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The Journal of Immunology, 2006, 177: 2601-2609.
Copyright © 2006 by The American Association of Immunologists

Inflammation-Responsive Transcription Factor SAF-1 Activity Is Linked to the Development of Amyloid A Amyloidosis

Alpana Ray1,*,{dagger}, Arvind Shakya*, Deepak Kumar*, Merrill D. Benson and Bimal K. Ray*,{dagger}

* Department of Veterinary Pathobiology and {dagger} Department of Orthopaedic Surgery, University of Missouri, Columbia, MO 65211; and {ddagger} Department of Pathology and Laboratory Medicine, Indiana School of Medicine and Roudebush Veterans Affairs Medical Center, Indianapolis, IN 46202


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Abundantly expressed serum amyloid A (SAA) protein under chronic inflammatory conditions gives rise to insoluble aggregates of SAA derivatives in multiple organs resulting in reactive amyloid A (AA) amyloidosis, a consequence of rheumatoid arthritis, Crohn’s disease, ankylosing spondylitis, familial Mediterranean fever, and Castleman’s disease. An inflammation-responsive transcription factor, SAF (for SAA activating factor), has been implicated in the sustained expression of amyloidogenic SAA under chronic inflammatory conditions. However, its role in the pathogenesis of AA amyloidosis has thus far remained obscure. In this paper we have shown that SAF-1, a major member of the SAF family, is abundantly present in human AA amyloidosis patients. To assess whether SAF-1 is directly linked to the pathogenesis of AA amyloidosis, we have developed a SAF-1 transgenic mouse model. SAF-1-overexpressing mice spontaneously developed AA amyloidosis at the age of 14 mo or older. Immunohistochemical analysis confirmed the nature of the amyloid deposits as an AA type derived from amyloidogenic SAA1. Furthermore, SAF-1 transgenic mice rapidly developed severe AA amyloidosis in response to azocasein injection, indicating increased susceptibility to inflammation. Also, during inflammation SAF-1 transgenic mice exhibited a prolonged acute phase response, leading to an extended period of SAA synthesis. Together, these results provide direct evidence that SAF-1 plays a key role in the development of AA amyloidosis, a consequence of chronic inflammation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Amyloidosis is a large group of incurable diseases in which deposits of fragmented or sometimes intact proteins referred to as amyloid fibrils accumulate in the extracellular spaces of organs, leading to dysfunction of the affected organs (1, 2). Amyloid fibrils are formed when normally soluble precursor proteins undergo conformational changes and polymerize as insoluble fibrils, with the beta-pleated sheet conformation visible under polarized light as yellowish-green birefringence (3). Although all organs could be affected, amyloid deposits are predominantly seen in the spleen, kidney, liver, and brain. In secondary or inflammation-associated amyloid A (AA)2 amyloidosis, inflammation plays a central role, as patients suffering from various chronic inflammatory diseases including rheumatoid arthritis, Crohn’s disease, ankylosing spondylitis, familial Mediterranean fever, and Castleman’s disease, frequently develop this condition as a secondary complication. In AA amyloidosis, the amyloid fibril is derived from a precursor protein identified as the acute phase serum AA (SAA) (2) protein (4). The SAA family is composed of a number of genes and proteins. The human SAA gene family contains two highly homologous SAA1 and SAA2 genes, a less related SAA4 gene, and a nonexpressible SAA3 gene (5, 6, 7, 8). Similarly as in humans, SAA1 and SAA2 isoforms in mice are highly homologous, whereas SAA3 and SAA4 are distinct (8, 9). Among these different isoforms, the SAA1 of both human and mice is predominant in AA amyloid deposits.

A major factor responsible for the development of AA amyloidosis is the increased synthesis and subsequent degradation of the precursor protein SAA1 under chronic inflammatory conditions. Under normal conditions a trace amount of this protein is present, but in response to inflammation its level can increase up to 1000-fold. Increased synthesis of the SAA protein is primarily due to the transcriptional induction of the SAA gene (reviewed in Ref. 10). Proinflammatory cytokines and mediators such as IL-1, IL-6, TNF-{alpha}, bacterial LPS, and PMA either alone or in combination induce SAA transcription (10). Multiple transcription factors, including C/EBP (11, 12), NF-{kappa}B (13, 14, 15), and SAF (SAA activating factor) (16, 17), have been defined in the regulation of SAA induction. Several other transcription factors, including YY1, SEF, AP-2, and Sp1, have also been implicated (reviewed in Ref. 10). Although C/EBP and NF-{kappa}B play critical roles in tissue-specific expression of amyloidogenic SAA1 and SAA2 during acute inflammatory conditions, SAF has been implicated in the expression under the chronic inflammatory condition that predisposes to AA amyloidosis (18). Normally, under acute inflammatory conditions such as septic shock, inflammatory signals are intense but short lived. Thus, the effect of inflammation subsides rapidly within 24 to 48 h. The amyloidogenic SAA level, which is highly increased during this condition, also rapidly declines to a normal level. In contrast, during chronic inflammatory conditions such as rheumatoid arthritis the effect of inflammation persists for an extended period of time, causing prolonged expression of amyloidogenic SAA with the potential consequence of developing AA amyloidosis. An earlier finding of increased SAF activity under chronic inflammatory conditions and its ability to promote expression of amyloidogenic SAA (18) suggests that SAF could be linked to AA amyloidosis. To test this possibility, we have examined amyloid tissues from human AA amyloidosis patients for evaluation of the level of this transcription factor. To further examine this potential causal link of SAF, we have developed a transgenic mouse model that overexpresses SAF-1, a major isoform of this transcription factor. In this paper we provide evidence that increased abundance of SAF-1 plays a critical role in the pathogenesis of AA amyloidosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animal studies

