Mallory bodies revisited
2000; Elsevier BV; Volume: 32; Issue: 4 Linguagem: Inglês
10.1016/s0168-8278(00)80233-0
ISSN1600-0641
AutoresHelmut Denk, Cornelia Stumptner, Kurt Zatloukal,
Tópico(s)Skin and Cellular Biology Research
ResumoSince their first description in 1911 by F.B. Mallory (1.Mallory FB Cirrhosis of the liver: five different lesions from which it may arise.Bull Johns Hopkins Hosp. 1911; 22: 69-75Google Scholar, 2.Denk H Franke WW Kerjaschki D Eckersdorfer R Mallory bodies in experimental animals and man.Int Rev Exp Pathol. 1979; 20: 77-121PubMed Google Scholar, 3.Jensen K Gluud C The Mallory body: morphological, clinical and experimental studies.Hepatology. 1994; 20 (part 1 of a literature survey): 1061-1077Crossref PubMed Scopus (80) Google Scholar, 4.Jensen K Gluud C The Mallory body: theories on development and pathological significance.Hepatology. 1994; 20 (part 2 of a literature survey): 1330-1342Crossref PubMed Scopus (44) Google Scholar, 5.Denk H Lackinger E Cytoskeleton in liver diseases.Semin Liver Dis. 1986; 6: 199-211Crossref PubMed Scopus (38) Google Scholar, 6.French SW Nash J Shitabata P Kachi K Hara C Chedid A et al.Pathology of alcoholic liver disease.Semin Liver Dis. 1993; 13: 154-169Crossref PubMed Scopus (124) Google Scholar), Mallory bodies (MBs) or alcoholic hyalin have stimulated the interest of hepatopathologists for several reasons:1.(i) MBs are characteristic cytoplasmic hyaline inclusions in hepatocytes and thus resemble a peculiar manifestation of liver cell injury.2.(ii) MBs are morphologic hallmarks particularly of alcoholic and non-alcoholic steatohepatitis, liver disorders with poorly understood pathogenesis but considerable clinical importance.3.(iii) MBs share morphologic and physicochemical features with inclusion bodies associated with chronic degenerative disorders of the central nervous system, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, diseases with etiology and pathogenesis still undetermined. Thus, elucidation of the mechanisms involved in MB formation and itsconsequences for the affected cell may throw light not only on a special type of liver cell injury but also on degenerative disorders affecting other organs and tissues. MBs are characteristically associated with alcoholic and non-alcoholic steatohepatitis (NASH), but are also present in benign and malignant hepatocellular neoplasms, and a diversity of metabolic, toxic and chronic cholestatic liver disorders (for review, see 6.French SW Nash J Shitabata P Kachi K Hara C Chedid A et al.Pathology of alcoholic liver disease.Semin Liver Dis. 1993; 13: 154-169Crossref PubMed Scopus (124) Google Scholar, 7.Hall P Pathological spectrum of alcoholic liver disease.in: Hall P Alcoholic Liver Disease. Pathology and Pathogenesis. Edward Arnold, London, Boston, Melbourne, Auckland1995: 41-68Google Scholar, 8.Ishak KG Zimmerman HJ Ray MB Alcoholic liver disease: pathologic, pathogenetic and clinical aspects. 15. Clin Exp Res, Alcoholism1991: 45-66Google Scholar, 9.Zimmerman HJ Ishak KG Non-alcoholic steatohepatitis and other forms of pseudo-alcoholic liver disease.in: Hall P Alcoholic Liver Disease. Pathology and Pathogenesis. Edward Arnold, London, Boston, Melbourne, Auckland1995: 175-198Google Scholar, 10.James OFW Day CP Non-alcoholic steatohepatitis (NASH): a disease of emerging identity and importance.J Hepatol. 1998; 20: 495-501Abstract Full Text PDF Scopus (357) Google Scholar, 11.Baptista A Bianchi L DeGroote J Desmet VJ Gedigk P Korb G et al.Alcoholic liver disease: morphological manifestations. Review by an international group.Lancet. 1981; i: 707-711Google Scholar, 12.Burt AD Mutton A Day CP Diagnosis and interpretation of steatosis and steatohepatitis.Semin Diagnostic Pathol. 