In Murine and Human Liver Revealed by Immunofluorescen

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Proc; Nati. Acad. Sci. USA Vol. 76, No. 8, pp. 4112-4116, August 1979 Medical Sciences Formation and involution of Mallory bodies ("alcoholic hyalin") in murine and human liver revealed by immunofluorescence microscopy with antibodies to prekeratin (alcoholic hepatitis/liver pathology/keratin/intermediate filaments/tonofilaments) HELMUT DENK*, WERNER W. FRANKEt, ROMANA ECKERSTORFER*, ERIKA SCHMIDt, AND DONTSCHO KERJASCHKI* *Division of Gastroenterologic Pathology and Hepatopathology (Hans Popper Laboratory), Department of Pathology, University of Vienna School of Medicine, Vienna, Austria; and tDivision of Membrane Biology and Biochemistry, Institute of Experimental Pathology, German Cancer Research Center, Heidelberg, West Germany Communicated by Hans Popper, May 29, 1979

ABSTRACT Antibodies raised against prekeratin intensely In the present communication the following findings are and specifically stain, in immunofluorescence microscopy, reported. (i) Antibodies t6 bovine prekeratin react with MBs Mallory bodies ("alcoholic hyalin") present in livers of human alcoholics and griseofulvin-treated mice. The high sensitivity of human origin, thus extending our original observation in of this method allows the identification of small distinct cyto- experimental animals (5) to human disease. (ii) Immunofluo- plasmic structures that are observed during early stages of rescence microscopy using such antibodies as well as antibodies Mallory body formation, especially frequent in the perinuclear to MBs of human origin allows the tracingof MB formation and cytoplasm, as well as during stages of Mallory body disinte- involution by detection of early (precursor) stages of MB for- gration and disappearance, such as after withdrawal of the drug. mation as well as MB fragments during involution. (iii) MBs of In the latter situation, the prekeratin-containing small particles exhibit a characteristic pattern of arrangement in the hepatocyte human liver differ from those in livers of griseofulvin-treated periphery. Electron microscopy illustrates that such smal bodies mice in that they immunologically crossreact with prekeratin are heap-like aggregates of typical Mallory body filaments. from desmosome-attached tonofilamepts of bovine muzzle but Immunofluorescence studies with antibodies to isolated pre- not with prekeratin polypeptide components of bovine hoof. keratin polypeptides from bovine hoof or muzzle epidermis (iv) Neither original nor colchicine- or griseofulvin-reinduced show that Mallory body filaments, in particular those in human murine MBs are related to the vimentin-type of intermedi- liver, are immunologically more closely related to prekeratin of tonofilaments from living epidermal cells (stratum spinosum). ate-sized filaments (9). The data indicate that Mallory bodies contain a pathologic form of prekeratin-like material. They also suggest that disorders of MATERIALS AND METHODS cytoskeletal structures of the intermediate-sized filament class Human Liver. Liver tissue from patients with alcoholic are associated with specific diseases and can be visualized and characterized by immunofluorescence microscopy by using hepatitis and cirrhosis was removed at autopsy. For comparison, antibodies to constitutive proteins of such filaments. samples of alcoholic fatty liver and of livers without pathologic alterations were examined. Mallory bodies (MBs) ("alcoholic hyalin") are unique cyto- Animals. Male Swiss albino mice (strain Him:OF 1 SPF; plasmic inclusions, mostly observed in hepatocytes (1). Their Institute for Laboratory Animal Research, University of Vienna major and most characteristic components are randomly oi- School of Medicine, Himberg, Austria) were fed a powdered ented, unbranched, rod-shaped filaments of 14-20 nm in di- standard diet (Altromin, LippeW West Germany) containing ameter, which often exhibit a dense fimbriated coat (2-5). They 2.5% griseofulvin (Qlaxo, Greenford, England) as de- are characteristic morphologic features of alcoholic hepatitis scribed (6). Animals were killed after 1, 2, 3, 4, 