Current Concepts of Amyloid1

Current Concepts of Amyloidl EDWARD C. FRANKLIN AND DOROTHEA ZUCKER-FRANKLIN Rheumatic Diseases Study Group, Deparlment of Medicine, New York University Medical Center, New York, New York

I. Introduction . . . . . . . . . . . . . . . 11. General Approaches to the Study of Amyloid 111. Morphological Studies in Sectioned Tissues . . . . IV. Morphological, Biochemical, Physicochemical, and Antigenic Properties of Amyloid Fibrils . . . . . . . A. Dye Binding . . . . . . . . . . . B. Morphological Studies . . . . . . . . C. Physicochemical and Biochemical Studies of Amyloid Fibrils and Their Subunits . . . . . . . . . D. Immunological Studies . . . . . . . . E. Comparative Studies of Different Amyloid Preparations . F. Discussion and Conclusions . . . . . . . . . . . V. Speculations on the Pathogenesis of Amyloid References . . . . . . . . . . . .

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I. Introduction

Amyloid appears in the light microscope as an amorphous, eosinophilic, hyaline, extracellular substance which is readily recognized by its unique green birefringence after staining with Congo red when viewed in the polarizing microscope and by its metachromatic properties with dyes such as Crystal Violet, Toluidine Blue, or Methyl Violet (Bennhold, 1922; Cohen, 1966b, 1967). When present, it is usually widely distributed in many organs of the body and frequently replaces the cellular elements present in these tissues. Although most commonly it accompanies a large number of apparently unrelated disorders, it is occasionally seen as a primary illness unassociated with any underlying pathological conditions. Although organs with the gross appearance of amyloid infiltrates have been recognized for over 300 years, the first clear-cut descriptions of the “lardaceous liver” and “sago spleen,” now recognized as being characteristic of this disorder, can be attributed to von Rokitansky (1846) and Virchow ( 1854), respectively. The substance which infiltrates these tissues and which is presumably responsible for this appearance was mis‘This work is supported by U. S. Public Health Service Grants #AM 02594, #AM 01431, and #AM 012274; the Health Research Council of the City of New York and U. S. Public Health Service Research Career Development Award #5-K03 A1 09572. 249

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named “amyloid by Virchow because it resembled starch in its staining properties with iodine and sulfuric acid. The name has remained, despite the fact that as early as 1859, Friedreich and Kekulb discovered the now generally accepted protein nature of amyloid. Progress in elucidating the pathogenesis of these disorders, the nature of the deposits, and their precise origin has been slow. Difficulties in defining the etiology of the disease arise mostly because in man, and many species of animals, amyloid is associated with a variety of diseases that appear to have few if any features in common. Confusion has been compounded by the plethora of agents and procedures, again with few obvious common properties, which can induce amyloid in experimental animals. As a result, it has proved difficult to identify any single mechanism responsible for this disorder. As mentioned below, it seems possible that many different factors may ultimately interact or initiate a limited number of stimuli that can induce the synthesis and deposition of amyloid. (For a review, see Mandema et al., 1968.) Nevertheless, during the past few years a number of significant observations have been made which have given us a clearer insight into the nature of this material and promise to provide us with a more unified concept of the pathogenesis of amyloid. Impetus for a new look at amyloid was provided by the demonstration in several laboratories of distinct and characteristic fibrils in what had been presumed previously to be a homogeneous structureless material (Cohen and Calkins, 1959; Spiro, 1959). The availability of purified fibrils free of all other soluble and insoluble tissue constituents then permitted direct chemical, physical, and immunological analyses of the major constituent of amyloid. As a result of these approaches we have gained knowledge of the ultrastructure of amyloid (Shirahama and Cohen, 19f37b; Pras et al., 1968) and have obtained antisera which appear to react with amyloid specifically. This may prove to be of great value diagnostically (Franklin and Pras, 1969; Glenner et al., 1969; Cathcart et al., 1970a). Most importantly, it has been clearly established that amyloid is often closely related structurally to immunoglobulins, in particular to the variable region of the light polypeptide chain (Glenner et al., 1970b, 1971b; Harada et al., 1971). If the fragments of immunoglobulins can, indeed, be shown to be present in all types of amyloid, it seems likely that the excessive production of such fragments may be the final common effector mechanism resulting in the deposition of amyloid. In spite of the promise that these studies will in time permit the development of an accurate and meaningful system of classification based primarily on the biochemical properties of different types of amyloid, analogous to that now known for the closely related immunoglobulins,

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these studies have not progressed sufficiently to permit the delineation of such a system at this time. As a consequence, we shall introduce this review with a composite classification which incorporates features of several of those currently employed. It seems quite likely that all of these may become obsolete and will be replaced eventually by a rational system based on pathogenetic mechanisms and chemical properties of different types of amyloid. Even in our present state of knowledge, it is apparent that none of the currently employed systems of classification permits a clear-cut separation of amyloid into different classes since there exists a great deal of overlap in the tissue distribution and staining characteristics of the several major types of amyloid. Consequently, although they are widely used and are of some clinical value, it is commonly felt that each one of these classifications is somewhat arbitrary and indistinct. The simplest and most widely used classification of amyloidosis is based on the four major categories introduced by Reimann (Reimann et al., 1935). ( 1 ) Primary amyloid occurs with no known antecedent or coexistent disease and usually involves mesodermal tissues, such as smooth or skeletal muscle, or the cardiovascular system. Generally there is variability in staining of the deposits with Congo red, iodine, and metachromatic dyes. ( 2) Secondary amyloid is usually associated with chronic diseases such as infections, neoplasms, neurological disorders, or connective tissue diseases especially rheumatoid arthritis. It generally involves spleen, liver, kidney, intestines, and adrenals and tends to have reproducible and characteristic staining properties with the abovementioned dyes. ( 3 ) Amyloid associated with myeloma tends to resemble the primary type but is invariably associated with a neoplasm involving plasma cells or lymphocytes, such as multiple myeloma, macroglobulinemia, or heavy chain disease. (4)Tumor-forming amyloid is characterized by small masses of amyloid in the skin, eye, bladder, urethra, respiratory tract, or other organs and is generally unassociated with any underlying disease. In addition to these four major classes of amyloid, a large number of familial types of amyloidosis, each involving a different organ system and characterized by a different form of inheritance, have been reported from many parts of the world ( Andrade et al., 1970). The most interesting, and probably also the most common one is the type of amyloid associated with familial Mediterranean fever (FMF), most often seen in Sephardic Jews, but also occasionally in other ethnic groups (Sohar et al., 1967). Although many of the clinical and pathological features of these familial types of amyloidosis might be included in the primary and secondary types, it appears better at this time to keep them separate since

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the basic mechanism responsible for the deposition of amyloid may not be the same as in the sporadic cases. Nevertheless, it seems appropriate to mention at this point that electron microscopically amyloid from all of these types is indistinguishable and that chemical studies of some of the proteins isolated from patients with the primary, secondary, myelomaassociated amyloid, and at least one familial type, that associated with FMF, have revealed similarities (Pras et al., 1969) and have recently been shown to consist of a fraction of immunoglobulin light chains (Glenner et al., 1970b, 1971a,b,c; Harada et al., 1971). Thus, on the basis of these studies it seems possible that the amyloid produced in all of these disorders may be chemically similar and that the characteristic tissue distribution may be related to other, as yet poorly defined, factors. However, one should bear in mind the recent observations of Benditt and Eriksen (1971), Franklin et al. (1972), and Pras et al. ( 1971) which have raised the possibility that some amyloids, especially those obtained from patients with the so-called secondary type of amyloid and the type associated with FMF, may be chemically different and consist of a major protein that can be isolated in dilute acid and which, on the basis of its amino acid analysis and size, does not appear to be related to any known immunoglobulin fraction. Rather detailed reviews of two large and well-studied series of patients with amyloidosis have been published recently (Brandt et al., 1968; Barth et al., 1969b). These two reports provide ample clinical descriptions of the various types of amyloidosis and also emphasize some similarities in the clinical and pathological features of the three major types of amyloidosis. This subject will not be discussed in detail in this review. A different type of classification, initially based on the positive birefringence of amyloid and its conversion to negative by phenol or glycerol, has divided amyloid into the perireticular and the pericollagenous types (Missmahl and Hartwig, 1953; Missmahl, 1957, 1968; Heller et al., 1964). In the perireticular form, there appears to be a generalized vascular disease resulting from the deposition of amyloid starting at the basement membrane of the vessels and spreading outward. This type of amyloid is seen in the secondary type, certain familial forms including FMF, and a number of instances of primary or idiopathic amyloidosis with the so-called “typical distribution.” The pericollagenous type is said to start in the connective tissue, beginning in the adventitia of the vessel from where it appears to spread inward, and is most commonly seen in the classic primary type, in amyloidosis associated with multiple myeloma, in the tumor-forming types of amyloid, and in certain other familial types of amyloidosis. Although this classification has been used

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in a number of recent studies, it appears to us to add little in terms of a greater understanding of the disease and is mentioned here only for the sake of completeness. Before proceeding to a detailed discussion of certain aspects of amyloidosis, a few words seem appropriate concerning the aims of this review. Rather than to provide an exhaustively documented, detailed, complete, historical coverage of the field, we shall discuss in a selective fashion current concepts and in many instances controversial and as yet unsettled questions related to the structure, chemistry, immunology, cellular origin, metabolism, and possibly pathogenesis of amyloid, citing only certain references that appear to us to be pertinent to these areas. The interested reader who wishes a more detailed documentation of the field might do well to read the reviews by Cohen (196613, 1967) and Mandema et al. (1968). I I . General Approaches to the Study of Amyloid

Two major approaches have been employed in recent years to characterize amyloid more definitively. One of these, the morphological approach, has used the electron microscope ( E M ) to study amyloid. Initially EM studies were limited to examination of infiltrated tissues from man and experimental animals. With the identification of characteristic fibrils as the major constituent of amyloid and the development of methods for their isolation, similar studies have now also been carried out on the isolated amyloid fibrils. As a result of studies of the partially purified fibrils, two major structural components have been identified: ( 1 ) the characteristic fibrillar component makes up more than 955%of amyloid and appears to be unique to this material (Shirahama et al., 196713; Pras et al., 1968; Glenner et al., 1969; Harada et al., 1971); (2) another structure, known as the “rod,” the “doughnut,” or the “P” component has not been identified in tissues but has been seen in partially purified preparations (Bladen et al., 1966; Cathcart et al., 1 9 6 7 ~ ) .It appears to represent a normal serum a,-globulin and makes up a much smaller fraction of the amyloid complex. It is largely removed during most fractionation procedures and its precise role in the pathogenesis of amyloid remains to be elucidated. The other approach to study the nature of amyloid has attempted to characterize it chemically and immunologically. Many investigators have stained amyloid tissues with fluorescent antisera to a variety of serum proteins in the hope of detecting components that may be important constituents of amyloid deposits. In this manner, a large number of different serum proteins have been identified in amyloid deposits. Of particular interest, in the light of the recent chemical studies and be-