1998; 15: 246-258PubMed Google Scholar). Their appearance is related to alterations of the cytokeratin (CK) intermediate filament (IF) cytoskeleton of hepatocytes (5.Denk H Lackinger E Cytoskeleton in liver diseases.Semin Liver Dis. 1986; 6: 199-211Crossref PubMed Scopus (38) Google Scholar, 13.Franke WW Denk H Schmid E Osborn M Weber K Ultrastructural, biochemical and immunologic characterization of Mallory bodies in livers of griseofulvin-treated mice. Fimbriated rods of filaments containing prekeratin-like polypeptides.Lab Invest. 1979; 40: 207-220PubMed Google Scholar, 14.Katsuma Y Swierenga SHH Khettry U Marceau N French SW Changes in the cytokeratin intermediate filament cytoskeleton associated with Mallory body formation in mouse and human liver.Hepatology. 1987; 7: 1215-1223Crossref PubMed Scopus (48) Google Scholar). The spectrum of alcoholic liver disease comprises steatosis, alcoholic hepatitis (alcoholic steatohepatitis), fibrosis and finally cirrhosis (6.French SW Nash J Shitabata P Kachi K Hara C Chedid A et al.Pathology of alcoholic liver disease.Semin Liver Dis. 1993; 13: 154-169Crossref PubMed Scopus (124) Google Scholar, 7.Hall P Pathological spectrum of alcoholic liver disease.in: Hall P Alcoholic Liver Disease. Pathology and Pathogenesis. Edward Arnold, London, Boston, Melbourne, Auckland1995: 41-68Google Scholar, 8.Ishak KG Zimmerman HJ Ray MB Alcoholic liver disease: pathologic, pathogenetic and clinical aspects. 15. Clin Exp Res, Alcoholism1991: 45-66Google Scholar, 11.Baptista A Bianchi L DeGroote J Desmet VJ Gedigk P Korb G et al.Alcoholic liver disease: morphological manifestations. Review by an international group.Lancet. 1981; i: 707-711Google Scholar, 12.Burt AD Mutton A Day CP Diagnosis and interpretation of steatosis and steatohepatitis.Semin Diagnostic Pathol. 1998; 15: 246-258PubMed Google Scholar). Steatosis, a common and reversible consequence of alcohol intoxication, appears within a relatively short time of abuse. More serious and life-threatening consequences, namely alcoholic hepatitis and cirrhosis, develop only in 20 to 40% of heavy and chronic drinkers (3.Jensen K Gluud C The Mallory body: morphological, clinical and experimental studies.Hepatology. 1994; 20 (part 1 of a literature survey): 1061-1077Crossref PubMed Scopus (80) Google Scholar, 6.French SW Nash J Shitabata P Kachi K Hara C Chedid A et al.Pathology of alcoholic liver disease.Semin Liver Dis. 1993; 13: 154-169Crossref PubMed Scopus (124) Google Scholar), which suggests a multifactorial pathogenesis, including environmental and genetic factors in addition to toxic effects of ethanol and its metabolites (3.Jensen K Gluud C The Mallory body: morphological, clinical and experimental studies.Hepatology. 1994; 20 (part 1 of a literature survey): 1061-1077Crossref PubMed Scopus (80) Google Scholar, 8.Ishak KG Zimmerman HJ Ray MB Alcoholic liver disease: pathologic, pathogenetic and clinical aspects. 15. Clin Exp Res, Alcoholism1991: 45-66Google Scholar, 15.Bosron WF Ehrig T Li TK Genetic factors in alcohol metabolism and alcoholism.Semin Liver Dis. 1993; 13: 126-135Crossref PubMed Scopus (164) Google Scholar, 16.Grove J Daly AK Bassendine MF Day CP Association of a tumor necrosis factor promoter polymorphism with susceptibility to alcoholic steatohepatitis.Hepatology. 1997; 26: 143-146Crossref PubMed Scopus (252) Google Scholar, 17.Lindros KO Alcoholic liver disease: pathobiological aspects.J Hepatol. 