Intralysosomal Formation of Amyloid Fibrils Tsuranobu Shirahama, MD, and Alan S. Cohen, MD
Unusual inclusions, which occurred in the reticuloendothelial cells intimately associated with fresh amyloid deposits. were analyzed by electron microscopy. The inclusions were located in the areas rich in the primary lysosome type of dense bodies and the cytoplasmic invaginations containing well-oriented amvloid fibrils. They were single-membrane-bounded, measured 0.3 to 0.8 in width and 0.5 to sev eral microns in length, and showed considerable variation in the electron density of their contents. The latter consisted of two different ultrastructural elements: fibrillar profiles and a homogeneous or finely- granular electron-dense substance. The fibrillar profiles were virtually identical in ultrastructure to the amyloid fibrils and were well-oriented parallel to the long axis of the inclusion. The homogeneous or finely granular electron-dense substance appeared to be comparable to that composing the dense body matrix. The inclusions were usually% acid phosphatase positive. but did not take up intrav enouslv injected Thorotrast particles. These data led us to conclude that these inclusions were transitional forms from the usual dense bodies to the deep cytoplasmic inv aginations containing well-oriented amyloid fibrils (which are accepted by most investigators as the sites of amyloid formation) and thus constitute direct ev idence for the involvement of lysosomes in amyloid fibril formation. (Am J Pathol 81:101-116, 1975)
DESPITE SIGN-IFICAN-T ACHIEVEMIENTS in research on amrvloid, its pathogenesis has not yet been fully clarified. The concept put forth bv Smetana in 1927 '-that the reticuloendothelial (RE) cells are intimately associated with the genesis of amyloid-has gained substantial stipport. 2 At the ultrastruictural level, in 1963 (a few years after the definition of amyloid as a unique fibrillar substance 7), Gueft and Ghidoni 8 described cytoplasmic invaginations which contained tufts of well-oriented amyloid fibrils at the cell-amvloid interface and designated them as the sites of amvloid formation. This notion has been supported by many investigations,26 including electron microscopic autoradiographic studies,9" 0 and has not vet met any serious challenge. Since a) the RE sy-stem may play an important role in amyloidogenesis, b) the system is intimatelv related to Ivsosomal activity," and c) the restults of recent From the Evans Memorial Department of Clinical Research. University Hospital. the Thorndike Memorial Laboratory. Boston City Hospital. and the Department of Medicine. Boston University School of Medicine. Boston. Massachusetts Presented in part at the International Symposium on Am%loidosis. Helsinki. Finland. August 1974 Suipported by Grants AM-04599 and Tl-AN1-5285 from the National Institute of Arthritis and Mletabolic Diseases. US Public Health Sen-ice. Grant RR-533 from the General Clinical Research Centers Branch of the Division of Research Resources. National Institutes of Health, and by grants from the Massachusetts Chapter of the Arthritis Foundation, the Arthritis Fouindation, the John A Hartford Foundation, and the Unisersity Hospital Accepted for publication Jine 14. 197. Address reprint requests to Dr T Shirahama. U niv ersity Hospital. Room E&37. 75 East Newton
Street. Boston. \MA 02 1 8
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biochemical studies of amyloid suggest the possibility that protein fragments resulting from enzymatic cleavage may constitute some type of amvloid,3'5', there has been speculation that Iysosomes might participate in the process of amyloid formation.5'15 We recently reported that unusual Ivsosomal activitv in the RE cells was closelv associated with earlv amvloid deposits.16 However, all the findings available to date concerning the lysosomal involvement in amyloid fibril formation have been circumstantial, and no direct evidence exists in support of the concept. The present paper reports our observations, which strongly suggest amyloid fibril formation in lysosome-originated inclusions and represent perhaps the first direct evidence for the participation of lysosomes in amvloid fibril formation. Materials and Methods Amyloidosis was induced in 8- to 10-week-old CBA J mice (Jackson Laboratory, Bar Harbor, Me ) by daily subcutaneous injections of 03a ml of 12% casein (Casein Hamersten, Control No 3320. Nutritional Biochemicals Company, Cleveland, Ohio) This schedule shortened the duration of amyloid induction to about two-thirds of that achieved with our routine schedule injecting 0.O ml of 10%c casein 17 In the present model. amy.loid was present in the spleen after 8 to 12 injections, in the liver after 12 to 16 injections, and in the kidney after 16 to 20 injections. After specific intervals of the amyloid induction regimen, animals were sacrificed on the day after the last casein injection, and spleen, liver, and kidney were processed for light and electron microscopy. Groups of the animals received intravenous injection of 0.1 ml of Thorotrast (Fellows Testagar, Detroit, Mich.) into the tail vein on the day after the last casein injection, and then sacrificed at 3, 10 minutes and 1, 2, or 24 hours after the Thorotrast injection Spleen, liver, and kidney were then prepared for electron microscopy For the electron microscopic studies, small tissue blocks were either double-fixed with 2 0% formaldehvde-2 3c% glutaraldehyde '" and 2%t osmium tetroxide in 0 1 Mt cacodylateHCI buffer or single-fixed with 2% osmium tetroxide in cacodylate or phosphate buffer l9 They were then dehydrated in graded ethanols and embedded in Epon or Araldite Thin sections were cut on an LKB Ultrotome with glass knives and stained w ith uranyl acetate 21 and, or lead citrate 2 when feasible Tissues were prefixed with the aldehyde " and cut into 30-M slices on a Sorvall TC-2 tissue sectioner ' and then treated for cvtochemical demonstration of acid phosphatase activity as described elsewhere.'. As controls, spleens, livers, and kidnevs from mice untreated or treated with intravenous injections of Thorotrast alone were also prepared in the same fashion as those from the amvloidotic mice The specimens were examined in a Siemens Elmiskop I at initial magnifications of 1000 to 40.000 X The observations concentrated on the foci of venr early small amyloid deposits rarely exceeding several microns in diameter. The present study was oriented towards summing up our observations conceming the peculiar inclusions and accordingly about 1000 new electron micrographs were processed In addition, because about 10 years have passed since the peculiar inclusions had first come to our attention. we have accumulated a large amount of data relating to them. These included a variety of preparations from variouis species and strains (humans, rabbits, guinea pigs, C3H HeJ mice. C37B10 6J mice, etc ) Those data were also reanalvzed recently and taken into consideration in the interpretation of the present results.
