggest that the determination of resistance or susceptibility to secondary amyloidosis could operate at the level of AEF production.     

Changes in Natural Killer Activities in Experimental Secondary Amyloidosis

Natural killer (NK) cell activity in experimental murine amyloidosis was studied. In CBA/J mice, which show a high incidence of amyloidosis, NK activity was significantly decreased after 1 week of casein treatment. In C3H mice, which show a low incidence of amyloidosis, NK activity was not changed by casein treatment. Pretreatment with lipopolysaccharide in vivo enhanced the NK activities in CBA/J and C3H mice. These increases were not observed after casein treatment. The lowered NK activity of cells from CBA/J mice after casein treatment was restored to the normal range by indomethacine in vitro. Depletion of adherent cells from the spleen cells treated with casein had no effect on NK activity. Single-cell assay showed that casein treatment impaired the killing but not the binding of NK cells to target cells. After casein treatment, the splenic serum amyloid A (SAA) level gradually increased in CBA/J mice but remained low in C3H mice. NK activity was suppressed by the addition of serum obtained from CBA/J mice treated with casein but not by normal control serum. And partially purified AA protein obtained from the spleen of CBA/J mice treated with casein also suppressed NK activity in vitro.     

Casein-induced experimental amyloidosis. VI. A pathogenic role for b cells in the murine model.

The relationship between cellular (B-cell) responses and the development of casein-induced amyloidosis was explored. The main findings are: (1) B-cell abnormalities are more pronounced in amyloid susceptible CBA/J than in amyloid resistant A/J mice; these include increased mitogen responses to SIII, PI:C and DXS, and enhanced primary immune responses to T-independent antigens; (2) CBA/J amyloid spleen cell suspensions appear to be enriched with antibody dependent killer cells and precursors of antibody-forming cells; these abnormalities are not seen in casein treated A/J mice. This hitherto unrecognized proliferation of immunoblasts in casein-treated CBA/J animals raises the possibility that B cell-macrophage interaction may play an important role in the pathogenesis of amyloid disease.     

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.     

Amyloid resorption in the spleen grafted into intact and amyloid recipients

Changes in amyloid spleens from CBA mice were studied when grafted into recipients of the following four groups: intact CBA mice, CBA mice with casein-induced amyloidosis, intact C57BL mice, and noninbred rats. Amyloid resorption in a syngeneic graft in recipients with amyloidosis was much less intensive than in intact animals. The intensity of amyloid resorption in intact recipients with syngeneic, allogeneic, and xenogeneic grafts increased with increasing heterogeneity of the transplanted material. The development of systemic amyloidosis (the so-called “transfer” of amyloidosis) was observed in some intact recipients after syngeneic transplantation of an amyloid spleen; the resorption of amyloid in the graft in these animals was less marked than in mice without “transfer.” Administration of hydrocortisone into animals with a syngeneic graft of an amyloid spleen completely inhibited amyloid resorption in the graft.     

Colchicine inhibition of the first phase of amyloid synthesis in experimental animals.

Colchicine was found to inhibit the first phase of casein-induced synthesis of murine amyloid. When mice were treated with colchicine during the first 7 days of an amyloid induction regimen or when colchicine was given to the donor mice in a transfer model, the amyloidogenic stimulus of casein was blocked completely. Amyloid synthesis was however, not interrupted by the administration of colchicine during the last 7 days of the casein regimen nor by colchicine treatment of recipient mice in a transfer model.     

Role of the acute phase response in the Shwartzman phenomenon.

Following elicitation of the local Shwartzman reaction by intradermal injection of Salmonella enteritidis lipopolysaccharide (LPS) in mice, there was a marked acute phase response which was monitored by measuring the serum levels of serum amyloid P component (SAP) and C3. Prednisolone therapy had no effect on either the cutaneous lesion or the accompanying acute phase response. Also, in vivo complement depletion with cobra factor did not affect the lesion or the SAP response despite gross reduction in serum C3 levels. In contrast, administration of colchicine at the same time as LPS suppressed both the acute phase response and the Shwartzman reaction. Inhibition of the cutaneous reaction by colchicine was abrogated by injecting mice with casein, and unrelated acute phase stimulus, the day before challenge with LPS. These observations suggest that acute phase proteins may participate in pathogenesis of the Shwartzman phenomenon.     