1995; 23: 7-15PubMed Google Scholar). Several risk factors, alone or in combination, may determine individual susceptibility. Alcoholic hepatitis is morphologically characterized by liver cell injury with macro- and/or microvesicular steatosis of various degrees, ballooning of hepatocytes, necrosis, cholestasis, occurrence of MBs, mostly neutrophil granulocytic inflammation and pericellular and perivenular fibrosis. A diversity of non-alcoholic liver disorders, collectively termed NASH, shares some or even all morphologic features with alcoholic hepatitis (9.Zimmerman HJ Ishak KG Non-alcoholic steatohepatitis and other forms of pseudo-alcoholic liver disease.in: Hall P Alcoholic Liver Disease. Pathology and Pathogenesis. Edward Arnold, London, Boston, Melbourne, Auckland1995: 175-198Google Scholar, 10.James OFW Day CP Non-alcoholic steatohepatitis (NASH): a disease of emerging identity and importance.J Hepatol. 1998; 20: 495-501Abstract Full Text PDF Scopus (357) Google Scholar, 11.Baptista A Bianchi L DeGroote J Desmet VJ Gedigk P Korb G et al.Alcoholic liver disease: morphological manifestations. Review by an international group.Lancet. 1981; i: 707-711Google Scholar, 12.Burt AD Mutton A Day CP Diagnosis and interpretation of steatosis and steatohepatitis.Semin Diagnostic Pathol. 1998; 15: 246-258PubMed Google Scholar, 18.Ludwig J Viggiano TR McGill DB Ott BJ Non alcoholic steatohepatitis. Mayo Clinic experiences with a hitherto unnamed disease.Mayo Clin Proc. 1980; 55: 434-438PubMed Google Scholar). Particularly morbid obesity, weight-reducing surgery and type II diabetes mellitus may closely mimick in their liver pathology alcoholic liver disease, including the tendency to progress to cirrhosis. Drugs and toxins (like amiodarone, perhexiline maleate, 4,4-diethylaminoethoxyhexestrol) are able to produce similar lesions in human liver, and so do griseofulvin (GF) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in mice (19.Denk H Gschnait F Wolff K Hepatocellular hyalin (Mallory bodies) in long term griseofulvin-treated mice: a new experimental model for the study of hyalin formation.Lab Invest. 1975; 32: 773-776PubMed Google Scholar, 20.Tsunoo C Harwood TR Arak S Yokoo H Cytoskeletal alterations leading to Mallory body formation in livers of mice fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine.J Hepatol. 1987; 5: 85-97Abstract Full Text PDF PubMed Scopus (20) Google Scholar). In contrast to earlier views, MBs do not seem to be signs of adverse prognosis and their presence does not significantly influence mortality rates (for review, see 3.Jensen K Gluud C The Mallory body: morphological, clinical and experimental studies.Hepatology. 1994; 20 (part 1 of a literature survey): 1061-1077Crossref PubMed Scopus (80) Google Scholar, 4.Jensen K Gluud C The Mallory body: theories on development and pathological significance.Hepatology. 1994; 20 (part 2 of a literature survey): 1330-1342Crossref PubMed Scopus (44) Google Scholar). Therefore, on the basis of clinico-pathologic experience MBs seem to be indicators of a special type of liver cell injury rather than active players in the necroinflammatory process; nevertheless, the analysis of their composition and pathogenesis is essential for our understanding of the pathogenetic principles involved in chronic alcoholic liver disease and, at least morphologically, related disorders. MBs are cytoplasmic inclusions in hepatocytes, ranging in size from small granules to large irregular masses (Fig. 1; see ref. 2.Denk H Franke WW Kerjaschki D Eckersdorfer R Mallory bodies in experimental animals and man.Int Rev Exp Pathol. 1979; 20: 77-121PubMed Google Scholar, 6.French SW Nash J Shitabata P Kachi K Hara C Chedid A et al.Pathology of alcoholic liver disease.Semin Liver Dis. 1993; 13: 154-169Crossref PubMed Scopus (124) Google Scholar, for further information). They display a predominantly filamentous ultrastructure consisting of 10–20-nm-thick filaments coated by fuzzy more electron-dense material2.Denk H Franke WW Kerjaschki D Eckersdorfer R Mallory bodies in experimental animals and man.Int Rev Exp Pathol. 