Clinical and Experimental Immunology

OR I GI NA L ART IC LE

doi:10.1111/cei.12615

Long-term kinetics of AA amyloidosis and effects of inflammatory restimulation after disappearance of amyloid depositions in mice

N. Muhammad,* T. Murakami,*† Y. Inoshima* and N. Ishiguro* *United Graduate School of Veterinary Sciences, Laboratory of Food and Environmental Hygiene, Department of Veterinary Medicine, Gifu University, Gifu, Japan, and †Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Tokyo, Japan

Accepted for publication 22 February 2015 Correspondence: N. Ishiguro, Laboratory of Food and Environmental Hygiene, Department of Veterinary Medicine, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan. E-mail: [email protected]

Summary Amyloid A (AA) amyloidosis is characterized by extracellular pathogenic deposition of insoluble fibril protein in various body organs. Deposited amyloid generally remains in a variety of organs for long periods, but its disappearance has been reported after the precursor protein is diminished. The kinetics of AA deposition are not completely understood and, in particular, the roles of cells and cytokines in the deposition and clearance of amyloid remain unclear. In this study, we investigated the disappearance of amyloid depositions in mice over a 1-year period. AA amyloidosis was induced experimentally in mice by injecting amyloidenhancing factor (AEF) and silver nitrate. Mice were killed at different time-points to examine the occurrence and disappearance of amyloid depositions. Maximum levels of amyloid depositions were observed at 20 days after inoculation. Clearance of amyloid depositions was observed from the 40th day onwards, with only minute traces of amyloid present by 240 days. A second inflammatory stimulus consisting of AEF and silver nitrate was given at 330 or 430 days, after amyloid depositions had disappeared almost completely. After that, serum amyloid A was overproduced and redeposition of amyloid was observed, indicating that all mice were primed for aggressive amyloid depositions. After administration of the inflammatory stimuli, the proinflammatory environment was found to have increased levels of interleukin (IL)-6, while anti-inflammatory conditions were established by IL-10 as regression of amyloid deposition occurred. These results suggest that the proinflammatory and anti-inflammatory status have key roles in both amyloid deposition and clearance. Keywords: AA-amyloid, cytokine, long-term kinetics, mice, SAA

Introduction Amyloid A (AA) amyloidosis is a systemic disorder characterized by extracellular pathogenic depositions of insoluble fibril amyloid protein in various body organs, in particular spleen, liver and kidneys [1,2]. AA amyloidosis occurs as a consequence of long-standing inflammation resulting in persistent elevation of a precursor protein of amyloid, serum amyloid A (SAA). SAA is an acute phase apolipoprotein reactant produced primarily by hepatocytes under the control of proinflammatory cytokines, including interleukins (IL)-1, -6 and tumour necrosis factor (TNF)-a [3,4]. Chronic inflammatory disease or trauma leads the proinflammatory cytokines to trigger an

increase in SAA levels to a thousand-fold of its basal level. SAA can be processed proteolytically, misfolded and hence can aggregate into highly structured protein fibrils with a cross b-pleated structure known as amyloid [5,6]. Murine AA amyloidosis can be induced experimentally by repeated inflammatory stimulation with casein or silver nitrate [7]. Pretreatment with amyloid enhancing factor (AEF), which can be extracted from amyloid-laden murine tissue, can considerably shorten the induction time of amyloidosis from months to days [8]. External amyloid fibril is thought to act as a seed that causes internal amyloid precursor protein to polymerize quickly into amyloid fibrils. The spleen is the primary predilection site

