Yonago Acta medica 2006;49:83–92

Mallory Bodies in Hepatocytes of Alcoholic Liver Diseaseand Primary Biliary Cirrhosis Contain-(Carboxymethyl)lysine-Modiﬁed Cytokeratin,but not those in Hepatic Carcinoma Cells
Masako Kato, Shinsuke Kato*, Seikoh Horiuchi†, Ryoji Nagai†, Yasushi Horie andKazuhiko Hayashi‡
Pathology Division, Tottori University Hospital, Yonago 683-8504, *Department of Neuropathology, Institute of Neurological Sciences, Tottori University Faculty of Medicine, Yonago 683-8504,† Department of Medical Biochemistry, Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Department of Biochemistry, Kumamoto University School of Medicine, Kumamoto 860-0811 and ‡Division of Molecular Pathology, Department of Microbiologyand Pathology, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8503
JapanMallory bodies (MBs) are intracytoplasmic bodies seen in hepatocytes of alcoholic liverdisease, primary biliary cirrhosis and hepatocellular carcinoma. However, the mechanism of MB formation has not been fully understood. Proteins could be modiﬁed to advanced glycation end products (AGEs) after long-term incubation with reducing sugar.
AGEs are known to accumulate in several tissues in aging and age-enhanced disorders.
To study the possible glycation process in the formation of MBs, hepatocytes of 80 humanliver tissues with MBs were subjected to immunohistochemical analyses with ﬁve AGEs,
two markers for oxidative stress proteins (OSPs) and four stress-response proteins (SRPs).
MBs in hepatocytes of primary biliary cirrhosis and alcoholic liver disease were strongly
positive for -(carboxymethyl)lysine (CML) and weakly positive for pyrraline. MBs in
hepatocellular carcinomas were negative for both CML and pyrraline. No significant
immunoreactivity was detected in MBs for other AGEs, such as -(carboxyethyl)lysine,pentosidine, and 3DG-imidazolone, or for OSPs and SRPs. Stainings for cytokeratin,a major protein component of MBs, and CML were co-localized. Furthermore, immunoblot analysis suggested that cytokeratin of MBs was modiﬁed to AGE, since a single
protein band detected by a monoclonal anti-CML had a molecular weight identical to cytokeratin. The absence of the CML signal in MBs of hepatocellular carcinoma cells could
be explained by scarce content of cytokeratin in carcinoma MBs.
Key words: advanced glycation end product; cytokeratin; immunohistochemistry; Mallorybody; -(carboxymethyl)lysine
Abbreviations: ABC, avidin-biotin-immunoperoxidase complex; AEC, 3-amino-9-ethylcarbazole; AGE, advanced glycation end product; BSA, bovine serum albumin; CEL, -(carboxyethyl)lysine; CML, -(carboxymethyl)lysine; DAB,3,3'-diaminobenzidine tetrahydrochloride; HCC, hepatocellular carcinoma; H&E staining, hematoxylin and eosin staining-; HNE, 4-hydroxy-2-nonenal; MB, Mallory body; PAS, periodic acid Schiff; PBC, primary biliary cirrhosis; SDS,sodium dodecyl sulfate; SRP, stress-response protein

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M. Kato et al.

Alzheimer’s disease (Smith et al., 1994). AGEmodiﬁcation is known to occur in many proteins,
especially long-lived proteins, which makes theminsoluble, thus enhancing the deposition of modiﬁed proteins inside the cells or in the extracellularspace (Makino et al., 1995; Giardino et al., 1996).
Finally, AGE-modiﬁed proteins that are accumulated in the tissues exhibit direct toxic effects oncells biologically (Vlassara et al., 1994).
AGE-structures reported so far include ﬂuorescent and cross-linking structures such as pentosidine (Sell and Monnier, 1990) and crossline(Nakamura et al., 1992), and nonﬂuorescent andnon-cross-linked structures such as imidazolone(Niwa et al., 1997), - (carboxymethyl)lysine(CML) (Ahmed et al., 1986) and pyrraline (Hayaseet al., 1989). The purpose of the present study
was to investigate whether AGE-modification
could play a role in MB formation. To do thisend, human liver tissues with MBs were examinedimmunohistochemically using antibodies against
CML, -(carboxyethyl)lysine (CEL), pyrraline,pentosidine and 3DG-imidazolone. Our results
revealed that chemical modiﬁcation of cytokeratin by CML, a major antigenic AGE-structure, isinvolved in MB formation in hepatocytes in alcoholic liver disease and primary biliary cirrhosis
in contrast to no involvement of CML in MBs inHCC.