1979; 20: 77-121PubMed Google Scholar,13.Franke WW Denk H Schmid E Osborn M Weber K Ultrastructural, biochemical and immunologic characterization of Mallory bodies in livers of griseofulvin-treated mice. Fimbriated rods of filaments containing prekeratin-like polypeptides.Lab Invest. 1979; 40: 207-220PubMed Google Scholar,21.Yokoo H Minick OT Batti F Kent G Morphologic variants of alcoholic hyalin.Am J Pathol. 1972; 69: 25-40PubMed Google Scholar,Fig. 2) . According to their ultrastructural appearance, MBs can be classified as type I (bundles of filaments in parallel arrays), type II (randomly oriented filaments) and type III (granular and amorphous material). Types II and III prevail and are often seen in combination of type II at the periphery and type III in the center (21.Yokoo H Minick OT Batti F Kent G Morphologic variants of alcoholic hyalin.Am J Pathol. 1972; 69: 25-40PubMed Google Scholar). MBs are usually present in enlarged, ballooned hepatocytes, most of them looking vital with large nuclei and nucleoli. Most MB-containing hepatocytes lack large fat vacuoles but may display microvesicular steatosis. Although MBs are not obligatory for the diagnosis of alcoholic hepatitis in hematoxylin-eosin stained sections, more sensitive immunohistochemical stainings using CK or ubiquitin antibodies disclose MBs in most cases of otherwise morphologically characteristic alcoholic hepatitis. Neutrophils predominantly, but not constantly, cluster around MB-bearing hepatocytes. Pericellular fibrosis seems to be more pronounced around MB-containing cells (8.Ishak KG Zimmerman HJ Ray MB Alcoholic liver disease: pathologic, pathogenetic and clinical aspects. 15. Clin Exp Res, Alcoholism1991: 45-66Google Scholar, 11.Baptista A Bianchi L DeGroote J Desmet VJ Gedigk P Korb G et al.Alcoholic liver disease: morphological manifestations. Review by an international group.Lancet. 1981; i: 707-711Google Scholar).Fig. 2Electron microscopy of MBs in human alcoholic hepatitis (a; Hu - AH), 2 months DDC (b; 2 m DDC-ML)- and 4 months GF (c; 4 m GF-ML)-fed mouse livers. The figures show the filamentous ultrastructure of MBs with filaments in irregular arrangement (type II). Note identical morphology of human and murine MBs. Bar, 500 nm.View Large Image Figure ViewerDownload (PPT) Animal models are indispensable tools for elucidation of pathogenetic principles involved in human disease, although usually not all features of human disease can be reproduced in a single model. Several animal models for human alcoholic hepatitis and NASH have been designed (2.Denk H Franke WW Kerjaschki D Eckersdorfer R Mallory bodies in experimental animals and man.Int Rev Exp Pathol. 1979; 20: 77-121PubMed Google Scholar, 19.Denk H Gschnait F Wolff K Hepatocellular hyalin (Mallory bodies) in long term griseofulvin-treated mice: a new experimental model for the study of hyalin formation.Lab Invest. 1975; 32: 773-776PubMed Google Scholar, 20.Tsunoo C Harwood TR Arak S Yokoo H Cytoskeletal alterations leading to Mallory body formation in livers of mice fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine.J Hepatol. 1987; 5: 85-97Abstract Full Text PDF PubMed Scopus (20) Google Scholar, 22.Lieber CS DeCarli LM Sorrell MF Experimental methods of ethanol administration.Hepatology. 1989; 10: 501-510Crossref PubMed Scopus (243) Google Scholar, 23.French SW Morimoto M Tsukamoto H Animal models of alcohol-associated liver injury.in: Hall P Alcoholic Liver Disease. Pathology and pathogenesis. Edward Arnold, London, Boston, Melbourne, Auckland1995: 279-296Google Scholar). In the alcohol-feeding models fatty liver, centrilobular necrosis, inflammation and fibrosis were markedly exaggerated if ethanol was administered together with a diet rich in polyunsaturated fat (22.Lieber CS DeCarli LM Sorrell MF Experimental methods of ethanol administration.Hepatology. 