AGE-modiﬁcation in Mallory bodies

1MBs were not stained with antibodies against
oxidative stress proteins (OSPs) such as acrolein
and HNE nor against stress response proteins(SRPs) (27 kDa, 32 kDa, 72 kDa or 90 kDa) (datanot shown). MBs in HCCs were also identified
by positive staining to anti-ubiquitin antibody(Fig. 3A). MBs in all cases of HCCs were weakly
stained by anti-cytokeratin antibody. By contrast,
MBs in HCCs were not stained to CML (Fig. 3B),
pyrraline, pentosidine, CEL, 3DG-imidazolone,
two types of OSPs (acrolein and HNE) nor fourtypes of SRPs (srp27, 32, 72 and 90). There wereno significant differences in AGE-expressions
among the histologically different specimens inHCCs.
The proportion of positively-immunostainedMBs varied from one sample to another, ranging from less than 10% to more than 50% of total
MBs (Table 3). Immunohistochemical stainings of CML of PBC and alcoholic liver disease
showed that 1 out of 20 cases was of category+++; 4 out of 20 cases were of category ++, and15 cases out of 20 cases were of category +. Immunohistochemical stainings of pyrraline of PBC
and alcoholic liver disease showed that one caseof PBC was of category +/– and nine of 17 casesof alcoholic liver disease were of category +/–.
MBs with diffuse distribution pattern were positive for CML or pyrraline more than MBs withsparse type. All cases of PBC and alcoholic liverdisease were of category ++ about cytokeratin.
All cases of HCC were of category +/– about cytokeratin.
Hepatocytes from 10 control individuals
were not stained by five anti-AGE antibodies
against either of CML, pyrraline, pentosidine,CEL or 3DG-imidazolone. Similarly, normal hepatocytes showed no immunoreactivities to OSPs,SRPs, B-crystallin nor ubiquitin. No staining
was detected when sections were incubated withPBS. The specificity and high affinity of these
antibodies were conﬁrmed by control tissues. Asexpected (Makino et al., 1995; Kume et al., 1995;
Horie et al., 1997), CML-immunoreactivities wereobserved in smooth muscle cells of atherosclerotic

2kDa
20066

Fig. 4. Western blot analysis
using monoclonal CML (withmonoclonal antibody 6D12) inliver tissues.
17

Lane 1: normal control liver.
Lane 2: primary biliary cirrhosis liver (Patient 2).

lesions, and immunoreactivities for pyrraline,pentosidine, CEL and 3DG-imidazolone were also
noted in the thickening intimae of arteries. As reported earlier (Uchida et al., 1995), macrophagederived foam cells in the atheromatous lesions
were positive for HNE. Anti-CML antibody pretreated with an excess amount of CML-modiﬁed
BSA did not stain smooth muscle cells in the atherosclerotic lesions. Similarly, anti-pyrraline, oranti-pentosidine antibody pretreated with an excess amount of pyrraline- or pentosidine-modiﬁed
BSA did not stain the thickening intimae of arteries.
Immunoblot analysis
The results of immunoblot analyses are shown inFig. 4. When the liver-tissue homogenate of PBC(Patient 2, a 61-year-old female), whose hepatocytes were demonstrated to contain CML-positive
MBs immunochemically, was subjected to immunoblotting with anti-CML antibody, a single bandwith a molecular weight indistinguishable fromthat of cytokeratin was detected (Fig. 4). Immunoblotting of the fresh autopsy liver specimen of
a normal individual (a 68-year-old female) did notshow any speciﬁc band (Fig. 4).
89