6, 8, and 12 (1) but have also been observed in diverse liver disorders not weeks of continuous griseofulvin feeding, and 1, 2, 3, and 10 related to alcoholism (3). Typical MBs can be experimentally days and 3, 8, and 12 weeks after withdrawal of the drug in produced in mouse liver by long-term griseofulvin feeding (6). order to trace development and involution of MBs. In addition, The major constituents of experimentally induced MB filaments some mice were kept on the following regimen: continuous are a group of polypeptides with apparent Mr values ranging griseofulvin treatment for 2.5 months, then 2.5 or 3 months on from 48,000 to 66,000 (5), and a similar composition has re- a drug-free diet, then on the griseofulvin-containing diet again cently been reported for MBs from human alcoholics (7). In this for 4 days prior to sacrifice. In another group of animals final respect, MBs resemble prekeratins from different sources that griseofulvin refeeding was replaced by the administration of all contain characteristic polypeptides in the 47,000- to colchicine for 8 days (10) by using a colchicine dosage of 0.5 68,000-Mr range (see ref. 8 for references). In addition, it has ,tg/g of body weight injected subeutaneously twice a day. been shown that antibodies to prekeratin from bovine hoof Antibodies. Antibodies were (i) guinea pig antisera to total horny layer, in immunofluorescence microscopy, specifically reconstituted, purified bovine hoof (including stratum cor- decorate murine MBs, which further strengthens the relation- neum) prekeratin (5, 8); (ii) IgG from i made monospecific for ship between MB filaments and intermediate-sized filaments prekeratin as described (11); (iii) guinea pig anitisera against of the prekeratin type present in most if not all epithelial cells electrophoretically separated individual polypeptides or ("cytokeratins") (8). On the basis of these findings it has been polypeptide fractions of bovine hoof prekeratin and desmo- proposed that MB formation represents a pathologic form of some-associated tonofilaments from bovine muzzle (11-13); hyperkeratosis (5). (iv) guinea pig IgG from antisera to prekeratin from human epidermal tissue obtained from autopsy skin material that was The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviations: MBs, Mallory bodies; DT, indicates desmosome-asso- vertisemennt" in accordance with 18 U. S. C. §1734 solely to indicate ciated tonofilament prekeratin component; PK, indicates prekeratin this fact. component from bovine hoof. 4112 Downloaded by guest on September 25, 2021 Medical Sciences: Denk et al. Proc. Natl. Acad. Sci. USA 76 (1979) 4113

prepared as described for bovine hoof prekeratin (8); (v) guinea Patterns of MB Formation and Involution. Mouse liver pig IgG from antisera to human MBs isolated from autopsy liver damaged by griseofulvin administration was examined by using according to French et al. (14) and further purified as described antibodies to bovine prekeratin, to individual prekeratin by Franke et al. (5); (vi) guinea pig antisera against murine components, and to MBs on consecutive sections of frozen liver vimentin (9). Controls included preimmune sera and sera ab- tissue. Agreement of immunolocalization was observed with sorbed with the specific antigens. the different antibody preparations. MB-like material was Immunofluorescence Microscopy. Small pieces of liver detected in some mice as early as 4 weeks after commencement were quickly frozen in isopentane cooled with liquid nitrogen. of griseofulvin feeding in the form of small granules that were Immunofluorescence microscopy was performed on frozen distributed either diffusely in the cytoplasm of scattered liver sections as described (5). cells or, more characteristically, in prominent perinuclear ar- Electron Microscopy. Mice were anesthetized with ether, rays. Such perinculear granules seem to be typical for early and the livers were perfused through the portal vein first with stages of MB formation (Fig. 1 a, d, and e and Fig. 2). After 6 Hanks' balanced salt solution and then with 2.5% glutaral- weeks of continuous griseofulvin administration, sporadic dehyde in 0.1 M sodium