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for amyloid deposition, followed by liver and other systemic organs [9,10]. Deposited amyloid generally persists in many organs for a long period of time with a poor turnover. Recent studies have demonstrated the significance of SAA in the occurrence of AA amyloidosis and the clearance of AA amyloid depositions [9,11,12]. Disappearance of deposited amyloid has been observed after precursor protein is diminished, or it may be depleted immunologically [13]. After amyloid regression, further administration of inflammatory stimulus can lead to enhanced amyloid depositions [14]. However, the relationship between SAA production, amyloid load and the outcome in amyloidosis is unclear [15]. The kinetics of amyloid deposition, roles of cytokines and other molecules in the deposition and disappearance of amyloid fibrils are not completely understood. Moreover, the time factor in amyloid depositions, SAA levels, redeposition following secondary inflammatory stimuli and disappearance of deposits are still enigmatic. Therefore, a longterm kinetic study was required to analyse the process of amyloid deposition. In this study, we investigated amyloid deposition and disappearance in experimentally induced amyloidosis in mice for over a 1-year period. After clearance of amyloid depositions, mice were restimulated with inflammatory treatments to observe the recurrence of AA amyloidosis. Restimulation of primed mice with either silver nitrate or silver nitrate and AEF was performed at two different time-points for assessing the animal response to either secondary inflammation or inflammation accompanied with further intake of amyloid seed. SAA levels and the serum cytokine profile were analysed after the first and second inoculations with AEF. After the first inoculation, maximum amounts of amyloid depositions around spleen follicles were observed on the 20th day and then amyloid depositions disappeared progressively, with a corresponding increase in the levels of IL-10. The second inflammatory stimulus resulted in further aggressive amyloid depositions in spleen, liver and kidneys. Amyloid depositions and disappearance appeared to be stimulated by both pro- and anti-inflammatory cytokines.

Materials and methods Animals Sixty male C57BL/6J 8-week-old mice weighing 20–25 g (Japan SLC, Shizuoka, Japan) were used. Animals were kept three to five per cage with ad libitum access to food and water. For adaptation to the laboratory environment, animals were kept for 4 weeks before the experiment began. All procedures used in the following experiments were approved by the Animal Care Committee at Gifu University (approval no.: 11104). 134

Preparation of murine amyloid-enhancing factor Amyloid was extracted from the spleens of mice confirmed previously by Congo red staining to contain amyloid deposits, according to the method of Pras et al. [16], and used as AEF. Briefly, amyloid-laden spleens were homogenized in 0
15 M NaCl and centrifuged at 40 000 g for 20 min at 4  C in an MLA-55 rotor using an Optima MAX-XP ultracentrifuge (Beckman Coulter, Fullerton, CA, USA). The supernatant was discarded and this process was repeated seven times. The resulting pellet was homogenized in chilled distilled water and centrifuged as described above. The supernatant was recovered and this step was repeated twice. Supernatants were pooled and centrifuged at 100 000 g for 1 h at 4  C. Pellets were stained with Congo red and examined by polarized light microscopy to confirm the presence of amyloid fibrils as described below. Pellets containing amyloid fibrils were dissolved in distilled water and stored at 280  C until use. A 2% solution of silver nitrate was used as the inflammatory stimulus.

Detection of amyloid by Western blot analysis Sodium dodecyl sulphate-polyacrylamide gel electrophoresis was performed to access the contents of the amyloid protein. These were visualized by Coomassie brilliant blue R250 staining. A duplicated part of the gel was used for Western blot analysis using anti-mouse SAA1 goat antibody (1 : 5000) (AF2948; R&D Systems, Minneapolis, MN, USA). Protein concentrations in the AEF extract were measured by DC Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA) and adjusted to 1 lg/ll for inoculation.

Experimental design Animals were divided into 11 groups (A–K) based on the treatment given, as shown in Table 1. Mice in groups A, D, E, F, H, I and J were given AEF and silver nitrate (AgNO3) on day 0. Groups B, G and K were given silver nitrate only and no treatment was given to group C mice. After disappearance of amyloid depositions, animal responses to second inoculation were observed at two different time–points, i.e. 340 and 440 days. Mice in groups F, G, J and K were given a second injection with both AEF and silver nitrate and mice in groups E and I were given a second injection with silver nitrate only. Mice in groups D and H were not given any additional inoculation and were kept until autopsy on days 340 or 440, respectively.

Induction of amyloidosis with AEF and silver nitrate Amyloidosis was induced by intraperitoneal injection with 0
3 ml AEF (1 lg/ll) and subcutaneous (s.c.) injection with 0
5 ml of 2% silver nitrate. Induction and disappearance of amyloid depositions were studied in organs

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Long-term kinetics of AA amyloidosis and effects of inflammatory restimulation Table 1. Inoculation schedule and amyloid depositions in spleen after inoculation with amyloid enhancing factor (AEF) and silver nitrate Amyloidotic mice after 1st inoculation

Amyloidotic mice after 2nd inoculation

Days post-inoculation (dpi) Group name A B C D E F G H I J K

No. of mice

Inoculum

0

2

4

10

20

40

60

80

120

240

Inoculum at 330 days

340 dpi

30 3 3 3 3 3 3 3 3 3 3

AEF/AgNO3* AgNO3 None AEF/AgNO3 AEF/AgNO3 AEF/AgNO3 AgNO3 AEF/AgNO3 AEF/AgNO3 AEF/AgNO3 AgNO3

