Published online before print February 19, 2008
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Departments of
* General Internal Medicine and
Gastroenterology and Hepatology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands;
Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA; and
National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA
1Correspondence: Department of General Internal Medicine (463), Radboud University Nijmegen Medical Centre, Geert Grooteplein 8, P. O. Box 9101, 6500 HB, Nijmegen, The Netherlands. E-mail: j.vanderhilst{at}aig.umcn.nl
ABSTRACT
Amyloid A (AA) amyloidosis is a severe complication of many chronic inflammatory disorders, including the hereditary periodic fever syndromes. However, in one of these periodic fever syndromes, the hyper IgD and periodic fever syndrome, amyloidosis is rare despite vigorous, recurring inflammation. This hereditary syndrome is caused by mutations in the gene coding for mevalonate kinase, an enzyme of the isoprenoid pathway. In this study, we used a cell culture system with human monocytes to show that inhibition of the isoprenoid pathway inhibits amyloidogenesis. Inhibition of the isoprenoid pathway by lovastatin resulted in a dose-dependent reduction of amyloid formed [53% at 10 µM (P=0.01)] compared with mononuclear cells that are exposed only to serum AA. The inhibitory effects of lovastatin are reversible by addition of farnesol but not geranylgeraniol. Farnesyl transferase inhibition also inhibited amyloidogenesis. These results implicate that the isoprenoid metabolism could be a potential target for prevention and treatment of AA amyloidosis.
Key Words: amyloidosis • SAA • hyper IgD syndrome • monocyte • isoprenoid
INTRODUCTION
Type amyloid A (AA), or reactive, amyloidosis is a serious, potentially life-threatening complication of chronic inflammatory conditions. The kidneys are most often affected, but other organs such as the intestines or heart can also be involved [1 ].
Amyloidosis is caused by the deposition of insoluble fibrils in the extracellular matrix of organs and tissues [2 , 3 ]. Amyloid fibrils in AA amyloidosis are derived from a C-terminally cleaved fragment of the acute-phase protein serum AA (SAA), which is an acute-phase protein produced in the liver in response to proinflammatory cytokines [4 ]. It is transported in the plasma as a component of high-density lipoprotein (HDL) [5 ]. Although the SAA protein produced by liver is 104 residues in length, amyloid fibrils consist mainly of fragments of SAA containing the 76 N-terminal residues [6 , 7 ]. The process of C-terminal cleavage and assembly into amyloid fibrils is referred to as amyloidogenesis. It is generally accepted that macrophages are central to this process [8 9 10 11 ]. After internalization by the macrophage, SAA trafficks from early to late endosomes and then to lysosomes, where proteases of the cathepsin family fully catabolize or partially degrade SAA [8 9 10 , 12 13 14 15 ].
Periodic fever syndromes, nowadays also designated as hereditary, autoinflammatory syndromes, are a group of disorders characterized by recurrences of fever accompanied by a vigorous, acute-phase response [16 ]. They include familial Mediterranean fever (FMF), Cryopyrin-associated periodic syndrome (CAPS), TNFR-associated periodic syndrome (TRAPS), and hyper IgD and periodic fever syndrome (HIDS). The latter disorder is an autosomal, recessive disorder characterized by recurring, inflammatory attacks with a combination of one or more of the following signs: fever, skin rash, abdominal pain, arthritis, and lymphadenopathy [17 ]. HIDS is caused by a defective mevalonate kinase, a major enzyme in the isoprenoid pathway, which eventually leads to the production of cholesterol and nonsterol isoprenoids (Fig. 1 ).
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Figure 1. Overview of the isoprenoidbiosynthesis. 3-Hydroxy-3-methylglutaryl (HMG) CoA is metabolized into mevalonate by HMG CoA reductase. Mevalonate is phosphorylated by mevalonate kinase.
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As HIDS patients all have the prerequisites for developing amyloidosis [22 ], we hypothesized that it is the underlying genetic defect that confers protection [23 ]. If the mevalonate kinase defect interferes with amyloidogenesis through a deficiency of isoprenoid products, then inhibiting the preceding enzyme, HMG CoA reductase, should also inhibit amyloid formation. To test this hypothesis, we used a human cell culture system that allows the quantitative and qualitative study of amyloidogenesis in vitro.
MATERIALS AND METHODS
SAA and amyloid-enhancing factor (AEF)
Mouse recombinant SAA (rSAA) 1.1 was produced in Escherichia coli and purified as described previously [24
]. Purified SAA was dissolved in 4 M urea at a concentration of 7.5 µg/µl. AEF was prepared as described previously [25
].