Major acute-phase reactant synthesis during chronic inflammation in amyloid-susceptible and -resistant mouse strains

Hepatic levels of mRNA specific for total serum amyloid A (SAA), the SAA1 and SAA2 isotypes, serum amyloid P (SAP), C-reactive protein (CRP), and fibronectin, as well as the plasma concentrations of SAA and SAP were examined in amyloid-resistant (A/J) and amyloid-susceptible (CBA/J) mice during azocaseininduced chronic inflammation. In both strains hepatic SAA and SAP mRNA levels and plasma SAA and SAP protein concentrations increased dramatically during the early stages of inflammation; this was followed by a decrease to concentrations that were maintained at levels considerably higher than background. The ratios of SAA1 and SAA2 mRNA and plasma protein were 1 1 throughout. This indicated that there was no preferential accumulation of mRNA specifying a particular isotype and no preferential synthesis or clearance of a particular isotype during chronic inflammation and the early stages of amyloidogenesis in either strain. Similarly, hepatic SAP mRNA levels in both strains increased dramatically during the early stages of inflammation and were subsequently maintained at elevated levels. Plasma SAP concentrations increased rapidly during the first three days of the study in both A/J and CBA/J mice; however, during the later stages of inflammation, A/J plasma SAP levels decreased to a steady-state concentration that was approximately half that observed in CBA/J mice. Our results identify differences in the hepatic mRNA and plasma protein levels of the major mouse acute-phase reactants (APR) in the amyloid-resistant A/J and amyloid-susceptible CBA/J mouse strains. These findings are consistent with circulating inflammatory APR concentrations contributing, together with other factors, to the onset and pathogenesis of secondary amyloidosis.     

Casein-induced experimental amyloidosis. IX. Alterations in marrow dependent function.

CBA/J mice receiving multiple injections of sodium caseinate (CAS) or bovine serum albumin (BSA) were assayed for marrow dependent functions by measuring their ability (i) to reject bone marrow allografts and (ii) to resist Friend virus (FV)-induced suppression of lymphocyte mitogenesis. Mice that developed amyloidosis following 25-30 injections completely lost the ability to reject allogeneic marrow cells, whereas nonamyloid BSA-treated mice had enhanced rejection of marrow allografts. There was increased resistance to the suppressive effects of FV in spleen cells from 'preamyloid' mice receiving CAS injections and nonamyloid mice receiving 10-40 BSA injections. Amyloid mice appeared to be as susceptible to the effects of FV-induced suppression as control (untreated) animals. These data indicate that alterations in marrow dependent function may be related to the pathogenesis of amyloid disease.     

Isolation and characterization of C-reactive protein and serum amyloid P component in the rat.

C-reactive protein (RP) and serum amyloid P component (SAP) have been identified for the first time in rat serum and isolated by calcium-dependent affinity chromatography. Rat CRP closely resembled human CRP in its amino acid composition, in having five subunits per molecule and in its electron microscopic appearance as a pentameric annular disc. It differed, however, from all other mammalian CRP's characterised hitherto in being a glycoprotein bearing a single complex oligosaccharide on each polypeptide subunit. Furthermore one pair of tis subunits per molecule was linked by a interchain disulphide bridges whereas in other animals the subunits of both CRP and SAP are all non-covalently associated. The serum concentration of CRP in normal healthy laboratory rats and in specific pathogen-free rats was 300-600 micrograms/ml which is much greater than has been described in any other species and exceeds even maximal acute phase levels of CRP in man. Following injections of casein or croton oil, serum CRP levels rose to a maximum of about 900 micrograms/ml. Rat CRP bound to pneumococcal C-polysaccharide (CPS( but, in marked contrast to the behaviour of CRP from man, rabbit and marine teleost fish, it did not precipitate with CPS solutions, agglutinate CPS-coated sheep erythrocytes or initiate complement activation. Rat SAP, like SAP of other species, was a glycoprotein but unlike them it was composed only of a single pentameric disc not two such discs interacting face-to-face. The normal level of SAP in rat serum was 20-50 micrograms/ml, very similar to the levels seen in man, and it did not behave as an acute phase reactant in response to casein or croton-oil injections. In this respect it resembled human SAP but differed from murine SAP which is a major acute phase reactant.     