cacodylate buffer, pH 7.4. Small cubes hepatocytes in centrolobular position with irregularly ramified of liver tissue were postfixed for at least 4 hr in the same fixative. MB-like structures and aggregates of confluent prekeratin- They were then osmicated and processed for embedding and containing granules were observed. At 8 weeks, MB-containing electron microscopy of ultrathin sections according to con- hepatocytes were already numerous and were arranged in ventional procedures. groups of 4-10 cells, mostly in the center of the lobules sur- rounding the central veins. At this stage, MBs in many hepatocytes were large and exhibited irregular, reticular contours RESULTS or coiled organization. They were often, but not always, in Reaction of Murine and Human MBs with Antibodies to paranuclear position, sometimes forming thick perinuclear rings Diverse Prekeratin Preparations and to Human MBs. Anti- (Fig. le). However, despite the presence of typical fully de- bodies to prekeratin from total bovine hoof epidermis and from veloped MBs in some cells,, adjacent, hepatocytes often still bovine muzzle desmosome-associated tonofilaments strongly exhibited small granules of MB material or showed arrange- ments suggestive of stages of confluence of sma'll granules and specifically decorated MBs in mouse liver (Fig. ig). When (Fig. 1 d and e), indicating continuous development of MBs under antisera to individual polypeptide fractions of bovine hoof sustained griseofulvin administration. Prolonged prekeratin (11) were examined, antibodies to polypeptide griseofulvin fractions PK 1-4-i.e., components with apparent Mr values feeding resulted in only quantitative changes of the pattern in that hepatocytes containing MBs in various stages of develop- of 54,000-63,000-showed stronger reaction with mouse MBs and than did antisera to with ment increased in number, and, consequently, the original (Figs. le, 2c, 3b) components centrolobular predominance of MB-containing lower molecular weights (Mr of 48,000-52,000). Antisera to the liver cells dis- from des- appeared. At such later stages, extracellular MBs were some- major polypeptides electrophoretically separated times detected by immunofluorescence microscopy in the si- mosome-attached tonofilaments of bovine muzzle epidermis nusoidal lumina or were taken up by Kupffer cells. MBs in also stained and antibodies to cells strongly MBs, again larger poly- of hyperplastic nodules (6) were identical in their immuno- peptides (Mr values: DT 1, 68,000; DT 2,60,000; DT 3,58,000) reactivity to those present in hepatocytes. were more effective (e.g., Fig. la) than antisera to the smaller After discontinuation of griseofulvin feeding, the intensity polypeptides (Mr values: DT 4, 52,000; DT 5, 51,000) (8, 12). of decoration of MBs with antibodies to prekeratin and to. It is noteworthy that some of the antibodies to hoof prekeratin human MBs progressively decreased, and often a nonfluorescent and desmosome-attached tonofilaments (especially PK 1-3 and core portion was visible (Fig. If). The number of fluorescing DT 2, 4, and 5) intensely reacted with tonofilament structures MBs gradually decreased. Concomitant with the disappearance in bile duct epithelial cells (Fig. la) (15), whereas other pre- of the typical large MBs, small granules strongly decoratable keratin antibodies (PK 4 and 5, and DT 1 and 3) exhibited no with antibodies to human MB-material and to prekeratin ap- significant reaction. peared at the periphery of the hepatocytes (Fig. 3). In some Antibodies to human epidermal prekeratin also specifically hepatocytes, mostly in centrolobular ones, these granules per- stained MBs in mouse liver (Fig. lb), and so did antisera raised sisted for up to 2-3 months after drug withdrawal. against isolated human MBs (Fig. 1 d, f, and h and Fig 3b). When mice, which originally had been fed a griseofulvin- However, our guinea pig antisera to human MBs did not sig- containing diet for 3 months, were kept on a griseofulvin-free nificantly decorate fibrils of bile duct epithelia of mouse liver diet for 1-3 months and then griseofulvin was readministered and cultured rat kangaroo (PtK2) cells but did