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cause their direct bearing on immunological factors thought to be of importance in the pathogenesis of the disorder, have been the immunoglobulins and certain complement components, demonstrated in amyloid by Mellors and Ortega (1956), Cathcart and Cohen ( 1966), Cathcart et al. (1967a), Lachmann et al. (1962), Milgrom et al. (1966), Muckle (1968a,b), Vazquez and Dixon (1956), Vogt and Kochem (1960), Williams et d . ( 1960), and others. Although these observations have at times been conflicting in nature and difficult to interpret they have greatly influenced our concepts of etiology and pathogenesis. A more direct approach, which during the past year has provided clear-cut evidence that some amyloid is composed of immunoglobulin light-chain fragments, has been to isolate the major constituents of amyloid and to subject them to chemical and immunological analysis. Such studies have clearly demonstrated that the major fibrillar component isolated from a number of different types of amyloid is composed of a subunit ranging in molecular weight from 5000 to 30,000 daltons which includes at least the first part of the variable region of the light chain of ,-globulin. The possibility that it encompasses additional parts of the 7-globulin molecule has not been ruled out and in certain instances appears likely (Glenner et al., 1970b, 1971a,b,c; Harada et al., 1971). The likelihood that not all amyloid is related to light chains was raised by the unusual protein devoid of cysteine described by Glenner et al. (1970b). This was also suggested by the recent finding of large amounts of a homogeneous protein which does not appear to be related to immunoglobulins in dilute acid extracts from amyloid tissues (Benditt and Eriksen, 1971) and pure amyloid fibrils (Pras and Reshef, 1972; Franklin et al., 1972). In contrast, the second constituent, known as the “doughnut” (Glenner et al., 1970a,b), is a serum a,-globulin that assumes a peculiar morphological appearance in amyloid (Bladen et al., 1966; Cathcart et al., 1967a,c). This protein is not unique to amyloid and its role in the formation and pathogenesis of amyloid deposits remains as yet unexplained. None of the other serum constituents that have been identified in amyloid tissue sections can be detected after further purification, and their specific role in the deposition of amyloid deposits, therefore, remains doubtful. Since the most meaningful studies in recent years have been carried out with isolated or partially purified fibrils, it seems appropriate to list a few of the methods most commonly employed in their purification prior to providing the detailed results from these studies. One of these, the method of Cohen and Calkins (1964) subjects the amyloid-laden tissue to homogenization and maceration in physiologic saline. The homogenate is then centrifuged at 12,OOOg for 30 minutes

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and the top layer, rich in amyloid fibrils, is subsequently subjected to repeated sucrose gradient centrifugation ( Cohen, 1966a) to obtain a relatively pure fibril preparation. The other, the method of Pras et al. (1968), is based on the observation that amyloid fibrils are insoluble in physiologic saline but that they are readily extracted in distilled water. Consequently, the tissue is repeatedly homogenized in solutions of physiologic saline and Centrifuged at about 9OOOg for 30 minutes. The contaminating proteins are discarded in the supernatant, and the pellets are rehomogenized and recentrifuged until the O.D. of the supernatant at 280 nm. is 0. The pellet is then repeatedly rehomogenized in distilled water and the resultant homogenate is centrifuged at 80,000 g for 1hour. After three or four such extractions, the supernatant becomes opalescent, contains Congo red-binding material, and can be shown to contain from 20 to 95%of the amyloid fibrils present in the starting tissue. Further purification can be achieved by column chromatography on Sepharose or Sephadex in solvents containing guanidine with a reducing agent in order to dissociate noncovalent and disulfide bonds ( Glenner et al., 1969). This step provides not only some additional purification but also dissociates the polymers into their basic subunits which can then be subjected to further chemical studies. For electron microscopy, the purified fibrils can be pelleted, fixed, dehydrated, and embedded by standard methods (Luft, 196l), following which they are sectioned and positively stained with lead (Millonig, 1961) and uranium salts (Watson, 1958). Alternatively, the purified fibrils can be placed directly on Formvar-covered EM grids and then negatively “stained with heavy metals ( Brenner and Horne, 1959). Ill. Morphological Studies in Sectioned Tissues

The fibrillar nature of amyloid tissue deposits was reported in 1959 (Cohen and Calkins, 1959; Spiro, 1959) and has since been illustrated repeatedly (Hjort and Christensen, 1961; Gueft and Ghidoni, 1963; Thiery and Caroli, 1961; Boere et al., 1965; Sorenson and Bari, 1968; to name a few). A typical example of an amyloid infiltrated lymph node is seen in Fig. 1. It is not surprising that the fibrils were not recognized earlier since even on electron microscopy at low magnification the material appears amorphous and mixed with cellular debris; only at higher resolutions are the fibrils clearly delineated (Figs. 2 to 4). In some tissues, particularly in those where parenchymal cells are held together in a loose fashion, such as the spleen, lymph nodes, and bone marrow, the fibrils are oriented at random and can be seen to crisscross in all directions. In such sections, individual fibrils measure about 120~4.in width and usually do not bend or branch. In other areas, amyloid fibrils may

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be seen in more organized arrays (Fig. 3 ) at right angles to or in parallel with adjacent cell membranes or bundles of collagen fibrils. To date no significance has been attributed to any particular organization the fibrils may display; rather it is tacitly assumed that parallel bundles of fibrils reflect the stresses and strains of the tissues in which they are located or, as the case may be, the deformability of the surrounding cells. In view of the close relation of some amyloid fibrils to immunoglobulin light chains, it becomes of utmost importance to delineate the mechanisms involved in the deposition of amyloid and to define the cells responsible for its synthesis. In the absence of detailed in oitro biosynthetic studies, some information bearing on these questions can be obtained from E M observations. As regards the deposition of the type of amyloid related to immunoglobulins, at least two possibilities, which are not mutually exclusive, must be considered. On the one hand, light chains may be synthesized and secreted by plasma cells and circulate in the blood. They may, by as yet unknown mechanisms, undergo the changes requisite for assuming the characteristic morphological appearance of amyloid and then interact with, as yet undefined, tissue constituents or receptors to form amyloid deposits. Evidence possibly in favor of such a mechanism was provided by the studies of Osserman (Osserman et al., 1964) which demonstrated the binding of fluoresceinated Bence-Jones proteins to tissue sections prepared from muscle, kidney, liver, and intestine. It is of interest that Bence-Jones proteins both from patients with and without amyloid bound to tissues, with possibly somewhat more intense staining by proteins from patients with amyloidosis. However, there was no significant degree of tissue specificity which paralleled the distribution of amyloid infiltrates in the cases from which the Bence-Jones proteins were obtained. Alternatively, amyloid may be deposited in close proximity to cells that synthesize a substance which assumes the typical fibrillar appearance in their immediate vicinity without first having to circulate in the bloodstream. Such a view is favored by the not infrequent existence of masses of amyloid in close proximity to plasma cells (see Fig. 1).Although the precise mechanism for the synthesis and the sites of assembly of the fibrils from the soluble precursor subunits remains to be estabFIG. 1. Thin section of a lymph node infiltrated with amyloid obtained from a patient with multiple myeloma. The amyloid deposit (Am) is surrounded by plasma cells ( P ), monocytoid cells ( M ), lymphocytes ( L ), and reticuloendothelial ( RE ) cells. Magnification: X 4000. ( From Zucker-Franklin and Franklin, 1970.) FIG. 2. Detail of a lymphocytoid plasma cell surrounded by amyloid fibrils (AM). Arrow points to intracytoplasmic fibrils. Nucleus ( N); rough endoplasmic reticulum ( E R ) . Magnification: X 23,000. (From Zucker-Franklin and Franklin, 1970.)

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lished, it seems possible that the fibrils are formed intracellularly and released by cellular dissolution or, alternatively, that a soluble precursor is released which polymerizes to form the fibrils either on the outer cell membrane or in the surrounding tissue fluid. Whereas EM studies have demonstrated fibrils intracellularly on occasion ( see below), examination of fixed tissues cannot provide kinetic evidence allowing a choice between the different pathways of fibril formation. The problem is compounded by the fact that the deposits are often found in such close association with surrounding cells that it is difficult to determine whether the fibrils are inside or outside the cell (Figs. 4 and 5 ) . This is further aggravated by the state of the cells concerned which are often damaged or necrotic (Fig. 5A and B). Even when the cells are well preserved, their plasmalemmas have often disappeared in the area adjacent to the amyloid deposit. When no fibrils are seen in the cytoplasm of such cells, there seems to be little question that the fibrils have indented the cell from the outside (Fig. 4). At a recent symposium on amyloidosis, such illustrations prompted Bywaters to draw an analogy between the appearance of these cells and San Sebastian transfixed with arrows: “He looks a bit sick too, but nobody has suggested that he was secreting the arrows” (Bywaters, 1968). Although San Sebastian adorns the cover of the Proceedings of this meeting, the final word on the subject has not been spoken. Ranlov ( 1968) clearly demonstrated intracellular fibrils which appear to form an integral part of the cytoplasm in the spleen of experimental mice, and in our own studies on human specimens, this impression has been amply corroborated ( Zucker-Franklin and Franklin, 1970). In organs heavily infiltrated with amyloid, cells are often seen to contain fibrils which are morphologically identical to those in the extracellular space (Figs. 2, 6, and 7 ) . In many instances, the cell appears otherwise normal and displays an intact surface membrane, at no time suggesting that penetration into the cell by the fibrils has taken place. Other cells show degenerative changes with displacement of organelles by the cytoplasmic fibrils. It should be emphasized that intracellular fibrils are only seen in a small percentage of specimens and then mostly in heavily infiltrated organs such as lymph nodes, spleen, and bone marrow. Ranlov (1968) and Ranlov and Wanstrup (1968) interpreted the intracellular amyloid precipitation as a “fatigue phenomenon” on the part of the cell, ~~

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FIG. 3. Amyloid fibrils seen in bundles parallel to cell membrane. Paired filaments may be distinguished within each fibril. Magnification: ~64,000. FIG. 4. Detail of a reticuloendothelial cell surrounded by amyloid (Am)fibrils. The arrows point to areas where the fibrils appear to invaginate the cell. Intracytoplasmic fibrils can also be seen. Magnification: X29,OOO. (From Zucker-Franklin and Franklin, 1970.)

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and postulated that it only takes place in cases where amyloid deposition is unusually fast. Indeed, it is easy to accept the hypothesis that intracellular polymerization or precipitation of the protein could occur if the excretory mechanism of the cells were impaired. Although the intracytoplasmic fibrils illustrated in Figs. 6 and 7 are morphologically similar to those in the extracellular space and may, on the basis of immunofluorescence studies, represent amyloid, one should not ignore the fact that structurally identical fibers are occasionally encountered in epithelial cells of normal subjects. Such fibrils are usually referred to as tono fibrils (Fawcett, 1966) or stress fibrils (Buckley and Porter, 1967). The biochemical nature or function of such fibrils in normal cells is entirely conjectural. However, their occurrence raises the possibility that small amounts of amyloid are synthesized by such cells under normal conditions. If this were true, extracellular amyloid would reflect overproduction of a normal cell constituent rather than an abnormal protein. Apart from the debate as to whether or not the intracellular fibrils represent amyloid is the controversy over the specific type of cells responsible for its synthesis. In early studies, Cohen et al. (1965) performed tissue culture studies on amyloid-infiltrated rabbit spleens and, with the help of isotope-labeled amino acids, showed that the label was incorporated in the reticuloendothelial ( R E ) cells before it appeared on the extracellular fibrils. Other investigators studying liver and splenic tissues (Heefner and Sorenson, 1962; Gueft and Ghidoni, 1963; Teilum, 1968; Laufer and Tal, 1967), also incriminated RE cells, and Battaglia ( 1961, 1962) even suggested that during the induction of experimental amyloidosis in mice, amyloid could be seen in Kupffer cells. He postulated that following rupture of these cells there was spillage of the fibrils into the space of Disse. In studies of bone marrow, Ben-Ishay and Zlotnick (1968) also found intracellular fibrils in RE cells. In the central nervous system, Terry (1963, 1964) described typical fibrils within socalled senile plaques. The amyloid nature was confirmed by staining with Congo red and birefringence on polarization microscopy. In this location the fibrils related closely to the surrounding glial cells-the nervous system’s counterpart of RE cells. However, other cells not belonging to the RE system (RES) in a strict sense, have also been considered to produce FIG. 5. ( A ) Degenerated lymphoid cell entirely surrounded by amyloid (Am) obtained from bone marrow specimen of a patient with IgA myeloma. The plasma membrane of this cell does not seem to constitute a barrier between the cytoplasm and the extracellular space (arrow). Nucleus ( N u ) . ( B ) Area in inset of Fig. 5A shown at a higher magnification. At this site the plasma membrane has disappeared; intra- and extracellular fibrils are seen in continuity. Magnification: x 120,000.