Circulating serum amyloid P component is the precursor of amyloid P component in tissue amyloid deposits.

Intravenous administration of 125I-labelled isolated mouse serum amyloid P component (SAP) to mice with systemic amyloidosis was followed by specific deposition of the labelled protein in amyloidotic organs. Although only a small proportion of the total injected dose became localized in this way, the amount correlated with the quantity of amyloid present in different organs and was greatest in the spleen. No such localization was detected in the organs of control, untreated mice or animals which had received inflammatory stimuli but did not have amyloidosis. The labelled SAP was found by autoradiography to be present in the same distribution within the tissues as the Congophilic amyloid deposits. These observations establish directly, for the first time, that circulating SAP is the precursor of the amyloid P component (AP) in systemic amyloidosis. They were confirmed by the further finding that intravenous injection into amyloidotic mice of human SAP, either in whole human serum or in isolated pure form, was followed by appearance of the human SAP in the mouse amyloid deposits. In addition to elucidating one aspect of the pathogenesis of amyloid deposition and strengthening the homology of functional behaviour between SAP of different species, the present results suggest a means for selective targeting of diagnostic tracers and/or effector agents to amyloid deposits in vivo.     

Experimental amyloidosis in the hamster: correlation between hamster female protein levels and amyloid deposition.

Reactive systemic AA amyloidosis was induced in female, male and castrated male hamsters either by repeated injection of casein or by injection of amyloid enhancing factor (AEF) followed by casein. The circulating concentrations of serum amyloid A protein (SAA), the putative precursor of the AA amyloid fibril protein, and of female protein (FP), the pentraxin homologue of serum amyloid P component (SAP) of other species, were measured and correlated with the speed and extent of amyloid deposition. The SAA responses of the three groups of hamsters were indistinguishable in both experiments but, in confirmation of previous reports, castrated males had FP levels higher than those of control males though still lower than in females. No differences were seen between groups in amyloid induction by casein injection alone. However, in the accelerated model using AEF, amyloid deposition occurred sooner and was more extensive in both females and castrated males than in unoperated males. These results strengthen the association between SAP, of which FP is the hamster counterpart, and the pathogenesis of amyloidosis.     

Effect of colchicine on experimental amyloidosis in two CBA/J mouse models. Chronic inflammatory stimulation and administration of amyloid-enhancing factor during acute inflammation

To investigate the mechanism of action of colchicine in blocking amyloid deposition, two model systems of amyloidosis in CBA/J mice were studied. In experimental chronic inflammation, daily injection of silver nitrate (AgNO3) resulted in the deposition of 667 +/- 68 ng of amyloid A protein (AA)/mg of spleen after 25 days. Treatment with 10 micrograms of colchicine daily decreased AgNO3-induced AA deposition to 12 +/- 1 ng of AA/mg of spleen (p less than 0.001). Colchicine diminished the acute phase serum amyloid A protein (SAA) response after 24 hours. Over a 25-day period, SAA concentrations declined and approached baseline both in colchicine-treated and (unexpectedly) in control mice. This suggested that suppression of SAA levels was not the primary event inhibiting amyloid deposition. In a model of accelerated amyloid deposition, injection of preformed amyloid-enhancing factor along with AgNO3 induced the deposition of 974 +/- 46 ng of AA/mg of spleen 48 hours later. Colchicine only partially decreased amyloid-enhancing factor-induced amyloid deposition to 578 +/- 91 ng of AA/mg of spleen, while blunting the acute phase SAA response. These results suggest that colchicine inhibits amyloidosis in the predeposition phase, possibly by blocking formation of amyloid-enhancing factor.     

Amyloid P-component in mice injected with casein: identification in amyloid deposits and in the cytoplasm of hepatocytes.