SAF-1 transgenic mice were developed as described earlier (19). The CMV immediate-early enhancer/promoter drives transcription of the SAF-1 gene in multiple organs of the transgenic mice that exhibit elevated levels of SAF-1 activity as revealed by promoter-binding activity. The identities of the transgenic and nontransgenic mice were determined by PCR analysis of genomic DNA isolated from tail biopsies as described before (19). Both SAF-1 transgenic and nontransgenic littermates belong to a hybrid strain called B6C3F1 that is derived from C57BL/6J and C3H/HeJ inbred lines; they are heterozygous for the unique loci and homozygous for the common loci for these two inbred lines.

Amyloid induction

Mice (8 wk old) were divided into two groups: SAF-1 transgenic and nontransgenic. These two groups were subdivided into five subgroups, and each subgroup contained seven animals. Animals received a daily s.c. injection of 300 µl of a 80 mg/ml solution of azocasein (Sigma-Aldrich) in 10 mM NaHCO3 for 7, 14, 21, 35, and 42 days. Control groups of transgenic and nontransgenic mice received 10 mM NaHCO3 only. The animals were euthanized by CO2 asphyxiation at the end of treatments. Tissues were collected, a portion was fixed in 10% buffered formalin, and the remaining tissues were stored in liquid nitrogen until further use. All procedures used for treatment of mice in this study were approved by the University of Missouri Institutional Animal Care and Use Committee (Columbia, MO).

Human amyloid tissue samples

Formalin-fixed and paraffin-embedded autopsy specimens of kidney from five patients, ranging in age from 35 to 66, were used in this study. These patients had suffered from AA amyloidosis due to an underlying disease of rheumatoid arthritis, and one was paraplegic due to spinal injury. Normal tissue specimens in formalin-fixed and paraffin-embedded blocks were obtained from the PathServe Autopsy and Human Tissue Bank.

Analysis of amyloid

Formalin-fixed tissues were paraffin embedded, and sections on slides were stained with alkaline alcoholic Congo red as described earlier (20). Briefly, tissue sections in microscope slides were deparaffinized in xylene and hydrated by stepwise submersion in 100, 95, 75, and 50% ethanol. The slides were incubated at room temperature (25°C) for 20 min in freshly prepared 80% ethanol containing 3% NaCl and 0.01% NaOH. The slides were then incubated at room temperature for 60 min in freshly prepared and filtered 0.2% Congo red in 80% ethanol containing 3% NaCl and 0.01% NaOH. Excess dye was removed by washing the slides in distilled water three times. The slides were dipped in hematoxylin solution for 10 min and washed three times in distilled water. Tissue sections in slides were then dehydrated in graded (50–100%) ethanol and rinsed in xylene, and a coverslip was placed with Permount. The Congo red-positive staining was observed under polarized light. Each stained section was evaluated histologically, and the severity scores of amyloid deposition were determined in a blinded manner and rated on a scale of 0–5 following a previously described method (21). The grades used for rating are as follows: 0, no detectable amyloid; 1, occasional amyloid specks; 2, mild amyloid deposits mainly in the perifollicular zone; 3, moderate deposits in perifollicular zone; 4, abundant deposits but an intact follicular structure; and 5, severe perifollicular and interfollicular deposits.

Immunohistochemical analysis

Immunohistochemical staining was performed using a monoclonal anti-human AA (clone mc1; DakoCytomation) and affinity-purified anti-SAF-1 rabbit IgG (22) as primary Abs. Details on the preparation of the anti-SAF-1 Ab have been described earlier (22). HRP-conjugated goat anti-rabbit IgG and goat anti-mouse IgG were used as the secondary Abs. Tissue sections were cut 5-µm thick, deparaffinized in xylene, and rehydrated in graded ethanol solutions followed by rinsing in wash buffer (50 mM Tris-HCl (pH 7.5) and 0.15 M NaCl). Endogenous peroxidase activity was quenched by immersion in 3% H2O2 in methanol for 20 min, followed by rinses in the wash buffer. The slides were then incubated in 0.1% trypsin solution with 0.1% CaCl2 for 60 min at 37°C to unmask Ags. Nonspecific binding was blocked for 30 min at 37°C with 10% normal goat serum. Slides were incubated overnight at 4°C with primary Abs. The slides were rinsed twice in the wash buffer containing 0.05% Tween 20 and then incubated with the secondary Ab. Bound primary Ab was detected using the HRP method with substrate-chromogen solution. Sections were counterstained with Mayer’s hematoxylin solution. In control experiments, the primary Ab was preabsorbed using purified SAF-1 protein.