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amyloid. In serial studies on renal amyloidosis, experimentally induced with casein in rabbits, Cohen and Calkins (1960) found the earliest deposits between endothelial cell cytoplasm and basement membrane. When deposits became more abundant, the epithelial side of the basement membrane also became involved. The function of the basement membrane as a barrier or accomplice in the development of the renal lesions has never been clearly defined. From such studies, one would have to conclude that the endothelial cell is also capable of synthesizing amyloid. However, Shibolet et al. ( 1967), who followed the development of amyloid in the kidneys of Leishmania-infected Syrian hamsters, concluded that fibroblasts infiltrating the interstitial space between capillary and tubular membranes were involved in the deposition of amyloid. Still a different opinion was expressed by Sorenson and Shimamura (1964) who induced renal amyloidosis in mice and found the first accumulations of fibrils in the mesangial area of the glomerulus. In the human only a limited number of serial studies on the deposition of amyloid in the kidney are available. However, particular attention has been drawn to the mesangial cell matrix and basement membrane as the earliest region involved in human renal amyloidosis (Suzuki et aZ., 1963). Furthermore, in light of recent chemical studies of amyloid and in view of the concepts that immunological mechanisms are important in the pathogenesis of amyloid, the most interesting cell that has been considered to play a role in the production of amyloid is the plasma cell. On the basis of histochemical techniques, Teilum (1956, 1964a,b) contends that it is this cell which is primarily responsible for the synthesis of this protein. His view has been shared by Caesar (1960). In our own studies ( Zucker-Franklin and Franklin, 1970), intracellular fibrils were seen in RE, “monocytoid” cells, and also in well-delineated plasma cells (Figs. 6 and 7 ) surrounding or immediately adjacent to extensive extracellular deposits. If it is difficult to consider the possibility that all the above-named cells are capable of elaborating amyloid or a soluble precursor substance, it may be helpful to recall that all these cells are of mesenchymal origin and that they may, therefore, be potentially capable

FIG. 6. Detail of a plasma cell showing intracytoplasmic fibrils obtained from a lymph node of a patient with multiple myeloma. Rough endoplasmic reticulum (ER) as well as free and clustered ribosomes are abundant. Mitochondria ( M ) . Magnification: X 37,000. (From Zucker-Franklin and Franklin, 1970.) FIG.7. Detail of a plasma cell with intracytoplasmic fibrils. Most of the fibrils are located around the Golgi zone ( G ) , but some bundles run more peripheral. Mitochondria ( M ) ; rough endoplasmic reticulum ( ER ) ; nucleus ( N ) ; extracellular amyloid (Am). Magnification: x20,OOO.

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of synthesizing the same protein, as a result of dedifferentiation or in response to a particular stimulus. IV. Morphological, Biochemical, Physicochemical, and Antigenic Properties of Amyloid Fibrils

Many controversial data have accumulated during the last 125 years concerning the nature of amyloid. This confusion persisted because until recently most of the analyses have been carried out on tissue extracts and homogenates which were contaminated to various extents by nonamyloid components. Only with the availability of the major fibrillar components of amyloid in pure form has it been possible to obtain reasonably firm data on the properties of amyloid. Even here, however, some confusion arose initially because the P component, which has a characteristic rodlike or doughnutlike appearance in the EM (see below) was initially thought by some to represent the fibrillar component (Glenner and Bladen, 1966). In this section we shall, therefore, concentrate primarily on the recent studies of the purified fibrils and summarize only briefly the limited biochemical studies on the P component. The interested reader can find complete historical reviews of the studies on the chemistry of amyloid elsewhere (Cohen, 1966b, 1967; Ashkenazi et al., 1968; Mandema et al., 1968).

A. DYEBINDING Probably the single most useful property of the amyloid fibrils, which has permitted their extraction from tissues in high yield and great purity, is their unusual behavior in the presence of salt (Pras et al., 1968). Unlike most other proteins the major fibrillar protein component of amyloid is completely insoluble and presumably highly polymerized even at low concentrations of salt (0.025 M NaCl) , thus permitting the removal of most contaminants by repeated extraction in 0.15 M NaCI. In the absence of salt, and following homogenization, amyloid changes its physical properties sufficiently so that it can be removed from the remaining insoluble substance by ultracentrifugation. The distilled water extract of amyloid is generally rather opalescent, quite unstable, and tends to precipitate on prolonged standing. Because of this and the presence of typical fibrils in the EM, it seems likely that amyloid in distilled water represents a suspension or colloidal solution rather than a true solution. Although the distilled water extract has a number of properties characteristic of amyloid, we do not know if it contains the intact fibril or a natural subunit which can polymerize to yield the fibril seen in the EM. The unusual solubility properties of amyloid are shown in Fig. 8.

CURRENT CONCEPTS OF AMYLOID

0005 0010 a015 0.0200025

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,,

0.075"0.15 SALT CONCENTRATION , rnole/liter

FIG. 8. Relation between the amount of amyloid left in solution and the concentration of added salt, NaCl or CaCL The amount of amyloid left in solution was determined in two ways: ( a ) by the percent of the initial absorbance at 280 mp. left in the supernatant solution after adding salt and removing precipitated amyloid by centrifugation, and ( b ) by measuring the percent of Congo red in saline not precipitated when added to the same supernatant solution. The solid Congo red line was obtained with the supernatant solutions after addition of NaCI. (From Pras et d., 1968.)

The binding of Congo red by amyloid and the associated green birefringence have long been recognized and used to detect amyloid in tissue (Bennhold, 1922; Gafni et al., 1968; Benditt et al., 1970). More recently, the ability of amyloid to combine with Congo red quantitatively and presumably stoicheometrically has proved of great value in following the purification of the amyloid material (Pras et al., 1968) and in studying the reconstitution of fibrils from soluble precursors. Another characteristic property of amyloid is metachromasia with a number of dyes, such as Crystal Violet or Toluidine Blue. Thus the addition of amyloid to these dyes causes a shift in the absorption peak from about 600 to 550 mp. which can be perceived grossly and measured accurately with a spectrophotometer (Pras and Schubert, 1968). It is of interest that the acid mucopolysaccharides responsible for the metachromasia are probably not an integral part of the fibril protein and that they can be extracted from it (Pras et al., 1971). The solubility and dyebinding properties appear to be characteristic of all amyloid fibrils studied to date; however, the quantitative aspects of the Congo red binding may vary for different specimens suggesting chemical differences between different amyloid preparations. The availability of purified fibrils has permitted a series of ultrastructural, biochemical, physicochemical, and immunological studies designed to characterize these substances and has, in recent years, provided significant insight into the possible nature of amyloid.

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B. MORPHOLOGICAL STUDIES When the isolated fibrils are embedded, sectioned, and examined in the EM, they appear identical to those in freshly fixed amyloid-laden tissues obtained at autopsy or on biopsy (Fig. 9A and B). Their width is about 100 to 150A and their length cannot be determined since they crisscross into and out of the plane of section. A large percentage of the fibrils consists of two longitudinal subunits or filaments equal in width and separated by a space of +25 A. The two subunits remain parallel throughout their course. Scattered among the fibrils are irregularly shaped dots which are assumed to represent tangential and cross sections of the fibrils. Frequently, the cross sections show a radiolucent core which is more apparent in some preparations than in others (Fig. 9B ) . When the “soluble” fibrils are salt-precipitated rather than precipitated by ultracentrifugation, they form thicker bundles which run parallel for short distances after which they twist, cross, or divert. Single subunits are more difficult to find in such preparations. However, the basic structure does not change suggesting that salt merely promotes

FIG.9. ( A and B ) Isolated fibrils that have been embedded and sectioned. Note cross sections of fibrils in Fig. 9B show hollow core (arrows) ; also fibrils appear different in quality and size. Both taken at same magnification: x102,OOO.

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aggregation andlor polymerization of the fibrils and that the material obtained in distilled water represents a finer dispersion of the basic subunits, When “soluble amyloid is negatively stained, i.e., when the fibrils are directly placed and stained on an EM grid, without prior fixation or embedding, greater resolution can be obtained. In such preparations the most common configuration seen is the paired filament (Fig. 10). Individual filaments measure 50-75A. in diameter and range from several millimicrons in length to the smallest resolvable fragments. The filaments have a beaded or helical substructure (Shirahama and Cohen, 1965). These may lend themselves to further structural analysis as smaller longitudinal subunits or protofibrils, measuring only 20-25 A. in width, can occasionally be seen alongside filaments in our preparations (Pras et al., 1968). Shirahama and Cohen (1967b) have attempted to define further subunits of the protofibril, but since these studies are still controversial, they will not be described in detail here. Thus, presently we use the following terminology: the amyloid fibril seen in thin tissue sections consists of a number of filaments, aggregated side by side, and often forms thick bundles. The filaments measure 50-75 A. in width and, in tissue sections as well as negatively stained preparations, are often found in pairs. The protofibril, which has only been seen in negatively stained material so far, measures 25-35A. in width. Its precise relationship to other protofibrils within the amyloid filament has not been established beyond reasonable doubt (Boere et al., 1965; Emesen et al., 1966). Morphological studies on the P component of amyloid (for a discussion, see Section IV,D) have been limited to negatively stained material (Fig. 11). No structure resembling either the doughnut or rod configuration of this component has ever been described in tissue sections derived from patients or experimental animals. This could be attributable to the fact that the P component is estimated to constitute only 5%of the total amyloid mass and could conceivably have been overlooked. However, in a review of our own material comprising several hundred micrographs, no structure reserribling the P component could be found. It is conceivable that the P component is extracted during the embedding procedure-a possibility made more likely by the claim that this small fraction of amyloid represents an @-globulin,a soluble serum protein. If this is true, it ought to be possible to perform EM studies on the component isolated from normal blood. The appearance of the negatively stained P component extracted from amyloid-laden tissues was first described by Bladen et (11. (1966; see, also, Fig. 11) and Benditt and Eriksen (1966) who equated this rod structure with the main

268

EDWARD C. FRANKLIN AND DOROTHEA ZUCKER-FRANKLIN

CURRENT CONCEPTS OF AMYLOID

269

FIG. 11. Negatively stained periodic rods representing the P component of amyloid. Arrow points to “doughnuts,” the unit structural components of the rod. (From Glenner and Bladen, 1966; electron micrograph obtained through courtesy of Dr. H. A. Bladen.)

amyloid fibril. Subsequently the structural appearance of the negatively stained P component was confirmed by Cathcart et al. (1967a,c) and compared and contrasted with the true amyloid fibril (Shirahama and Cohen, 1967b). Since the P component, at least as defined by biochemical and immunological techniques, is found in the serum of normal subjects, it may not play a significant role in the pathogenesis of amyloidosis but may merely represent another serum component, nonspecifically adsorbed to this peculiar extracellular protein. Another way of studying the physical structure of the amyloid fibril is by X-ray diffraction, and these studies on various types of human amyloid as well as on mouse and duck amyloid have been carried out by three different groups with virtually identical results (Eanes and Glenner, 1968; Bonard et al., 1969; Shmueli et al., 1969). All X-ray diffraction patterns have shown a sharp meridional arc at about 4.7 A. and a more diffuse equatorial arc at 9.8A. These characteristic reflections have been interpreted as indicative of a cross-p or a pleated-sheet configuration by two groups (Eanes and Glenner, 1968; Bonard et al., 1969) and as an ordered polymer of globular protein subunits by another (Shmueli et aZ., 1969). All three consider that this rather unusual appearance is characteristic of amyloid in tissue and not the result of denaturation or preparative artifacts. FIG. 10. Isolated purified amyloid negatively stained with phosphotungstic acid. Arrows point to single filaments, Note that most filaments are paired and end together (half-circles). Some thicker bundles ( B ) in which filaments are twisting around each other can also be seen. Magnification: X96,OOO.

