Vol.
179,
No.
September
2, 1991
16,
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1991
1134-1140
OCCURRENCE OF AUTOIMMUNE ANTIBODIES TO LIVER MICROSOMAL PROTEINS IN ASSOCIATION WITH FULMINANT HEPATITIS IN THE LEC STRAIN OF RATS Sekio Nagayama, Ryuji Kitamura. Tsuyoshi Yokoi, Yasuro Kawaguchil. Noriyuki Kasaia, Noritoshi Takeichiap Hiroshi Kobayashis and Tetsuya Kamataki Division
of Analytical Biochemistry, Hokkaido University, IResearch 2Institute
Laboratory. Tokushima
August
Taiho Pharmaceutical 77 1-O 1, Japan
for Animal Experimentation, Hokkaido University, Sapporo
SLaboratory
Received
Faculty of Pharmaceutical Sapporo 060, Japan
13,
Sciences,
CO.,
School of Medicine, 060, Japan,
of Pathology, Cancer Institute of Hokkaido University, Sapporo 060, Japan 1991
Summary: The Long Evans Cinnamon (LEC!) rat, which has been established as a strain showing hereditary hepatitis and hepatic carcinoma, was found to possess autoimmune antibodies to liver microsomal proteins, particularly to a protein with the molecular weight of 56kD. The antibodies also recognized a protein(s) in liver microsomes from Long Evans Agouti and Sprague-Dawley rats, About 42 and 15 percent of respective female and male LEC rats died within a week after acute hepatitis: sera from all of the animals contained the antibodies. About 43 and 0 percent of the surviving female and male LEC rats possessed the antibodies, respectively. These results suggest that the autoantibodies occur in association with acute lethal hepatitis in the LEC Q1991AcademicPress, Inc. rats.
There
are a number
autoantibodies occurrence whether diseases.
(1).
Copyright All rights
Recent
studies
of autoantibodies,
As regards
autoantibodies
of antibodies
which
are believed
have accumulated
although
or not the autoantibodies
appearance 0006-291X/91
of diseases
it has not been
are causes against
to mitochondria,
$1.50
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
1134
to be caused knowledge fully
or consequences cytoplasmic smooth
on the
understood of such
organelles. muscle,
by
liver
the and
Vol.
179,
No.
kidney
BIOCHEMICAL
2, 1991
microsomes
antibodies that against
and ribosomes
to liver
patients
with
tienilic
against
liver
microsomes;
human
cytochrome
induced
hepatitis
(LKMl)
were
P-450, that
recognized these
in
human
only
active
limited
also
IA2
sera
cytochrome seems
clinical
of children
with
P-450
Although
information
is available
correlation
between
the occurrence
necessary
to establish
an animal
Studies
using
experimental
immunochemical Sasaki,
animals
mechanisms Yoshida
responsible
and their
Long Evans
Cinnamon
of their
nature
in showing
genetical
months
after
jaundice,
a bleeding
of the serum 40 percent onset
levels
clinical
tendency,
between
one and
of infection
virus
(11). Genetic
analysis
the control
of a single
symptoms
To confirm
the it is
phenomena.
information
on the
have established
demonstrated
are characteristic at three
to four
of hepatitis
include
severe
transaminase to die within
recessive
and elevation
activities.
such
the hepatitis
gene (13).
the
spontaneously
of age (12). agents
About
a week after
carcinoma
years
that
a strain
hepatitis
hepatocellular
1135
of
as yet. since
loss of body weight
of transmissible
autosomal
of
progression
shows similar
fulminant
one-and-a-half
no evidence
the
(LEC) rats, which
are reported
been
hepatitis
and autoantibodies.
provide
and hepatic
In survivors,
(9)
microsomes
be confirmed
(10,ll)
oliguria,
of bilirubin
of the animals
of jaundice.
appears
The
et al.
for such hepatitis.
associates
of rats, namely
birth.
may
of
the appearance
at present.
which
to a form
kidney
with
of hepatitis model
dihydralazine-
autoimmune
to be associated cannot
recognized
Guegen
and
that
autoantibodies
antibodies
to liver
this possibility
autoantibodies
with
addition,
dbl.
on
it was reported
specifically patients
In
Focusing
et al. (7) reported
to contain
(8).