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Results Gew Apecb
Although amyloidosis was induced more rapidly in the present study, the fine structure was comparable to that described in the animals in which amyloidosis was induced by other techniques,2,4,6 8-0,15-17 except for the frequent appearance of the peculiar inclusions which will be described in detail. In addition, the formation of deep cytoplasmic invaginations containing tufts of well-oriented amyloid fibrils and the frequent lysosomal and vesicular profiles in the peripheral cytoplasm of the RE cells closely associated with amyloid deposits 16 were even more prominent in the present specimens. Pecular luskok and Their Relation to Des Boces and Amy Fblb
In the present study, the RE cells intimately associated with small (less than several microns in diameter) amyloid deposits often displayed unusual cytoplasmic inclusions (designated as dense fibrillar inclusions hereafter) (Figure 1). The inclusions were observed most frequently in the Kupffer cells of the liver, less frequently in the fixed reticular cells and sinus lining cells of the spleen, and seldom in the glomerular mesangial cells of the kidney. They were round to long-fusiform in shape, measured 0.3 to 0.8 M in width and 0.5 to several microns in length, and varied considerably in the electron density of their contents. Some of them with lighter electron density displayed an inner structure as described below. These inclusions were usually closely associated with the ordinary dense bodies and the single-membrane-bounded well-oriented amyloid fibrils (which probably represented either cross or grazing sections of amyloid fibrils in the cytoplasmic invaginations or truly intracellular singlemembrane-bounded amyloid fibrils). At higher resolution (Figures 2A and 2B), the inclusions were revealed to be single membrane bounded and containing well-organized fibrillar profiles embedded in homogeneous or finely granular electron-dense substance. The well-organized fibrillar profiles were always oriented in parallel to the long axis of the inclusion and were comparable to amyloid fibrils; they were about 100 A in diameter, rigid, and nonbranching. The homogeneous or finely granular electron-dense substance constituting the background was comparable, ultrastructurally, to the dense body matrix. Furthermore, there were sufficient data to suggest a continuous transition from the usual primary lysosome type of dense bodies (by ultrastructural definition) to the dense fibrillar inclusions and further to the singlemembrane-bounded well-oriented amyloid fibrils or the well-oriented amyloid fibrils in deep cytoplasmic invaginations. The following sequence
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suggested itself: 1) tvpical primary-lysosome-type dense bodies which contained homogeneous or finely granular electron-dense matrix, 2) elongated single-membrane-bounded particles with a matrix similar to the dense bodies, 3) further-elongated particles containing a slightly less electron-dense matrix in which the presence of well-organized fibrillar profiles was suggested, 4) long fusiform single-membrane-bounded inclusions exhibiting clearer fibrillar profiles embedded in a homogeneous or finely granular electron-dense substance, 5) inclusions containing prominent fibrillar profiles and traces of the electron-dense background substance, and 6) single-membrane-bounded well-organized fibrillar profiles, i.e., amyloid fibrils, with virtually no background substance. Giant cells, which displayed multiple nuclei, rich ribosomes, welldeveloped endoplasmic reticulum, large multiple Golgi complexes, frequent mitochondria and abundant dense bodies, were occasionally observed-presumably macrophage polykaryons.2' Although none of those cells were attached to the extracellular amyloid deposits, they demonstrated an abundance of the dense fibrillar inclusions in the various stages described above (Figures 3-5). Findings suggesting the fusion of Golgi-associated vesicles to the dense fibrillar inclusions, standard dense bodies to the inclusions, and the dense fibrillar inclusions to each other were not unusual in the RE cells as well as in the macrophage polykaryons (Figures 1-5). Acid
Ph
Activit in the Dns FBincusions
The RE cells apparently related to amyloid production showed high acid phosphatase activity in this study, as was observed in the previous study.16 The enzyme reaction products were localized in the usual dense bodies and small vesicles, and were occasionally found diffusely in the free cytoplasm. The dense fibrillar inclusions often contained deposits of the acid phosphatase reaction product (Figures 6 and 7). The deposits tended to be frequent and heavier in the small inclusions which demonstrated high electron density and fewer fibrillar profiles, and to be less frequent and sparse in the larger ones which showed clearer fibrillar profiles.
Dstiuon d Injectd Thoorst Partdes in Reation to the Des Fbrw lsin Shortly after injection (3 to 10 minutes) (Figure 8), Thorotrast particles were primarily localized in the extracellular spaces, i.e., in the vascular lumen, in the space in between contacting cells, and in the connective tissue. It appeared in some cytoplasmic vesicles mostly in the luminal
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peripheral cvtoplasm of endothelial cells. The Thorotrast particles were distributed sparsely and fairly evenly among the extracellular amyloid fibrils including those in the deep cytoplasmic invaginations and, on occasion, among the single-membrane-bounded well-oriented amyloid fibrils. These, however, probably were the amyloid fibrils in the cytoplasmic invaginations observed in cross or grazing sections. The dense fibrillar inclusions were virtually always free of the Thorotrast particles. Later (1 to 24 hours after the injection) (Figure 9), the Thorotrast particles disappeared with time from the vascular space and were concentrated in the classic large phagocytic vacuoles of the RE cells. They still remained sparsely distributed among the extracellular amyloid fibrils and some of the apparently single-membrane-bounded amyloid fibrils, but often showed a considerable concentration near the boundary to the RE cell membrane. The dense fibrillar inclusions were largely free of the Thorotrast particles. The apparent fusion of the Thorotrast-containing vacuoles or vesicles with the inclusions was occasionally observed.
Dimsn Data accumulated after Smetana's report in 1927 1 have lent strong support to the concept that the RE cells play an important role in amyloid formation.2" Many investigators (including us) had indeed long deliberated on the possibility of the involvement of lysosomes or lysosomal enzymes in amyloidogenesis. For example, Uchino in 1967 15 strongly endorsed this concept on the basis of his electron microscopic study. In addition, studies such as those of Osserman and co-workers which suggested a close relation of plasma cell dyscrasias to amyloidosis 2 would on reconsideration be a good background for the hypothesis. Until recently, however, investigations on amyloid had not produced concrete evidence in support of the
hypothesis.2'6 The discovery of homology in amino acid sequence of proteins derived from certain amyloids (mainly primary and myeloma-associated) to the amino terminal fragments of immunoglobulin light chains by Glenner and his co-workers '" and subsequent studies which created amyloid-like fibrils from Bence Jones proteins by proteolysis 28 31 raised the possibility of proteolytic enzyme processing in amyloid fibril formation, and thus have shed new light on the hypothesis suggesting direct involvement of lysosomes in amyloidogenesis. 5 The other major amyloid fibril protein, protein AA (according to the nomenclature established at the International Symposium on Amyloidosis in Helsinki, Finland in 1974), was first sequenced by Benditt and his co-workers,32 is known to be the major constituent of secondary amyloid (including casein-induced guinea pig amyloid), and