0/3† – – – – – – – – – –

0/3 – – – – – – – – – –

3/3 – – – – – – – – – –

3/3 – – – – – – – – – –

3/3 – – – – – – – – – –

3/3 – – – – – – – – – –

3/3 – – – – – – – – – –

3/3 – – – – – – – – – –

3/3 – – – – – – – – – –

2/3 0/3 0/3 – – – – – – – –

None AgNO3 AEF/AgNO3 AEF/AgNO3 – – – –

0/3 3/3 3/3 3/3

Inoculum at 430 days

440 dpi

None AgNO3 AEF/AgNO3 AEF/AgNO3

0/3 3/3 3/3 3/3

*AgNO3, silver nitrate; †Number of amyloid-deposited mice/number of mice tested.

recovered at 0, 2, 4, 10, 20, 40, 60, 80, 120 and 240 days after the first inoculation and on 340 or 440 days after the second inoculation. Liver, spleen, kidney, lung, heart and small intestine were collected at each autopsy and fixed in 10% neutral buffered formalin and embedded in paraffin.

Detection of amyloid depositions and immunohistochemical experiments Deparaffinized tissue sections were stained with Congo red and haematoxylin [17] and evaluated for amyloid depositions by conventional and polarized light microscopy using BX 43 (Olympus, Tokyo, Japan). The intensity of deposited amyloid load in immunohistochemically stained tissue sections was scored using NIH Image J software [18]. Another set of 3-mm thick deparaffinized tissue sections was processed for immunohistochemistry (IHC) using steam/heat treatment for antigen retrieval. Sections were washed with phosphate-buffered saline (pH 7
4), blocked with 3% H2O2 and incubated with anti-mouse SAA1 goat antibody (1 : 200) (AF2948; R&D Systems) for 2 h at room temperature. The sections were incubated further with anti-goat immunoglobulin (Ig)G, F(ab’)2 horseradish peroxidaseconjugated donkey antibody (1 : 1000) (SC3851; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 30 min before colour development with 3, 3diaminobenzidine-4HCl (DAB) for approximately 40 s. For confirmation of amyloid depositions, tissue sections were stained with Congo red and checked for emerald green birefringence under polarized light. Relative amyloid depositions in tissues were scored as follows: 2, no amyloid depositions; 1/2, may or may not be positive for amyloid depositions; 1, traces of amyloid depositions; 11, low amount of amyloid depositions; 11/111, low to moderate amount of amyloid depositions; 111, moderate

amount of amyloid depositions; and 1111, large amount of amyloid depositions.

SAA measurement and serum cytokine profile analysis Blood samples were collected from autopsied mice by cardiac puncture and retro-orbital bleeding from negative control groups under an inhalant ether anaesthesia. The collected blood samples were centrifuged at 7000 g for 10 min at 4  C in a TMA-29 rotor using an MX-150 centrifuge (Tomy, Tokyo, Japan). Serum samples were stored at 230  C until assayed for SAA levels. The SAA concentrations in sera were measured using a mouse SAA enzyme-linked immunosorbent assay test kit (Life Diagnostics, West Chester, PA, USA). Experimental procedures were performed according to the manufacturer’s instructions and the serum concentration of SAA was expressed as absorbance values. The amount of cytokines in sera was measured using a mouse T helper type 1 (Th1)/Th2/Th17 cytokine cytometric bead array (CBA) kit [IL-2, IL-4, IL-6, IL-10, IL17A, TNF-a and interferon (IFN)-g] (BD Biosciences, Franklin Lakes, NJ, USA), following the manufacturer’s instructions. Data were acquired on a BD fluorescence activated cell sorter (FACS)CantoII flow cytometer (BD Biosciences) and analysed with FACSDiva software (BD Biosciences).