(2-5).
possessed
Sera from
1 antibodies
the
hepatitis,
Beaune
autoantibodies
shown
COMMUNICATIONS
possessed
hepatitis
IICS.
type
autoantibodies
chronic
the
P-450
the
present
(6).
acid-induced
P-450
RESEARCH
to hepatitis,
hepatitis
organelle
patients
reported
in relation
drug-induced
the cytoplasmic
BIOPHYSICAL
has been reported
microsomes
with
cytochrome
AND
There
has
as hepatitis occurs
under
Vol.
179,
No.
In the present autoantibodies that
BIOCHEMICAL
2, 1991
study,
we found
to a protein(s)
the autoantibodies
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
that sera from some LEC rats contained
in liver
appeared
microsomes.
in individuals
The which
results
showed
died by fulminant
hepatitis. MATERIALS
AND METHODS
LEC rats and Long Evans Agouti (LEA) rats were supplied by the Center for Experimental Plants and Animals of Hokkaido University, Japan. Male and female Sprague-Dawley (SD) rats (7 weeks) were obtained from SLC, Chemicals were obtained from the following sources: Japan. Streptavidine-biotinylated horseradish peroxidase complex (ABC), biotinylated anti-rat immunoglobulin (Ig) (from sheep) from Amersham Japan, 3,3’-diaminobenzidine tetrahydrochloride (DAB) from Sigma, St. Louis, bovine serum albumin (Fraction V) from Boehringer Mannheim, Mannheim. West-Germany, and Immobilon-P filter from Millipore, Bedford. Blood was taken from the tail vein of rats. Sera were obtained by centrifugation and diluted with phosphate-buffered saline (PBS). Subcellular fractions, such as nuclei, mitochondria, microsome and of liver homogenates were prepared by the method cytosol fractions, reported by de Duve et al (14). Each subcellular fraction was washed once with 0.25 M sucrose and used as a preparation. Protein was determined by the method of Lowry et al. (15) with bovine serum albumin as a standard. Activities of glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) were measured by the W method using Kit from Kokusai Shiyaku, Ltd., Japan. Sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) was performed according to the method of Laemmli (16) with a 12.5% concentration of acrylamide. The separated proteins were transferred by the method of Bjerrum and Schafer-Nielsen (17) to an Immobilon-P filter using Trans-Blot (BIO-BAD) with a transfer buffer (48 mM Tris, 39 mM glycine and 20 % methanol). The filter was shaken gently in 3 % bovine serum albumin (BSA) in PBS The excess BSA solution was removed, and then the filter overnight. paper was incubated with serum from LEC, LEA or SD rats, which had been previously diluted at 1:lOO with PBS, for 1 hr. The filter paper was washed three times with PBS (10 min/each wash), followed by incubation with biotinylated sheep anti-rat Ig solution (diluted 1:400 with PBS) for 1 hr. The blots were washed three times with PBS and incubated with the ABC solution (diluted 1:400 with PBS) for 1 hr. To wash out the ABC solution, the blots were washed three times with PBS, and then allowed to colorize in PBS containing 0.06% DAB and 0.03% hydrogen peroxide. The reaction was terminated by washing the stained blots several times in water. Prestained molecular size markers (Sigma, St. Louis) were used as a standard. RESULTS Efforts
have been paid
Despite
these studies
it has
been
occurrence
reported. of hepatitis
AND DISCUSSION
to clarify
performed
the causes
so far, no strong
To define
one
of the
in LEC rats, we examined 1136
of hepatitis evidence causes
in LEC rats. to understand
involved
the possibility
in
the
that
the
Vol.