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has a unique amino acid sequence unrelated to immunoglobulin.35 Although the parent proteins or the precursors to the protein AA have not yet been identified, the variations existing in the amino acid sequences, especially at their amino terminals, indicate the possibility that the protein AA may also have been affected by enzymatic cleavage.5 Meanwhile, in other areas of investigation, recent biochemical studies have clearly established the roles of proteolytic processes in the biosynthesis of certain proteins.& Despite these recent biochemical developments, the authentication of the direct engagement of lysosomes in amyloidogenesis in vivo would require additional information. Our recent study which revealed the close ultrastructural relationship of lysosomes to early deposits of amyloid fibrils in an animal model 16 can be viewed as additional, though still indirect morphologic support. In 1970, Kawaoi and Utsuki -" described spindle-, rod-, or diamondshaped dense bodies in reticular cells of rabbits and mice in which amyloidosis had been induced with immune complexes or heat-denatured DNA. Those bodies appear to be comparable to the dense fibrillar inclusions reported here. More recently, Kazimierczak3 also reported the presense of numerous dense bodies and cytoplasmic inclusions containing amyloid fibrils and favored their participation in amyloid formation. On careful review of our data, we soon found that the peculiar dense fibrillar inclusions could be found more frequently and reproducibly in the livers, spleens, and kidneys in the animals (especially mice) in which amyloidosis had been induced very rapidly. Thus, by using such animals, we could analyze their ultrastructure in detail and conduct experiments to obtain additional information concerning their nature. The present study has, in essence, revealed the dense fibrillar inclusions in the RE cells intimately associated with early amyloid deposits. They are considered not only on the purely ultrastructural basis, but also from their content of enzyme activity, to represent transitional forms linking the ordinary primary lysosome type of dense bodies and the deep cytoplasmic invaginations containing well-oriented amyloid fibrils-the latter having been accepted as sites of amyloid formation. The reasons for which the ultrastructural events described here should be considered as amyloid production but not as amyloid absorption, despite the involvement of the RE cells and lysosomal system, have previously been discussed in detail.16 In brief, they depended on a) abundant supporting literature for the involvement of RE cells in amyloid production, b) ultrastructural differences between these events and the phagocytosis of amyloid fibrils observed in in vitro conditions, and c) the experimental conditions. In addition, the
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present experiment with Thorotrast adds one more dimension. Although the injected Thorotrast particles were distributed sparsely amongst the extracellular amvloid fibrils shortly after the injection and showed concentration near the border of the cells with time, the inclusions did not, as a rule, contain significant Thorotrast particles at any of the observed intervals. This virtually rules out the possibilitv of the direct engagement of the inclusions in phagocytosis of the extracellular amyloid fibrils. Moreover, the present results utilizing Thorotrast and the cytochemical studv suggest that the inclusions, especially those with clear fibrillar profiles, were indeed of intracellular existence by eliminating the possibility that they were cvtoplasmic invaginations with amvloid fibrils seen in cross or grazing sections. Thus, the present findings are best interpreted as representing a series of events in the development of amvloid fibrils in the single-membrane-bounded intracellular compartments originating from the primary Iysosome type of dense bodies and, accordinglv, as direct evidence linking the Ivsosomal system to amvloidogenesis. In summary: We observed peculiar dense fibrillar inclusions in the RE cells closely related to the initial amyloid deposits in a rapidlv induced amyloid model in the mouse, as well as in other species. They were considered, from the evidence of ultrastructural and cytochemical studies, to be transitional forms linking the primary Iysosome type of dense bodies and the cytoplasmic invaginations containing well-oriented amyloid fibrils. Supported by evidence accumulated to date 2-6 and the results of the present experiments, we interpreted these inclusions as representing a series of events engaged in amyloid fibril formation, thus providing direct evidence for the role of the lysosomal system in amyloidogenesis. Referenes 1. Smetana H: The relation of the reticulo-endothelial system to the formation of amyloid. J Exp Med 45:619-632, 1927 2. Cohen AS: The constitution and genesis of amyloid. Int Rev Exp Pathol 4:139-243, 1965 3. Cohen AS, Cathcart ES: Amvloidosis and immunoglobulins. Adv Intern Med 19:41-56, 1974 4. Franklin EC, Zucker-Franklin D: Current concepts of amyloid. Adv Immunol 15:249-304, 1972 3. Glenner GG, Terr WD, Isersky C: Amyloidosis: Its nature and pathogenesis. Semin Hematol 10:65-86, 1973 6 Mandema E, Ruinen L, Scholten JH, Cohen AS (Editors): Amvloidosis. Amsterdam, Excerpta Medica Foundation, 1968 7 Cohen AS, Calkins E: Electron microscopic observations on a fibrous component in amyloid of diverse origins. Nature 183:1202-1203, 1959 8. Gueft B, Ghidoni JJ: The site of formation and ultrastructure of amvloid. Am J Pathol 43:837-854, 1963
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9. Bari 'A. Pettengill OS. Sorenson GD: Electron microscopy and electron microscopic autoradiography of splenic cell cultures from mice with amrnloidosis Lab Invest 20:234-242, 1969 10. Cohen AS. Gross E, Shirahama T: The light and electron microscopic autoradiographic demonstration of local amyloid formation in spleen explants Am J Pathol 47:1079-1112, 1965 11 de Duve C. 'attiaux R: Functions of lysosomes Ann Rev Physiol 28:435-492. 1966 12 Pearsall NN. WNeiser RS: The Macrophage. Philadelphia. Lea & Febiger. 1970 13. V'emon-Roberts B: The Macrophage Cambridge. Cambridge University Press. 1972 14 Skinner NI Cathcart ES, Cohen AS. Benson NID: Isolation and identification by- sequence analysis of experimentally induced guinea pig amyloid fibrils. J Exp Med 140:871-876. 1974 13. Uchino F: Pathological study on amyloidosis: Role of reticuloendothelial cells in inducing arnyloidosis. Acta Pathol Jap 1 7:49-82. 1967 16. Shirahama T, Cohen AS: An analysis of the close relationship of lysosomes to early deposits of amvloid: Ultrastructural evidence in experimental mouse amryloidosis Am J Pathol 73:97-114, 1973 17 Cohen AS, Shirahama T: Animal model for human disease: Spontaneous and induced amyloidosis. Am J Pathol 68:441 444. 1972 18. Karnovsky MJ: A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 27:137A-138A. 1965 (Abstr) 19. Millonig G: Advantages of a phosphate buffer for osmium tetroxide solutions in fixation. J Appl Phys 32:1637, 1961 (Abstr) 20 Luft JH: Improvement in epoxy resin embedding methods J Biophys Biochem Cvtol 9:409-414, 1961 21 WN7atson NIL Staining of tissue sections for electron microscopy with heavy metals J Biophys Biochem Cy-tol 4:475-478, 1958 22. Venable JH. Coggeshall R: A simplified lead citrate stain for use in electron microscopy. J Cell Biol 25:407-408, 1965 23. Smith RE, Farquhar NIG: Preparation of thick sections for cytochemistrv and electron microscopy by a non-freezing technique. Nature 200:691. 1963 24 Shirahama T. Cohen AS: Lysosomal breakdown of amyloid fibrils by macrophages Am J Pathol 63:463-486, 1971 25 Nlariano N. Spector WG: The formation and properties of macrophage polykarvons (inflammatory giant cells). J Pathol 113:1-19. 1974 26 Osserman EF. Takatsuki K, Talal N: The pathogenesis of "amyloidosis": Studies on the role of abnormal gamma globulins and gamma globulin fragments of the Bence Jones (L-polypeptide) type in the pathogenesis of "primary" and "secondary amyloidosis," and the "amyloidosis" associated with plasma cell myeloma. Semin Hematol 1: 3-86, 1964 27 Glenner GG, Temr W, Harada NI, Isersky C. Page D: Amyloid fibril proteins: Proof of homology with immunoglobulin light chains by sequence analyses Science 172:1150-1131. 1971 28 Glenner GG, Ein D, Eanes ED, Bladen HA. Terry W. Page DL: Creation of ramvloid" fibrils from Bence Jones proteins in vitro. Science 174:712-714. 1971 29. Shirahama T, Benson MD, Cohen AS, Tanaka A: Fibrillar assemblage of variable segments of immunoglobulin light chains: An electron microscopic study J Immunol 110:21-30,1973 30. Linke RP, Zucker-Franklin D, Franklin EC: NMorphologic, chemical, and immunologic studies of amyloid-like fibrils formed from Bence Jones proteins by proteolysis. J Immunol 111:10-23, 1973
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:33.