Results Amyloid depositions in spleen at first inoculations Two days after the first injections with AEF and silver nitrate, all the autopsied mice were negative for amyloid depositions (Fig. 1a). However, between days 4 and 120 after injection, all autopsied mice were positive for

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Fig. 1. Representative immunohistochemistry (IHC) and Congo red staining of amyloid depositions in spleen. (a) IHC of spleen after the first inoculation using anti-mouse SAA1 goat antibodies. The presence of amyloid was confirmed by IHC and Congo red staining under polarized light. The intensity of deposited amyloid load was scored from large amount of amyloid deposition (1111) to no amyloid depositions (2) using NIH Image J software. Peak amyloid depositions were observed at 20 days after the first inoculation. (b) A large (1111) amount of amyloid deposition in group F was observed after the second inoculation. Bar = 200 lm.

amyloid depositions. At autopsy on day 240, two of three mice were positive for amyloid depositions. The results of IHC revealed that there was no AA amyloid deposition in the spleen until 2 days postinoculation (dpi). On the 4th day, low (11) amounts of amyloid depositions were observed in the spleen of one mouse, while traces (1) of amyloid deposition were found in the other two mice. On the 10th day, low and moderate (111) amounts of amyloid deposition were observed in two mice and one mouse, respectively. Moderate amyloid depositions were observed in all three mice at 20 dpi. A progressive decrease in amyloid depositions around spleen follicles was observed from the 40th day onwards. On day 60, amyloid deposition had reduced to mild in two mice and traces in one mouse (Fig. 1a). To estimate the severity of amyloidosis, we measured the intensity of amyloid deposition in spleen around follicles using Image J software after IHC of tissue sections (Fig. 2a,b). In mice given only one injection, the maximum intensity of amyloid deposition around spleen follicles was estimated to be a mean score of 6
8 on 20 dpi (Fig. 2c). Maximum amyloid depositions were observed in spleen on this day after persistently elevated concentrations of SAA for more than 10 days. A reduction in splenic amyloid depositions was observed at 40 dpi. On the 60th day, bands of amyloid deposition around spleen follicles were not as intense as on the 20th day. Scattered patches of amyloid depositions were observed by IHC staining with a mean intensity score of 2
4 at 120 dpi (Fig. 2c). By 240 dpi minor depositions were observed around spleen follicles. As the effect of the inflammatory stimuli reduced, SAA levels returned back to normal basal levels and amyloid depositions disappeared progressively. 136

Amyloid depositions in spleen at 340 days At 330 dpi mice were reinoculated with AEF and silver nitrate and were divided into four groups, D, E, F and G (Table 1). The IHC findings in mice autopsied at 340 dpi revealed that amyloid deposition was at quite a high intensity in groups E and F, and was more severe than that observed in group A after the first inoculation (Fig. 1a,b). In group D amyloid depositions disappeared until 340 days. In group G, the severity of amyloid depositions is similar to the depositions observed after first-time inoculation (Fig. 1b). In group E, amyloid deposition in mice inoculated with only silver nitrate showed a mean intensity score of 8
3. In group F, mice were inoculated twice with both silver nitrate and AEF and a mean intensity of amyloid deposition was scored at 10
7, which was the highest intensity among all groups (Fig. 2c).

Amyloid depositions in spleen at 440 days In another set of experiments, the second inoculation was given at 430 dpi and autopsies were performed 10 days later. The IHC findings of mice autopsied on the 440 dpi revealed that the maximum amount of amyloid depositions were found in group J followed by group I (Fig. 2a). In group I, there were massive typical splenic marginal zone and interfollicular amyloid depositions. In group J, intrafollicular amyloid deposition was prominent and accompanied by massive marginal zone and interfollicular amyloid depositions. Representative raw scores for depositions in one stained tissue section from groups H, I, J and K were 0
6, 9
4, 10
8 and 7
1, respectively (Fig. 2a,b). The mean intensity of amyloid depositions of three mice in groups H, I, J and K is shown in Fig. 2c.

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Long-term kinetics of AA amyloidosis and effects of inflammatory restimulation