Amyloid P-component was sought by immunofluorescence in the tissues of CBA mice receiving repeated subcutaneous injections of casein. P-component appeared in a perifollicular distribution in the spleen after about twenty casein injections, and correlated precisely with amyloid deposits identified by staining with Congo red. In mice which had received at least fourteen injections of casein, P-component also became detectable within the cytoplasm of periportal hepatocytes. This is the first demonstration of P-component in murine amyloid and of a possible site for in vivo synthesis of P-component.     

Lymphoid cells in endotoxin-induced production of the amyloid-related serum amyloid A protein.

Endotoxin-treated mice exhibit a rapid rise in the level of the serum amyloid A (SAA) protein, but this effect is not observed in endotoxin-resistant C3H/HeJ mice. To evaluate the role of lymphoid cells in the production of SAA protein, C3H/HeJ mice were adoptively transfused with endotoxin-sensitive (C3H/HeN) bone marrow cells. After such adoptive transfer, endotoxin treatment of C3H/HeJ mice resulted in high serum levels of SAA protein. The ability of chimeric mice to make SAA protein correlated with the presence of endotoxin-sensitive B lymphocytes and macrophages. These findings suggest that lymphocytes and/or macrophages play an important role in initiating SAA protein synthesis after endotoxin treatment and suggest a possible mechanism by which chronic infection or inflammation leads to amyloidosis.     

Serum protein concentrations during Schistosoma mansoni infection in intact and T-cell deprived mice. I. The acute phase proteins, C3 and serum amyloid P-component (SAP).

Normal mice developed marked elevation of two acute phase plasma proteins, C3 and serum amyloid P-component (SAP), between 40 and 50 days after percutaneous infection with cercariae of Schistosoma mansoni. The high levels persisted until the end of the experiment (day 106). The onset of this acute phase response corresponds with the reported time at which granulomata develop in the liver. In mice deprived of T cells by thymectomy and anti-thymocyte serum, the granulomata were significantly smaller and all the animals died between days 70 and 80. These mice had normal C3 levels throughout although there was a rise in SAP concentration. C3 and SAP levels in infected control mice, which had been thymectomized but not deprived of T cells with anti-thymocyte serum, were the same as in intact infected animals. The different behaviour of C3 and SAP in infected T-cell deprived mice may reflect the alteration in specific schistosomal pathology and/or a role for T cells in mediation of the acute phase production of some proteins.     

Amyloid resistance in A/J mice is not determined by genetic variants at, or close to, the serum amyloid P component locus.

An experimental model to assess the utility of variants of the serum amyloid P component (SAP) gene in predicting resistance to amyloidosis resulting from chronic inflammation was developed. F2 mice were generated from amyloid resistant (A/J) and amyloid susceptible (C57BL/6J) progenitor strains. Mice were injected daily with azocasein for 30 days, a treatment protocol determined to produce little amyloid deposition in the A/J progenitor strain and substantial amyloid deposition in the C57BL/6J progenitor strain. In a blind study the F2 spleens were subjectively scored for amyloid deposition and DNA was isolated from F2 livers and subjected to Southern blot analysis to determine the inheritance of progenitor strain specific SAP restriction fragment length polymorphisms (RFLPs). No correlation between relative amyloid resistance and SAP genotype was observed, indicating that SAP RFLPs have no value in predicting predisposition to amyloid disease in the mouse model studied.     

EXPERIMENTAL MURINE AMYLOIDOSIS — Evaluation of Induction Methods and Strain Difference —

Several experimental methods inducing murine amyloidosis were tested using six strains of mice including congential asplenic mice and athymic nude mice. Traditional induction method of amyloidosis by casein injections failed to cause amyloidosis in C₃H/He mice. Single injection with complete Freund's adjuvant reinforced with Mycobacterium butyricum was also unable to induce amyloidosis in C₃H/He, CBA, and BALB/c mice. Six or four injections with complete Freund's adjuvant at an interval of once a week successfully induced amyloidosis in CBA mice with high incidence but not in C₃H/He, ICR/SLC, and BALB/c mice. Athymic nude mice with genetic background of BALB/c and congential asplenic mice, cross-bred with G₅₇BL/6 × C₃H, were free from amyloidosis after six or fourteen injections with the adjuvant. Experimental amyloidosis in mice, therefore, might mainly depend on the strain of mice used and the induction method chosen.