179,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
43kD-
01
’ *” 2~.: I
02
123456
COMMUNICATIONS
12
3
Figure 1. Immunoblot analysis for the antigenic 56 kD protein in liver subcellular fractions. Liver subcellular fractions (19 ).tg protein) such as total homogenates (lane 1). nuclei [lane 2). mitochondria (lane 3). lysosome (lane 4). microsome (lane 5) and cytosol (lane 6) were applied to SDS-PAGE, and analyzed for the antigenic 56 kD protein using serum from 20-week-old LEC rats as the antibody preparation. Immunoblot analysis for the antigenic 56 kD protein in liver Figure 2. microsomes of LEC. LEA and SD rats using sera from LEC rats as an antibody preparation. The amount of liver microsomes from LEC (lane 1). LEA (lane 2) and SD rats (lane 3) was 19 pg.
sera of LEC
rats
fractions
of livers.
fractions
as antigens
seen clearly, antigenic
might
The results
protein(s) (lane
intensive
band
very faint
band
a small
amount
excluded
that
1).
was seen with detectable.
the faint
autoantibodies it may
proteins
and
(data
shown),
proteins
in sera of LEC rats. 1137
with
most only a
to contain
(14). it cannot
that
in plasma
plasma
that
human
be
anti-
membranes.
membranes
were not contained
indicating
the
is due to a contaminated
(18) reported
liver
An
were applied
is supposed
and the membranes
The antibodies
specifically
nuclei
that
LEC rats.
by nuclei
fraction
recognized
protein(s),
subcellular
applied,
as contaminants
et al.
fraction.
present
followed
seen with
also be possible
not
using
homogenates
fractions
microsomes,
Loeper
the antigenic
liver
Since nuclear
band
in subcellular
are shown in Fig. 1. As can be
when
in the nuclear SD rats
blot analysis
subcellular
of microsomal
liver/kidney
contain
Among
to proteins
in sera of 20-week-old
was detectable
protein(s).
rats
of Western
autoantibodies
microsomal
Therefore,
antibodies
and sera as antibodies
we found
as antigens
contain
of LEC
contaminated in sera of LEA
the autoantibodies
are
Vol.
179,
No.
2, 1991
BIOCHEMICAL
The presence
of the antigenic
microsomes
from
assume
the antigenic
that
in LEC rats.
LEC rats.
This
all
The results
preparations
antigenic
estimated
band
shown
blot
sera
significance
of the
experiments.
Blood
taken
of serum
values
animals examined; shown
in Table
according period. each
with was thus
from
showed
died.
24 female
weeks.
varied
The
from results
the animals status
of the autoantibodies
All serum
the of
LEC, LEA and SD rats was by SDS-PAGE.
The titer
of
since the positively
stained
if the sera from
LEC rats
protein
Although
in liver that
severity
not
microsomes the antigenic
and
appeared
of hepatitis.
examined
in the
13 male
considerably
or survived
a positive
the
weight
The following
LEC rats were
15 to 22 weeks of age, and were analyzed
1. In the Table,
The presence
consecutive
produce
or not the autoantibodies
to be suffered
animals
that
in
species.
and GPT
to decreased
rat
to
GOT and GPT and for the autoantibodies.
judged
some
cells
dogs, suggesting
association
samples
of GOT
were
the antigenic
autoantibodies
once a week from
activities peak
in
preparation
was present
PBS (data not shown).
to know whether
rats
from
as judged
and beagle
LEC, LEA
The molecular
was detectable
over animal
of LEC
hepatic
to be very high,
we found
It was of interest
of hepatitis
from
protein(s)
in LEC rats.
analysis
monkeys
is present
factor
in
one can
it can be assumed
from
in liver microsomes
only
then
liver microsomes
Thus,
is released
paper,
humans,
is true,
the antigenic
up to 1 : 1600 with
in this
protein
in
that
was supposed
were diluted
from
using
to be 56 kD in common
in Western
be restricted
can be the causal
characteristically
the antibodies
postulation
protein(s)
showed
protein
may
COMMUNICATIONS
and sera from LEC rats as an antibody
protein(s)
the antigenic
RESEARCH
protein(s)
of microsomes.
autoantibodies
BIOPHYSICAL
If this
was examined
and SD rats as antigens (Fig. 2).
AND
1138
hepatitis on
the
from
Although
the
animals,
period are
into two groups
at the end of the
band
all
autoantibodies
were divided
the
individuals,
within
was judged
immunostained samples
among
for the
when at
sera from
56kD
which
indicated
for
two
died during
Vol.