Analysis of amyloid fibril protein

Spleen and kidney tissues were homogenized in ice-cold saline using a Polytron homogenizer (Brinkmann Instruments). The homogenates were centrifuged at 10,000 x g for 30 min at 4°C. The pellets were washed in ice-cold saline twice and once with a buffer containing 10 mM Tris-HCl (pH 8.0) and 0.05 M sodium citrate. The pellets were resuspended by homogenization in ice-cold saline and centrifuged at 10,000 x g for 30 min at 4°C. The pellets were resuspended in ice-cold distilled water and centrifuged at 20,000 x g for 3 h at 4°C. The final pellets were resuspended in a buffer containing 250 mM Tris-HCl (pH 8.0) and 6 M guanidine HCl. Aliquots were fractionated in a 16% polyacrylamide gel, and the protein bands were visualized by Coomassie brilliant blue staining. Unstained proteins in the duplicate lanes were excised from the gel, subjected to tryptic digestion, and analyzed by MALDI-TOF mass spectrometry (Perceptive STR; Applied Biosystems). The peptides were identified using the MS-Fit search program.

Induction of acute phase response and analysis of plasma SAA level

Four groups of mice (n = 8 per group) consisting of young (8 wk old) and aged (14 mo old) SAF-1 transgenic or nontransgenic mice were given a single s.c. injection of 1 ml of a 80 mg/ml solution of azocasein (Sigma-Aldrich) in 10 mM NaHCO3. Blood samples (200 µl) were collected from each mouse before azocasein injection and on days 1, 2, 3, 4, 7 and 21 after injection, and plasma was isolated by centrifugation. SAA level in the plasma before and after azocasein injection was measured using an ELISA method following the manufacturer’s protocol (BioSource International). Each sample was assayed in duplicate. The average OD reading from the duplicates sample was then plotted against a standard curve to obtain the concentration of SAA.

Northern blot analysis

Total RNA was extracted from multiple tissues by the guanidinium thiocyanate method (23). Fifty micrograms of each RNA sample was fractionated in a 1% agarose gel containing 2 M formaldehyde and transferred onto a nylon membrane using capillary transfer method (24). Transferred RNA in the membrane was hybridized to a 32P-labeled oligonucleotide probe specific for mouse SAA1 (25). The same membrane was stripped and rehybridized to 32P-labeled GAPDH cDNA probe to evaluate the relative amount of each RNA sample on the membrane.

RT-PCR analysis

Total RNA from Formalin-fixed, paraffin-embedded human kidney tissue samples was isolated using the RecoverAll total nucleic acid isolation kit for formaldehyde- or paraformaldehyde-fixed, paraffin-embedded tissues obtained from Ambion. RT-PCR was performed using an RT-PCR kit following the manufacturer’s protocol (Invitrogen Life Technologies). One microgram of DNase-treated RNA was used in the reverse transcription reaction with a human SAA1-specific extension primer located in exon 4 (5'-CCTGCCCCATTC ATCGGCAGCCTGATC-3'). PCR was performed with two SAA1-specific primers, one located in exon 3 and the other in exon 4 (5'-GAGAGCCTACTCTGACATGAGAGAAG-3' and 5'-GCCAGTGAGTCCTCCGCACCATG-3', respectively). DNA products were resolved in a 2% agarose gel, and the identity of the amplified DNA was verified by DNA sequence analysis.

Western immunoblot assay

Tissues from nontransgenic and transgenic mice were homogenized in lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1% Nonidet P-40, and 1% Brij96) using a Polytron homogenizer (Brinkmann Instruments). The cell lysates were centrifuged at 12,500 rpm in a microcentrifuge at 4°C. Clear supernatants were used as the source of proteins whose concentrations were measured using the Bradford method (26). Fifty micrograms of protein from each sample was fractionated on 12% SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane using an electroblotter. After transfer, relative amounts of proteins in each lane were verified by staining with Ponceau S solution (Sigma-Aldrich). Immunoblotting was performed with an anti-SAF-1 Ab. Bands were detected using a chemiluminiscence detection system (Amersham Biosciences).

EMSA

Nuclear extracts were prepared from various tissues of mice following the method described earlier (17). Protein content was measured by the Bradford method (26). EMSA was performed with equal amounts of proteins using a method described previously (17). A radiolabeled probe containing the SAF-binding element of the SAA promoter was prepared by using [{alpha}-32P]dCTP as the substrate to label the double-stranded oligonucleotide probe (17). Two complementary oligonucleotides, 5'-CCCTTCCTCTCCACCCACAGCCCC-3' and 3'-GGGAAGGAGAGGTGGGTGTCGGGGG-5', representing sequences from –254 to –230 of the rabbit SAA2 promoter, were annealed to prepare the double-stranded SAF-binding element. Oligonucleotides containing nucleotide sequences from –190 to –150 and –98 to –78 of the mouse SAA1 promoter (9) were used as C/EBP- and NF-{kappa}B-binding elements, respectively, in the EMSA. These DNA molecules were radiolabeled using [{alpha}-32P]dCTP as the substrate. DNA-protein complexes were separated in a nondenaturing 6% polyacrylamide gel. For competition analysis, 25- and 50-fold molar excesses of the unlabeled double-stranded SAA SAF-1 oligonucleotide (–254/-230) were used. As a nonspecific oligonucleotide, an Epstein-Barr nuclear Ag-1 DNA-binding element containing the sequence 5'-TATCTGGGTAGCATATGCTATCCTAAT-3' was used. For an Ab interaction study, anti-SAF1 and anti-SAF2 Abs were added to the reaction mixture during a preincubation period of 30 min on ice.