1989; 10: 501-510Crossref PubMed Scopus (243) Google Scholar, 23.French SW Morimoto M Tsukamoto H Animal models of alcohol-associated liver injury.in: Hall P Alcoholic Liver Disease. Pathology and pathogenesis. Edward Arnold, London, Boston, Melbourne, Auckland1995: 279-296Google Scholar, 24.Tsukamoto H French SW Benson N Delgado G Rao GA Larkin EC et al.Severe and progressive steatosis and focal necrosis in rat liver induced by continuous intragastric infusion of ethanol and low fat diet.Hepatology. 1985; 5: 224-232Crossref PubMed Scopus (195) Google Scholar, 25.Tsukamoto H Gad K French SW Insights into the pathogenesis of alcoholic liver necrosis and fibrosis: status report.Hepatology. 1990; 12: 599-608Crossref PubMed Scopus (161) Google Scholar, 26.Tsukamoto H Horne W Kamimura S Niemelä O Parkkila S Yiä-Herttuala S et al.Experimental liver cirrhosis induced by alcohol and iron.J Clin Invest. 1995; 96: 620-630Crossref PubMed Scopus (452) Google Scholar, 27.Tsukamoto H Cheng S Blander WS Effects of dietary polyunsaturated fat on ethanol-induced Ito cell activation.Am J Physiol. 1996; 270: G581-G586PubMed Google Scholar). MBs and associated liver cell changes, such as ballooning and pericellular accumulation of polymorphonuclear leukocytes ("satellitosis"), are, however, not evoked in animal livers under these conditons. On the other hand, chronic GF and DDC feeding of mice leads to the appearance of MBs mostly in enlarged hepatocytes, but also to liver cell necrosis, steatosis, fibrosis and ductular reaction, in addition to protoporphyrin accumulation. Satellitosis is occasionally seen but less pronounced than in human alcoholic hepatitis (19.Denk H Gschnait F Wolff K Hepatocellular hyalin (Mallory bodies) in long term griseofulvin-treated mice: a new experimental model for the study of hyalin formation.Lab Invest. 1975; 32: 773-776PubMed Google Scholar, 20.Tsunoo C Harwood TR Arak S Yokoo H Cytoskeletal alterations leading to Mallory body formation in livers of mice fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine.J Hepatol. 1987; 5: 85-97Abstract Full Text PDF PubMed Scopus (20) Google Scholar, 28.Denk H Eckerstorfer R Gschnait F Konrad K Wolff K Experimental induction of hepatocellular hyalin (Mallory bodies) in mice by griseofulvin treatment. I. Light microscopic observations.Lab Invest. 1976; 35: 377-382PubMed Google Scholar; see also 2.Denk H Franke WW Kerjaschki D Eckersdorfer R Mallory bodies in experimental animals and man.Int Rev Exp Pathol. 1979; 20: 77-121PubMed Google Scholar, for review). Thus, although the latter models are obviously not models for alcohol toxicity in the strict sense, they provide important morphologic features of alcoholic hepatitis and NASH and allow studies of specific aspects of their pathogenesis. The common pathogenetic pathways of alcoholic hepatitis and NASH remain to be uncovered. However, radical injury, believed to be of central importance in alcoholic liver disease, also seems to be involved in NASH and related models (29.Ortiz de Montellano PR Beilan HS Kunze KL N-alkylprotoporphyrin IX formation in 3,5-dicarbethoxy-1,4-dihydrocollidine-treated rats. Transfer of the alkyl group from the substrate to the porphyrin.J Biol Chem. 1981; 256: 6708-6713Abstract Full Text PDF PubMed Google Scholar, 30.Knasmüller S Parzefall W Helma C Kassie F Ecker S Schulte-Hermann R Toxic effects of griseofulvin: disease models, mechanisms, and risk assessment.Crit Rev Toxicol. 