:34.
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Epstein NWV. Tan N. WN'ood IS: Formation of "amvloid" fibrils in vitro by action of huiman kidnev 1 -sosomal enzy-mes on Bence Jones proteins. J Lab Clin Med 84:107-110, 1974 Benditt EP, Eriksen N, Hermodson MA, Ericsson LH: The major proteins of huiman and monkey amy-loid substance: common properties including uinusual Nterminal amino acid sequences. Fed Eur Biochem Soc Letters 19:169-173. 1971 Lian JB. Benson ID, Skinner NI. Cohen AS: A 25.000 dalton protein constituent of human amyloid fibrils related to protein AA. (Unpublished data) Steiner DF, Kemmler W, Tager HS, Peterson JD: Proteoly-tic processing in the biosynthesis of insulin and other proteins. Fed Proc 33:2105-2115. 1974 Kawaoi A, Utsuki Y: Experimental amyloidosis induced by- immtine complex or heat denatured DNA. I. An electron microscopic observation. Acta Pathol Jap 20:47-57, 1970 Kazimierczak J: Ultrastructural observations on the first amyloid to be found in the spleen of casein treated mice. Acta Pathol N icrobiol Scand [.A 2:33:141-150. 1972
Acknowledgments The auithors express their appreciation for the excellent technical assistance of Or ille Rodgers and Be%-erl%- Greer
Legends for Figures Figure 1-Electron micrograph of CBA/J mouse liver after 14 daily injections of 0.5 ml of 12% casein. A Kupffer cell (Ku) abutting small amyloid deposits measuring less than a few microns in diameter (Am) displays peculiar dense fibrillar inclusions (/). They are located in the area rich in dense bodies (solid arrows). Although some of them resemble the dense bodies in their electron density, the inclusions are generally larger and more elongated than dense bodies and show considerable variation in the electron density of their contents. Some of them display well-organized fibrillar profiles. An adjacent Kupffer cell demonstrates numerous cytoplasmic invaginations containing well-oriented amyloid fibrils, some opening to the extracellular amyloid deposit and others apparently in cross or grazing sections (open arrows). The ultrastructure of the Kupffer cells is otherwise unremarkable. (Single-fixed with osmium tetroxide in phosphate buffer and stained with uranyl acetate and lead citrate, x 28,000) Figure 2A and 28-Higher power electron micrographs of portions of the area shown in Figure
1. The inclusions are single-membrane-bounded, and contain two different structural elements; fibrillar profiles and homogeneous or finely granular electron-dense substance. The fibrillar profiles are well organized in parallel to the long axis of the inclusion and are about 100-A wide, rigid, and nonbranching, so as to resemble amyloid fibrils. The homogeneous or finely granular substance appears to be of the same nature as that composing the dense body matrix. The numbers labeled on the cytoplasmic particles and inclusions correspond to those used in the text for the description of the probable transition or relations of these structural elements to one another. (x 64,000)
Figure 3-Low-power electron micrograph showing a portion of a giant cell, macrophage polykaryon, observed in a mouse liver after 14 daily injections of 0.5 ml of 12% casein. The cell displays multiple nuclei (N), rich ribosomes, well-developed endoplasmic reticulum, large multiple Golgi complexes (G), many mitochondria, numerous dense bodies, and above all an abundance of the "dense fibrillar inclusions." This giant cell nowhere contacted the extracellular amyloid deposits. Ku = Kupffer cells, Hep = hepatocytes. (Fixed in osmium tetroxide alone and stained with uranyl acetate and lead citrate, x 7000)
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Figure 4-A portion of the giant cell shown in Figure 3 at a moderate magnification for clearer demonstration of various cell organelles and inclusions. The former are very well developed but unremarkable, and the dense fibrillar inclusions are abundant. (x 25,000) Figure 5-High-power electron micrograph of
a portion of Figure 3 for analysis of the ultrastructure of the inclusions. The inclusions have the same features as those seen in other RE cells. The numbers on the inclusions correspond to those used in the text for description of the probable sequence of transition. Some inclusions (2 and 3) appear to be in cross sections. (x 64,000).
Figue 6-A portion of a Kupffer cell from a CBA/J mouse that received 14 daily casein injections treated for the demonstration of acid phosphatase activity. Several "dense fibrillar" inclusions (1-/4) are observed, and some of them (/l and /2) show a heavy deposition of the enzyme reaction products. Although their internal structure is not clear because of the reaction products, their size and shape suggest that they are in an earlier stage of the development of the inclusions, i.e., having higher electron density and fewer fibrillar profiles (see text). The enzyme reaction products are deposited sparsely in other inclusions (/3 and /4), one of these (/4) demonstrates clearly the well organized fibrillar profiles. Some cytoplasmic granules (solid arrows) also show acid phosphatase activity. Am = amyloid fibrils in cytoplasmic invaginations. (x 56,000)
Figure 7-Preparation similar to Figure 6. In a Kupffer cell, a "dense fibrillar" inclusion (/), which demonstrates moderate electron density and fibrillar profiles (arrow), displays modest deposition of the acid phosphatase reaction product. Am = amyloid fibrils in cytoplasmic invaginations. (x 56,000) Figure 8-CBA/J mouse liver after 14 casein injections and 3 minutes after injection of Thorotrast. Except for those in a few vesicles in the peripheral cytoplasm (solid arrow), the Thorotrast particles are localized in the extracellular space. They are distributed sparsely and fairly evenly among the extracellular amyloid deposits (Am). They are also located among the well-oriented amyloid fibrils-possibly in the cytoplasm surrounded by single membranes but more likely in the cytoplasmic invaginations seen in cross or grazing sections (open arrows). The dense fibrillar inclusions (I) are free of Thorotrast particles. Ku = Kupffer cell, Hep = hepatocyte. (x 25,000)
Fgue 9-CBA/J mouse liver after 14 casein injections and 1 hour after injection of Thorotrast.