Statistical analysis

Incidence of amyloids in different groups of mice and the histological amyloid grades described in Table I were assessed by using the Mann-Whitney U test. A two-way ANOVA was performed to determine the significant differences between the transgenic and nontransgenic mice in the azocasein-treated and untreated groups.


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  		Table I. Histologic evaluation of amyloid deposits in the spleens of 8-wk-old mice

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Presence of SAF-1 protein in human AA amyloidotic kidney tissue

Serial sections of amyloid deposit-containing kidney tissue from human AA amyloidosis patients were examined using Congo red dye stain followed by visualization under polarized light. A representative sample revealed the presence of amyloid deposits in the kidney of an AA amyloidosis patient (Fig. 1Aa). Immunohistochemical analysis using an anti-AA Ab characterized the amyloid deposits as derivatives of amyloidogenic SAA (Fig. 1Ac). A high level of SAF-1 was also detected in the AA amyloid-laden tissues (Fig. 1Ae). However, both AA and SAF-1 levels were found to be much lower in the normal tissues (Fig. 1A, d and f). Similar high levels of AA protein and SAF-1 were detected in all other specimens of human AA amyloidosis patients. Preabsorption of anti SAF-1 Ab with purified SAF-1 protein completely abrogated the staining of the tissue sections (data not shown), indicating that the staining in Fig. 1A, e and f, was SAF-1-specific. To assess whether the kidney tissue expressed SAA in the pathogenic condition of amyloidosis, RNA was extracted from the paraffin-embedded tissues and analyzed for the presence of SAA transcripts by RT-PCR. A DNA product of predicted 180 bp, based upon the two PCR primers chosen, was detected (Fig. 1B). DNA sequence analysis of the PCR product further verified the identity of the DNA as SAA1. As shown in Fig. 1B, compared with a normal kidney the amyloid-laden kidney contained higher levels of SAA1, suggesting that SAA1 is overexpressed in the amyloidotic kidney tissue.


Figure 1
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  		FIGURE 1. Presence of SAF-1 protein in the kidney tissue of a human AA amyloidosis patient. A, Kidney tissue sections from an AA amyloidosis patient (AA) and normal cadaver (N) were stained with Congo red and viewed under polarized light (a and b, respectively). The bright stains in a represent highly abundant amyloid deposits. In c and d immunohistochemical staining with anti-AA Abs for amyloidosis and normal kidney tissue sections, respectively, is shown. In e and f is shown IHC staining with anti-SAF-1 Abs for amyloidosis and normal kidney tissue sections, respectively. Magnification, x200. B, RT-PCR analysis of kidney for SAA expression. RNA was isolated from the paraffin-embedded kidney tissues as described in Materials and Methods. Following RT-PCR, the products were fractionated in a 2% agarose gel. Lanes 1 and 2 contain samples derived from two amyloidotic kidney tissues, and lanes 3 and 4 contain those derived from normal kidney tissues. A major DNA product of ~180 bp in size is identified. Lane M contains DNA size markers.

 
Development and characterization of SAF-1 transgenic mice

A high level of SAF-1 in the affected tissues of AA amyloidosis patients raises the possibility that SAF-1 might play a critical role in the pathogenesis of the disease. To further investigate this possibility, SAF-1 transgenic mice were constructed in which a full-length SAF-1 cDNA was placed under the control of CMV immediate-early enhancer/promoter (Fig. 2A). The presence of the SAF-1 gene in the founder mice was verified by PCR amplification of DNA obtained from tail clippings using primers specific for the SAF-1 cDNA and the CMV enhancer/promoter DNA. A band 574 bp in size was seen in transgenic mice (Fig. 2B). As the CMV enhancer/promoter is capable of directing expression in multiple tissues, the SAF-1 transgene was found to be expressed in various organs, albeit at different levels (19).


Figure 2
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  		FIGURE 2. Configuration of SAF-1 transgene and identification of SAF-1 transgenic mice. A, Schematic showing CMV-SAF-1 transgene. A full-length SAF-1 cDNA was placed under the control of a CMV promoter using the pCI-Neo mammalian expression vector (Promega). The arrows indicate the positions of transgene specific primers used for PCR to identify the SAF-1 transgene. B, PCR assay to identify the transgenic animals. A 574-nucleotide product is amplified only when the animals contain the SAF-1 transgene. Lanes 1 and 3 show PCR-amplified products from SAF-1 transgenic animals, while lanes 2 and 4 show the absence of such DNA in nontransgenic animals. Lane M shows molecular size markers.

 
SAF-1 transgenic mice spontaneously develop AA amyloid in multiple organs

To examine whether SAF-1 transgenic mice contain amyloid deposits in different organs, both SAF-1 transgenic and nontransgenic littermates were sacrificed, and liver, kidney, and spleen tissues were tested. Tissue sections of mice of different ages, from 4 wk to 18 mo, were stained with Congo red dye and visualized under cross-polarized light. Up to the age of 6 mo there was no noticeable amyloid deposit in either the nontransgenic or the SAF-1 transgenic mice (data not shown). However, 12- to 14-mo-old SAF-1 transgenic mice were found to contain amyloid deposits in spleen, kidney, and liver (Fig. 3, b, d, and f), whereas no such deposits were seen in the age-matched nontransgenic animals (Fig. 3, a, c, and e). The spleens of SAF-1 transgenic mice appeared to contain the most abundant amyloid deposits.