1997; 27: 495-537Crossref PubMed Scopus (62) Google Scholar). The first evidence of a relationship between MBs and the CK cytoskeleton was provided by observations that MBs react with CK antibodies and contain polypeptides closely resembling CKs of hepatocytes (Fig. 3, Fig. 4). An additional non-CK protein component with a molecular weight between 62–65 kDa has been found in MBs but not in CKs of normal hepatocytesFig. 4,2.Denk H Franke WW Kerjaschki D Eckersdorfer R Mallory bodies in experimental animals and man.Int Rev Exp Pathol. 1979; 20: 77-121PubMed Google Scholar,13.Franke WW Denk H Schmid E Osborn M Weber K Ultrastructural, biochemical and immunologic characterization of Mallory bodies in livers of griseofulvin-treated mice. Fimbriated rods of filaments containing prekeratin-like polypeptides.Lab Invest. 1979; 40: 207-220PubMed Google Scholar). The predominantly filamentous ultrastructure of MBs together with diminution of hepatocellular cytoplasmic CK-IF immunostaining was originally (and erroneously) interpreted to indicate that MBs result from collapse of the CK-IF network. However, poly- and monoclonal CK antibodies, including those which recognize conformation-dependent epitopes on CKs, revealed that the MB-associated CK polypeptides are not organized as in normal IFs (2.Denk H Franke WW Kerjaschki D Eckersdorfer R Mallory bodies in experimental animals and man.Int Rev Exp Pathol. 1979; 20: 77-121PubMed Google Scholar, 13.Franke WW Denk H Schmid E Osborn M Weber K Ultrastructural, biochemical and immunologic characterization of Mallory bodies in livers of griseofulvin-treated mice. Fimbriated rods of filaments containing prekeratin-like polypeptides.Lab Invest. 1979; 40: 207-220PubMed Google Scholar, 31.Denk H Franke WW Eckerstorfer R Schmid E Kerjaschki D Formation and involution of Mallory bodies (alcoholic hyalin) in murine and human liver revealed by immunofluorescence microscopy with antibodies to prekeratin.Proc Natl Acad Sci USA. 1979; 76: 4112-4116Crossref PubMed Scopus (96) Google Scholar, 32.Denk H Franke WW Dragosics B Zeiler I Pathology of cytoskeleton of liver cells: demonstration of Mallory bodies (alcoholic hyalin) in murine and human hepatocytes by immunofluorescence microscopy using antibodies to cytokeratin polypeptides from hepatocytes.Hepatology. 1981; 1: 9-20Crossref PubMed Scopus (113) Google Scholar, 33.Denk H Krepler R Lackinger E Artlieb U Franke WW Immunological and biochemical characterization of the keratinrelated component of Mallory bodies: a pathological pattern of hepatocytic cytokeratins.Liver. 1982; 2: 165-175Crossref PubMed Scopus (43) Google Scholar, 34.Okanoue T Ohta M Ou O Kachi K Kagawa K Yuki T et al.Relationship of Mallory bodies to intermediate filaments in hepatocytes. A scanning electron microscopy study.Lab Invest. 1985; 53: 534-540PubMed Google Scholar, 35.Hazan R Denk H Franke WW Lackinger E Schiller DL Change of cytokeratin organization during development of Mallory bodies as revealed by a monoclonal antibody.Lab Invest. 1986; 54: 543-553PubMed Google Scholar, 36.Zatloukal K Boeck G Rainer I Denk H Weber K High molecular weight components are main constituents of Mallory bodies isolated with a fluorescence activated cell sorter.Lab Invest. 1991; 64: 200-206PubMed Google Scholar, 37.Zatloukal K Spurej G Rainer I Lackinger E Denk H Fate of Mallory body-containing hepatocytes: disappearance of Mallory bodies and restoration of the hepatocytic intermediate filament cytoskeleton after drug withdrawal in the griseofulvintreated mouse.Hepatology. 