Mallory first described cytoplasmic hyaline degeneration in hepatocytes of alcoholic liver cirrhosis (Mallory, 1911). This cytoplasmic hyaline
inclusion has been called Mallory body (MB).
MB had been thought as speciﬁc to alcoholic liverdisease (Edmondson, 1986). However, they arealso associated with a number of non-alcoholic
hepatobiliary diseases, such as Indian childhood
cirrhosis, primary biliary cirrhosis (PBC), Wilson’s disease, hepatocellular carcinoma (HCC)and adenomatous hyperplasia (Jensen and Gluud,
1994a; Terada et al., 1989). Although several
different theories have been proposed for theformation of MBs (Jensen and Gluud, 1994b),
the mechanism of MB formation as well as theirdevelopmental and pathological significance hasremained unknown.
Ultrastructurally MBs consist of aggregatesof ﬁlaments (Yokoo et al., 1972), and aberrant intermediate ﬁlaments of cytokeratin polypeptides(Katsuma et al., 1987). Recently, hyperphosphorylation of cytokeratin 8 and 18 (Stumptneret al., 2000) or ubiquitination of cytokeratin proteins was revealed in MB formation (Yuan et al.,1996). MBs were also known to be positive for
B-crystallin immunohistochemically (Lowe et al.,1992). Namely, MBs contain protein components
such as cytokeratin, ubiquitin or B-crystallin.
Furthermore, the other chemical analyses demonstrated that MBs possess carbohydrates (Lyonand Christoffersen, 1971) and reducing sugar(Luisada-Opper et al., 1977) in addition to cytokeratin as the major protein component.
Long-term incubation of proteins with glucose
leads, through the formation of early products
such as Schiff base and Amadori rearrangement
products, to the formation of advanced glycation end products (AGEs). Since monoclonal or
polyclonal antibodies against these AGEs havebeen newly produced, immunological studies using anti-AGE antibodies have demonstrated accumulation of AGE-modiﬁed proteins in several
human tissues in association with aging (Arakiet al., 1992; Kimura et al., 1996) and several disorders such as diabetic complications (Makino etal., 1995), atherosclerosis (Kume et al., 1995) andMaterials and Methods
Patients
Eight hundred liver tissues from autopsy, biopsy
and surgical ﬁles kept in our Department and Division were surveyed from 1973 to 2000, and MBs
were found histologically in 80 cases (8 autopsy
cases, 11 biopsy cases and 61 surgical cases). The
80 cases with MBs included three PBC cases (females, aged 37 to 68 years), 17 cases of alcoholic
liver disease (15 males and 2 females, aged 33 to66 years) and 60 cases of HCC (52 males and 8females, aged 29 to 80 years). Surgical and autopsy specimens of normal liver tissues and other84

AGE-modiﬁcation in Mallory bodies

Table 1. Sources of primary antibodies and dilutions
Antibody

Clonality

Clone

Anti-MB-component protein antibody
Cytokeratin, broad
Monoclonal
B-crystallin
Polyclonal
Ubiquitin
Polyclonal
Anti-AGE antibody
CML

6D12
CMS10Dilution

Source (reference)

Ready-to-use
1:2501:1000

Nichirei (Tokyo, Japan)
J. E. Goldman (Iwaki et al., 1989)
S. H. Yen (Lee et al., 1989)

0.5 μg/mL
0.5 μg/mL
1.0 μg/mL
1.0 μg/mL
0.6 μg/mL
0.5 μg/mL

S. Horiuchi (Ikeda et al., 1996)
Kumamoto Immunochem. Lab. (Kumamoto, Japan)
S. Horiuchi (Hayase et al., 1989)
S. Horiuchi (Miyata et al., 1996)
Kumamoto Immunochem. Lab. (Kumamoto, Japan)
Kumamoto Immunochem. Lab. (Kumamoto, Japan)

Pyrraline
Pentosidine
CEL
3DG-imidazolone

Monoclonal
Monoclonal
Polyclonal
Polyclonal
Monoclonal
Monoclonal

Anti-OSP antibody
Acrolein
4-HNE

Monoclonal
Monoclonal

0.5 μg/mL
0.5 μg/mL

NOF (Tokyo, Japan)
JICA (Fukuroi, Japan)

Anti-SRP antibody
SRP27
SRP32
SRP72
SRP90

Monoclonal
Monoclonal
Monoclonal
Monoclonal

Ready-to-use
1:200
1:500
1:5000

BioGenex (San Rmon, CA)
Santa Cruz (Santa Cruz, CA)
Amersham (Buckinghamshire, United Kingdom)
Afﬁnity BioReagent (Neshanic Station, NJ)