Figure 3
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  		FIGURE 3. Histopathological analysis of the tissues from aged (14 mo old) SAF-1 transgenic (SAF-1 Tg) mice for amyloid deposits. Tissue sections were stained with Congo red dye, and the amyloid deposits were viewed under polarized light as bright white stains in the picture. a, c, and e, typical sections from spleen, kidney, and liver tissues of nontransgenic (Non-Tg) mice, and b, d, and f represent spleen, kidney, and liver tissues of SAF-1 transgenic mice, respectively. Eight mice in each group were examined. The aged SAF-1 transgenic mice contained amyloid deposits in the spleen, kidney, and liver tissues, whereas no such deposition was seen in the age-matched nontransgenic mice. Magnification, x200

 
To assess whether the amyloid deposits are composed of AA, serial sections of spleen and kidneys from 14-mo-old SAF-1 transgenic and age-matched nontransgenic mice were further examined using a monoclonal anti-AA Ab. Results shown in Fig. 4, a, b, e, and f, revealed that the amyloid deposits in the spleen and kidneys of SAF-1 transgenic mice are indeed composed of AA amyloid protein. Immunohistochemistry with anti-SAF-1 Ab showed higher levels of SAF-1 protein in the amyloid deposit-containing tissues of SAF-1 transgenic mice (Fig. 4, d and h). Preabsorption of anti-AA and anti-SAF1 Abs abrogated staining, indicating AA and SAF1-specific staining in Fig. 4, b, d, f, and h (data not shown). Together, the data correlated well with the results obtained from human AA amyloidosis patients shown in Fig. 1.


Figure 4
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  		FIGURE 4. Immunohistochemical analysis of the amyloid-laden spleen and kidney tissues of aged (14 mo old) SAF-1 transgenic mice (SAF-1 Tg) in comparison with age-matched nontransgenic (Non-Tg) mice. Thin sections from spleen (a–d) and kidney (e–h) of nontransgenic (a, c, e, and g) and SAF-1 transgenic (b, d, f, and h) mice were immunostained using anti-AA (a, b, e, and f) and anti-SAF-1 (c, d, g, and h) Ab, respectively. Magnification is x200. Intense dark stains (indicated by arrows) depict positive staining for amyloid A and SAF-1.

 
The AA amyloid protein in the amyloid fibril was further analyzed by fractionation of the protein and subsequent mass spectrometry analysis using MALDI-TOF. A peptide corresponding to the amino-terminal region of mouse SAA1 was identified, indicating that the AA amyloid is derived from the SAA1 isoform (data not shown). No SAA2-specific peptides were detected in the mass spectrometry analysis.

SAF-1 and SAA1 levels are high in the affected organs of SAF-1 transgenic mice

To test whether the increased level of SAF-1 detected in the AA amyloid tissue might play a role in the increased SAA1 expression in the amyloidotic organs of aged SAF-1 transgenic mice, we examined the activity level of SAF-1 and the expression of SAA1, which is the predominant SAA isoform in the AA amyloid. The functional activity of SAF-1 in different tissues was determined by using DNA-binding assay. Spleens, kidneys, and liver tissues from transgenic mice showed high levels of DNA-binding activity (Fig. 5A), which was characterized as SAF-1 by competition analysis and Ab ablation assay (Fig. 5B). Among the three tissues, spleens showed the highest level of SAF-1 activity. SAF-1 protein level was also the highest in the spleen tissue, whereas its level in nontransgenic mice was much lower (Fig. 5C). It is worthy of mention in this context that the spleen tissue of SAF-1 transgenic mice displayed the most amyloid deposition. Northern blot analysis revealed high levels of SAA1 mRNA presence in the spleen, kidney, and liver tissues of SAF-1 transgenic mice (Fig. 5D). These data correlated well with the mass spectrometry analysis of purified amyloid fibril protein, which identified it as SAA1 (described above). It was interesting to note that although the nuclear extracts from nontransgenic mice contained noticeable DNA-binding activity of SAF (Fig. 5A), it did not contribute toward SAA1 expression because there was no detectable SAA1 mRNA in nontransgenic mice tissues (Fig. 5D). This finding could be due to the binding of SAF-2, a splice variant that inhibits the transactivation potential of SAF-1 (27), to the SAF-binding element of the SAA promoter. This possibility was evaluated in a DNA-binding assay. Ablation of the DNA-protein complex by SAF-2 Ab (Fig. 5B, lane 18) indicated the presence of SAF-2 in the spleen nuclear extract of nontransgenic mice. Similar levels of SAF-2 were present in other tissues (data not shown). Overexpression of SAF-1 in the SAF-1 transgenic mice significantly increased the nuclear pool of SAF-1 (Fig. 5C), thus overcoming the inhibitory effect of SAF-2 and activating SAA1 expression.