1990; 11: 652-661Crossref PubMed Scopus (33) Google Scholar). This is in line with data obtained by biochemical and immunochemical analyses of isolated and purified MBs which showed that in MBs the equimolar ratio of type I and type II CKs (i.e. CK18 and CK8), which is essential for normal IF assembly, is not maintained and that CK8 prevails over CK18 (35.Hazan R Denk H Franke WW Lackinger E Schiller DL Change of cytokeratin organization during development of Mallory bodies as revealed by a monoclonal antibody.Lab Invest. 1986; 54: 543-553PubMed Google Scholar, 36.Zatloukal K Boeck G Rainer I Denk H Weber K High molecular weight components are main constituents of Mallory bodies isolated with a fluorescence activated cell sorter.Lab Invest. 1991; 64: 200-206PubMed Google Scholar). Interestingly, in DDC-fed mice already in early stages of intoxication a shift of the CK8 to CK18 ratio towards CK8 occurred when tested by immunoblots in liver homogenates (Fig. 5).Fig. 4One-dimensional SDS-polyacrylamide gel electrophoresis of MBs isolated and semipurified from the livers of 2 months DDC-fed mice. Note the presence of three major polypeptide bands (I, II, III). Bands II and III react in immunoblotting with CK antibodies and thus are of CK nature. Band II corresponds to CK8 and band III to CK18. Band I is a non-CK component. In addition, poorly soluble high molecular material remains at the interphase between stacking and resolving gels.View Large Image Figure ViewerDownload (PPT)Fig. 5The upper panel shows relative amounts of CKs 8 (K8) and 18 (K18) protein as revealed in mouse liver homogenates by immunoblotting with a polyclonal antibody directed to CKs 8 and 18. Note that in mice fed a normal diet CKs 8 and 18 are present in a 1:1 ratio. Already after 1 week of DDC feeding (1 w DDC) CK8 clearly prevails over CK18 and this situation is maintained after a 2.5-month (2.5 m DDC) DDC feeding period. The lower CK protein levels after prolonged DDC intoxication (2.5 m DDC) may reflect the higher number of hepatocytes with diminished intermediate filament cytoskeleton. The increased amount of CK8 resembles unassembled polypeptide. The lower panel shows mRNA levels in mouse liver homogenates. DDC intoxication (for 1 week and 2.5 months) leads to a conspicuous increase of CKs 8 (K8) and 18 (K18) mRNA levels. A peak level is already reached after 1 week (1 w DDC) of DDC feeding.View Large Image Figure ViewerDownload (PPT) In addition to CKs a variety of non-CK components were identified in MBs. They include the MM120-1-, SMI 31- and MPM-2-reactive antigens, ubiquitin and high and low molecular weight heat shock proteins as revealed by immunohistochemistry and immunoblot analysesFig. 3,38.Lowe J Blanchard A Morell K Lennox G Reynolds L Billet M et al.Ubiquitin is a common factor in intermediate filament inclusion bodies of diverse type in man, including those of Parkinson's disease, Pick's disease, and Alzheimer's disease, as well as Rosenthal fibers in cerebellar astrocytomas, cytoplasmic bodies in muscle, and Mallory bodies in alcoholic liver disease.J Pathol. 1988; 155: 9-15Crossref PubMed Scopus (470) Google Scholar,39.Ohta M Marceau N Perry G Manetto V Gambetti P Autilio-Gambetti L et al.Ubiquitin is present on the cytokeratin intermediate filaments and Mallory bodies of hepatocytes.Lab Invest. 1988; 59: 848-856PubMed Google Scholar,40.Zatloukal K Denk H Spurej G Lackinger E Preisegger KH Franke WW High molecular weight component of Mallory bodies detected by a monoclonal antibody.Lab Invest. 1990; 62: 427-434PubMed Google Scholar,41.Preisegger KH Zatloukal K Spurej G Riegelnegg D Denk H Common epitopes of human and murine Mallory bodies and Lewy bodies as revealed by a neurofilament antibody.Lab Invest. 