M. Kato et al.

was suggested that CML-combined protein might
be cytokeratin.
Glycation is one of biochemical reactions andit occurs when proteins were incubated with reducing sugars. Finally, CML, CEL or pyrralinecombined proteins through several steps by glycation form AGEs. Although oxidation is necessary
for the formation of AGEs in vitro (Nagai et al.,1997), the intention level of oxidation, the natureof oxidative processes, and the period of oxidative
stresses are of different in vivo. At the cellularlevel, living cells can induce a diverse group ofSRPs in response to different types of biologicalstresses, including oxidative damage (Morimotoet al., 1990). Since OSPs and SRPs in MBs were
not detected in the present immunohistochemicalstudies, the amounts of these compounds in MBs
might be, if any, negligible, suggesting that theoxidative stress that generates these marker compounds does not contribute to the process of MB
formation in vivo.
Modification by glycation occurs in many
proteins in relation to the pathogenesis of diseases such as atherosclerosis (Kume et al., 1995),
diabetic complications (Makino et al., 1995),
Alzheimers’ disease (Smith et al., 1994). Furthermore, AGEs are common to be long-lived,
insoluble molecules, readily deposited in cells
that have a direct cytotoxic effect (Vlassara et al.,1994). Although several hypotheses of MBs havebeen discussed (Jensen and Gluud, 1994a, 1994b)
the results of the present study clariﬁed that AGEmodiﬁcation of cytokeratin, a major protein component of MBs, plays an important role in the formation of MBs in hepatocytes in the PBC and thealcoholic liver disease. Taken together with abnormal cytokeratin aggregation toxicity, it is conceivable that the AGE modiﬁcation of cytokeratinin MBs could amplify the aggregation of cytokeratin and that the formation of the AGEs could
result in greater toxicity in hepatocytes-bearing
MBs in patients with the PBC and alcoholic liverdisease. Considering the facts that MBs of HCCs
contain less amount of cytokeratin protein, HCC
cells form MBs for a short disease duration of tu-Discussion
Although the frequency and the distribution ofMBs that were detected by H&E staining werevarious among 80 cases examined, all of the MBs
were positive for ubiquitin, B-crystallin and cytokeratin immunohistochemically. MBs in HCCs
were weakly positive for cytokeratin, but MBs
in PBC and alcoholic liver disease were strongly
positive for cytokeratin. The present immunohistochemical results coincide with the previous
reports which demonstrated that MBs consistedof aggregates of cytokeratin ﬁlaments (Yokoo etal., 1972) or that cytokeratin protein of MBs wasubiquitinated (Yuan et al., 1996). In the present study, the facts that MBs contain cytokeratin,
ubiquitin and B-crystallin as protein components
were conﬁrmed immunohistochemically.
The novel finding of the present study isthat MBs in PBC and alcoholic liver disease werepositive for CML and pyrraline in contrast to
those of HCCs. In cases of PBC and alcoholic
liver disease, immunohistochemical results ofthe same paraffin sections using anti-CML andanti-cytokeratin antibodies, revealed that bothCML and cytokeratin were co-localized on thesame MBs. These results suggest that MBs in
PBC and alcoholic liver disease have epitopes ofCML addition to cytokeratin. No CML-positive
reactivities except MBs were found in the liver
sections of PBC, alcoholic liver disease, HCCs
and normal controls. Furthermore, immunoblot
analysis with anti-CML antibody supported theimmunohistochemical ﬁndings. Single band witha molecular weight about 55 kDa was detectedin the liver-tissue homogenate of PBC. This result demonstrated that liver tissue-homogenateof PBC contain CML-combined protein withmolecular weight about 55 kDa. Considering thefact that the molecular weight of ubiquitin, one ofprotein components of MBs is about 8 kDa (Leeet al., 1989; Lowe and Mayer, 1990) and that ofB-crystallin is also 22 kDa (Iwaki et al., 1989), it90

AGE-modiﬁcation in Mallory bodies

morigenesis or have abnormal biological metabolism, AGE-modiﬁcation do not contribute to theformation of the MBs in HCC cells. To elucidate
the differences between AGE-expressions of alcoholic liver disease or PBC and AGE-expressionsof HCCs, a further complete understanding of themolecular mechanisms of MB formation in hepatocytes will be necessary. Our results suggest thatthe formation of AGEs might be cytotoxic to MBcontaining hepatocytes in PBC and alcoholic liverdisease in contrast to HCCs.

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19 Lyon H, Christoffersen P. Histochemical study ofAcknowledgments: The authors express their appreciation to Dr. James E. Goldman (Department of Pathology, Columbia University), for providing the antibody to
B-crystallin and to Dr. S.-H. Yen (Department of Neuroscience, Mayo Clinic), for donating the antibody to ubiquitin.
This study was supported in part by a Grant-in-Aidfor Scientiﬁc Research (c) from the Ministry of Education,Culture, Sports, Science and Technology of Japan (SK:17500229), a Grant From Research on Psychiatric and Neurological Disease and Mental Health (SK) and a Research
Grant on Measures for Intractable Diseases from the Ministry of Health, Labour and Welfare of Japan (SK).

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Corresponding author: Masako Kato, MD92