Fig. 2. Intensity scoring of amyloid depositions in spleen using Image J. (a) Representative immunohistochemistry (IHC) of amyloid deposition around spleen follicles. (b) Image J was used to measure the intensity of amyloid depositions around spleen follicles. Images of IHC-stained spleen sections in (a) were processed with Image J to obtain the raw score in (b). Representative raw score of depositions in one stained tissue section from groups H, I, J and K was 0
6, 9
4, 10
8 and 7
1, respectively. (c) Long-term kinetics of intensity score of amyloid depositions around spleen follicles. Yellow arrows indicate the first inoculation on day 0 and second inoculations on 330 or 430 days. Yellow circles indicate the mice treated with both silver nitrate and amyloid enhancing factor (AEF) at first inoculation and then killed at different time-points from zero to 240 days in group A. Groups B and C represented by light green diamonds were served as control. Blue diamonds indicate groups D and H treated with silver nitrate, and AEF at first inoculation and no treatment at second inoculation. Purple circles indicate groups G and K treated with only silver nitrate at first inoculation and with both silver nitrate and AEF at second inoculation. Red triangles indicate groups E and I treated with silver nitrate and AEF at first inoculation, treated subsequently with only silver nitrate at second inoculation. Yellow diamonds indicate groups F and J treated with both silver nitrate and AEF at first and second inoculations. The mean intensity score in group F was estimated as 10
7. Mean intensity scores of 8
3 and 6
8 were observed in groups E and G, respectively. In group D, amyloid deposition had almost disappeared by 340 days with a mean intensity score of 0
8.

Amyloid depositions in liver and kidney at first and second inoculations Amyloid depositions in liver varied in mice given first or second inoculations. Low to moderate amyloid depositions were observed in liver after a single inoculation. The second injections resulted in severe amyloid deposition in the liver. After the inoculation with both AEF and silver nitrate in group G, amyloid depositions were seen around sinusoids in the portal zone. In mice in group E injected with only silver nitrate at the second inoculation, amyloid depositions were seen concentrated mainly around blood vessels and veins (Fig. 3). In group F, mice were inoculated twice with both silver nitrate and AEF; amyloid depositions were seen concentrated at sinusoids

and blood vessels (central veins). Amyloid depositions disappeared progressively in group D with time. The presence of amyloid fibrils in liver was confirmed by Congo red staining (Fig. 3). Amyloid depositions in groups H, I, J and K at 440 days were the same as those observed in groups D, E, F and G at 340 days, respectively. Amyloid depositions were not observed in the renal tissue of mice after first inoculation (data not shown). After the second inoculation, the representative amyloid depositions in kidney of groups H, I, J and K at 440 days were shown in Fig. 4. Severe amyloid depositions in renal tissue were observed in the groups E and F at 340 days, and in groups I and J at 440 days. In mice groups E and I treated

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Fig. 3. Immunohistochemistry (IHC) and Congo red staining of amyloid depositions in liver. Amyloid depositions are seen around sinusoids in the portal zone of livers from mice in group G. Amyloid depositions are seen concentrated mainly around blood vessels and veins in group E mice inoculated only with silver nitrate at day 330. In group F, amyloid depositions are seen concentrated at sinusoids and blood vessels (central veins). Amyloid depositions cleared progressively in group D. The presence of amyloid was confirmed using Congo red staining.

with only silver nitrate at second inoculation, renal corpuscles were mainly affected. In groups F and J treated with both silver nitrate and AEF at second inoculation, renal corpuscles were affected leading to marked deformation of renal parenchyma. Minute traces of AA amyloid were observed in renal tissue of groups G and K by IHC, but they could not be verified by Congo red staining.

Cytokine profile assay Concentrations of serum cytokines were measured and are shown in Fig. 5a. In group A, AEF and silver nitrate inoculation significantly stimulated the production of proinflammatory cytokine IL-6 within 24 h of inoculation (Fig. 5a). IL-6 peak levels in sera of the mice group inoculated with both AEF and silver nitrate (group A) were reached earlier than those inoculated with only silver nitrate (group B). A decrease in the level of IL-6 was observed from 48 h onwards after the first inoculation. The serum cytokine profile assay after the second inoculation on day 330 showed a high level of IL-10 (antiinflammatory cytokine) in groups D, E and F. In group D, animals maintained high levels of anti-inflammatory IL-10 until 340 days, attained after the first inoculation. In group E, reinoculated with silver nitrate only, the concentration of IL-10 was remarkably increased. In group F, reinoculated with both AEF and silver nitrate, the mean concentrations of IL-10 and IL-6 increased to 149 pg/ml and 25
55 pg/ml, respectively. In group G, the level of IL6 was high and that of IL-10 was low. The concentrations of IL-2, IL-4, IL-17A, TNF-a and IFN-g were not detected, possibly because of their low concentrations. Results of the cytokine profile assay on the second inoculation at 430 days was the same as that observed at the second inoculation at 330 days.