In the extracellular amyloid deposits (Am), Thorotrast particles show considerable concentration in the area near the cell-amyloid border (open arrows), while they are distributed sparsely all over the amyloid deposits. In the Kupffer cell (Ku), Thorotrast particles are localized mainly in typical large phagocytic vacuoles. The dense fibrillar inclusions (/) are virtually free of Thorotrast. Probable fusion between a dense fibrillar inclusion and a phagocytic vacuole containing Thorotrast particles is observed (solid arrow). Hep = hepatocyte. (x 25,-
000)
br)t
Ji
2A
2B
1:
Unusual inclusions, which occurred in the reticuloendothelial cells intimately associated with fresh amyloid deposits. were analyzed by electron microscopy. The inclusions were located in the areas rich in the primary lysosome type of dense bodies and the cytoplasmic invaginations containing well-oriented amvloid fibrils. They were single-membrane-bounded, measured 0.3 to 0.8 in width and 0.5 to sev eral microns in length, and showed considerable variation in the electron density of their contents. The latter consisted of two different ultrastructural elements: fibrillar profiles and a homogeneous or finely- granular electron-dense substance. The fibrillar profiles were virtually identical in ultrastructure to the amyloid fibrils and were well-oriented parallel to the long axis of the inclusion. The homogeneous or finely granular electron-dense substance appeared to be comparable to that composing the dense body matrix. The inclusions were usually% acid phosphatase positive. but did not take up intrav enouslv injected Thorotrast particles. These data led us to conclude that these inclusions were transitional forms from the usual dense bodies to the deep cytoplasmic inv aginations containing well-oriented amyloid fibrils (which are accepted by most investigators as the sites of amyloid formation) and thus constitute direct ev idence for the involvement of lysosomes in amyloid fibril formation. (Am J Pathol 81:101-116, 1975)
DESPITE SIGN-IFICAN-T ACHIEVEMIENTS in research on amrvloid, its pathogenesis has not yet been fully clarified. The concept put forth bv Smetana in 1927 '-that the reticuloendothelial (RE) cells are intimately associated with the genesis of amyloid-has gained substantial stipport. 2 At the ultrastruictural level, in 1963 (a few years after the definition of amyloid as a unique fibrillar substance 7), Gueft and Ghidoni 8 described cytoplasmic invaginations which contained tufts of well-oriented amyloid fibrils at the cell-amvloid interface and designated them as the sites of amvloid formation. This notion has been supported by many investigations,26 including electron microscopic autoradiographic studies,9" 0 and has not vet met any serious challenge. Since a) the RE sy-stem may play an important role in amyloidogenesis, b) the system is intimatelv related to Ivsosomal activity," and c) the restults of recent From the Evans Memorial Department of Clinical Research. University Hospital. the Thorndike Memorial Laboratory. Boston City Hospital. and the Department of Medicine. Boston University School of Medicine. Boston. Massachusetts Presented in part at the International Symposium on Am%loidosis. Helsinki. Finland. August 1974 Suipported by Grants AM-04599 and Tl-AN1-5285 from the National Institute of Arthritis and Mletabolic Diseases. US Public Health Sen-ice. Grant RR-533 from the General Clinical Research Centers Branch of the Division of Research Resources. National Institutes of Health, and by grants from the Massachusetts Chapter of the Arthritis Foundation, the Arthritis Fouindation, the John A Hartford Foundation, and the Unisersity Hospital Accepted for publication Jine 14. 197. Address reprint requests to Dr T Shirahama. U niv ersity Hospital. Room E&37. 75 East Newton
Street. Boston. \MA 02 1 8
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biochemical studies of amyloid suggest the possibility that protein fragments resulting from enzymatic cleavage may constitute some type of amvloid,3'5', there has been speculation that Iysosomes might participate in the process of amyloid formation.5'15 We recently reported that unusual Ivsosomal activitv in the RE cells was closelv associated with earlv amvloid deposits.16 However, all the findings available to date concerning the lysosomal involvement in amyloid fibril formation have been circumstantial, and no direct evidence exists in support of the concept. The present paper reports our observations, which strongly suggest amyloid fibril formation in lysosome-originated inclusions and represent perhaps the first direct evidence for the participation of lysosomes in amvloid fibril formation. Materials and Methods Amyloidosis was induced in 8- to 10-week-old CBA J mice (Jackson Laboratory, Bar Harbor, Me ) by daily subcutaneous injections of 03a ml of 12% casein (Casein Hamersten, Control No 3320. Nutritional Biochemicals Company, Cleveland, Ohio) This schedule shortened the duration of amyloid induction to about two-thirds of that achieved with our routine schedule injecting 0.O ml of 10%c casein 17 In the present model. amy.loid was present in the spleen after 8 to 12 injections, in the liver after 12 to 16 injections, and in the kidney after 16 to 20 injections. After specific intervals of the amyloid induction regimen, animals were sacrificed on the day after the last casein injection, and spleen, liver, and kidney were processed for light and electron microscopy. Groups of the animals received intravenous injection of 0.1 ml of Thorotrast (Fellows Testagar, Detroit, Mich.) into the tail vein on the day after the last casein injection, and then sacrificed at 3, 10 minutes and 1, 2, or 24 hours after the Thorotrast injection Spleen, liver, and kidney were then prepared for electron microscopy For the electron microscopic studies, small tissue blocks were either double-fixed with 2 0% formaldehvde-2 3c% glutaraldehyde '" and 2%t osmium tetroxide in 0 1 Mt cacodylateHCI buffer or single-fixed with 2% osmium tetroxide in cacodylate or phosphate buffer l9 They were then dehydrated in graded ethanols and embedded in Epon or Araldite Thin sections were cut on an LKB Ultrotome with glass knives and stained w ith uranyl acetate 21 and, or lead citrate 2 when feasible Tissues were prefixed with the aldehyde " and cut into 30-M slices on a Sorvall TC-2 tissue sectioner ' and then treated for cvtochemical demonstration of acid phosphatase activity as described elsewhere.'. As controls, spleens, livers, and kidnevs from mice untreated or treated with intravenous injections of Thorotrast alone were also prepared in the same fashion as those from the amvloidotic mice The specimens were examined in a Siemens Elmiskop I at initial magnifications of 1000 to 40.000 X The observations concentrated on the foci of venr early small amyloid deposits rarely exceeding several microns in diameter. The present study was oriented towards summing up our observations conceming the peculiar inclusions and accordingly about 1000 new electron micrographs were processed In addition, because about 10 years have passed since the peculiar inclusions had first come to our attention. we have accumulated a large amount of data relating to them. These included a variety of preparations from variouis species and strains (humans, rabbits, guinea pigs, C3H HeJ mice. C37B10 6J mice, etc ) Those data were also reanalvzed recently and taken into consideration in the interpretation of the present results.