1992; 66: 193-199PubMed Google Scholar). The MM120-1 antigen is associated with a still uncharacterized high molecular weight protein that in vivo is an exclusive MB component (40.Zatloukal K Denk H Spurej G Lackinger E Preisegger KH Franke WW High molecular weight component of Mallory bodies detected by a monoclonal antibody.Lab Invest. 1990; 62: 427-434PubMed Google Scholar). In vitro studies revealed that MM120-1-protein can be induced in tissue culture cells by stress treatments (e.g., Ca-ionophore, sodium arsenite, or heat shock) but also by transfection with a human CK18 gene construct (Fig. 6), suggesting a relationship to stress proteins (42.Stumptner C Fuchsbichler A Denk H Zatloukal K Overexpression of cytokeratin induces accumulation of the Mallory body-specific component MM120-1 in vitro.Hepatology. 1996; 24: 129AAbstract Full Text PDF Scopus (119) Google Scholar). The antibody SMI 31, which is directed against a phosphorylated epitope present on neurofilaments and on abnormal tau protein associated with Alzheimer's neurofibrillary tangles, recognizes the non-CK MB component with an apparent molecular mass of 62 to 65 kDa and very acidic isoelectric pH around pH 4.5 (41.Preisegger KH Zatloukal K Spurej G Riegelnegg D Denk H Common epitopes of human and murine Mallory bodies and Lewy bodies as revealed by a neurofilament antibody.Lab Invest. 1992; 66: 193-199PubMed Google Scholar). Although the nature of the SMI 31-reactive MB component is as yet unclear, the apparently common phospho-epitope in MBs and neurofibrillary tangles implies that similar protein kinases (i.e. proline-directed kinases) are responsible for hyperphosphorylation in both instances (43.Nukina N Kosik KS Selkoe DJ Recognition of Alzheimer paired helical filaments by monoclonal neurofilament antibodies is due to crossreaction with tau protein.Proc Natl Acad Sci USA. 1987; 84: 3415-3419Crossref PubMed Scopus (165) Google Scholar, 44.Lichtenberg-Kraag B Mandelkow EM Biernat J Steiner B Schröter C Gustke N et al.Phosphorylation - dependent epitopes of neurofilament antibodies on tau protein and relationship with Alzheimer tau.Proc Natl Acad Sci USA. 1992; 89: 5384-5388Crossref PubMed Scopus (179) Google Scholar; for review, see 45.Mandelkow EM Mandelkow E Tau as a marker for Alzheimer's disease.Trends Biol Sci. 1993; 18: 480-483Abstract Full Text PDF PubMed Scopus (114) Google Scholar). The similarity in the phosphorylation state of MB and neurofibrillary tangle proteins is further underlined by the observation that the 62–65-kDa MB protein reacted in immunoblots with MPM-2 antibodies. The MPM-2 antibody also reacts with neurofibrillary tangles and is directed to hyperphosphorylated epitopes generated on diverse proteins by mitotic kinases in the M-phase of the cell cycle. Therefore, M-phase specific kinases, like p34 cdc2- and MAP-kinase, seem to create this epitope (46.Westendorf JM Rao PN Gerace L Cloning of cDNAs for Mphase phosphoproteins recognized by the MPM2 monoclonal antibody and determination of the phosphorylated epitope.Proc Natl Acad Sci USA. 1994; 91: 714-718Crossref PubMed Scopus (239) Google Scholar, 47.Vincent I Rosado M Davies P Mitotic mechanisms in Alzheimer's disease.J Cell Biol. 1996; 132: 413-425Crossref PubMed Scopus (352) Google Scholar, 48.Kondratick CM Vandre DD Alzheimer's disease neurofibrillary tangles contain mitosis-specific phosphoepitopes.J Neurochem. 1996; 67: 2405-2416Crossref PubMed Scopus (59) Google Scholar). This SMI 31/MPM-2-reactive MB component closely resembles in its electrophoretic coordinates p62 which has recently been identified by our group as the major protein constituent of intracellular hyaline bodies (not identical with MBs!) present in hepatocellular carcinoma cells (49.Stumptner C Heid H Fuchsbichler A Hauser H Mischinger HJ Zatloukal K et al.Analysis
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