decorate cyto- for 4 days, large typical MBs reappeared within 4 days (cf. ref. plasmic fibrils present in human (HeLa) and bovine (BMGE; 6). A similar effect of fast reinduction is observed when col- cf. ref. 8) epithelial cells as well as in frozen-sectioned bovine chicine is injected for a total of 8 days (10). Such reinduced MBs muzzle epidermis (data not shown). reacted with antibodies to prekeratin and to human MBs but Antibodies to bovine hoof prekeratin polypeptides stained not with antibodies to vimentin (Fig. 1 g-i). Typical MBs can human MBs only poorly, if at all, in contrast to intensive dec- be reinduced with griseofulvin as well as with colchicine in oration of human MBs with the antibodies to the large poly- livers that still contain hepatocytes with peripheral granules of peptides of desmosome-associated tonofilament prekeratin (DT MB-like material but also in livers in which no trace of MB- 1-3; Fig. 1c). Hepatocytes of normal human liver and of alco- material is detected in the control animals. holic fatty liver exhibited only very poor, if any, reaction with Electron Microscopy. When early stages of MB forma- antibodies to prekeratin, as described in rodent liver (15), most tion-i.e., when only a few hepatocytes show granules positively probably reflecting the small amounts of tonofilaments reacting with antibodies to prekeratin or human MB-mate- present. rial-were examined by electron microscopy, small heaps MBs of murine and human origin were not stained to a sig- (diameters ranging from 0.1 to 1 um) of randomly oriented nificant extent by antibodies to murine vimentin (Fig. li) and typical fimbriated filaments were seen in locations, often to actin. around the nucleus (Fig. 2 a and b), corresponding to those Downloaded by guest on September 25, 2021 4114 Medical Sciences: Denk et al. Proc. Natl. Acad. Sci. USA 76 (1979) described by immunofluorescence microscopy (Fig. 2c). A 2b). MB material arranged in the hepatocyte periphery after conspicuous association of these small aggregates of MB fila- withdrawal of the drug showed an identical filamentous ul- ments with tufts of typical tonofilament bundles was seen (Fig. trastructure.

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strletlyFIG. 2.coWr(a)OleElectronotoeasginacnlcnpxsAnavrmicrograph of a hepatocyte showing small (precursor) paranuclear aggregates of MB filaments (outlined area and arrowheads).1nm.Bar denotes (b) Higher magnification from the area outlined in a. Note the occurrence of typical tonofilament bundles in the immediate vicinity of small MBs (arrows). Bar denotes 1 am. (c) Immunofluorescence microscopy of a corresponding stage of MB formation with antibodies to bovine hoof stratum corneum prekeratin fraction PK 1, showing perinuclear MB granules. Bar denotes 20im. DISCUSSION murine and human MBs reflects different composition of the or the MBs in these species is not known. In our conexperimenthally MBrformationemicina y umanlveredg cprekeratin addition, strictly conform to those arising in alcoholic hepatitis and var- cmlrated in the filarmetious compnsMBsr(f.rof e ma riprsean ious non-alcohol-associated liver disorders in their light and mulated inclamentous assonents of MBs (3,e5). electron microscopic appearance (5, 6). Moreover, human and an abnormal assembly of normal hepatocyte prekeratin fila- murineMesextMBsperimnallare closelypormoduerelatedibnormiceimmunologicallybygrk iseoulibausefheefidingboth aditrmionsments or istobheclrfewhtrcomposed ofprekeratin-likeprtisoheprekeratin-like proteinsmaterialsspecificallyac-ibl react with antibodies to tonofilament prekeratin of bovine synthesized during treatment with griseofulvin in mice or muzzle epidermis and to isolated human MBs. This substantiates during excessive chronic alcohol ingestion in humans. In this our concept (5) that MB formation, also in human liver, rep- context it is, however, worth stating that MBs can contain, in resents a special form of abnormal hyperkeratosis. The finding addition to the prekeratin-like proteins, other materials, possibly that material contained in MBs of different species crossreacts, enriched in the granular portions of MBs (cf. ref. 3) and in small which agrees with data of French et al. (16), appears to reflect membranous inclusions often associated with MBs (3, 5). the immunologic relatedness observed with prekeratins from Therefore, although our data show that MBs in general contain various vertebrates (8, 11, 17). Whether the observed difference prekeratin-like proteins in their filamentous components, they of reaction of antibodies to bovine hoof prekeratin between do not exclude the occurrence of other materials (i.e., non-