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Results Gew Apecb
Although amyloidosis was induced more rapidly in the present study, the fine structure was comparable to that described in the animals in which amyloidosis was induced by other techniques,2,4,6 8-0,15-17 except for the frequent appearance of the peculiar inclusions which will be described in detail. In addition, the formation of deep cytoplasmic invaginations containing tufts of well-oriented amyloid fibrils and the frequent lysosomal and vesicular profiles in the peripheral cytoplasm of the RE cells closely associated with amyloid deposits 16 were even more prominent in the present specimens. Pecular luskok and Their Relation to Des Boces and Amy Fblb
In the present study, the RE cells intimately associated with small (less than several microns in diameter) amyloid deposits often displayed unusual cytoplasmic inclusions (designated as dense fibrillar inclusions hereafter) (Figure 1). The inclusions were observed most frequently in the Kupffer cells of the liver, less frequently in the fixed reticular cells and sinus lining cells of the spleen, and seldom in the glomerular mesangial cells of the kidney. They were round to long-fusiform in shape, measured 0.3 to 0.8 M in width and 0.5 to several microns in length, and varied considerably in the electron density of their contents. Some of them with lighter electron density displayed an inner structure as described below. These inclusions were usually closely associated with the ordinary dense bodies and the single-membrane-bounded well-oriented amyloid fibrils (which probably represented either cross or grazing sections of amyloid fibrils in the cytoplasmic invaginations or truly intracellular singlemembrane-bounded amyloid fibrils). At higher resolution (Figures 2A and 2B), the inclusions were revealed to be single membrane bounded and containing well-organized fibrillar profiles embedded in homogeneous or finely granular electron-dense substance. The well-organized fibrillar profiles were always oriented in parallel to the long axis of the inclusion and were comparable to amyloid fibrils; they were about 100 A in diameter, rigid, and nonbranching. The homogeneous or finely granular electron-dense substance constituting the background was comparable, ultrastructurally, to the dense body matrix. Furthermore, there were sufficient data to suggest a continuous transition from the usual primary lysosome type of dense bodies (by ultrastructural definition) to the dense fibrillar inclusions and further to the singlemembrane-bounded well-oriented amyloid fibrils or the well-oriented amyloid fibrils in deep cytoplasmic invaginations. The following sequence
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suggested itself: 1) tvpical primary-lysosome-type dense bodies which contained homogeneous or finely granular electron-dense matrix, 2) elongated single-membrane-bounded particles with a matrix similar to the dense bodies, 3) further-elongated particles containing a slightly less electron-dense matrix in which the presence of well-organized fibrillar profiles was suggested, 4) long fusiform single-membrane-bounded inclusions exhibiting clearer fibrillar profiles embedded in a homogeneous or finely granular electron-dense substance, 5) inclusions containing prominent fibrillar profiles and traces of the electron-dense background substance, and 6) single-membrane-bounded well-organized fibrillar profiles, i.e., amyloid fibrils, with virtually no background substance. Giant cells, which displayed multiple nuclei, rich ribosomes, welldeveloped endoplasmic reticulum, large multiple Golgi complexes, frequent mitochondria and abundant dense bodies, were occasionally observed-presumably macrophage polykaryons.2' Although none of those cells were attached to the extracellular amyloid deposits, they demonstrated an abundance of the dense fibrillar inclusions in the various stages described above (Figures 3-5). Findings suggesting the fusion of Golgi-associated vesicles to the dense fibrillar inclusions, standard dense bodies to the inclusions, and the dense fibrillar inclusions to each other were not unusual in the RE cells as well as in the macrophage polykaryons (Figures 1-5). Acid
Ph
Activit in the Dns FBincusions
The RE cells apparently related to amyloid production showed high acid phosphatase activity in this study, as was observed in the previous study.16 The enzyme reaction products were localized in the usual dense bodies and small vesicles, and were occasionally found diffusely in the free cytoplasm. The dense fibrillar inclusions often contained deposits of the acid phosphatase reaction product (Figures 6 and 7). The deposits tended to be frequent and heavier in the small inclusions which demonstrated high electron density and fewer fibrillar profiles, and to be less frequent and sparse in the larger ones which showed clearer fibrillar profiles.
Dstiuon d Injectd Thoorst Partdes in Reation to the Des Fbrw lsin Shortly after injection (3 to 10 minutes) (Figure 8), Thorotrast particles were primarily localized in the extracellular spaces, i.e., in the vascular lumen, in the space in between contacting cells, and in the connective tissue. It appeared in some cytoplasmic vesicles mostly in the luminal
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peripheral cvtoplasm of endothelial cells. The Thorotrast particles were distributed sparsely and fairly evenly among the extracellular amyloid fibrils including those in the deep cytoplasmic invaginations and, on occasion, among the single-membrane-bounded well-oriented amyloid fibrils. These, however, probably were the amyloid fibrils in the cytoplasmic invaginations observed in cross or grazing sections. The dense fibrillar inclusions were virtually always free of the Thorotrast particles. Later (1 to 24 hours after the injection) (Figure 9), the Thorotrast particles disappeared with time from the vascular space and were concentrated in the classic large phagocytic vacuoles of the RE cells. They still remained sparsely distributed among the extracellular amyloid fibrils and some of the apparently single-membrane-bounded amyloid fibrils, but often showed a considerable concentration near the boundary to the RE cell membrane. The dense fibrillar inclusions were largely free of the Thorotrast particles. The apparent fusion of the Thorotrast-containing vacuoles or vesicles with the inclusions was occasionally observed.
Dimsn Data accumulated after Smetana's report in 1927 1 have lent strong support to the concept that the RE cells play an important role in amyloid formation.2" Many investigators (including us) had indeed long deliberated on the possibility of the involvement of lysosomes or lysosomal enzymes in amyloidogenesis. For example, Uchino in 1967 15 strongly endorsed this concept on the basis of his electron microscopic study. In addition, studies such as those of Osserman and co-workers which suggested a close relation of plasma cell dyscrasias to amyloidosis 2 would on reconsideration be a good background for the hypothesis. Until recently, however, investigations on amyloid had not produced concrete evidence in support of the
hypothesis.2'6 The discovery of homology in amino acid sequence of proteins derived from certain amyloids (mainly primary and myeloma-associated) to the amino terminal fragments of immunoglobulin light chains by Glenner and his co-workers '" and subsequent studies which created amyloid-like fibrils from Bence Jones proteins by proteolysis 28 31 raised the possibility of proteolytic enzyme processing in amyloid fibril formation, and thus have shed new light on the hypothesis suggesting direct involvement of lysosomes in amyloidogenesis. 5 The other major amyloid fibril protein, protein AA (according to the nomenclature established at the International Symposium on Amyloidosis in Helsinki, Finland in 1974), was first sequenced by Benditt and his co-workers,32 is known to be the major constituent of secondary amyloid (including casein-induced guinea pig amyloid), and