Figure 5
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  		FIGURE 5. DNA-binding activity of SAF-1 and SAA levels in SAF-1 transgenic (SAF-1 Tg) and nontransgenic (nonTg) mice. A, Measurement of SAF-1 DNA-binding activity. Nuclear extracts (10 µg of protein) prepared from kidney (lanes 1 and 2), liver (lanes 3 and 4), and spleen (lanes 5 and 6) from 14-mo-old SAF-1 transgenic mice and age-matched nontransgenic mice were incubated with a 32P-labeled SAF-binding element of rabbit SAA2 promoter. The resultant DNA-protein complexes were resolved in a 6% native polyacrylamide gel. Asterisk denotes a nonspecific band that was only seen to be formed with spleen nuclear extract. B, Characterization of the DNA-protein complex. Nuclear extracts (10 µg of protein) prepared from kidney (lanes 7–11) and spleen (lanes 12–16) tissues of SAF-1 transgenic mice were incubated with the 32P-labeled SAF-binding probe. In addition, lanes 8, 9, 13, and 15 contained 25- and 50-fold molar excess of unlabeled SAF-binding oligonucleotide (SAF-1 oligo), lanes 10 and 15 contained 100-fold molar excess of nonspecific oligonucleotide (nonsp. oligo), and lanes 11 and 16 contained anti-SAF-1 Ab. In lanes 17 and 18 nuclear extracts of nontransgenic mice spleen tissues were used. Lane 18 also contained anti-SAF-2 Ab. C, Western blot analysis. Fifty micrograms of protein prepared from the indicated tissues of nontransgenic and SAF-1 transgenic animals as shown in A were fractionated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-SAF-1 Ab. D, Northern blot analysis. Total RNA (50 µg) from spleen, kidney, and liver tissues of nontransgenic and SAF-1 transgenic mice, as indicated, was separated in a 1.0% agarose gel containing 2 M formaldehyde, transferred to a nylon membrane, and probed with a 32P-labeled, SAA1-specific oligonucleotide. The same blot was stripped and rehybridized using 32P-labeled GAPDH cDNA.

 
Because SAA1 expression is known to be induced by C/EBP and NF-{kappa}B, EMSA was performed to measure the level these transcription factors in the liver, kidney, and spleen tissues of SAF-1 transgenic mice. Neither C/EBP nor NF-{kappa}B activities were induced in any of these tissues of the SAF-1 transgenic mice (data not shown). This result is consistent with an earlier finding, which revealed that chronic inflammatory condition is not associated with NF-{kappa}B or inducible C/EBP activities (18). To the contrary, SAF activity was found to remain high and contributed to persistent SAA expression (18). Together, the data indicate that SAF-1 is a major inducer of SAA1 expression in the amyloidotic tissues of the transgenic mice.

SAF-1 transgenic mice are highly susceptible to develop AA amyloidosis in response to inflammatory stimulus

Our finding that only aged SAF-1 transgenic mice spontaneously develop AA amyloidosis (Figs. 3 and 4) raises the question of whether younger SAF-1 transgenic mice are resistant or whether they might develop AA amyloidosis if exposed to additional inflammatory stimulus. To test these possibilities, we injected azocasein, a known inflammatory stimulus (28, 29), into the SAF-1 transgenic mice and compared their response with the nontransgenic mice. Amyloid-enhancing factor, in conjunction with an inflammatory stimulus, is traditionally used for the rapid induction of experimental amyloidosis in mice. We chose not to use amyloid-enhancing factor, because it markedly enhances the process and therefore holds the possibility of blurring the rate of amyloid development. Eight-week-old SAF-1 transgenic and nontransgenic mice received daily azocasein injections for different lengths of time, namely 7, 14, 21, and 42 days. At the end of each treatment period mice were sacrificed, and serum, spleen, kidney, liver, and other tissues were collected and stored at –70°C. Paraffin-embedded sections of kidney and spleen tissues were stained with Congo red dye and examined for amyloid deposition (Fig. 6). As shown in Fig. 6A,amyloid deposits were not detected in nontransgenic mice until day 21 of azocasein treatment, and the deposits were much milder compared with that of SAF-1 transgenic mice. The rate of amyloid deposition was highly accelerated in SAF-1 transgenic mice and, although both groups of mice developed amyloids in multiple organs (Fig. 6, A and B), at 42 days the severity score in the SAF-1 transgenic group was much higher (Table I). Immunohistochemical analysis indicated that amyloid-laden tissues contained AA amyloids that were highly prevalent in SAF-1 transgenic mice (Fig. 6C).


Figure 6
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  		FIGURE 6. Histopathological analysis of amyloid deposits in azocasein-injected SAF-1 transgenic (SAF-1 Tg) mice. Eight-week-old nontransgenic (Non-Tg) and SAF-1 transgenic mice were given daily s.c. injection of azocasein. Mice were sacrificed at different days as indicated, and the tissues were processed for detection of amyloid deposition. A, Paraffin-embedded sections of kidney tissues from SAF-1 transgenic and nontransgenic mice were collected at specified time points, stained with Congo red dye, and visualized under cross-polarized and UV light. Magnification, x200. The presence of amyloid deposits is revealed by the appearance of bright white color streaks. B, Paraffin-embedded sections of spleen tissue from SAF-1 transgenic and nontransgenic mice collected at day 42 of azocasein treatment were stained with Congo red dye and visualized under cross-polarized and UV light as indicated. Magnification, x200. Presence of amyloid deposits is revealed by the bright white streaks. C, Typical sections of spleen and kidney tissues of azocasein-injected nontransgenic and SAF-1 transgenic mice at day 42 were immunostained using anti-AA Ab. Intense dark stains indicate positive staining for amyloid A.

