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Annals of Clinical & Laboratory Science, vol. 44, no. 3, 2014 249 Accumulation and Absorption of Serum A and E Fragments in the Course of AA : a Study in a Mouse Model

Jyunji Sato, Kazuhiko Kotani, and Toshiyuki Yamada

Department of Clinical Laboratory Medicine, Jichi Medical University, Japan

Abstract. Reactive AA amyloidosis develops secondary to chronic inflammatory disorders. Serum amyloid A (SAA) and its degradation products, named AAs, are the main components of amyloid deposits, while (apoE) fragments are the minor components. To further understand the molecu- lar mechanism of AA amyloidosis, we examined SAA/AAs moieties and apoE in the spleen and plasma throughout the amyloid-generating and amyloid-absorbing phases in a mouse model. SAA and four AA species (8.5kDa, 7.8kDa, 7.0kDa, and 6.2kDa) were detected in the spleen. SAA and the 8.5 kDa and 7.8 kDa AAs were prominent in the acute phase, whereas the 7.0kDa AA, the second smallest AA correspond- ing to the most common form in the human disease, was prominent in the chronic phase. These results indicate that the higher molecular weight species first constituted the fibril,we follo d by the 7.0kDa species, which were finally absorbed. ApoE was a component of the amyloid deposits at a degradation size from the beginning and was absorbed without being converted to another size. Degradation products, either from SAA or apoE, did not appear in the plasma during the course of the disease. A more detailed understanding of the moieties of amyloid-related peptides may help in the development of a method that can indicate the disease activity of AA amyloidosis.

Keywords: amyloidosis, serum amyloid A, apolipoprotein E, fragment, mouse

Introduction Reactive amyloidosis has long been considered pro- gressive. However, recent advances in the methods Reactive amyloidosis is a serious complication of used to sufficiently and simultaneously suppress in- chronic inflammatory diseases including rheuma- flammatory activity and the production of SAA re- toid arthritis and tuberculosis. The disease is char- vealed that amyloid accumulation could be arrested acterized by the deposition of amyloid A (AA), and the amyloid load could be reduced [4,5]. A which forms insoluble fibrillar aggregates in the ex- generally accepted concept is that accumulated am- tracellular spaces of miscellaneous organs [1,2]. AA yloids can be absorbed. However, the mechanism is the amino-terminal segment of serum amyloid A by which accumulated are absorbed has (SAA) resulting from the proteolytic removal of its yet to be confirmed, and if it is elucidated it would carboxyl terminal portion [3]. The production of be helpful for understanding the disease’s activity. SAA is mainly induced in the by inflammatory such as -6. Prolonged inflam- To address this issue, we adopted a murine model mation results in the accumulation of SAA in the of reactive amyloidosis. The disease can be generat- blood and potentially in the tissues. SAA may be ed in mice by inflammatory stimuli. Amyloid de- converted to AA during or after the formation of posits have also been shown to be absorbed when fibrils, which may then be abundant in stable amy- the animal does not receive inflammatory stimula- loid deposits. tion for a prolonged period of time [6]. Using this model, we examined SAA and AA moieties in the Address correspondence to Toshiyuki Yamada, MD., Department spleen, in which amyloid deposition is the most of Clinical Laboratory Medicine, Jichi Medical University, 3311 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan; phone: 0285 58 abundant, during the disease’s course. Since AAs are 7386; fax: 0285 44 9947; e mail: [email protected] heterogeneous in size because of multiple

0091-7370/14/0300-249. © 2014 by the Association of Clinical Scientists, Inc. 250 Annals of Clinical & Laboratory Science, vol. 44, no. 3, 2014