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has a unique amino acid sequence unrelated to immunoglobulin.35 Although the parent proteins or the precursors to the protein AA have not yet been identified, the variations existing in the amino acid sequences, especially at their amino terminals, indicate the possibility that the protein AA may also have been affected by enzymatic cleavage.5 Meanwhile, in other areas of investigation, recent biochemical studies have clearly established the roles of proteolytic processes in the biosynthesis of certain proteins.& Despite these recent biochemical developments, the authentication of the direct engagement of lysosomes in amyloidogenesis in vivo would require additional information. Our recent study which revealed the close ultrastructural relationship of lysosomes to early deposits of amyloid fibrils in an animal model 16 can be viewed as additional, though still indirect morphologic support. In 1970, Kawaoi and Utsuki -" described spindle-, rod-, or diamondshaped dense bodies in reticular cells of rabbits and mice in which amyloidosis had been induced with immune complexes or heat-denatured DNA. Those bodies appear to be comparable to the dense fibrillar inclusions reported here. More recently, Kazimierczak3 also reported the presense of numerous dense bodies and cytoplasmic inclusions containing amyloid fibrils and favored their participation in amyloid formation. On careful review of our data, we soon found that the peculiar dense fibrillar inclusions could be found more frequently and reproducibly in the livers, spleens, and kidneys in the animals (especially mice) in which amyloidosis had been induced very rapidly. Thus, by using such animals, we could analyze their ultrastructure in detail and conduct experiments to obtain additional information concerning their nature. The present study has, in essence, revealed the dense fibrillar inclusions in the RE cells intimately associated with early amyloid deposits. They are considered not only on the purely ultrastructural basis, but also from their content of enzyme activity, to represent transitional forms linking the ordinary primary lysosome type of dense bodies and the deep cytoplasmic invaginations containing well-oriented amyloid fibrils-the latter having been accepted as sites of amyloid formation. The reasons for which the ultrastructural events described here should be considered as amyloid production but not as amyloid absorption, despite the involvement of the RE cells and lysosomal system, have previously been discussed in detail.16 In brief, they depended on a) abundant supporting literature for the involvement of RE cells in amyloid production, b) ultrastructural differences between these events and the phagocytosis of amyloid fibrils observed in in vitro conditions, and c) the experimental conditions. In addition, the
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present experiment with Thorotrast adds one more dimension. Although the injected Thorotrast particles were distributed sparsely amongst the extracellular amvloid fibrils shortly after the injection and showed concentration near the border of the cells with time, the inclusions did not, as a rule, contain significant Thorotrast particles at any of the observed intervals. This virtually rules out the possibilitv of the direct engagement of the inclusions in phagocytosis of the extracellular amyloid fibrils. Moreover, the present results utilizing Thorotrast and the cytochemical studv suggest that the inclusions, especially those with clear fibrillar profiles, were indeed of intracellular existence by eliminating the possibility that they were cvtoplasmic invaginations with amvloid fibrils seen in cross or grazing sections. Thus, the present findings are best interpreted as representing a series of events in the development of amvloid fibrils in the single-membrane-bounded intracellular compartments originating from the primary Iysosome type of dense bodies and, accordinglv, as direct evidence linking the Ivsosomal system to amvloidogenesis. In summary: We observed peculiar dense fibrillar inclusions in the RE cells closely related to the initial amyloid deposits in a rapidlv induced amyloid model in the mouse, as well as in other species. They were considered, from the evidence of ultrastructural and cytochemical studies, to be transitional forms linking the primary Iysosome type of dense bodies and the cytoplasmic invaginations containing well-oriented amyloid fibrils. Supported by evidence accumulated to date 2-6 and the results of the present experiments, we interpreted these inclusions as representing a series of events engaged in amyloid fibril formation, thus providing direct evidence for the role of the lysosomal system in amyloidogenesis. Referenes 1. Smetana H: The relation of the reticulo-endothelial system to the formation of amyloid. J Exp Med 45:619-632, 1927 2. Cohen AS: The constitution and genesis of amyloid. Int Rev Exp Pathol 4:139-243, 1965 3. Cohen AS, Cathcart ES: Amvloidosis and immunoglobulins. Adv Intern Med 19:41-56, 1974 4. Franklin EC, Zucker-Franklin D: Current concepts of amyloid. Adv Immunol 15:249-304, 1972 3. Glenner GG, Terr WD, Isersky C: Amyloidosis: Its nature and pathogenesis. Semin Hematol 10:65-86, 1973 6 Mandema E, Ruinen L, Scholten JH, Cohen AS (Editors): Amvloidosis. Amsterdam, Excerpta Medica Foundation, 1968 7 Cohen AS, Calkins E: Electron microscopic observations on a fibrous component in amyloid of diverse origins. Nature 183:1202-1203, 1959 8. Gueft B, Ghidoni JJ: The site of formation and ultrastructure of amvloid. Am J Pathol 43:837-854, 1963
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9. Bari 'A. Pettengill OS. Sorenson GD: Electron microscopy and electron microscopic autoradiography of splenic cell cultures from mice with amrnloidosis Lab Invest 20:234-242, 1969 10. Cohen AS. Gross E, Shirahama T: The light and electron microscopic autoradiographic demonstration of local amyloid formation in spleen explants Am J Pathol 47:1079-1112, 1965 11 de Duve C. 'attiaux R: Functions of lysosomes Ann Rev Physiol 28:435-492. 1966 12 Pearsall NN. WNeiser RS: The Macrophage. Philadelphia. Lea & Febiger. 1970 13. V'emon-Roberts B: The Macrophage Cambridge. Cambridge University Press. 1972 14 Skinner NI Cathcart ES, Cohen AS. Benson NID: Isolation and identification by- sequence analysis of experimentally induced guinea pig amyloid fibrils. J Exp Med 140:871-876. 1974 13. Uchino F: Pathological study on amyloidosis: Role of reticuloendothelial cells in inducing arnyloidosis. Acta Pathol Jap 1 7:49-82. 1967 16. Shirahama T, Cohen AS: An analysis of the close relationship of lysosomes to early deposits of amvloid: Ultrastructural evidence in experimental mouse amryloidosis Am J Pathol 73:97-114, 1973 17 Cohen AS, Shirahama T: Animal model for human disease: Spontaneous and induced amyloidosis. Am J Pathol 68:441 444. 1972 18. Karnovsky MJ: A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 27:137A-138A. 1965 (Abstr) 19. Millonig G: Advantages of a phosphate buffer for osmium tetroxide solutions in fixation. J Appl Phys 32:1637, 1961 (Abstr) 20 Luft JH: Improvement in epoxy resin embedding methods J Biophys Biochem Cvtol 9:409-414, 1961 21 WN7atson NIL Staining of tissue sections for electron microscopy with heavy metals J Biophys Biochem Cy-tol 4:475-478, 1958 22. Venable JH. Coggeshall R: A simplified lead citrate stain for use in electron microscopy. J Cell Biol 25:407-408, 1965 23. Smith RE, Farquhar NIG: Preparation of thick sections for cytochemistrv and electron microscopy by a non-freezing technique. Nature 200:691. 1963 24 Shirahama T. Cohen AS: Lysosomal breakdown of amyloid fibrils by macrophages Am J Pathol 63:463-486, 1971 25 Nlariano N. Spector WG: The formation and properties of macrophage polykarvons (inflammatory giant cells). J Pathol 113:1-19. 1974 26 Osserman EF. Takatsuki K, Talal N: The pathogenesis of "amyloidosis": Studies on the role of abnormal gamma globulins and gamma globulin fragments of the Bence Jones (L-polypeptide) type in the pathogenesis of "primary" and "secondary amyloidosis," and the "amyloidosis" associated with plasma cell myeloma. Semin Hematol 1: 3-86, 1964 27 Glenner GG, Temr W, Harada NI, Isersky C. Page D: Amyloid fibril proteins: Proof of homology with immunoglobulin light chains by sequence analyses Science 172:1150-1131. 1971 28 Glenner GG, Ein D, Eanes ED, Bladen HA. Terry W. Page DL: Creation of ramvloid" fibrils from Bence Jones proteins in vitro. Science 174:712-714. 1971 29. Shirahama T, Benson MD, Cohen AS, Tanaka A: Fibrillar assemblage of variable segments of immunoglobulin light chains: An electron microscopic study J Immunol 110:21-30,1973 30. Linke RP, Zucker-Franklin D, Franklin EC: NMorphologic, chemical, and immunologic studies of amyloid-like fibrils formed from Bence Jones proteins by proteolysis. J Immunol 111:10-23, 1973
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:33.