Measurement of SAA levels Concentrations of SAA in the collected mouse sera were measured and are shown in Fig. 5b. Following the first 138

injection with AEF and silver nitrate, elevated SAA levels were observed within the first 24 h. The elevated SAA level persisted up to 10 dpi and then fell sharply to baseline levels by 20 days. Inflammatory stimuli resulted in an increase in circulating concentrations of SAA after the first and second inoculations. SAA levels surged in all groups treated either with silver nitrate or AEF and silver nitrate. Irrespective of this surge, amyloid depositions were observed only in groups given AEF and silver nitrate. After the second inoculation, the persistently elevated SAA level returned to basal level within only 10 days. The SAA level elevated after administration of inflammatory stimuli and then returned to normal values.

Discussion The objective of this study was to examine the longterm kinetics of amyloid deposition and clearance, serum cytokine status and SAA levels in mice with experimentally induced AA amyloidosis using AEF and silver nitrate. In this study, an early amyloid deposition on the fourth day was detected in all autopsied mice given a single injection of AEF and s.c. silver nitrate. Subcutaneous silver nitrate is a strong inducer of the acute phase response and the maximum SAA level was attained in a short period of time. In-vitro experiments have demonstrated that amyloidogenic proteins from homologous animal species can potentiate the protein aggregation process more efficiently than those from heterologous animal species [19,20]. Therefore, administration of AEF obtained from mice might have reduced the length of the lag phase and resulted in the quick sequestration of SAA into amyloid in all amyloidotic mice. The progressive disappearance of visually detectable amyloid after peak depositions was in accordance with previous studies [9,15]. Our observations demonstrated that the first inoculation led to peak amyloid depositions in spleen on 20 dpi followed by liver, while the second

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Long-term kinetics of AA amyloidosis and effects of inflammatory restimulation

Fig. 4. Immunohistochemistry (IHC) and Congo red staining of amyloid depositions in kidney. Amyloid deposition in kidney was not observed in group H at 440 days after first inoculation. Amyloid deposition in kidney was not detected in group K, even if mice were inoculated with AEF and silver nitrate at 430 days. In kidney in groups I and J, the presence of amyloid was confirmed using Congo red staining.

inoculation led to more aggressive amyloid depositions in the spleen and liver. Aggressive amyloid depositions after the second inoculation were due to the remnants of first-time amyloid depositions acting as an amyloid seed [21]. Intrafollicular amyloid depositions might occur because of a more aggressive and systemic form of amyloidosis after the second inoculation. Amyloid depositions were observed surrounding sinusoids in the liver after the first inoculation, and around blood vessels and central veins at the second inoculation [9]. This type of hepatic amyloid deposition was observed in group A mice and in mice from groups E and I after the second inoculation with only silver nitrate. We observed that two rounds of simultaneous administration of AEF and silver nitrate in groups F and J caused amyloid depositions both around sinusoids and in central veins. This sort of AA deposition in liver in response to inflammatory treatment requires further investigation. The results of this study showed that detectable levels of some key cytokines in the blood were induced in mice. The level and types of these cytokines were altered with the prognosis of AA amyloidosis. Amyloid deposition was accompanied by increased SAA production, up-regulated by proinflammatory cytokine activity [22]. SAA synthesis can be stimulated by both external and internal factors. External factors include inflammation due to administration of a drug or inflammatory stimulus, and internal factors can be bacterial lipopolysaccharide, proinflammatory cytokines and glucocorticoids [23]. In this study, inoculation of inflammatory stimuli resulted in the increased release of proinflammatory cytokine IL-6 leading to SAA production. Mihara et al. [24] demonstrated that IL-6 is a key cytokine for the induction of AA amyloidosis, because the administration of IL-6 receptor antibody to amyloidogenic-treated mice inhibited development of AA amyloidosis. The appearance of the anti-inflammatory cytokine IL-10 during the disappearance of amyloid depositions was observed with a reduction in IL-6 expression to basal levels. The increased level of IL-10 during the recovery period may be an important phase, because it inhibits the production of IL-6 by Th1 cells and down-regulates the expression of major histocompatibility complex (MHC) class molecules [25]. An increased level of IL-10 after the