270

EDWARD C. FRANKLIN AND DOROTHEA ZUCKER-FRANKLIN

C. PHYSICOCHEMICAL AND BIOCHEMICAL STUDIESOF AMYLOID F m w AND THEIRSU~UNITS Previous studies measuring the binding of certain dyes to amyloid (Carnes and Forbes, 1956; Goldberg and Deane, 1960) suggested that the isolectric point of amyloid was between 4.5 and 5. Free electrophoresis of a single sample of the water-soluble protein in unbuffered Tris at pH 10 showed the material to be negatively charged, but it was not possible to obtain an exact electrophoretic mobility in the absence of salt (Pras et al., 1968). Ultracentrifugation studies of intact fibrils have also been difficult to interpret since they had to be carried out in distilled water in the absence of any ions. Most preparations, when examined shortly after extraction, were homogeneous and had a major component with a sedimentation coefficient of about 45 S. However, if extraction proved more difficult or after prolonged standing, larger polymers with sedimentation coefficients of 7 5 s and higher appeared. Within a short time, they begin to sediment rapidly even at low centrifugal forces, and after a while these larger aggregates have generally shown a tendency to precipitate from solution spontaneously. These precipitates could generally by redispersed to the 45 S component by vigorous homogenization. Unfortunately, it has not yet been possible to correlate the ultrastructure of the fibrils or their subunits with the sedimentation rates obtained in the ultracentrifuge since EM studies are always carried out on material which has been precipitated either by salt or dehydration. As mentioned below, a few of the preparations contained a more slowly sedimenting component with sedimentation coefficients ranging from 8 to 15s the nature of which remains as yet unknown (Pras et al., 1968, 1969). From the start, it seemed likely that the 45s component extracted in distilled water was composed of smaller subunits and, consequently, a number of attempts have been made to define chemically homogeneous subunits and to isolate the constituent polypeptide chains. With some of the initial preparations studied by us, urea, guanidine, and reducing agents aIone and in combination brought the material out of solution and could not be used as such for chemical studies. Because of these difficulties we employed 0.1 M NaOH to produce a smaller subunit which was soluble in 0.15 M NaCl and which we referred to as degraded amyloid (DAM) (Pras et al., 1969). This material has a sedimentation coefficient of 1 to 2 s and an estimated molecular weight of about 30,000 to 40,000 daltons based on peptide maps and the content of arginine and lysine. This subunit, produced by as yet undefined mechanisms, still contained some small fragments of fibrils on E M (Fig, 12). Although this material did not lend itself to detailed chemical analysis,

CURRENT CONCEPTS OF AMYLOID

271

it proved to be very useful in immunological and some superficial chemical studies to be described below. Use of alkali to degrade amyloid is not new. Prior studies by Hass (Hass and Schulz, 1940) and by Cohen’s group ( Newcombe and Cohen, 1964) had already investigated the effect of alkali; Hass had probably extracted amyloid from tissues as DAM using sodium hydroxide, whereas Newcombe (Newcombe and Cohen, 1964) and Shirahama (Shirahama and Cohen, 1967a) probably stopped short of the pH required to dissociate the molecule into a subunit when they studied the effect of pH on the solubility and extractability of amyloid. Several other attempts at dissociation have been reported recently. Miller et al. ( 1968) employed cyanogen bromide designed to cleave at methionine residues, urea, a reducing agent, and acid. All of these procedures yielded smaller fragments. Degradation was almost complete with CNBr, whereas urea, alkali, and acid yielded 50% or less of a smaller subunit. Although all the agents appeared to produce subunits with similar behavior on Sephadex filtration and polyacrylamide electrophoresis, these subunits were not sufficiently well characterized to permit any estimates as to their size or nature. An unusual feature, which has severely hampered the biochemical analysis of amyloid and which may be of some biological significance in terms of long-term persistence of amyloid in tissues, is the resistance of the native undenatured fibrils to many proteolytic enzymes used in the biochemical analysis of other proteins (Sorenson and Binington, 1964; Emeson et al., 1966; Ruinen et al., 1967; Pras et al., 1969; Kim et al., 1969). Incubation of native undenatured amyloid with papain, pepsin, and trypsin failed to produce identifiable subunits and left much of the residual fibrillar protein in tact, Only pronase seemed to be effective in degrading the fibrils more or less completely. Following denaturation, the fibrils become more susceptible to several of these proteolytic agents. Probably the single most important observation dealing with the nature of amyloid came through the efforts of Glenner and his collaborators (1970b, 1971a,b,c; Harada et al., 1971). These investigators treated several purified preparations of amyloid fibrils with 6 M guanidine and a reducing agent-a procedure that destroyed the fibrillar appearance of amyloid, the characteristic appearance on X-ray diffraction, and eliminated the capacity to bind Congo red. Following filtration on Sephadex and Sepharose, they isolated a smaller subunit from these fibrils ranging in molecular weight from 5000 to 31,000 daltons which, in each instance, appeared relatively homogeneous on SDSZdisc electro-

* Sodium

dodecyl sulfate.

272

EDWARD C. FRANKLIN AND DOROTHEA ZUCKER-FRANKLIN

CURRENT CONCEPTS OF AMYLOID

273

phoresis. Chemical studies of several of these preparations by Glenner et al. (1970b, 1971a,b) showed these small subunits to resemble immunoglobulin chains in having either pyroglutamic acid (PCA), Asp, or Glu as the N-terminal residue, a finding reported also for the intact fibril by Skinner and Cohen (1971). In a series of short notes which promise to be followed by additional full length reports in the near future, Glenner et al. (1970b, 1971a,b) subjected two of these fragments having unblocked N termini to analysis in the automatic sequencer and demonstrated in each of them a striking homology to the first 30 residues of the variable region of the k light chain. In one instance, the major subunit appeared similar to the whole light chain present in the patient’s urine by peptide mapping (Glenner, 1972). This finding has led to the exciting conclusion that the major protein component of amyloid is a portion of an immunoglobulin polypeptide chain. Recently, as might be expected, these workers have also demonstrated structural identity of the major amyloid protein and the light chain derived from the same subject (Glenner et al., 1972). At the present time the precise structure of the amyloid proteins remains unknown since the molecular weight of a light chain is approximately 22,500 daltons, whereas the major amyloid protein ranges from 5000 to 30,000 daltons. It seems possible that the amyloid protein may represent a part of a light chain, primarily the variable region, either with an internal deletion or with degradation and consequent loss of varying amounts of the constant and possibly the variable region. The ability to make amyloid fibrils, consisting primarily of the variable region from selected Bence-Jones proteins, with a variety of proteolytic enzymes strongly favors the second alternative. It is worth noting that structural studies of this type have so far been limited to k-like proteins since these have unblocked N termini, but that h-type proteins with blocked N termini are far more commonly found in amyloid. To date the published information is not sufficient to decide whether amyloid consists of a series of identical subunits or if the molecule is composed of more than one type of polypeptide chain, nor is there any estimate of the

FIG. 12. Fragments seen in denatured amyloid following treatment of isolated amyloid fibrils with 0.1 M NaOH for 1 hour. Fragments range from 200 to 1500A. in length. In specimens digested for 3 hours, there is an increase in smaller-sized fragments. Magnification: X 75,000. ( From Franklin and Zucker-Franklin, 1972.) FIG. 13. Precipitate obtained by pepsin digestion of a A-type Bence Jones protein, negatively stained with 1%phosphotungstic acid at pH 5.4. The fibrils in this case are indistinguishable from those of negatively stained isolated amyloid. Magnification: x75,OOO.

274

EDWARD C. FRANKLIN AND DOROTHEA ZUCKER-FRANKLIN

number of subunits of the major 4 5 s component. The chemical homogeneity of amyloid and the ability to make amyloid fibrils form purified Bence Jones proteins (see below) favor the presence of only a single type of subunit. In a parallel, but so far less precise approach to the study of amyloid subunits, we have noted the presence of about 6 to 8 characteristic common peptides in the peptide maps of similarly prepared amyloid subunits (Levin et al., 1972a). Most of these are present also in k and A Bence Jones proteins and appear to be derived from the variable region of the molecule. The constant occurrence of these peptides in all preparations studied and also in “synthetic amyloid prepared by proteolysis of Bence Jones proteins (see below) suggests that certain structural features are required for all amyloid proteins but that similar sequences may be found also in proteins from patients without amyloidosis. The conclusions obtained from the studies of amyloid fibrils regarding the role of light chains in amyloid have derived significant support from a parallel line of investigations which has demonstrated that treatment of many Bence Jones proteins, regardless of their origin, with pepsin or trypsin can give rise to fibrils which show green birefringence when stained with Congo red and which have the characteristic appearance of amyloid in the EM and on X-ray diffraction (Glenner et al., 1971c; Linke et al., 1972). Detailed studies of one such prepar.‘1t’ion revealed complete homology of the 4600 daltons molecular weight fragment with a Bence Jones protein from residue 3 to 22. It is of interest that, in this protein, pepsin appears to have removed the first two residues thus making the protein susceptible to analysis in the sequencer. Peptide maps revealed, as expected, absence of the constant region peptides ( Glenner et al., 1 9 7 1 ~ )Studies . in our laboratory (Linke et al., 1972) of 1 8 X and 13k Bence Jones proteins have yielded a fragment from four of these proteins having a molecular weight of about 8 to 12,000 daltons which no longer has any of the antigenic properties of the native Bence Jones proteins, yet which on acidification can give rise to fibrils resembling amyloid on EM (Fig. 13). Preliminary chemical studies suggest that this fragment lacks most of the common region peptides and yet contains the same peptides that are characteristic of the major component of amyloid isolated from tissues. Several clues that the situation may be somewhat more complex and that exceptions to these findings may occur have recently been noted by three groups of investigators. Glenner has described a single subunit having a molecular weight of 5000 daltons which lacked cysteine residues (Glenner et al., 1970a). Further analysis of this protein revealed complete lack of homology to any known immunoglobulin polypeptide

275

CURRENT CONCEPTS OF AMYLOID

chain (Ein et al., 1972)-a finding which clearly indicates that other proteins may also be involved in the formation of amyloid. In a series of studies dealing with the major constituents of amyloid, Benditt et aZ. (1962, 1968, 1970; Benditt and Eriksen, 1964, 1966) have presented evidence for the existence of several low molecular weight subunits extractable from amyloid tissues with acid and urea. In a recent study, Benditt and Eriksen (1971) have partly characterized the A protein obtained from amyloid-containing tissues of patients with secondary amyloidosis and from a patient with FMF. The A protein extracted from amyloid tissues has the same electrophoretic mobility as the major protein found in purified amyloid fibrils. It has a molecular weight of about 7000, and amino acid analyses of three of these were quite similar. The absence of cysteine and threonine and the low values of proline have led these observers to conclude that the A protein is not related to the variable portion of an ordinary immunoglobulin. This initial conclusion was strengthened by a subsequent report from the same group which presented the amino acid sequence of the first 24 residues of the A protein isolated from the liver of a patient with secondary amyloidosis accompanying tuberculosis and clearly demonstrated the lack of homology of this fragment to any known immunoglobulin (Benditt et al., 1971). In an independent study, Pras and Reshef (1971) have succeeded in extracting a homogeneous protein with a molecular weight of about 8000 with 0.02 M HC1 from purified amyloid fibrils isolated from patients with FMF, rheumatoid arthritis, bronchiectasis, Hodgkin’s disease, and tuberculosis. This acid soluble component constituted up to 608 of the weight of the purified fibrils and could be reconstituted into a fibrillar shape. Amino acid analyses of three of these preparations showed them to lack cysteine. They resembled the unusual protein of Glenner and were virtually identical to the A protein of Benditt and Eriksen. Preliminary sequence studies (Franklin et al., 1972) show no homology to any immunoglobulins. The complete amino acid sequence of one of these proteins from a patient with FMF (Levin et al., 197213) is as follows: 10

5

1

15

Arg-Ser-Phe-Phe-Ser-Phe-Leu-Gly-Glu-Ala-Phe-Asp-Gly-Ala-Arg-~p-Met-Trp-Arg 20

25

30

35

Ala-Tyr-Ser-Asp-Met-Arg-Glu-Ala-Asn-Tyr-Ile-Gly-Ser-Asp-Lys-Tyr-Phe-H~-Ala 45

40

*

50

55

Arg-Gly-Asn-Tyr-Asp-Ala-Ala-Lys-Arg-Gly-Pro-Gly-Gly-Ala-Arg-Ala-Ala-Glu-Val 60

65

70

76

Ile-Ser-Asn-Ala-Arg-Glu-Asri-Ile-Gln-Arg-Leu-Thr-Gly-Arg/Gly-Ala-Glu-Asp-Ser

* Uncertain.