179,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Table 1. Summary of the results of immunoblot analysis for the autoimmune antibodies against the microsomal 56 kD protein status of LEC rats*
No. of sera examined
No. of sera with autoantibodies**
Female Died Survived
10 14
10 6
(100) I 43)
Male Died Survived
2 11
2 0
(100) I 0)
I%)
* The survival of LEC rats was judged at the end of the 23rd week of age. ** Serum samples which showed a positive immunostained band at 56 kD in Western blot analysis for two consecutive weeks were judged to contain the autoantibodies.
the
period
contrast, and
examined,
the autoantibodies
none
of
autoantibodies Supporting lethal
showed
eleven appeared
the
idea
before
shown).
et al.
Kenna
halothane-induced
al.
with
serum
these
(19.20)
individual
lines
cause
with rat.
was of
Possible
of acute
the
have
rats which
died
occures
in
symptom
of
that
drug,
since
this
shown
which by which
is under
laboratory. 1139
play
(data
patients
with
Vergani
et
in the
has been known
in this
paper
strongly
of LEC rats appear to the death
examination
to
Together
in
of the
the autoantibodies
current
not
against
a role
in vitro.
leads
with
rats as early
patients.
in the serum
hepatitis,
association
from
may
the
hepatitis.
of autoantibodies
of hepatocytes
the results
females when
jaundice
sera
autoantibodies
of this
By
of
in LEC
from halothane-treated
mechanisms hepatitis
to know
5 forms
autoantibodies lethal
interest
were detectable
killing
of evidence,
acute
of
reported
these
of the reaction
the idea that
association
the
showing
that
of autoantibodies. in six of fourteen
autoantibodies
lymphocyte-mediated
support
the
in
microsomes
(21) suggested
induce
It
hepatitis
in liver
pathogenesis
males.
that
as 7-week-old,
presence
were detectable
the autoantibodies
hepatitis,
proteins
the
act as in
this
Vol.
179,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Acknowledgment A part of this study was supported of Education,
Science
and Culture,
by a Grant-in-Aid
from the Ministry
Japan.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Pohl. L. R., Satoh, H. and Kenna, J. G. (1988) Annu Rev. Pharmacol. Toxicol. 28:367-387. Homberg, J., Abuaf, N., Helmy-Khalil, S., Biour, M., Poupon, P., Islam, S. and Benhamou, J. (1985) HepatoIogy 5:722-727. Berg, P., Doniach, D. and Roitt, I. (1967) J. Exp. Med. 126:277-290. Homberg, J.. Rizzetto, M. and Doniach. D. (1974) Clin. Exp. ImrnunoZ. 17:6 19-630. Smith, M., Williams, R. and Walker, G. (1974) Br. Med. J. 2:80-84. Whittingham. S., Irwin, J. and Mackay, I. (1965) Gastroenterology 51: 499-505 Beaune. P., Dansette, P., Mansuy, D., Kiffel, L.. Finck, M., Amar, C., Leroux, J. and Homberg, J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84:551-555. Bourdi, M., Larrey, D., Nataf, J., Bemuau, J., Pessayre, D., Iwasaki, M.. Guengerich. F. and Beaune. P. (1990) J. Clin. Invest. 85:1967-1973. Guegen. M.. Yamamoto, A., Bernard, 0. and Alvarez. F. (1989) Biochem Biophys. Res. Corm-nun. 159:542-547. Sasaki. M., Yoshida, M. and Kagami, K. (1985) Rat News Letter 14:4-6. Yoshida. M., Masuda, R. and Sasaki, M. (1987) J. Heredity 78:361-365. Masuda, R., Yoshida, M. and Sasaki, M. (1988) Jpn. J. Cancer Res. 79:828-835. Masuda, R., Yoshida, M. and Sasaki, M. (1988) Lab Anim 22:166- 169. de Duve. C. D., Pressman, B. C.. Gianetto, R, Wattiaux, R. and Appelmans, F. (1955) Biochcm J. 60:604-617. Lowry, O.H.. Rosebrough, N.J.. Farr, A. and Randall, R. (1951) J. Biol. Chem. 193:265-275. Laemmli, U. K. (1970) Nature 227:680-685. Bjerrum. 0. J. and Schafer-Nielsen, C. (1988) Analytical Electrophoresis (M.J. Dunn, Ed.) pp.315-350 Verlag Chcmie, Wein heim. Loeper, J., Descatoire, V.. Maurice, M., Beaune. P., Feldmann, G.. Larrey, D. and Pessayre, D. (1990) Hepatology 11:850-858. Kenna, J. G., Neuberger, J. and Williams, R. (1987) J. Pharmacol. Exp. Ther. 242~733-740. Kenna, J. G., Satoh, H., Christ, D. and Pohl, L. R. (1988) J. PhcmnacoZ. i&p. Ther. 245:1103-l 109. Vergani. D.. Miely-Vergani, G.. Alberti. A., Neuberger, J., Eddleston. A., Davis, M. and Williams, R. (1980) N. Engl. J. Med. 303:66-71.