Figure 1. Immunohistochemical detection of SAA/AA (A) and apoE (B) in the spleens of amyloidotic mice. Specimens were taken on day 2 (upper left), day 5 (upper right), day 21 (lower left), and day 55 (lower right). degradation sites on the SAA molecule, we analyzed formalin and embedded in paraffin for histochemical their moieties by size. We also attempted to find studies. The research protocol was approved by the AA-like components in the plasma under the ex- Animal Experiment Ethic Committee of Jichi Medical pectation that the detection of such components University. may indicate the disease activity. Antibodies. Monoclonal anti-mouse SAA antibodies were generated in our laboratory by immunizing rats In addition, the search for degradation products in with recombinant mouse SAA1, followed by the fusion the spleen and plasma was performed on apolipo- of their spleen cells with mouse myeloma cells [11]. protein E (apoE). ApoE, specifically its fragments, Among the several clones obtained, clone BI-2 was cho- is known to be a component of amyloid deposits, sen for this study based on its reactivity with many AA regardless of the amyloidosis type, and loses its ami- species on immunoblots. The goat anti-human apoE no-terminal segment in these deposits [7,8]. antibody was a kind gift from Eiken Chemical, Inc. (Japan). Materials and Methods Immunoblot analysis. Removed spleens were homoge- nized at 100 mg/ml in 6M urea, 0.1M Tris-HCl, pH Mice and induction of amyloidosis. A human apoE 8.5. Ten microliters of homogenate or 1 μl of plasma was knock-in mouse, which was generated to express human subjected to sodium dodecyl sulfate polyacrylamide gel apoE2 instead of murine apoE [9], was used in the pres- electrophoresis, followed by electrotransferral to a poly- ent study. To induce amyloidosis, 15- to 20-week-old vinyl difluoride membrane. The membranes reacted female mice were injected intraperitoneally with amy- with the anti-SAA antibody BI-2 or anti-apoE and then loid-enhancing factor (AEF), which was prepared from with peroxidase-conjugated secondary antibodies. Color the amyloidotic spleen as previously described [10]. This was developed using a commercially available substrate was followed by a subcutaneous injection of 0.5 ml of (ATTO, USA). The molecular size of each band was es- 1% silver nitrate. AEF can shorten the period required timated by a commercial molecular weight marker (GE for amyloid deposition, probably due to its seed effects Healthcare, USA). Bands were analyzed with the soft- [2]. Mice were sacrificed on day 0 (no treatment), day2, ware image J (ver. 1.4) to calculate their relative day 5, day 21, and day 55. Each group, referred as the intensity. amyloidotic group, consisted of 3 or 4 animals. The same protocol was performed without the initial injection of -linked immunosorbent assay (ELISA). SAA in AEF on two animals each, referred to as the control the plasma was quantified by an ELISA utilizing a newly group. Blood and spleen were obtained at autopsy, while established antibody (BI-2) [11]. To quantify SAA/AA plasma and a part of the spleen were kept at -70°C until in the spleen, a piece of the tissue was homogenized at use. Another part of the spleen was fixed with 10% SAA and apoE fragments in AA amyloidosis 251

100 mg/ml in 4M guanidine HCl, pH8.2, ac- cording to the method by which human AA was measured in biopsy materials [12], and was then subjected to the same ELISA. ApoE in the plasma was quantified by an ELISA as human apoE ac- cording to a previously described method [13].

Immunohistochemistry. After being deparaf- finized, tissue specimens were subjected to im- munostaining. Monoclonal BI-2 or polyclonal anti-apoE was used as the primary antibody, fol- lowed by the avidin-biotin-peroxidase system us- ing a commercial kit (Vectastain, USA).

Results

Immunohistochemical detection of SAA/ AA and apoE in the spleen. SAA/AA immu- noreactivity was observed in the white pulp of spleens in the amyloidotic group on day 2, its staining intensity increased on day 5, was maintained on day 21, and then markedly decreased on day 55 (Figure 1A). These re- sults agreed with immunochemical measure- ments for SAA/AA in that tissue (Figure 2C). In contrast, apo E immunoreactivity Figure 2. Immunoblot analysis of spleen tissues using anti-SAA (A) and anti-apoE (B). Representative patterns from each experi- was negative on day 2, faintly positive on day mental day were shown. The estimated molecular size was indi- 5, moderately positive on day 21, and weakly cated. No intact apoE corresponding to 24kDa was detected. The positive on day 55 (Figure 1B). SAA/AAs total amount of SAA/AA, determined by ELISA, and amount of each band, calculated by the total amount and intensity of each and apoE were not detected in the control band, were shown (C). The mean and S.E. were shown. group at any time (data not shown).