276

EDWARD C. FRANKLIN AND WROTHEA ZUCKER-FRANKLIN

Partial amino acid sequences for four other proteins studied in our laboratory and one reported by Benditt et al. (1971) were identical in the regions examined (1152 residues). To date, we have failed to extract this component from the amyloid of two patients with macroglobulinemia and multiple myeloma who had immunoglobulin-like subunits. The ultrastructure of the acid soluble fraction (Fig. 13A) bears no resemblance to the structure of the native amyloid fibrils. It is vermiforni in appearance, varies in thickness between 350-450 A depending on the method used for negative staining, and does not seem to have any resolvable substructure.

FIG. 13A. Electron micrograph of acid soluble fraction (ASF) prepared from isolated amyloid fibrils, negatively stained with uranyl acetate. Magnification: X 96,000.

CURRENT CONCEPTS OF AMYLOID

277

D. IMMUNOLOGICAL STUDIES Attempts to characterize the major constituents of amyloid immunologically have proceeded in two major directions. On the one hand, there has been a search for a variety of serum proteins within amyloidcontaining tissues and, later, when they became available, in preparations of purified fibrils. On the other hand, numerous attempts have been made to induce antibodies specifically reactive with amyloid by immunization with extracts of amyloid tissues. More recently purified amyloid fibrils, degraded amyloid fibrils, and the protein subunit have been used for this purpose. When used as antigens these preparations seem to have given rise to two distinct types of antibodies. One of these, that produced by immunization with the amorphous protein subunit, appears to react with idiotypic antigens unique for each amyloid and with k or h Bence-Jones proteins. The other, produced to NaOHor guanidine-degraded partially fibrillar amyloid, reacts only with amyloid (and with no other serum or tissue protein) and may detect some conformational antigenic site common to all amyloid fibrils (Franklin and Zucker-Franklin, 1972). The first type of antibody, that directed against the light-chain determinants, has proved of great value in further elucidating the nature of amyloid, whereas the antibodies to partially fibrillar amyloid have proved to be useful as diagnostic reagents for detecting amyloid in tissues ( Zucker-Franklin and Franklin, 1970). Over the years immunological mechanisms have often been implicated in the pathogenesis of amyloid. These ideas are based in part on the frequent association of the disease with multiple myeloma (Magnus Levi, 1956; Osserman et al., 1964; Pick and Osserman, 1968) and, in part, on a number of experimental as well as naturally occurring instances of amyloidosis associated with the administration of large amounts of foreign protein which, in turn, result in a state of hyperimmunization ( Teilum, 1968; Muckle, 1968a,b,c). Because of these theoretical considerations and the ready availability of antisera to y-globulins, the major effort has usually been directed toward the demonstration of immunoglobulins and complement components in amyloid-containing tissues and in association with amyloid fibrils. In spite of much contradictory data and difficulties in interpretation of some experimental observations, most of these studies suggested that y-globulin is usually present in amyloid deposits, possibly in amounts greater than in control tissues (Vazquez and Dixon, 1956; Mellors and Ortega, 1956; Schultz et al., 1966, 1968) and that complement components are also frequently found in this location. In the light of current knowledge dealing with the relation of amy-

278

EDWARD C. FRANKLIN AND WROTHEA ZUCKER-FRANKLIN

loid to y-globulins and the antigenic properties of amyloid, many of these studies are difficult to evaluate critically. On the one hand, as mentioned above, the basic aniyloid protein frequently appears to be similar to a light-chain fragment. On the other hand, in spite of these structural features, few if any antisera to y-globulin or light chains react with purified amyloid fibrils (Franklin and Pras, 1969) or the basic protein subunit (Isersky et al., 1972; Franklin and Levin, 1972). In fact, synthetic aniyloid fibrils prepared from Bence-Jones proteins often fail to react with antisera to Bence-Jones proteins, even when these are prepared to the same protein used to make amyloid (Linke and Franklin, 1972). Thus, because of these unusual properties, it is not surprising that much contradictory immunological data has accumulated. Probably some of the differences are related in part to the antisera employed, but others probably reflect the interaction of substances other than amyloid with antisera to various y-globulin fragments. y-Globulin was first detected with fluorescent antisera to amyloid by Mellors and Ortega (1956) and by Vazquez and Dixon (1956). The latter observers attempted to quantitate the amount of y-globulin by measuring the uptake of radioactively labeled antisera to 7-globulin and estimated that % 12- X as much antiserum was absorbed by amyloid tissues as by normal control tissue. Vogt and Kochem (1960) and 2 years later, Lachmann et al. (1962) demonstrated the presence of complement (PIC in particular) in amyloid deposits from 3 patients. Then, Schultz et a2. (1964, 1966) using mixed agglutination and antiglobulin consumption tests detected y-globulin in some but not all amyloidcontaining tissues and in partially purified amyloid fibrils and concluded that there were more heavy than light chains in amyloid tissue. In spite of these positive findings, however, some questions were raised by several observers about the amounts of y-globulin and its specificity in amyloid deposits. In 1958, Calkins et al. using the antiglobulin consumption test, concluded that amyloid from several individuals contained less than 1% y-globulin and that, consequently, amyloid cannot represent simply an antigen-antibody complex. Similarly, Benditt et al. (1962) failed to demonstrate a reaction between a urea extract of human amyloid and antisera to y-globulin; this treatment failed to alter the reactivity of y-globulin with antisera. Additional data pointing away from a specific association of y-globulin and amyloid were subsequently obtained by studies with isolated amyloid fibrils. Paul and Cohen ( 1963) , using ferritin-conjugated antisera to 7-globulin to detect y-globulin in purified preparations of amyloid fibrils, found no adherence of ferritin to the amyloid fibrils and concluded that y-globulin was not present as an integral part of the fibrils. These studies were

CURRENT CONCEPTS OF AMYLOID

279

extended by Cathcart and Cohen (1966) who demonstrated that purified amyloid fibrils, solubilized at p H 9.5, failed to react with antisera to yG, M, and A and also with antisera against k and h chains and Fc fragments. Consistent with this finding was the observation that antisera against some of these amyloid fibril preparations failed to react with YG-globulin and k and h chains and that absorption of antisera to y-globulins with amyloid fibril preparations failed to remove significant antibody activity. This series of studies, therefore, concluded that little, if any, y-globulin was associated with the major constituent of amyloid, the amyloid fibril. Similar conclusions have been arrived at by Cagli et al. (1962) who failed to demonstrate antibodies to 7-globulin in a rabbit immunized with an extract from amyloid and by Muckle (1964) who did not detect antibodies to y-globulin in antisera to extracts from 1 of 2 subjects. In a subsequent study, Muckle (1968a,b,c) interpreted the finding of immunoglobulins and other serum proteins in amyloid as the result of nonspecific binding. However, due to their varying nature and amount, he concluded that they are not essential to the fundamental nature of amyloid. More recently, using highly purified amyloid and smaller fibrillar subunits, we have been unable to demonstrate a reaction with antisera to each of the four major classes of immunoglobulins and their light chains ( Franklin, 1970). A clearer insight into some of these apparently contradictory observations has resulted from the chemical studies cited above and from immunological studies with antisera prepared against either the amorphous major protein subunit or against partially degraded amyloid which still retains some of the characteristic structural features of amyloid. Two types of results, depending on the antigens used for immunization have been obtained, Immunological studies with antisera produced to the basic protein subunit known to be related to the variable region of k or h BenceJones proteins have, as expected, demonstrated a strong, presumably idiotypic reaction with the protein used for immunization and varying degrees of cross-reaction with other amyloid proteins and Bence-Jones protein belonging to the same light-chain class ( k or h ) and variable region subclass. Since the antigens used for immunization were pure and in many instances chemically well-defined, these studies offer strong support for the concept that some amyloid is closely related to immunoglobulin light chains (Glenner et al., 1971a,b,c). It seems likely that these antisera will prove of great value in studying the primary structural relationships between different amyloid fractions. In contrast, and in direct support of prior studies indicating that amyloid failed to react with antisera to immunoglobulins, is the observa-

280

EDWARD C. FRANKLIN AND W R O T H E A ZUCKER-FRANKLIN

tion that the basic subunit, and even the synthetic amyloid fibrils prepared from Bence Jones proteins by proteolysis, generally fail to react with most antisera to light chains and 7-globulins. This finding is consistent with the known poor immunogenicity of the variable region of light chains and suggests that the light-chain determinants associated with amyloid may be hidden in the molecule and not readily reactive with antisera. On the one hand, it seems likely that many of the reported reactions of amyloid tissues with antisera to y-globulin may have reflected the presence of y-globulin contaminants. On the other hand, the possibility that the antisera reacted with immunoglobulin precursors of amyloid deserves serious consideration and may explain the frequently noted positive results. The other type of antibody, which is apparently directed to some structural feature common to all amyloid and, possibly, recognizes some conformational sites characteristic of the fibril, is of interest since it fails to react with any antigenic determinant on light chains or other serum proteins. It has been long recognized that amyloid fibrils are poorly immunogenic (Ram et al., 1968; Franklin and Pras, 1969; Glenner et al., 1969). In our own experience, for example, none of five rabbits immunized with purified fibrils produced antibodies. The reason for the poor immunogenicity of the intact fibrils may be their large size (Ram et al., 1968) and their marked resistance to phagocytosis (ZuckerFranklin, 1970) and proteolysis ( Sorenson and Binington, 1964; Kim et al., 1969; Pras et al., 1969). As a result, it may be difficult for the fibrils to be taken up by cells involved in processing antigens and to be degraded to a form that can induce an active immune response manifested by the formation of antibodies. Recently, this difficulty has been overcome by using various partially or completely degraded forms of amyloid for immunization since these may be more readily processed by cells involved in the immune response. Initially, we (Franklin and Pras, 1969) used alkali-degraded amyloid, a substance which still retains small fragments of fibrils to induce antibodies specifically reactive with amyloid. By using this substance as the immunizing agent, it was possible to induce antibodies that reacted with amyloid and failed to react with normal tissues or serum proteins. Although most of these antisera reacted only with DAM and amyloid and not with normal serum or tissue extracts by precipitin analyses and even by complement fixation, they were always absorbed with a marked excess of NHS and tissue extracts so as to avoid any possible confusion with cxtraneous antigens, Since this initial report, three other groups have succeeded in producing similar antisera using various partially degraded types of amyloid. Glenner et al. (1969) used urea- or guanidine-treated amyloid

CURRENT CONCEPTS OF AMYLOID

281

fibrils as the immunogens. Shapira et al. (1970) immunized with reduced and alkylated samples, whereas Cathcart et al. (1970a) used both NaOH and 5 M guanidine to produce potent immunogens. Treatment with sodium hydroxide, guanidine, and urea is relatively mild and alters the molecule sufficiently to make it immunogenic while retaining at least some of the fibrillar structure in the EM (Glenner et al., 1969). In support of the contention that these antisera pick up the tertiary structure of the fibril is our recent finding that reduction and alkylation in 6 A4 guanidine abolishes the reactivity of DAM with these antisera, whereas similar treatment of Bence Jones proteins does not abolish their reaction with antisera to Bence Jones proteins (Franklin and ZuckerFranklin, 1972) . An interesting finding, which is not entirely consistent with this interpretation, is the failure of synthetic amyloid fibrils prepared from Bence Jones proteins to react with this antibody. It seems possible, however, that these synthetic fibrils may not have exactly the same conformation as naturally occurring amyloid fibrils.