1140
179,
No.
September
2, 1991
16,
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1991
1134-1140
OCCURRENCE OF AUTOIMMUNE ANTIBODIES TO LIVER MICROSOMAL PROTEINS IN ASSOCIATION WITH FULMINANT HEPATITIS IN THE LEC STRAIN OF RATS Sekio Nagayama, Ryuji Kitamura. Tsuyoshi Yokoi, Yasuro Kawaguchil. Noriyuki Kasaia, Noritoshi Takeichiap Hiroshi Kobayashis and Tetsuya Kamataki Division
of Analytical Biochemistry, Hokkaido University, IResearch 2Institute
Laboratory. Tokushima
August
Taiho Pharmaceutical 77 1-O 1, Japan
for Animal Experimentation, Hokkaido University, Sapporo
SLaboratory
Received
Faculty of Pharmaceutical Sapporo 060, Japan
13,
Sciences,
CO.,
School of Medicine, 060, Japan,
of Pathology, Cancer Institute of Hokkaido University, Sapporo 060, Japan 1991
Summary: The Long Evans Cinnamon (LEC!) rat, which has been established as a strain showing hereditary hepatitis and hepatic carcinoma, was found to possess autoimmune antibodies to liver microsomal proteins, particularly to a protein with the molecular weight of 56kD. The antibodies also recognized a protein(s) in liver microsomes from Long Evans Agouti and Sprague-Dawley rats, About 42 and 15 percent of respective female and male LEC rats died within a week after acute hepatitis: sera from all of the animals contained the antibodies. About 43 and 0 percent of the surviving female and male LEC rats possessed the antibodies, respectively. These results suggest that the autoantibodies occur in association with acute lethal hepatitis in the LEC Q1991AcademicPress, Inc. rats.
There
are a number
autoantibodies occurrence whether diseases.
(1).
Copyright All rights
Recent
studies
of autoantibodies,
As regards
autoantibodies
of antibodies
which
are believed
have accumulated
although
or not the autoantibodies
appearance 0006-291X/91
of diseases
it has not been
are causes against
to mitochondria,
$1.50
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
1134
to be caused knowledge fully
or consequences cytoplasmic smooth
on the
understood of such
organelles. muscle,
by
liver
the and
Vol.
179,
No.
kidney
BIOCHEMICAL
2, 1991
microsomes
antibodies that against
and ribosomes
to liver
patients
with
tienilic
against
liver
microsomes;
human
cytochrome
induced
hepatitis
(LKMl)
were
P-450, that
recognized these
in
human
only
active
limited
also
IA2
sera
cytochrome seems
clinical
of children
with
P-450
Although
information
is available
correlation
between
the occurrence
necessary
to establish
an animal
Studies
using
experimental
immunochemical Sasaki,
animals
mechanisms Yoshida
responsible
and their
Long Evans
Cinnamon
of their
nature
in showing
genetical
months
after
jaundice,
a bleeding
of the serum 40 percent onset
levels
clinical
tendency,
between
one and
of infection
virus
(11). Genetic
analysis
the control
of a single
symptoms
To confirm
the it is
phenomena.
information
on the
have established
demonstrated
are characteristic at three
to four
of hepatitis
include
severe
transaminase to die within
recessive
and elevation
activities.
such
the hepatitis
gene (13).
the
spontaneously
of age (12). agents
About
a week after
carcinoma
years
that
a strain
hepatitis
hepatocellular
1135
of
as yet. since
loss of body weight
of transmissible
autosomal
of
progression
shows similar
fulminant
one-and-a-half
no evidence
the
(LEC) rats, which
are reported
been
hepatitis
and autoantibodies.
provide
and hepatic
In survivors,
(9)
microsomes
be confirmed
(10,ll)
oliguria,
of bilirubin
of the animals
of jaundice.
appears
The
et al.
for such hepatitis.
associates
of rats, namely
birth.
may
of
the appearance
at present.
which
to a form
kidney
with
of hepatitis model
dihydralazine-
autoimmune
to be associated cannot
recognized
Guegen
and
that
autoantibodies
antibodies
to liver
this possibility
autoantibodies
with
addition,
dbl.
on
it was reported
specifically patients
In
Focusing
et al. (7) reported
to contain
(8).