 
Acute phase response is prolonged in SAF-1 transgenic mice

To examine whether increased and/or prolonged acute phase response is accountable for the increased propensity of AA amyloid deposition in SAF-1 transgenic mice, we measured the plasma level of total SAA after a single injection of azocasein (n = 8 in each group). Results of this experiment indicated that SAF-1 transgenic mice express higher levels of SAA than the nontransgenic mice (Fig. 7). Moreover, whereas the SAA level in the nontransgenic mice almost returned to control values on day 21, SAF-1 transgenic mice continued to have higher levels. When compared between old and young SAF-1 transgenic and nontransgenic animals, 14-mo-old animals exhibited more robust SAA expression than young 8-wk-old mice. This result suggested that aged SAF-1 transgenic mice experience a much more increased and prolonged acute phase response, which accounts for the increased SAA levels in the circulations of these mice.


Figure 7
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  		FIGURE 7. Plasma SAA concentration of young and aged nontransgenic and SAF-1 transgenic mice after acute phase induction. Four groups of mice (n = 8) consisting of young (8 wk old) and aged (14 mo old) SAF-1 transgenic or nontransgenic mice were given a single s.c. injection of 1.0 ml of 80 mg/ml azocasein solution. Blood samples were collected before azocasein injection and subsequently 1, 2, 4, 7, and 21 days after the injection. Concentration of SAA was measured by ELISA in duplicate for each sample, and the average was calculated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Secondary or reactive AA amyloidosis is a fatal consequence of many chronic inflammatory diseases. Increased synthesis due to transcriptional induction of the amyloidogenic SAA1 gene during chronic inflammatory condition plays a central role in the development of AA amyloidosis. The transcriptional induction mechanism of SAA has therefore been intensely investigated. These studies led to the identification of the SAF-1 transcription factor, which is one of the primary regulators of SAA expression. To investigate whether SAF-1 plays any role in the pathogenesis of AA amyloidosis, we have developed a SAF-1 transgenic mouse model. Here, we show that >75% of aged SAF-1 transgenic mice spontaneously develop AA amyloidosis. Furthermore, compared with their nontransgenic counterparts, SAF-1 transgenic mice more rapidly and with increased severity develop experimentally induced AA amyloidosis in response to amyloid-inducing agents. We also observed that amyloid A deposits are highly prevalent in organs where SAA1 expression is very high. This latter finding correlated well with that for human AA amyloidosis patients, where amyloid-laden kidney tissue was found to overexpress amyloidogenic SAA1. Together, these findings indicate that: 1) SAF-1 is a potential risk factor whose overexpression predisposes host animals to develop AA amyloidosis; 2) persistent expression of SAF-1, endured by aged SAF-1 transgenic mice, also leads to spontaneous development of AA amyloidosis; and 3) increased overexpression of amyloidogenic SAA1 in different organs has a discrete and significant effect on the severity of amyloidosis developed in the affected organ. This latter aspect is further discussed below.

Numerous studies on SAA induction indicate that the regulatory process is highly complex as evidenced by variable inducibility ranging from 10- to 1000-fold, depending upon the signaling agent. Variations in the induction level of SAA are due to the activation of distinct pathways regulated by the involved transcription factors, including SAF-1 (18, 30). Multiple in vitro studies indicate that the activation of SAF-1 in response to cytokines in the cells results in increased SAA transcription in both hepatic and nonhepatic cells (16, 17, 31). Constitutive synthesis of amyloidogenic SAA1 at detectable levels in multiple organs of SAF-1 transgenic mice, but not in the nontransgenic littermates, correlated with the higher levels of the SAF-1 protein and the increased DNA-binding activity to the SAA promoter (Fig. 5, A and C), confirming the role of SAF-1 as a regulator of SAA1 expression in vivo (Fig. 5D). Consistent with this finding, the amyloid-laden kidney tissue of human amyloid patients was also found to overexpress amyloidogenic SAA (Fig. 1B). Together, these data showed that, during the pathogenic condition of AA amyloidosis, SAA is expressed in the affected organs such as the kidney. It is not known yet whether such local synthesis of SAA plays any significant role in amyloid deposition. Contrarily, earlier studies have shown that amyloid deposits are derived from circulating SAA in complex with high-density lipoprotein and that these SAA molecules are synthesized in the liver (32, 33). In the present study, the circulating level of SAA in the amyloidosis-affected, aged SAF-1 transgenic mice in the absence of any inflammatory stimulus was found to be ~100 µg/ml, which is only 2-fold higher than that of age-matched nontransgenic mice (see Fig. 7, bars marked 0d for day 0). It is possible that such an increase in the circulating level of SAA is at least partly responsible for the amyloidosis detected in the transgenic mice (Fig. 3). Another attractive speculation is that overexpression of SAA in the affected tissue may have contributed to the severity of amyloidosis. Our finding of the SAA overexpression in spleen and kidney tissues in addition to the liver (Fig. 5) raises such a possibility. It is noteworthy to mention in this regard that previous reports have also shown overexpression of SAA in extrahepatic tissues in pathogenic conditions (34, 35). Compelling evidence of local synthesis of SAA in amyloid deposition is provided by the finding of brain-specific expression of mouse SAA1 (36, 37). Our data, presented here, do not rule out the contribution of circulating SAA in amyloid formation. The answer to the question of whether both local production and increased circulation of SAA are necessary for amyloid deposition is yet to be established. The present finding, however, suggests that local production most likely increases the propensity, because SAF-1 transgenic mice spontaneously developed amyloidosis in organs overexpressing SAA. Together, our findings of high levels of SAF-1 along with AA amyloid protein deposits in amyloid-laden human kidney tissue (Fig. 1) are highly consistent with the proposed role of SAF-1 in the pathogenesis of AA amyloidosis.