FIG. 14. ( A ) Ouchterlony double-diffusion study of antiserum to denatured amyloid (DAM) (center well) reacting with ( a ) DAM; ( b ) normal serum, ( c ) spleen extract; ( d ) yG-globulin; ( e ) Fab fragment; and ( f ) Fc fragment. ( B ) Immunoelectrophoresis of DAM (top) and normal serum (bottom). Antiserum to DAM is in the trough. (From Franklin and Pras, 1969.)

282

EDWARD C. FRANKLIN AND WROTHEA ZUCKER-FRANKLIN

Antisera reactive specifically with amyloid can be tested in three ways. ( 1 ) By precipitation techniques either in agar or in the fluid phase using DAM as antigen-Fig. 14A and B shows an Ouchterlony plate and immunoelectrophoretic pattern. A single broad arc moving to the anode is clearly visible. Figure 15A shows a series of quantitative precipitin curves with one antiserum and a number of DAM preparations. Intact amyloid could not be studied in the same fashion because it is insoluble in physiologic saline and too large to diffuse through the agar. ( 2 ) By complement fixation-since the antigen need not be soluble to be tested by complement fixation, DAM and amyloid proved suitable for such analyses. Figure 15B shows the reaction with a number of DAM preparations and the native amyloid fibril corresponding to the immunogen. ( 3 ) By absorption studies-amyloid fibrils proved ideal for 140

-

E100

s

g 60

s

20 40

80

120

160

200

40

80

120 160 c/g ANTIGEN

I 200

FIG. 15. ( a ) Precipitin curve with an antiserum to denatured amyloid ( D A M ) reacting with eight DAM preparations. (0-0) DAM used for immunization. ( b ) Complement fixation with anti-DAM and five DAMS including the homologous and the homologous amyloid ( 0-0). Significant cross-reaction is DAM (0-0) apparent in all. (From Franklin and Pras, 1969.)

283

CURRENT CONCEPTS OF AMYLOID

such studies since, being insoluble in salt, they had all the properties of an insoluble immunoabsorbent. Thus addition of amyloid to antisera followed by an assay of the residual antibody left after absorption provided an excellent estimate of the cross-reactivity. Table I shows the results of such an absorption study demonstrating the generally marked cross-reaction of antisera prepared against DAM with amyloid. Nevertheless, in most instances it appeared that additional determinants were exposed in DAM which were not readily detectable in amyloid. Detailed studies using these antisera with a number of different amyloid preparations will be cited later. Suffice it to say here that the antisera to DAM generally reacted best with the antigen used for immunization but that they cross-reacted with most other DAMS (the degree of cross-reaction varied from significant to minimal with different antisera) and, in general, they reacted well also with the native amyloid fibrils. Since it usually required larger amounts of amyloid fibrils to reach equivalence and since frequently it was not possible to absorb the antisera fully with intact amyloid fibrils, it seems likely that some of the conformational determinants are hidden in the molecule or that additional determinants are exposed during the denaturation procedure necessary to convert the fibrils into a good immunogen. In the one instance where the fibrils could absorb all of the antibody, 5 times more amyloid was required to absorb the antiserum fully. Because many of these antisera reacted well with all amyloids tested, they were considered useful as general reagents reactive with amyloid in tissue. A pool of several antisera was conjugated with fluorescein isothiocyanate and shown to react specifically with amyloid-containing tissues ( Zucker-Franklin and Franklin, 1970). In fact, preliminary CROSS-RE.4CTION

TABLE I BETWEEN

AMYLOIDAND DENATURED AMYLOIDQ

~

Amyloid

A B C 1)

Residual ppt. after absorption with AM (%)b 25 0 30

80

% fixedc 25 100 100 AC

C fixation, ratio AM/DAM at peak 10 5 6

Adapted from Table I of Franklin and Pras, 1969. (ppt.) left (% of total) after absorption of antisera prepared against degraded amyloid (DAM) with amyloid (AM). CHWfixed at peak by AM c yo Fixed = x 100 CHso fixed at peak by DAM a

* Precipitate

284

EDWARD C. FRANKLIN AND DOROTHEA ZUCKER-FRANKLIN

studies suggest that these antisera may be more sensitive than Congo red and that they may detect amyloid present in cells. In view of the poor immunogenicity of amyloid fibrils and the frequent use of impure preparations for immunization, many attempts to induce antibodies unique to the fibrils have resulted only in antibodies to other protein contaminants. Among these are certain serum constituents, such as y-globulins, fibrinogen (Horowitz et al., 1965), lipoproteins, and certain p- and al-,and a,-globulins (Cathcart et al., 1967a; Kim et al., 1967; Milgrom et al., 1966; Muckle, 1964) some of which appear to be selectively concentrated in amyloid. As mentioned earlier, of particular interest and worthy of some comment appears to be an al-globulin, termed the P component, which was most carefully characterized by Glenner ) ~ Ram et al. ( 1968). This and Bladen ( 1966), Cathcart et al. ( 1 9 6 7 ~and P component may well be identical to an a,-globulin detected by Dixon (1965) and to one of the two components noted by Milgrom et al. (1966). Although it is carried along with the fibrils in certain preparative procedures, the P component is not an integral part of the major fibrillar constituent of amyloid. This material, which has been shown to be a normal serum constituent by Cathcart et al. (1967c), appears to be identical to the rodlike structure consisting of a stacked array of pentagonal doughnut-like discs and can be seen either lengthwise or in cross section in the EM (Fig. 11) (Bladen et al., 1966). This P component has been found, when looked for, in all partially purified amyloid fibril preparations, but can be removed by further purification and is absent from similar extracts prepared from normal tissues. However, in all instances, it made up only a small fraction of the amyloid preparation solubilized by treatment with glycine ( Glenner et al., 1968). Although the precise chemical nature of the P component remains to be elucidated, preliminary studies by density gradient ultracentrifugation have yielded a sedimentation coefficient of 10.2 S and an approximate molecular weight of 200,000 (Cathcart and Cohen, 1968; Cathcart et al., 1 9 6 7 ~).According to Glenner and Bladen (1968), the Type I1 fibers, which correspond to the rods, are larger and have a molecular weight of 800,000 and an elution volume on Sephadex G-200 consistent with this estimate. On acrylamide gel electrophoresis, the P component migrates as a single band (Cathcart et al., 1967c), but it can be dissociated into at least five bands by guanidine (Glenner and Bladen, 1966). The concentration of the P component in serum does not seem to be increased in patients with amyloidosis and other pathological states ( Cathcart et al., 1967b), although, according to Muckle ( 1969), the possibility exists that levels may be increased in the actively progressive form of the disease,

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285

It is of interest that when amyloid tissues were stained with antisera to P component and fibrils, fluorescence was located on the same region for both, thus indicating the close association of these two constituents of amyloid (Cathcart et al., 1970a).

E. COMPARATIVE STUDIESOF DIFFERENT AMYLOID PREPARATIONS In view of the different clinical and pathological types of amyloid, and some of the differences already alluded to, it was of interest to compare different amyloids by immunological, physical, and chemical methods in the hope of correlating some of these properties with the clinical classification. While differences have definitely been documented, it is unfortunate that most of these studies are as yet too limited to permit any definite conclusions as to chemical differences between amyloid belonging to the currently recognized clinical classes. However, preliminary chemical studies done in several laboratories suggest strongly that the major constituents of amyloid derived from patients with secondary amyloidosis and amyloidosis associated with FMF may not be identical to those derived from primary or myeloma-associated amyloid (Benditt et al., 1971; Benditt and Eriksen, 1971; Pras and Reshef, 1971; Franklin et al., 1972). Comparative studies have been attempted on* the ultrastructural, biochemical, and immunological level. In the EM, with some of the exceptions noted above, most of the amyloid preparations appear identical. Similarly, X-ray diffraction studies of a limited number of samples have failed to detect differences. On the other hand, biochemical and immunological analyses have yielded clear-cut evidence that, on this level, variations among different amyloid preparations may be recognized. Table I1 lists the sedimentation coefficients and Congo red binding capacities of nine amyloid preparations examined by us in detail. Although initially we felt that some of the quantitative differences in Congo red binding reflected the presence of impurities, it is now our opinion that these differences may in reality reflect differences among various amyloids. Most of the variations in sedimentation coefficients probably represent the existence of different sized polymers, which could usually be dissociated to a 45s subunit by repeated homogenization. However, two of the preparations had 8 and 13 S peaks which may represent a different type of fibril. In one instance, the EM appearance seemed to correlate with the physical properties (see Fig. 9). The results of carbohydrate analyses are even more difficult to interpret in view of lack of data concerning the degree of purity of many

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EDWARD C. FRANKLIN AND WROTHEA ZUCKER-FRANKLIN

TABLE I1 PHYSICAL AND DYE-BINDING PROPERTIES OF AMYLOIDS ISOLATED FROM NINESUBJECTS' Congo red bound (mg)

Sedimentation coeff, (major component)c

No.