(2-5).
possessed
Sera from
1 antibodies
the
hepatitis,
Beaune
autoantibodies
shown
COMMUNICATIONS
possessed
hepatitis
IICS.
type
autoantibodies
chronic
the
P-450
the
present
(6).
acid-induced
P-450
RESEARCH
to hepatitis,
hepatitis
organelle
patients
reported
in relation
drug-induced
the cytoplasmic
BIOPHYSICAL
has been reported
microsomes
with
cytochrome
AND
There
has
as hepatitis occurs
under
Vol.
179,
No.
In the present autoantibodies that
BIOCHEMICAL
2, 1991
study,
we found
to a protein(s)
the autoantibodies
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
that sera from some LEC rats contained
in liver
appeared
microsomes.
in individuals
The which
results
showed
died by fulminant
hepatitis. MATERIALS
AND METHODS
LEC rats and Long Evans Agouti (LEA) rats were supplied by the Center for Experimental Plants and Animals of Hokkaido University, Japan. Male and female Sprague-Dawley (SD) rats (7 weeks) were obtained from SLC, Chemicals were obtained from the following sources: Japan. Streptavidine-biotinylated horseradish peroxidase complex (ABC), biotinylated anti-rat immunoglobulin (Ig) (from sheep) from Amersham Japan, 3,3’-diaminobenzidine tetrahydrochloride (DAB) from Sigma, St. Louis, bovine serum albumin (Fraction V) from Boehringer Mannheim, Mannheim. West-Germany, and Immobilon-P filter from Millipore, Bedford. Blood was taken from the tail vein of rats. Sera were obtained by centrifugation and diluted with phosphate-buffered saline (PBS). Subcellular fractions, such as nuclei, mitochondria, microsome and of liver homogenates were prepared by the method cytosol fractions, reported by de Duve et al (14). Each subcellular fraction was washed once with 0.25 M sucrose and used as a preparation. Protein was determined by the method of Lowry et al. (15) with bovine serum albumin as a standard. Activities of glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) were measured by the W method using Kit from Kokusai Shiyaku, Ltd., Japan. Sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) was performed according to the method of Laemmli (16) with a 12.5% concentration of acrylamide. The separated proteins were transferred by the method of Bjerrum and Schafer-Nielsen (17) to an Immobilon-P filter using Trans-Blot (BIO-BAD) with a transfer buffer (48 mM Tris, 39 mM glycine and 20 % methanol). The filter was shaken gently in 3 % bovine serum albumin (BSA) in PBS The excess BSA solution was removed, and then the filter overnight. paper was incubated with serum from LEC, LEA or SD rats, which had been previously diluted at 1:lOO with PBS, for 1 hr. The filter paper was washed three times with PBS (10 min/each wash), followed by incubation with biotinylated sheep anti-rat Ig solution (diluted 1:400 with PBS) for 1 hr. The blots were washed three times with PBS and incubated with the ABC solution (diluted 1:400 with PBS) for 1 hr. To wash out the ABC solution, the blots were washed three times with PBS, and then allowed to colorize in PBS containing 0.06% DAB and 0.03% hydrogen peroxide. The reaction was terminated by washing the stained blots several times in water. Prestained molecular size markers (Sigma, St. Louis) were used as a standard. RESULTS Efforts
have been paid
Despite
these studies
it has
been
occurrence
reported. of hepatitis
AND DISCUSSION
to clarify
performed
the causes
so far, no strong
To define
one
of the
in LEC rats, we examined 1136
of hepatitis evidence causes
in LEC rats. to understand
involved
the possibility
in
the
that
the
Vol.