Immunoblot analysis of SAA and apoE frag- ApoE was observed in the spleen as an 11kDa frag- ments. The anti-SAA antibody BI-2 detected 5 ment and not as its intact size (Figure 2B). It SAA/AA bands in the spleen from amyloidotic reached its highest amount on day 21, which is mice (Figure 2A). The molecular sizes of each band consistent with the results obtained by immunohis- were estimated to be 10.5kDa (intact SAA), tochemistry. However, based on the strength of its 8.5kDa, 7.8kDa, 7.0kDa, and 6.2kDa, respective- stained band, the amount of apoE was low through- ly. The amount of each band was calculated by the out the experimental period. ratio of each band to the total SAA/AA concentra- tion in the tissue. The kinetics of the total SAA/AA Intact SAA and apoE, possibly derived from the concentration and the amount of each band were blood, were weakly observed in the control group shown in Figure 2C. The amounts of the 10.5kDa, and degraded bands were not detected (data not 8.5kDa, and 7.8kDa species were the highest on shown). day 5 and then decreased toward day 55. The amount of the 7.0kDa species was less on day 5, Presence of SAA and apoE in plasma. Plasma SAA increased on day 21, and then decreased. The concentrations were the highest on day 2, decreased amount of the 6.2kDa species was less throughout on day 5, reached normal levels by day 21, and the experimental period, but was the second most were maintained at these levels on day 55 (Figure abundant on day 2. 3A). No significant difference was observed in SAA 252 Annals of Clinical & Laboratory Science, vol. 44, no. 3, 2014 The manner by which SAA or AA accumu- lated as amyloid deposited in the spleen was demonstrated in this study. Intact SAA and 4 AA species are the components of amyloid deposits. These four AA species are all con- sidered to be the amino-terminal segments of SAA, and this was supported by the find- ing that another monoclonal antibody BG- 10, which should react with the carboxyl- terminus of mouse SAA, did not react with these 4 bands (data not shown). The 7.0kDa band may be a counterpart of the human component AA76, which was shown to be the most prominent band in human AA de- posits during any disease course [12]. In the present study using mice, the intact SAA and two high molecular weight AA species were abundant at the beginning (day 2 and day 5). The amounts of these species de- creased toward day 21, while the amount of the 7.0 kDa species was low on day 5 and Figure 3. Plasma concentrations of SAA (A) and apoE (B) in was the highest on the day 21. The total amyloidotic and control mice. The mean and S.E. were shown. No significant difference was observed in either concentration SAA/AA concentration in the deposits was between both groups. virtually unchanged from day 5 to day 21, and plasma SAA, the source of tissue SAA/ AAs, might not be elevated. Taken together, concentrations between the amyloidotic groups these results indicate that the 7.0 kDa AA should be and control groups. Plasma apoE concentrations generated from intact SAA and the two higher mo- appeared to remain constant throughout the course lecular weight AAs. The process by which the most of the disease without any significant differences common form of AA is generated during the stable being observed between the amyloidotic and con- phase as above has been proposed eapreviously [14] trol groups (Figure 3B). Immunoblot analysis re- and is consistent with the findings of the present vealed that intact SAA or intact apoE was promi- study. Amyloid deposits were subjected to absorp- nent without any specific degradation products tion after the stable phase; therefore, shortening (data not shown). this phase may accelerate the absorption of amyloid deposits. The fate of amyloid deposits in this phase Discussion should be clarified in more detail. The 6.2 kDaAA, the smallest AA, appeared in the acute phase and Amyloid was detected in the spleen as early as day 2 was unchanged in the stable phase. It is unknown in this model. Its amount increased up to day 5, why this segment appeared prior to the larger 7.0 and, although we did not examine its accumulation KDa AA, but this detail implies resistance from from day 5 to day 21, it appears that the amount proteolysis. measured on day 5 was maintained on day 21, as estimated by SAA/AA quantification. A decrease in As was shown in our previous study [15], the mouse the amount of amyloid was not observed even strain in which human apoE was knocked in, but though a large reduction in plasma SAA, the source which lacked apoE in the plasma, did not develop of amyloid, was reported during this period (Figure AA amyloidosis. Previous studies have also indicat- 3). Thus, we regarded the acute phase as being from ed the important contribution of apoE in amy- day 0 to day 5, the stable phase from day 5 to day loidogenesis [16, 17]. ApoE was detected in the 21, and the absorption phase from day 21 to day spleen from day 5 in our present study, which also 55. supports its contribution to amyloidogenesis. SAA and apoE fragments in AA amyloidosis 253 However, the amount of apoE in amyloid deposits References appeared to be markedly less than that of SAA/AAs, based on the output of immunological detection in 1. Benson MD. Amyloidosis. 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