KNH-30
JNH-27

AGE, advance glycation end product; CEL,-(carboxyethyl)lysine; CML,-(carboxymethyl)lysine; 4-HNE, 4-hydroxy-2nonenal; MB, Mallory body; OSP, oxidative stress protein; SRP, stress response protein.

enous peroxidase activity was quenched for 30
min with 0.3% H2O2. Sections were then washedin phosphate-buffered saline (PBS), pH 7.4. Normal sera isologous with each secondary antibody
were used as blocking reagents. Sections wereincubated with one of the primary antibodies
or PBS for 18 h at 4°C. As positive controls for
CML-immunostaining, atherosclerotic lesions in
the wall of aortae from control individuals wereused, biopsy specimens of diabetic kidney forpyrraline or pentosidine staining and atherosclerotic tissues also served as positive controls foracrolein and HNE stainings. Some sections wereincubated with anti-CML antibody that had beenpreabsorbed with 1 mg/mL of CML-modiﬁed bovine serum albumin (BSA). With respect to thepreabsorption test, some sections were incubatedwith anti-CML, anti-pyrraline, or anti-pentosidine
antibody that had been preabsorbed with excess
CML-, pyrraline- or pentosidine-modified BSA,
respectively. Bound antibodies were visualized
by the avidin-biotin-immunoperoxidase complex(ABC) method using the appropriate Vectastainorgan tissues from 10 individuals (7 males and 3females; aged 19 to 68 years) were also examined
as controls. The protocols were approved by theEthics Committee in Tottori University Faculty ofMedicine (No. 761).
Histology and immunohistochemistry
After ﬁxation in 10% buffered formalin, the specimens were embedded in parafﬁn, cut into 4-μmthick sections and examined by light microscopy.
Liver sections were stained by the following
routine methods: hematoxylin and eosin (H&E),
periodic acid-Schiff (PAS), digestive PAS, silver
and azan stainings. The distribution patterns of
MBs were classiﬁed according to the method ofNakanuma and Ohta (1986). HCCs were graded
into grades I, II and III according to the histological grading system of Edmondson and Steiner(1954). Serial sections were used for immunohistochemical analysis. The sources of primary
antibodies and their dilutions used are listed inTable 1. Sections were deparafﬁnized and endog85

M. Kato et al.

heated at 100°C for 5 min. Soluble protein extracts from the samples were separated on a SDSpolyacrylamide gel (10%–20% gradient, Bio-Rad)and transferred by electroblotting onto Immobilon
PVDF (Millipore, Bedford, MA). After blocking
with 5% skimmed milk for 30 min at room temperature, the blots were incubated with anti-CML
antibody (6D12) overnight at 4°C and visualizedwith the Vectastain ABC kit and DAB. Appropriate molecular weight markers (Bio-Rad) wereincluded in each run.

ABC kit (Vector Laboratories, Burlingame, CA)
and 3,3'-diaminobenzidine tetrahydrochloride(DAB) (Dako, Glostrup, Denmark) as the final
chromogen. For labeling multiple antigens inthe same tissue section, normal and abnormalstructures in H&E-stained sections were identiﬁed, mapped and photomicrographed. The H&E
sections were then decolorized in 70% ethanol
containing 1% HCl, after which the sections wererehydrated, quenched for 30 min with 0.3% H2O2,
rinsed in PBS and incubated with the ﬁrst primary antibody for 18 h at 4°C. Bound antibody wasvisualized by the ABC method using 3-amino-9ethylcarbazole (AEC; Vector Laboratories) as thechromogen, yielding a red product. After photographing the immunoreactive structures, AEC
sections were decolorized using 100% ethanol,rinsed twice in PBS and incubated for 60 min atroom temperature with glycine-HCl buffer (pH2.2) to elute the immunoreactive products. The
completeness of the elution process was verified by obtaining a negative reaction after reapplication of the appropriate ABC kit including asecondary antibody and AEC on the eluted sections. The sections were subsequently incubatedwith the second primary antibody for 18 h at 4°
C, and immunoreactivity was visualized by theABC method using DAB as the chromogen. The
proportion of positively-immunostained MBs wasclassiﬁed into ﬁve different categories; category –means negative staining, category +/– a few MBs
were stained weakly, category + less than 10%,
category ++ 10–50% and category +++ more than50%.