179,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
43kD-
01
’ *” 2~.: I
02
123456
COMMUNICATIONS
12
3
Figure 1. Immunoblot analysis for the antigenic 56 kD protein in liver subcellular fractions. Liver subcellular fractions (19 ).tg protein) such as total homogenates (lane 1). nuclei [lane 2). mitochondria (lane 3). lysosome (lane 4). microsome (lane 5) and cytosol (lane 6) were applied to SDS-PAGE, and analyzed for the antigenic 56 kD protein using serum from 20-week-old LEC rats as the antibody preparation. Immunoblot analysis for the antigenic 56 kD protein in liver Figure 2. microsomes of LEC. LEA and SD rats using sera from LEC rats as an antibody preparation. The amount of liver microsomes from LEC (lane 1). LEA (lane 2) and SD rats (lane 3) was 19 pg.
sera of LEC
rats
fractions
of livers.
fractions
as antigens
seen clearly, antigenic
might
The results
protein(s) (lane
intensive
band
very faint
band
a small
amount
excluded
that
1).
was seen with detectable.
the faint
autoantibodies it may
proteins
and
(data
shown),
proteins
in sera of LEC rats. 1137
with
most only a
to contain
(14). it cannot
that
in plasma
plasma
that
human
be
anti-
membranes.
membranes
were not contained
indicating
the
is due to a contaminated
(18) reported
liver
An
were applied
is supposed
and the membranes
The antibodies
specifically
nuclei
that
LEC rats.
by nuclei
fraction
recognized
protein(s),
subcellular
applied,
as contaminants
et al.
fraction.
present
followed
seen with
also be possible
not
using
homogenates
fractions
microsomes,
Loeper
the antigenic
liver
Since nuclear
band
in subcellular
are shown in Fig. 1. As can be
when
in the nuclear SD rats
blot analysis
subcellular
of microsomal
liver/kidney
contain
Among
to proteins
in sera of 20-week-old
was detectable
protein(s).
rats
of Western
autoantibodies
microsomal
Therefore,
antibodies
and sera as antibodies
we found
as antigens
contain
of LEC
contaminated in sera of LEA
the autoantibodies
are
Vol.
179,
No.
2, 1991
BIOCHEMICAL
The presence
of the antigenic
microsomes
from
assume
the antigenic
that
in LEC rats.
LEC rats.
This
all
The results
preparations
antigenic
estimated
band
shown
blot
sera
significance
of the
experiments.
Blood
taken
of serum
values
animals examined; shown
in Table
according period. each
with was thus
from
showed
died.
24 female
weeks.
varied
The
from results
the animals status
of the autoantibodies
All serum
the of
LEC, LEA and SD rats was by SDS-PAGE.
The titer
of
since the positively
stained
if the sera from
LEC rats
protein
Although
in liver that
severity
not
microsomes the antigenic
and
appeared
of hepatitis.
examined
in the
13 male
considerably
or survived
a positive
the
weight
The following
LEC rats were
15 to 22 weeks of age, and were analyzed
1. In the Table,
The presence
consecutive
produce
or not the autoantibodies
to be suffered
animals
that
in
species.
and GPT
to decreased
rat
to
GOT and GPT and for the autoantibodies.
judged
some
cells
dogs, suggesting
association
samples
of GOT
were
the antigenic
autoantibodies
once a week from
activities peak
in
preparation
was present
PBS (data not shown).
to know whether
rats
from
as judged
and beagle
LEC, LEA
The molecular
was detectable
over animal
of LEC
hepatic
to be very high,
we found
It was of interest
of hepatitis
from
protein(s)
in LEC rats.
analysis
monkeys
is present
factor
in
one can
it can be assumed
from
in liver microsomes
only
then
liver microsomes
Thus,
is released
paper,
humans,
is true,
the antigenic
up to 1 : 1600 with
in this
protein
in
that
was supposed
were diluted
from
using
to be 56 kD in common
in Western
be restricted
can be the causal
characteristically
the antibodies
postulation
protein(s)
showed
protein
may
COMMUNICATIONS
and sera from LEC rats as an antibody
protein(s)
the antigenic
RESEARCH
protein(s)
of microsomes.
autoantibodies
BIOPHYSICAL
If this
was examined
and SD rats as antigens (Fig. 2).