Amyloid induction
PBMC from normal human donors were isolated on Ficoll-Hypaque gradients and cultured in 96-well plates. Wells were rinsed after 4 h to select for adherent mononuclear cells and maintained in 100 µl RPMI supplemented with 15% FCS, 1 mM pyruvate, and 1% gentamicin at 37°C in an atmosphere with 5% CO2. Amyloid production was initiated by the addition of 2 µl SAA and 2 µl AEF, leading to a final concentration of 150 µg/ml and 12 µg/ml. Delipidated rSAA rapidly associates with HDL in the FCS [26
]. Where applicable, medium was supplemented with lovastatin (Sigma-Aldrich, St. Louis, MO, USA) at indicated concentrations, with or without 5 µM farnesol or geranylgeraniol (Sigma-Aldrich). Amyloid induction was also performed with supplementation of 200 nM farnesyl protein transferase (FPT) inhibitor II (Calbiochem, Nottingham, UK). Medium and supplements were replaced every other day. All conditions were tested in three independent experiments. Results are expressed as percentage of amyloid production versus cell without supplementation (100%).
Amyloid quantification
After the indicated time period, cells and cell-associated amyloid were collected and spun down on glass slides with a cytospin centrifuge for 10 min at 500 rpm. Cells were fixed in 10% neutral-buffered formalin and stained for 45 min in Congo red prepared in 80% alkaline ethanol. After quick dips in water, cells were counterstained with Gill’s hematoxylin solution (Sigma-Aldrich). They were dipped in acidified 70% ethanol, several times in water, and once in ammonium solution. After dehydration in alcohol, slides were cleared in xylene, and coverslips were applied with Permount.
The presence of amyloid fibrils was confirmed by visualization of apple-green birefringence of Congo red material on polarized light microscopy.
The amount of amyloid was determined with a digital image analysis protocol that was described previously [27 ] with minor modification. Briefly, the slides were analyzed using a charged-coupled device RGB camera (Sony 950 P) mounted on top of a light microscope (Axioskop 2 plus) and attached to a KS400 image analysis system (both from Karl Zeiss, Weesp, The Netherlands). A 20x objective with a numerical aperture of 0.5 was used for image acquisition, resulting in pixels with a dimension of 0.39 µm2. In each slide, 20 randomly selected fields were digitized for analysis. In each digitized RGB image, the red component was used to define the area covered with Congo red-bound amyloid. The amount of background, as determined by the area covered with red material in cell cultures treated with AEF but without SAA, was less that 0.3%. In addition, the number of cell nuclei in the selected fields was automatically assessed.
To study if lovastatin influenced the uptake of SAA, mononuclear cells were incubated with 150 µg/ml SAA, with or without 5 µM lovastatin. Aliquots of supernatant at 0, 24, and 48 h were subjected to Tris-tricine SDS-PAGE. Proteins were visualized by staining with Coomassie brilliant blue. Uptake of SAA was determined by comparing the amount of SAA remaining after 0 (100%), 24, and 48 h of incubation using a densitometer (Umax, Uden The Netherlands) and Totallab TL100 software (Nonlinear Dynamics, Newcastle, UK). The experiment was performed in triplicate, and results are expressed as mean percentage of SAA taken up ± SEM.
Statistical analysis
Data were compared with unpaired Student’s t-test. A P value <0.05 was considered to be significant.
RESULTS
We were able to induce in vitro amyloidogenesis in a reproducible and consistent manner. We found that amyloidosis induction was initiated by the addition of SAA (150 µg/ml) and AEF to human monocytes. With this concentration, which is well within the range found during inflammatory attacks of HIDS patients, the first extracellular amyloid deposits appearas early as 3 days after the start of induction. The deposits stain with Congo red and show typical apple-green birefringence under polarized light microscopy, indicative of amyloid fibrils (Fig. 2
). When induction of amyloidogenesis is prolonged, there is a gradual increase in the amount of amyloid as shown in Figure 3
. For example, Congo red material covers 
10% of the culture surface after a 9-day incubation with SAA.
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Figure 2. Extracellular Congo red deposits after 5 days of incubation with 150 µg/ml SAA and AEF (A). The Congo red deposits show typical apple-green birefringence under polarized light (B).
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Figure 3. Deposition of amyloid during time. Mononuclear cells were incubated with 150 µg/ml SAA and AEF. There is a linear increase in the amount of amyloid depositions during 9 days of incubation.
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Figure 4. Effect of lovastatin on amyloidogenesis. Mononuclear cells were incubated with 150 µg/ml SAA and AEF for 7 days. Addition of lovastatin gives a dose-dependent reduction of amyloid deposition. Error bars represent SEM. *, P < 0.05; **, P = 0.01.