TYP&

protein (mg)

AM

DAM

1 2 3 5 6 8 9 10 15

P S P MM MM MM S S S

0.32 0.26 0.21 0.34 0.22 0.33 0.11 0.13 0.24

45 40 7.6 74d 7.9 41 150d 8 . 6 ; 95d 44

1.3-2.8 1.7 2.1 1.1 2.5 2.1 2.4 2.7 2.4

Adapted from Pras et al., 1969. P-primary ; S--secondary; MM-myeloma. AM-amyloid; DAM-degraded amyloid. P o l y m e r 4 0 S component.

of the preparations studied. Although the nature of the carbohydrate group remains uncertain, it seems likely that the carbohydrate moiety may be trapped by the protein rather than being an integral part of it (Pras et al., 1971); nevertheless it appears to be responsible for the binding of the various metachromatic dyes (Pras and Schubert, 1968). Recent evidence suggests that heparitin sulfate rather than chondroitin sulfate is responsible for these properties (Bitter and Muir, 1966; Mowry and Scott, 1967; Dalferes, 1968; Muir and Cohen, 1968). The technique of peptide mapping too has demonstrated similarities and differences among different preparations. Figure 16 shows maps of several amyloid subunit preparations. In general, all of these were strikingly similar, and contained certain common peptides. Nevertheless they differed from one another in certain peptides, thus indicating clear-cut chemical differences among them ( Levin et al., 1972a). Similar results have recently also been reported by Glenner's group (Glenner et al., 1970a,b). They demonstrated, in addition, the virtual identity of amyloid extracted from two organs of the same individual and the amyloid and immunoglobulin light chain prepared from the same patient. More definitive evidence not only of microheterogeneity but also for the possible existence of at least two chemically distinct types of amyloid have been obtained from end-group analyses, amino acid analy-

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287

FIG. 16. Peptide maps of ( A ) amyloid No. 1 and ( B ) its denatured amyloid derivative, ( C ) amyloid No. 6, and ( D ) low molecular weight subunit obtained from a secondary amyloid. (From Pras et al., 1969.)

ses, and partial amino acid sequence studies of a number of amyloid fibril subunits. On one hand, many of the amyloid subunits resembled light chains, in particular the variable region. Thus NH, terminal analyses of eight subunit preparations by Glenner et al. (197Oa) and of twelve amyloid fibrils by Skinner and Cohen (1971) showed the majority to be unreactive, presumably due to the presence of PCA, whereas a small number had glutamic or aspartic acid as the NH, terminal residues. Amino acid analyses also showed striking differences among different subunit preparations (Table 111) (Glenner et al., 1970a). The reason for the existence of gross similarities as well as some structural differences among certain amyloid preparations has been provided by the demonstration by Glenner and his collaborators that the major protein constituent of many amyloid fibrils consists of a fragment of light chains of immunoglobulins, in particular the variable region. Detailed chemical studies of many homogeneous light chains have clearly demon-

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EDWARD C. FRANKLW AND DOROTHEA ZUCI(ER-FRANKLIN

TABLE I11 AMINOACID ANALYSIS, N-TERMINAL GROUP,AND MOLECULAR PROTEINS‘ WEIQHTOF MAJORAMYLOID Amyloid protein: Amino acid*

IIIa

IIIb

IV

VI

VIIa

VIIb

IX

XIV

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan

5.51 5.85 8.18 7.77 5.70 6.09 6.71 1.71 5.44 0.23 2.18 6.12 2.31 1.60 3.93 0.85 1.81 1.00

5.98 5.24 7.87 6.85 6.11 6.33 6.55 1.29 4.86 0.33 2.79 6.27 2.09 1.81 3.39 0.60 2.05 1.00

6.71 0 3.07 2.18 0 4.47 7.76 0 0 1.89 0.82 1.09 3.58 4.96 1.09 0.89 4.80 1.00

4.30 5.30 9.51 7.60 4.85 8.36 6.46 1.80 4.50 0 3.16 6.26 3.40 1.85 4.65 1.30 3.20 1.00

9.20 6.82 9.23 9.99 4.98 6.17 5.62 1.81 5.28 0.75 4.06 7.14 3.50 4.09 4.95 1.15 3.50 1.00

8.73 7.10 8.72 9.35 6.48 7.47 6.12 1.33 5.50 0.63 5.29 6.65 4.08 5.36 4.97 1.03 3.65 1.00

7.10 6.18 7.98 7.81 5.60 7.02 4.34 1.50 3.22 0.80 3.47 4.34 3.76 3.30 3.18 0 2.38 1.00

6.91 6.15 10.95 9.02 7.32 11.13 8.26 1.64 6.20 0 4.68 7.79 3.68 3.22 2.29 1.23 1.11 1.00

N-terminusc: U u u u Asx Asx Asx u Molecular weight: 27,600 13,700 5000 15,400 31,200 18,300 7500 14,600 a

From Glenner et al., 1970a.

c

U-unreactive to fluorodinitrobenzene.

* Expressed m micromoles per micromole of tryptophan. strated that all light chains have certain class specific characteristics in the “common” region, but that they differ in amino acid sequence in the variable region. However, even in the variable region there exist common subclass properties ( Milstein and Pink, 1970). While all the immunoglobulin-related amyloid subunits seem to bear a striking chemical resemblance to each other, another unrelated substance has recently been isolated from several amyloid preparations from patients with secondary amyloidosis and amyloidosis with FMF (Benditt and Eriksen, 1971; Benditt et al., 1971; Pras et al., 1971). Amino acid analyses (Table IV) and partial amino acid sequence studies of these proteins indicate that they are identical to each other and unrelated to any known immunoglobulin (Benditt et al., 1971; Franklin et al., 1972). While this material represents the major corn-

289

CURRENT CONCEPTS OF A M Y M I D

TABLE IV AMINOACIDCOMPOSITION OF ACID-SOLUBLE FRACTION FROM Two HUMAN AMYLOID FIBRILS(RESULTEXPRESSED AS No. RESIDUES FOUND)"

Amino acid

FMF

Hodgkins

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-Cystine Valine Methionineb Isoleucine Leucine Tyrosine Phenylalanine Tryptophanb

2.0 1.1 7.0 9.1 0.9 4.6 6.3 1 .o 7.6 11.6

2.3 1.8 6.6 7.3 0.2 4.0 5.4 0.8 6.6 10.0

0.9 (2) 2.0 2.2 3.7 4.7

1.1 (2) 2.0 1.2 3.4 5.0

-

+

-

= Mean of three analyses after 21 hr. hydrolysis. Identified from amino acid sequence analysis (p. 275).

b

ponent of these fibrils, it seems likely on the basis of preliminary observations that they may also contain small amounts of light chain related material (Levin et al., 1972). Similarly, the possibility that this protein may exist in trace amounts in primary or myeloma-associated amyloid cannot be excluded at this time.

F. DISCUSSION AND CONCLUSIONS On the basis of the morphological, chemical, and immunological studies, the following tentative conclusions can be reached. It would appear that amyloid in man and all other species studied consists primarily of a fibril with a characteristic ultrastructural appearance. A second component, the precise significance of which remains to be elucidated, and which makes up a small percent of the total, is a serum a,-globulin which assumes a rodlike or a doughnutlike appearance. A number of other proteins have been identified in amyloid deposits but they are not present in purified fibrils and their role in the pathogenesis of amyloid remains dubious. As a result of the studies reported during the past year, there can be little doubt that in many subjects the fibrils consist of subunits rang-

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EDWARD C. FRANKLIN AND WROTHEA ZUCKER-FRANKLIN

CURRENT CONCEPTS OF AMYLOU)

291

ing in size from 5000 to 30,000 daltons which seem to represent primarily the variable portion of immunoglobulin light chains. Although their precise structure remains to be elucidated, preliminary sequence studies of several proteins have indicated that, with one possible exception, these proteins are not complete light chains and that they may either have internal deletions of varying lengths or that they may have lost varying amounts of the constant region. Sufficient numbers of these immunoglobulin-related proteins have not been investigated in proper detail to accurately define the number of classes of amyloid, to determine if k and proteins have certain common features necessary for fibril formation, or if other immunoglobulin chains may play a role. In view of some of these structural features and circumstantial evidence that under some conditions plasma cells may be involved in the synthesis of amyloid, it is tempting to speculate that the protein could be a primitive variant of y-globulin, the synthesis of which is induced under certain conditions (see below). A particularly promising lead, since it seems to suggest chemical differences among various types of clinically defined amyloidosis, is the description of an apparently non-immunoglobulin-related substance from patients with secondary and FMF-related amyloidosis. This observation suggests the existence of several different, and possibly unrelated, proteins all of which, under certain conditions, can give rise to the material we define as amyloid and raises the hope that in time it will be possible to develop a rational classification of amyloidosis based on the chemical properties of the amyloid fibrils. The precise origin, mode of synthesis, and assembly of the amyloid related to immunoglobulins remains to be defined. As mentioned before, several mechanisms may be responsible for the synthesis and assembly of such amyloid fibrils. On the one hand, the deposition of large masses of amyloid in the vicinity of plasma cell tumors, coupled with the finding in certain instances of intracellular fibrils, suggest the possibility that amyloid may be locally produced and that fully formed fibrils can be assembled from the precursor in the plasma cell or, alternatively, immediately after secretion as is the case in some instances of macroFIG. 17. Peripheral blood cells incubated with amyloid. No phagocytosis was evident after 30 minutes. Cell in upper left is a monocyte (Mono); cell in lower right a neutrophile. Amyloid ( A m ) . Magnification: X7000. (From Zucker-Franklin, 1970. ) FIG. 18. Peripheral blood neutrophile incubated with antiserum-treated amyloid. Note abundance of phagocytic vacuoles containing amyloid ( Am). The cell is almost completely degranulated. Ultrastructure of amyloid within various phagosomes seems to have undergone alteration. Compare fine structure of inclusions L, L, and 13. Nucleus ( N ) . Magnification: X 14,000. (From Zucker-Franklin, 1970.)

292

EDWARD C. FRANKLIN AND DOROTHEA ZUCKER-FRANKLIN

globulinemia (Buxbaum et al., 1971). The unusual insolubility of the amyloid fibrils may explain the deposition of the fibrils in direct proximity to the cells synthesizing them. On the other hand, the demonstration of light-chain fragments of immunoglobulins as the major component of some types of amyloid raises the possibility that amyloid forms as the result of the interaction of circulating Bence-Jones proteins, immunoglobulins, or possibly antigenantibody complexes with certain tissue receptors. Preceding or following this, there may he some proteolytic degradation of these molecules which causes them to assume the characteristic fibrillar appearance, In support of this view is the demonstration by Osserman et al. (1964) that fluoresceinated Bence-Jones proteins, especially those derived from patients with amyloidosis, can bind to tissue sections, and the recent demonstration by Glenner et aZ. ( 1 9 7 1 ~ )and by Linke in our laboratory

FIG. 19. Monocyte obtained from specimen incubated with antiserum-treated amyloid fibrils. The phagocytic vacuoles containing fibrils (Am) vary in size and are irregular in circumference, presumably because of coalescence with other phagosomes. The arrow points to site where such coalescence may have taken place. Lysosome ( L ) . Magnification: X 13,000. ( From Zucker-Franklin, 1970.)

CURRENT CONCEPTS OF AMYLOID

293

FIG.20. Reticuloendothelial cell from a bone marrow specimen of a patient with multiple myeloma. The fibrils occupy membrane-bound spaces reminiscent of coalesced phagocytic vacuoles. Arrow points to site where amyloid (Am) appears to be engulfed by the cell. Location of amyloid in this cell is in marked contrast to intracytoplasmic fibrils illustrated in Figs. 6 and 7. Magnification: X6000.