An important result of the current study is the surprising appearance of significant AA deposits in >75% of the aged SAF-1 transgenic mice (Figs. 3 and 4). Amyloidosis in aged SAF-1 transgenic mice was not due to normal age-related changes in the cytokine profile or metabolic processes, because age-matched nontransgenic littermates showed very few amyloid deposits. An explanation for this apparent age-related phenomenon in spontaneous AA amyloid development can be obtained from the results presented in Fig. 7. Acute phase response, as assessed by the persistent higher plasma SAA level following an inflammatory stimulus, was prolonged in the SAF-1 transgenic mice compared with nontransgenic mice. Based on this finding, we hypothesize that periodic and extended acute phase responses throughout the lifetime of the mice lead to an increased and extended synthesis of amyloidogenic SAA that crosses the threshold level sanction deposition of amyloid fibrils. Interestingly, old nontransgenic mice contained the same plasma level of SAA as young transgenic mice at the early stages of acute phase response up to day 4, but the level rapidly declined thereafter in the old nontransgenic mice as compared with young transgenic mice, where the higher level persisted for extended period (Fig. 7). A persistent higher level of SAA appears to be a key factor in the development of AA amyloidosis, and SAF-1 plays a role in this process (18). The rapid decline of SAA levels in aged nontransgenic mice may explain why these mice did not develop AA amyloidosis.

In reactive AA amyloidosis, although the contribution of a precursor SAA1 protein is clear, it remains unknown why some patients develop amyloidosis while others with similar high levels of SAA1 in the system remain free of this pathology. It has been speculated that additional inflammation-responsive molecular components are necessary for the amyloid deposition. In correlation, SAA transgenic mice overexpressing high levels of SAA1 did not develop AA amyloidosis without an additional inflammatory stimulus (36, 37). Conversely, no such stimulus was needed for amyloid development in the aged SAF-1 transgenic mice. We postulate that in addition to the increased level of amyloidogenic SAA via transcriptional induction of this gene, inflammation-responsive stimulus provides an additional component(s) for the onset of amyloid deposits. The increased propensity for amyloid development in the young SAF-1 transgenic mice in response to the amyloid-inducing stimulus provided by azocasein injection supports this notion. Consistent with our finding, AA amyloidosis development in IL-6 transgenic mice was found to be age-dependent, with AA deposits being first evident at 3 mo of age and increasing over the next 6 mo (38). Interestingly, IL-6 has been shown to activate SAF-1 (16, 17, 18), which raises the possibility that one of the effects of this cytokine in the IL-6 transgenic mice could be the activation of endogenous SAF-1. Thus, SAF-1 appears to be a crucial molecule responsible for the development of AA amyloidosis.

Tissue-specific expression of SAA has been implicated as playing a determining role in the pathogenesis of a number diseases, including Alzheimer’s disease, arthritis, and atherosclerosis, where SAA is overexpressed in the specific cell types of the brain (39), synovium (40, 41, 42), and artery (43, 44). The regulation of SAA genes in these diverse cell types most likely involves different combinations of transcription factors that are active in the cells of the affected tissues. A paradigm is the elucidation of the tissue-specific expression of SAA1 and SAA2 in hepatic and epithelial cells (45) and SAA1 expression in aortic smooth muscle and hepatic cells (46). High levels of C/EBP activity in the liver have been found to be beneficial for SAA expression in the hepatic cells (11, 12, 14, 46), and such action of C/EBP is further potentiated by NF-{kappa}B activity (13, 14, 15, 47). Interestingly, SAF-1 is found to be very active in many tissue-specific cells, including synoviocytes and chondrocytes of the arthritic joint (22, 48) and macrophage cells of the atherosclerotic plaque (49). Recent evidence also implicates SAF-1 in the human SAA1 and SAA2 expression in the trophoblast cells during early fetal development (50).

The data presented in this paper establish that SAF-1 is involved in the development of AA amyloidosis by inducing amyloidogenic SAA1 expression in multiple tissues. Further studies will determine the potential impact of tissue-specific expression mechanisms in controlling the severity of the pathogenesis.


   Acknowledgments
 
We thank Howard Wilson for assistance in preparing photomicrographs and figures. We also thank Brad Wagner (University of Cincinnati, Transgenic Core Facility, Cincinnati, OH) for help in the development of SAF-1 transgenic founder mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Alpana Ray, Department of Veterinary Pathobiology, University of Missouri, 126A Connaway Hall, Columbia, MO 65211. E-mail address: rayal{at}missouri.edu Back

2 Abbreviations used in this paper: AA, amyloid A; SAA, serum AA; SAF, SAA activating factor. Back

Received for publication November 22, 2005. Accepted for publication May 11, 2006.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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