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Epstein NWV. Tan N. WN'ood IS: Formation of "amvloid" fibrils in vitro by action of huiman kidnev 1 -sosomal enzy-mes on Bence Jones proteins. J Lab Clin Med 84:107-110, 1974 Benditt EP, Eriksen N, Hermodson MA, Ericsson LH: The major proteins of huiman and monkey amy-loid substance: common properties including uinusual Nterminal amino acid sequences. Fed Eur Biochem Soc Letters 19:169-173. 1971 Lian JB. Benson ID, Skinner NI. Cohen AS: A 25.000 dalton protein constituent of human amyloid fibrils related to protein AA. (Unpublished data) Steiner DF, Kemmler W, Tager HS, Peterson JD: Proteoly-tic processing in the biosynthesis of insulin and other proteins. Fed Proc 33:2105-2115. 1974 Kawaoi A, Utsuki Y: Experimental amyloidosis induced by- immtine complex or heat denatured DNA. I. An electron microscopic observation. Acta Pathol Jap 20:47-57, 1970 Kazimierczak J: Ultrastructural observations on the first amyloid to be found in the spleen of casein treated mice. Acta Pathol N icrobiol Scand [.A 2:33:141-150. 1972
Acknowledgments The auithors express their appreciation for the excellent technical assistance of Or ille Rodgers and Be%-erl%- Greer
Legends for Figures Figure 1-Electron micrograph of CBA/J mouse liver after 14 daily injections of 0.5 ml of 12% casein. A Kupffer cell (Ku) abutting small amyloid deposits measuring less than a few microns in diameter (Am) displays peculiar dense fibrillar inclusions (/). They are located in the area rich in dense bodies (solid arrows). Although some of them resemble the dense bodies in their electron density, the inclusions are generally larger and more elongated than dense bodies and show considerable variation in the electron density of their contents. Some of them display well-organized fibrillar profiles. An adjacent Kupffer cell demonstrates numerous cytoplasmic invaginations containing well-oriented amyloid fibrils, some opening to the extracellular amyloid deposit and others apparently in cross or grazing sections (open arrows). The ultrastructure of the Kupffer cells is otherwise unremarkable. (Single-fixed with osmium tetroxide in phosphate buffer and stained with uranyl acetate and lead citrate, x 28,000) Figure 2A and 28-Higher power electron micrographs of portions of the area shown in Figure
1. The inclusions are single-membrane-bounded, and contain two different structural elements; fibrillar profiles and homogeneous or finely granular electron-dense substance. The fibrillar profiles are well organized in parallel to the long axis of the inclusion and are about 100-A wide, rigid, and nonbranching, so as to resemble amyloid fibrils. The homogeneous or finely granular substance appears to be of the same nature as that composing the dense body matrix. The numbers labeled on the cytoplasmic particles and inclusions correspond to those used in the text for the description of the probable transition or relations of these structural elements to one another. (x 64,000)
Figure 3-Low-power electron micrograph showing a portion of a giant cell, macrophage polykaryon, observed in a mouse liver after 14 daily injections of 0.5 ml of 12% casein. The cell displays multiple nuclei (N), rich ribosomes, well-developed endoplasmic reticulum, large multiple Golgi complexes (G), many mitochondria, numerous dense bodies, and above all an abundance of the "dense fibrillar inclusions." This giant cell nowhere contacted the extracellular amyloid deposits. Ku = Kupffer cells, Hep = hepatocytes. (Fixed in osmium tetroxide alone and stained with uranyl acetate and lead citrate, x 7000)
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Figure 4-A portion of the giant cell shown in Figure 3 at a moderate magnification for clearer demonstration of various cell organelles and inclusions. The former are very well developed but unremarkable, and the dense fibrillar inclusions are abundant. (x 25,000) Figure 5-High-power electron micrograph of
a portion of Figure 3 for analysis of the ultrastructure of the inclusions. The inclusions have the same features as those seen in other RE cells. The numbers on the inclusions correspond to those used in the text for description of the probable sequence of transition. Some inclusions (2 and 3) appear to be in cross sections. (x 64,000).
Figue 6-A portion of a Kupffer cell from a CBA/J mouse that received 14 daily casein injections treated for the demonstration of acid phosphatase activity. Several "dense fibrillar" inclusions (1-/4) are observed, and some of them (/l and /2) show a heavy deposition of the enzyme reaction products. Although their internal structure is not clear because of the reaction products, their size and shape suggest that they are in an earlier stage of the development of the inclusions, i.e., having higher electron density and fewer fibrillar profiles (see text). The enzyme reaction products are deposited sparsely in other inclusions (/3 and /4), one of these (/4) demonstrates clearly the well organized fibrillar profiles. Some cytoplasmic granules (solid arrows) also show acid phosphatase activity. Am = amyloid fibrils in cytoplasmic invaginations. (x 56,000)
Figure 7-Preparation similar to Figure 6. In a Kupffer cell, a "dense fibrillar" inclusion (/), which demonstrates moderate electron density and fibrillar profiles (arrow), displays modest deposition of the acid phosphatase reaction product. Am = amyloid fibrils in cytoplasmic invaginations. (x 56,000) Figure 8-CBA/J mouse liver after 14 casein injections and 3 minutes after injection of Thorotrast. Except for those in a few vesicles in the peripheral cytoplasm (solid arrow), the Thorotrast particles are localized in the extracellular space. They are distributed sparsely and fairly evenly among the extracellular amyloid deposits (Am). They are also located among the well-oriented amyloid fibrils-possibly in the cytoplasm surrounded by single membranes but more likely in the cytoplasmic invaginations seen in cross or grazing sections (open arrows). The dense fibrillar inclusions (I) are free of Thorotrast particles. Ku = Kupffer cell, Hep = hepatocyte. (x 25,000)
Fgue 9-CBA/J mouse liver after 14 casein injections and 1 hour after injection of Thorotrast.
In the extracellular amyloid deposits (Am), Thorotrast particles show considerable concentration in the area near the cell-amyloid border (open arrows), while they are distributed sparsely all over the amyloid deposits. In the Kupffer cell (Ku), Thorotrast particles are localized mainly in typical large phagocytic vacuoles. The dense fibrillar inclusions (/) are virtually free of Thorotrast. Probable fusion between a dense fibrillar inclusion and a phagocytic vacuole containing Thorotrast particles is observed (solid arrow). Hep = hepatocyte. (x 25,-
000)
br)t
Ji
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2B
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