Results
Histology and immunohistochemistry
Three cases clinically diagnosed as PBC showedhistopathologically liver cirrhosis, biliary (Table 2).
The histopathological ﬁndings of 17 cases clini-Table 2. Clinicopathological ﬁndings of 20 cases of PBC and ALD with MBs
Case Age
number1
234567891011121314151617181920

Immunoblot analysis
This analysis was carried out on one fresh liver
sample of a patient with PBC (Patient 2, a 61-yearold female) and on a liver tissue of age-matchednormal individual (a 68-year-old female). Inbrief, specimens were homogenized in Laemmli
sample buffer (Bio-Rad, Hercules, CA) containing
2% sodium dodecyl sulfate (SDS), 25% glycerol,
10% 2-mercaptoethanol, 0.01% bromophenol blueand 62.5 mM Tris-HCl, pH 6.8. The sample was37
61
68
56
44
54
47
61
37
47
4957
65
66
37
56
59
70
33
61

Sex

Clinicaldiagnosis

HistopathologyFFFM
M
M
M
M
M
M
F
M
M
M
F
M
M
M
M
M

PBC
PBC
PBC
ALD
ALD
ALD
ALD
ALD
ALD
ALD
ALD
ALD
ALD
ALD
ALD
ALD
ALD
ALD
ALD
ALD

LC/biliary
LC/biliary
LC/biliary
Fatty liver
AH
AH
AH
AH
Fibrosis
Fibrosis
Fibrosis
Fibrosis
Fibrosis
Fibrosis
LC/septal
LC/septal
LC/septal
LC/septal
LC/septal
LC/septal

AH, alcoholic hepatitis; ALD, alcoholic liver disease; F, female; LC/biliary, liver cirrhosis of biliary type (by Havana
classiﬁcation) (Sherlock, 1956); LC/septal, liver cirrhosis ofseptal type; M, male; MB, Mallory body; PBC, primary biliary cirrhosis.

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AGE-modiﬁcation in Mallory bodies

cally diagnosed as alcoholic liver disease, showed
fatty liver (1 case), alcoholic hepatitis (4 cases),
liver fibrosis (6 cases) and liver cirrhosis (septal type, 6 cases) by routine stainings (Table 2).
Sixty cases of HCCs were classiﬁed as the grade
I (15 cases), the grade II (41 cases) and the grade
III (4 cases). MBs were found predominantly inperipheral areas of regenerative nodules of livercirrhosis in PBC (Fig. 1A) and alcoholic liverdisease, and also in periportal areas of ﬁbrotic alcoholic liver disease. MBs were scattered in fatty
liver and in alcoholic hepatitis. MBs in HCCs
were found in the cancer tissues (Fig. 1B). The
ratio of MB-bearing hepatocytes to total hepatocytes varied from a few to ~10 percent in liver ﬁbrosis, liver cirrhosis and HCCs. The distribution
patterns of MBs of PBC were classiﬁed as diffuse
type (2 cases) and sparse type (1 case). The patterns of MBs of alcoholic liver disease were classiﬁed as diffuse type (2 cases) and as sparse type(15 cases). The patterns of MBs of HCCs were asclustering type (21 cases), diffuse type (26 cases)
and sparse type (13 cases) (Table 3).
The shape of MBs in PBC and alcoholic
liver disease showed granular (Fig. 2A), staghorn,or circlar pattern (Fig. 2D). In contrast, the shapeof MBs in HCCs was irregular (Fig. 1B). MBsof alcoholic liver disease, PBC and HCCs were1A

1BFig. 1. Hematoxylin and eosin staining of liver tissue ofPatient 2 with primary biliary cirrhosis (PBC) (A) andthe patient with hepatocellular carcinoma (B), showing
Mallory bodies (MBs) (arrows). Bar = 100 μm.

Table 3. Histopathological and immunohistochemical ﬁndings of 80 cases of PBC, ALD and HCCwith MBs

PBC

Alcoholic liver disease

Hepatocellular carcinoma

Distribution
pattern

Numberof cases

CML(6D12)

CML
Pyrraline Pentosidine CEL Cyto(CMS10)
keratin

Clustering
DiffuseSparse021+~+++++~+++++/–~–
––
–

–
–++
++

Clustering
DiffuseSparse0215+~++++~++++/–+/–~–––––++++

Clustering
DiffuseSparse2126
13––––––––––+/–+/–

CEL, -(carboxyethyl)lysine; CML, -(carboxymethyl)lysine; MB, Mallory body; PBC, primary biliary cirrhosis.
Distribution patterns of MBs were divided according to Nakanuma and Ohta (1986).
Proportion of MBs stained positively: +++, > 50%; ++, 10–50%; +, < 10%; +/–, only minor portion.

87