FIG. 1 (on preceding page). Immunofluorescence microscopy of frozen sections of murine (a, b, d-i) and human (c) liver with antibodies against total prekeratin (g) or isolated prekeratin polypeptides (a, c, e) of bovine origin, antibodies to total human epidermal prekeratin (b) or antibodies to MB material isolated from autopsy liver of human alcoholics (d, f, h). a and c show decoration of MBs with antibodies to tonofilament prekeratin component DT 2; in e, antibodies to prekeratin fraction PK 1 have been used (cf. refs. 11 and 13 and text). Note that bile duct epithelia are also stained with DT 2 antibodies (left side of a). As a control, the reaction of antibodies to murine vimentin is shown (i); these antibodies specifically stain mesenchymal cells, particularly Kupffer cells, but not MBs (arrowheads denote some MMs). The murine livers are from mice fed a griseofulvin-containing diet for 2 months (a, e) and 2.5 months (b, d). The liver shown in f is from a mouse fed griseofulvin for 2.5 months, followed by a griseofulvin-free diet for another week. (g-i) Livers of animals that have received griseofulvin for 2.5 months, then a griseofulvin-free diet for another 2.5 months, then, for reinduction of MBs, again the griseofulvin-containing diet for 4 days. Note specific and strong decoration of MBs with antibodies to prekeratin and isolated human MBs (g, h) but not with antibodies to murine vimentin (i). MBs in different stages of development from finely granular to coarse (a, d, e) and in stages of confluence up to the typical, often perinuclear MB (d, e) are demonstrated. These different stages can occur simultaneously in adjacent liver cells (d, e). f shows late stages of MBs (MB involution) with weakly staining centers. Bars denote 20 Aim. Downloaded by guest on September 25, 2021 4116 Medical Sciences: Denk et al. Proc. Natl. Acad. Sci. USA 76 (1979) light microscopic observations, to occur in livers of human alcoholics. The lack of decoration of MB aggregates, reinduced in the griseofulvin-pretreated liver by griseofulvin and by colchicine, by antibodies to murine vimentin refutes the possibility that these reinduced MBs are aggregates of filaments of the vimentin-type, which are well known to form perinuclear whorls in various cells growing in vitro under the influence of colchicine (9, 15, 24). Recently, antibodies to constitutive proteins of cytoskeletal intermediate-sized filaments have been produced in various laboratories and have been used for identifying, differentiating, and localizing different types of intermediate-sized filaments (9, 17, 24-26). With this method the following major filament types have been distinguished: (i) the type containing desmin, specific for myogenic cells; (ii) the type containing prekeratin-like proteins characteristic of epithelial cells; (iii) neurofi- laments specific for neuronal cells; and (iv) the filament type containing vimentin, which is characteristic of all mesenchymal cells but has also been observed in epithelial cells and myogenic cells grown in vitro. Our study on MB formation in liver pre- sents an example of the application of such antibodies to the examination of possible pathologic disorders of component proteins of intermediate-sized filaments, in this case of the prekeratin type. This study was supported in part by Fonds zur Forderung der Wissenschaftlichen Forschung, Vienna, Austria, Grant 3580. 1. Mallory, F. B. (1911) Bull. Johns Hopkins Hosp. 22, 69-75. 2. Biava, C. (1964) Lab. Invest. 13,301-320. 3. Yokoo, H., Minick, 0. T., Batti, F. & Kent, G. (1972) Am. J. Pathol. 69, 25-40. 4. Wiggers, K. D., French, S. W., French, B. A. & Carr, B. N. (1973) Lab. Invest. 