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Figure 5. Effect of farnesol and geranylgeraniol on inhibitory effect of lovastatin. Addition of farnesol (5 µM) but not geranylgeraniol (5 µM) reverses the inhibitory effect of lovastatin. FPT inhibitor (200 µM) inhibits amyloidogenesis. *, P < 0.05, compared with control. Error bars represent SEM. NS, Not significant.
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Using an in vitro system with human monocyte-derived macrophages, we show that lovastatin is able to inhibit amyloidogenesis in a dose-dependent manner. This inhibition can be reversed by the addition of farnesol but not geranylgeraniol and can be replicated by use of a FPT inhibitor, which suggests that the inhibitory effect of lovastatin on amyloidogenesis is dependent on farnesyl-derived isoprenoids.
Clinical amyloidosis is a complex, step-wise process, which depends primarily on the extent and duration of SAA elevation. Steps in the amyloidogenic mechanism include SAA internalization by macrophages, C-terminal cleavage of SAA to AA, intracellular initiation of fibril formation, deposition of fibrils in the extracellular space, and association of SAA/AA fibrils with glycosaminoglycans. Previous studies have provided evidence that monocyte-derived macrophages carry out the aforementioned steps [14 , 26 , 29 ]. On this basis, we have chosen to use the cell culture model for testing our hypothesis regarding the potential involvement of isoprenoid compounds in amyloid development.
Farnesyl precedes geranylgeranyl in the isoprenoid pathway (Fig. 1) . Farnesyl is a nonsterol isoprenoid that binds to a specific amino acid sequence in a huge array of proteins, a process that is catalyzed by FPT [30 ]. These proteins, including Ras, Rho, and HDJ2, require this prenylation for their proper membrane localization and activity [31 , 32 ]. Isoprenylated proteins are involved in many cellular functions including cell proliferation, chaperone function, and apoptosis, besides their direct effect on membrane composition and fluidity.
Farnesol, but not geranylgeraniol, reversed the antiamyloidogenic effect of lovastatin. Furthermore, inhibition of FPT reduced amyloid formation. This suggests that one of the farnesylated proteins has a role in the amyloidogenesis in mononuclear cells. The exact role of farnesylated proteins in amyloidogenesis remains unknown, but we can speculate on a possible mechanism through the regulation of intralysosomal enzyme expression. A critical step in amyloidogenesis is C-terminal cleavage of SAA, which may occur in the lysosomes of macrophages. In patients who develop amyloidosis, impaired degradation of SAA leads to the accumulation of 76 amino acid fragments that can adapt a β-sheet structure typical of all the amyloid fibrils. The enzymes that are capable of degrading SAA belong to the cathepsin family of proteases. Cathepsin L, B, and D have been implicated in the pathogenesis of amyloidosis through degradation of SAA [33 34 35 36 37 ]. In vitro studies demonstrated that cathepsin B can generate potentially deleterious, intermediate degradation products found in amyloid fibrils [33 , 35 ]. In contrast, cathepsin D protects against amyloidosis by degrading SAA in the N-terminal portion, thereby preventing the formation of amyloidogenic intermediates [36 , 38 ].
As the expression of cathepsins is regulated by the farnesylated protein Ras [39 40 41 42 43 ], decreasing farnesylation could alter the expression of these proteases and result in reduced activity in lysosomes, creating a microenvironment favorable to AA protein formation.
A link between amyloid fibril production and isoprenoid metabolism is also seen in another form of amyloidosis, i.e., Alzheimer’s disease, in which the cerebral plaques are composed of amyloid fibrils formed from Aβ protein. This link is not only suggested by epidemiological studies [44 45 46 47 48 49 ] but also in an in vitro study with similarities to ours, by Gellermann and colleagues [50 ], who found that lovastatin can reduce the formation of amyloid-like Aβ plaques by human macrophages by 35%.
The results presented here have two important implications. First, they point to the possibility that statins are an effective, therapeutic option for the treatment and/or prevention of AA amyloidosis. Second, these results offer a possible explanation for the initial observation that HIDS patients are less vulnerable to amyloidosis than the other periodic fever syndromes [22 ].
In conclusion, blocking the isoprenoid pathway reduces the capacity of monocytes to produce amyloid. This could be a potential target for prevention in patients at risk for AA amyloidosis and possibly also for treatment of patients with amyloidosis.
Received November 2, 2007; revised December 20, 2007; accepted January 3, 2008.
REFERENCES
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