second inoculation might be due to the body’s reaction to the previous amyloid induced trauma. A secondary inflammatory stimulus with silver nitrate or AEF or both resulted in a boost to the existing higher level of IL-10. The proinflammatory or anti-inflammatory environment may have played a role by modifying the function of macrophages in amyloidosis induction and the disappearance of amyloid depositions [26]. IL-1 has been reported to impair proteolysis of SAA, while phagocytic clearance of amyloid b is enhanced by the anti-inflammatory environment [27,28]. The peak of IL-6 attained after the second inoculation was lower than that attained after first inoculation. It may be an effect of the exiting higher level of IL-10, because IL-10 inhibits the release of proinflammatory mediators from monocytes or macrophages [29]. Elevated IL-10 has also been reported in Alzheimer’s disease studies [30,31]. Peripheral blood mononuclear cells derived from Alzheimer’s disease patients produced higher levels of IL-10, compared to healthy elderly cells [32]. The production of anti-inflammatory cytokine might balance the overproduction of proinflammatory cytokine. The immunological function of IL-10 for AA amyloid deposition in long-term amyloidosis remain unsolved. A recent study demonstrated the sustainable production of SAA using a dose-dependent, doxycycline-inducible transgenic expression of SAA in mice [11]. Sustained SAA levels induced by a transactivator gene led to AA amyloidosis in the absence of inflammation. In transgenic mice, sustainable production of SAA resulted in AA amyloidosis without any inflammation, but in our experiment its inflammation characterized by immune-regulators such as cytokines or chemokines seem to control SAA levels. The sharp fall in persistently elevated SAA levels to basal level by day 20 might be related to its sequestration into AA amyloid. After the second inoculation persistently elevated SAA levels returned to the basal level within 10 days. This could be due to rapid sequestration of SAA into amyloid depositions, as ready-to-use seed might be available from clearance of previous amyloid depositions. Conversely, any secondary inflammatory stimulus further boosts the levels of IL-10. Moreover, IL6 provided a proinflammatory environment for the induction of AA amyloidosis, while IL-10 provided an

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Fig. 5. Cytokine profile and serum amyloid A (SAA) concentration after first and second inoculations. (a) Cytokine concentrations of interleukin (IL)-6 and IL-10 were measured at 1 day and 331 days after first inoculation and second inoculation, respectively. In group A, significant production of IL-6 cytokine was observed within 24 h after first inoculation. After the second inoculation, elevated level of IL-10 was observed in groups D, E and F. The concentration of IL-6 in group G was higher compared to IL-10. (b) An increase in the SAA levels was observed after the first and second inoculations. Yellow arrows indicate the first inoculation on day 0 and second inoculations on day 330 or 430. The elevated SAA level returns to basal level until 20 and 10 days after the first and second inoculations, respectively.

anti-inflammatory environment for the clearance of amyloid depositions. The most common outcome of systemic AA amyloidosis is irreversible damage to various organs, in particular the kidneys [33]. Amyloid depositions were observed in the kidneys of animals having had a second exposure to inflammatory stimuli after regression of previous amyloid depositions. This observation is similar to the data from transgenic mice reporting significant depositions in kidney when mice were re-exposed to elevated SAA levels [11]. Previous studies have shown that glomerular amyloid was rare in nuclear magnetic resonance imaging (NMRI) mice, while reinduction resulted in amyloid appearance in renal papilla [9]. In CBA/J mice renal amyloid developed slightly later than in spleen or liver [21], and glomerular amyloid is found rarely in NIH Swiss white mice [34]. These apparent variations may be due to strain differences or varied animal responses to the inflammatory stimuli. Possible pathogenesis of renal parenchyma deformation might be renal papillary amyloid depositions leading to necrosis. However, assessment of renal papillary necrosis by detecting non-invasive biomarkers might have been of scientific value during clearance of amyloid depositions. The factors determining the anatomical site of amyloid depositions are not completely understood. Aggressive amyloid depositions with altered patterns after regression might be due to differences in the distribution of amyloid template. Moderate to severe amyloid depositions in kidneys were observed after the second inoculation, therefore systemic AA amyloidosis may be the result of recurring inflammation. There is still a need for thorough research to uncover the IL-10 responses during recurring inflammation in AA amyloidosis. Macrophages are observed frequently in close prox140

imity to amyloid deposits, and play an important role in AA amyloid clearance [10,12]. The mechanisms by which proinflammatory or anti-inflammatory cytokines modulate the function of macrophages in AA amyloidosis require further investigation.

Acknowledgements This research work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (Project Code no. 22380165, 25292171).

Disclosure The authors report no conflicts of interest.

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