29,652-658. 5. Franke, W. W., Denk, H., Schmid, E., Osborn, M. & Weber, K. (1979) Lab. Invest., 40, 207-220. 6. Denk, H., Eckerstorfer, R., Gschnait, F., Konrad, K. & Wolff, K. (1976) Lab. Invest. 35,377-382. FIG. 3. Small peripheral MB filament aggregates subjacent 7. Tinberg, H. M., Regan, R. J., Geier, E. A., Peterson, G. E. & to the liver cell membrane characteristic for MB dispersion after French, S. W. (1978) Lab. Invest. 39, 483-490. withdrawal of griseofulvin from mice as revealed by immunofluo- 8. Franke, W. W., Weber, K., Osborn, M., Schmid, E. & Freuden- rescence microscopy with antisera to isolated human MBs (a) and stein, C. (1978) Exp. Cell Res. 116,429-445. bovine hoof stratum corneum prekeratin fraction PK 2 (b). Bars de- 9. Franke, W. W., Schmid, E., Osborn, M. & Weber, K. (1978) Proc. note 20,pm. Natl. Acad. Sci. USA 75,5034-5038. 10. Denk, H. & Eckerstorfer, R. (1977) Lab. Invest. 36,563-565. 11. Freudenstein, C., Franke, W. W., Osborn, M. & Weber, K. (1978) prekeratinous substances) in some MBs or in MBs in general. Cell Biol. Int. Rep. 2,591-600. 12. Drochmans, P., Freudenstein, C., Watson, J. C., Laurent, L., In several other epithelial cells, abnormal hyperkeratosis has Keenan, T., Stadler, J., Leloup, R. & Franke, W. W. (1978) J. Cell been correlated with vitamin A deficiency (cf. ref. 18). In Biol. 79, 427-443. preliminary biochemical determinations of vitamin A content 13. Franke, W. W., Appelhans, B., Schmid, E., Freudenstein, C., of mouse liver under griseofulvin treatment and during sub- Osborn, M. & Weber, K. (1979) Eur. J. Cell Biol., in press. 14. French, S. W., Ihrig, M. J. & Norum, M. L. (1972) Lab. Invest. sequent drug withdrawal, a correlation of appearance and size 26,240-244. of MBs with lowered hepatic vitamin A levels has been observed 15. Franke, W. W., Schmid, E., Kartenbeck, J., Mayer, D., Hacker, (unpublished results). For example, we have found hepatic H. J., Bannasch, P., Osborn, M., Weber, K., Denk, H., Wanson, vitamin A contents of 30% and 13% of that present in control J. C. & Drochmans, P. (1979) Biol. Cell. 34, 99-110. 12 of 16. French, S. W., Sim, J. S. & Caldwell, M. G. (1977) in Membrane animals after and 60 days, respectively, griseofulvin Alterations as Basis of Liver Injury, eds. Popper, H., Bianchi, treatment. These findings, together with clinical and patho- L. & Reutter, W. (MTP, St. Leonardgate, Lancaster, England), logical observations of reduced vitamin A contents in livers and pp. 311-325. blood sera of patients with various forms of liver damage, al- 17. Sun, T. T. & Green, H. (1977) Cell 14, 469-476. coholic included 19 and see also ref. 21 for 18. Hicks, R. M. (1976) in Progress in Differentiation Research, ed. hepatitis (refs. 20; Miiller-B3rat, N. (North-Holland, Amsterdam), pp. 339-353. review), as well as in other conditions known to result in MB 19. Popper, H., Steigman, F., Meyer, K. A. & Zevin, S. S. (1943) Arch. formation (22), are compatible with the notion that vitamin A Int. Med. 72,439-460. deficiency may be involved in the initiation of MBs as it is in 20. Morrison, J. A., Russel, R. M., Carney, E. A. & Oaks, E. V. (1976) hyperkeratotic deviations in other epithelia. Gastroenterology 71, 922. 21. Mezey, E. (1978) Gastroenterology 74,770-783. Immunofluorescence microscopy with antibodies to purified 22. Scott, H. W., Law, D. H., Sandstead, H. H., Lanier, V. C. & prekeratin is a very sensitive and specific method for studying Younger, R. K. (1970) Ann. Surg. 171,770-782. the formation and involution of MBs. It is evident that MBs 23. Christoffersen, P. (1972) Acta Pathol. Microbiol. Scand. Sect. develop by coalescence of small precursor granules of preker- A 80, 705-712. 24. Bennett, G. S., Fellini, S. A., CroopJ. M., Otto, J. J., Bryan, J. & atin-rich filamentous material. Such a mode of development Holtzer, H. (1978) Proc. Natl. Acad. SVd. USA 75,4364-4368. from small aggregates of MB material has already been sug- 25. Hynes, R. 0. & Destree, A. T. (1978) Cell 13, 151-163. gested by Mallory (1) and Christoffersen (23), on the basis of 26. Lazarides, E. & Balzer, D. R. (1978) Cell 14, 429-438. Downloaded by guest on September 25, 2021