294

EDWARD C. FRANKLIN AND DOROTHEA ZUCKER-FRANKLIN

that amyloid can be formed in the test tube from Bence-Jones proteins. It is of some interest that in amyloid deposits as well as in the production of synthetic amyloid from Bence-Jones proteins there is an inordinately high frequency of proteins related to h light chains-a finding suggesting that this type of protein has some structural feature that makes it more susceptible to the deposition as amyloid. The recent chemical and immunological studies and others likely to derive from them in the near future, as well as a more precise characterization of the amyloid proteins not related to immunoglobulins, may well bring us closer to an answer to some of these questions. Another problem which so far has defied a clear-cut explanation is the long persistence of amyloid, often even after removal of the inciting stimulus, A partial explanation for this may be provided by the marked resistance of native amyloid fibrils to proteolysis and their failure to be ingested by peripheral blood phagocytes under conditions where these cells are able to engulf bacteria, foreign particles, and denatured serum proteins ( Fig. 17) ( Zucker-Franklin, 1970). Only fibrils treated with specific antiserum to amyloid were avidly phagocytized by these cells (Figs. 18 and 19). However, once the antibody-treated fibrils were seen within phagocytic vacuoles, they appeared to undergo ultrastnictural changes suggesting that Iysosomal enzymes may be capable of degrading amyloid intracellularly (Fig. 18). It is quite likely that in vivo, under some conditions, when the stimulus for the formation of amyloid has ceased, the fibrils can also be removed slowly by phagocytosis and intracellular degradation. Fifty years ago, Waldenstrom ( 1928 ) observed by means of serial splenic biopsies that excision of tuberculous fistulas or the amputation of limbs afflicted with osteomyelitis was accompanied by a reduction of splenic amyloidosis. More recently, isolated reports concerned with the resorption of amyloid in experimental animals (Richter, 1954) and in a few patients have also appeared (Lowenstein and Gallo, 1970). The RE cell depicted in Fig. 20 may bear witness to the phagocytosis of amyloid which may occur in the living organism. V. Speculations on the Pathogenesis of Amyloid

To the best of our knowledge, there is no other disease known to man which occurs naturally with as many associated disorders as amyloid. In addition, there is no other disorder which can be induced as readily in a variety of species with as many different agents and procedures as amyloid. Consequently, over the years there has accumulated a great mass of often contradictory information, all of which osten-

CURRENT CONCEPTS OF AMYLOID

295

sibly bears on the pathogenesis of the disease. It is obviously beyond the scope of this review to summarize all of this material in detail. We shall, therefore, discuss some of our own thoughts on this interesting question and cite only those experimental studies that have direct bearing on them. The theories published by some investigators, e.g., Teilum ( 1964a,b, 1968), Muckle ( 1968a,b), Janigan and Druet (1966, 1968a,b), and Janigan (1969) are primarily based on experimental results and, therefore, have the greatest appeal to us. For a more widely rounded discussion of the problem of pathogenesis, the reader is referred to the book “Amyloidosis” ( Mandema et al., 1968). In recent years, discussions related to pathogenesis of amyloid have dealt primarily with immunological mechanisms and have implicated the RES as the responsible organ system. Chemical and immunological studies demonstrating the relation of amyloid to immunoglobulin light chains provide direct proof for concepts based on the association of amyloid in man with a variety of chronic infectious diseases and plasma cell dyscrasias and support the idea that a hyperactive stimulated RES, frequently accompanied by excessive y-globulin production, may play a role in the pathogenesis (Teilum, 1968). However, in contrast to these conditions where the deposition of amyloid is associated with a hyperactive immunological system, amyloid also occurs with a fairly high frequency in certain of the immune deficiency diseases such as agammaglobulinemia-to date, 9 cases of amyloid complicating agammaglobulinemia have been reported (Teilum, 1968; Mawas et al., 1969). Amyloid also complicates disorders such as Hodgkin’s disease and other lymphomas where impairment in delayed hypersensitivity occurs. In animals, too, a similar association of amyloid with states of prolonged antigenic stimulation as well as immunological incompetence has been noted. It is a frequently cited fact that horses given prolonged antigenic stimulation for the production of commercial antisera often die with amyloidosis. These animals have very high levels of 7-globulin and high titers of antibodies to the immunogens used for immunization. Similarly, in the various experimental models, stimulation of the RES accompanied by active antibody synthesis is a common feature. Thus, casein and other substances (Druet and Janigan, 1966a,b) are given in large amounts, and both Freund’s adjuvant and endotoxin ( Barth et al., 1969a), now widely used to induce amyloid, have a profound stimulating effect on the RES. Nevertheless, the studies by Pierpaoli and Clerici (1964; Clerici et al., 1965) clearly demonstrated that antibody synthesis is not necessary and that animals made tolerant to casein are able to produce amyloid as readily as control animals. Contrary results were subsequently reported by Letterer and Kretschmer (1966).

296

EDWARD C. FRANKLIN AND DOROTHEA ZUCKER-FRANKLIN

Although most regimens employed to induce amyloid stimulate the

RES and present it with a profound antigenic challenge, according to

Druet and Janigan (1966a), these may, at the same time, cause thymic atrophy and peripheral lymphopenia. In addition, there appear to be a number of factors that accelerate amyloid formation seemingly by virtue of the fact that they inhibit the immune system. Thus steroids, immunosuppressive agents, and X-irradiation ( Hultgren et al., 1967; Teilum, 1968; Bradbury et al., 1964) have all been found to accelerate significantly the deposition of amyloid, as have thymectomy and bursectomy (Druet and Janigan, 1966b; Clerici et al., 1969)-a11 these procedures have in common the ability to decrease one or another phase of the immune response. Nevertheless, certain exceptions to this general rule have recently been reported since, in the hands of Ranlov (1968), both antilymphocyte serum and nitrogen mustard appear to inhibit cellular immunity and casein-induced amyloid. Thus we are faced by an apparent paradox. On the one hand, amyIoid occurs in a number of diseases which superficially have a hyperactive immune system and which are accompanied by excess y-globulin synthesis. On the other hand, amyloid often is found in association with diseases accompanied by various immune deficiency states and can be accelerated in experimental animals by a variety of procedures and agents which are generally used to inhibit the immune response. Furthermore, a recent report suggests that if lymphocyte transformation is used as a measure of delayed hypersensitivity, then patients with amyloidosis have a diminished response to herpes simplex virus but not to two other antigens nor to phytohemagglutinin (Lehner et al., 1970). We have recently confirmed the normal response to phytohemagglutinin (Friedman et d.,1970), but we have not investigated other antigens. The results presented above suggest the possibility that in all instances amyloid occurs in a situation where the antigenic stimulus is excessive for the available immune system or where, because of neoplastic change, it has escaped from normal control mechanisms. This may cause the immune system to respond with an exaggerated or an alternative response, possibly one existing during a stage of evolution before the development of classic antibodies and which may again be elicited under unusual stress. There is abundant evidence from phylogenetic studies that light chains represent the most primitive precursor of immunoglobulins. Impairment of the immune system in immune deficiency diseases and under the influence of immunosuppressives, X-rays, or steroids is obvious. Similarly, this impairment has been recognized in diseases affecting lymphopoietic tissues, such as Hodgkin’s disease and lym-

CURRENT CONCEPTS OF AMYLOID

297

phomas. Even in neoplastic disorders affecting plasma cells, such as myeloma and macroglobulinemia, despite the abundance of plasma cells and the excessive synthesis of y-globulins, it has been recognized for a long time that these cells are largely committed and that responsiveness to antigenic challenge is diminished or even absent. Thus, the presence of a plasma cell tumor or excess y-globulin may be accompanied by impaired immediate and delayed hypersensitivity. An analogous situation may well exist in chronic infections, rheumatoid arthritis, hyperimmunized horses, and casein-treated animals, all of which produce excessive amounts of antibodies. In all of these instances, it seems possible that the RES is committed to the synthesis of a specific type of antibody and, thus is immunologically deficient when a new and possibly weaker stimulus appears. In the case of casein-stimulated rabbits, Rodey and Good (1969) have recently demonstrated that mouse spleen cells from animals, pretreated with azocasein or Freund’s adjuvant, have a diminished response to phytohemagglutinin, possibly by prior activation and consequent “removal” of thymus-dependent cells. The immune response to other antigens in chronic infections has not been studied in detail; however, defects in certain aspects of the immune response have been observed in rheumatoid arthritis and systemic lupus. Particularly suggestive are the recent tissue culture studies of Herman et a?. (1971) which indicate that synovial cells removed from patients with rheumatoid arthritis who had been previously stimulated with tetanus toxoid produced very large amounts of y-globulin but that tetanus antitoxin constituted a much smaller fraction than lymphoid tissues from normals or synovia from other diseases. This finding clearly suggests that these tissues, although rich in plasma cells and actively synthesizing antibodies, are markedly defective in their response to another new antigenic stimulus. It would be of interest to examine these tissues for the presence of amyloid or if possible for their ability to synthesize amyloid in uitro. Consistent with such a view are the observations of Teilum (1954, 1964a,b, 1968) who, on the basis of morphological studies of tissues during the evolution of amyloid, has proposed a two-stage mechanism. Initially, there is a pronounced stimulation of pyroninophilic antibodyproducing plasma cells and RE cells which occurs prior to the elaboration of amyloid. As the antibody-producing system is exhausted, there is a decrease in the pyroninophilic cells and a concomitant diminution in the production of y-globulin. This is accompanied by a proliferation of periodic acid-Schiff (PAS )-positive RE cells which appear to be involved in the synthesis of amyloid. To date there are no studies of the immune response in many of the experimental models used to induce amyloidosis,

298

EDWARD C. FRANKLIN AND WROTHEA ZUCKER-FRANKLIN

but the phenomenon of antigenic competition makes it appear possible that the response to a new antigenic challenge might be decreased. A similar situation might be found in amyloidosis commonly associated with aging (Schwartz, 1968; Wright et al., 1969). It has long been recognized that small amounts of amyloid can be found at postmortem in many elderly individuals. This material has all the morphological attributes of amyloid and is estimated to be present in at least 50%of individuals over the age of 70. Although the immune response in man has not been carefully correlated with age, there has been the general impression that it diminishes and that this diminution may be one of the possible reasons for the increased incidence of neoplasia with age (Calkins, 1968). Thus it appears likely that amyloid in the aged accompanies a less active immune system than was available in youth. What then can we postulate as a possible pathogenetic mechanism? In its simplest form we might say that amyloid is likely to occur in situations where the size of the antigenic stimulus is excessive compared to the capacity of the immune system or where the immune system has escaped from normal control mechanisms. Thus, if the immune system is normal it can tolerate an enormous antigenic load (e.g., casein) before amyloid is produced. If it is defective, as in agammaglobulinernia, a smaller stimulus is effective. Whether only a specific type of antigen is capable of initiating this process or whether any antigen can do so, whether the nature of the stimulus predetermines the type of amyloid, and whether only living infectious agents might be capable of inciting it remains to be established. Since the material deposited is obviously heterogeneous, it is difficult to propose a unified concept. The possibility that amyloid is a consequence of imbalanced immunoglobulin chain synthesis or, possibly, even represents a vestigial response of some component of the immune system, which is latently present and called upon only when it is overwhelmed or dedifferentiated, is suggested by the light-chain nature of the basic protein subunit and by the presence of amyloid fibrils in plasma cells and lymphocytes under certain circumstances. Obviously, pursuit of this line of investigation and further chemical characterization of the other types of amyloid will prove most fruitful in unraveling this difficult problem. Although not directly related to the above concepts, one other aspect of experimental amyloid research deserves mention for the sake of completeness and because of its potential interest. This deals with a model in which amyloid enhancing factor (AEF) can be recovered from tissues, subcellular fractions, and, possibly, serum of animals that are developing amyloidosis. As assayed in the recipients, AEF markedly

CURRENT CONCEPTS OF AMYLOID

299

shortens the induction time for amyloid incited with casein or casein derivates. Although AEF has not yet been precisely characterized, it has been reported by one group to occur in serum and to be related to the 7-globulin fraction ( Janigan and Druet, 1968a,b). Others have fractionated cells and recovered it in the fraction containing cell nuclei after fractionation, probably deoxyribonucleic acid protein ( Ranlov, 1968) and also in the cytoplasm (Janigan and Druet, 1968a,b). Several reports suggest that AEF can cross species lines since such fractions from human amyloid can enhance the induction of the disease in mice. Whether the material transferred is an amyloid precursor, antigen per se, or antigen coupled to some cell constituents so as to make it more potent remains to be determined. A recent report by Rimon et al. (1972) has characterized this factor as a low molecular glycoprotein, unrelated to the amyloid fibrils. At any rate, it appears that AEF acts on the RE cells of the host and causes them to produce amyloid at a more rapid rate than normally. Although the model has not provided any definite answers, it appears at this time worthy of further study,

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