AND
1138
hepatitis on
the
from
Although
the
animals,
period are
into two groups
at the end of the
band
all
autoantibodies
were divided
the
individuals,
within
was judged
immunostained samples
among
for the
when at
sera from
56kD
which
indicated
for
two
died during
Vol.
179,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Table 1. Summary of the results of immunoblot analysis for the autoimmune antibodies against the microsomal 56 kD protein status of LEC rats*
No. of sera examined
No. of sera with autoantibodies**
Female Died Survived
10 14
10 6
(100) I 43)
Male Died Survived
2 11
2 0
(100) I 0)
I%)
* The survival of LEC rats was judged at the end of the 23rd week of age. ** Serum samples which showed a positive immunostained band at 56 kD in Western blot analysis for two consecutive weeks were judged to contain the autoantibodies.
the
period
contrast, and
examined,
the autoantibodies
none
of
autoantibodies Supporting lethal
showed
eleven appeared
the
idea
before
shown).
et al.
Kenna
halothane-induced
al.
with
serum
these
(19.20)
individual
lines
cause
with rat.
was of
Possible
of acute
the
have
rats which
died
occures
in
symptom
of
that
drug,
since
this
shown
which by which
is under
laboratory. 1139
play
(data
patients
with
Vergani
et
in the
has been known
in this
paper
strongly
of LEC rats appear to the death
examination
to
Together
in
of the
the autoantibodies
current
not
against
a role
in vitro.
leads
with
rats as early
patients.
in the serum
hepatitis,
association
from
may
the
hepatitis.
of autoantibodies
of hepatocytes
the results
females when
jaundice
sera
autoantibodies
of this
By
of
in LEC
from halothane-treated
mechanisms hepatitis
to know
5 forms
autoantibodies lethal
interest
were detectable
killing
of evidence,
acute
of
reported
these
of the reaction
the idea that
association
the
showing
that
of autoantibodies. in six of fourteen
autoantibodies
lymphocyte-mediated
support
the
in
microsomes
(21) suggested
induce
It
hepatitis
in liver
pathogenesis
males.
that
as 7-week-old,
presence
were detectable
the autoantibodies
hepatitis,
proteins
the
act as in
this
Vol.
179,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Acknowledgment A part of this study was supported of Education,
Science
and Culture,
by a Grant-in-Aid
from the Ministry
Japan.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Pohl. L. R., Satoh, H. and Kenna, J. G. (1988) Annu Rev. Pharmacol. Toxicol. 28:367-387. Homberg, J., Abuaf, N., Helmy-Khalil, S., Biour, M., Poupon, P., Islam, S. and Benhamou, J. (1985) HepatoIogy 5:722-727. Berg, P., Doniach, D. and Roitt, I. (1967) J. Exp. Med. 126:277-290. Homberg, J.. Rizzetto, M. and Doniach. D. (1974) Clin. Exp. ImrnunoZ. 17:6 19-630. Smith, M., Williams, R. and Walker, G. (1974) Br. Med. J. 2:80-84. Whittingham. S., Irwin, J. and Mackay, I. (1965) Gastroenterology 51: 499-505 Beaune. P., Dansette, P., Mansuy, D., Kiffel, L.. Finck, M., Amar, C., Leroux, J. and Homberg, J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84:551-555. Bourdi, M., Larrey, D., Nataf, J., Bemuau, J., Pessayre, D., Iwasaki, M.. Guengerich. F. and Beaune. P. (1990) J. Clin. Invest. 85:1967-1973. Guegen. M.. Yamamoto, A., Bernard, 0. and Alvarez. F. (1989) Biochem Biophys. Res. Corm-nun. 159:542-547. Sasaki. M., Yoshida, M. and Kagami, K. (1985) Rat News Letter 14:4-6. Yoshida. M., Masuda, R. and Sasaki, M. (1987) J. Heredity 78:361-365. Masuda, R., Yoshida, M. and Sasaki, M. (1988) Jpn. J. Cancer Res. 79:828-835. Masuda, R., Yoshida, M. and Sasaki, M. (1988) Lab Anim 22:166- 169. de Duve. C. D., Pressman, B. C.. Gianetto, R, Wattiaux, R. and Appelmans, F. (1955) Biochcm J. 60:604-617. Lowry, O.H.. Rosebrough, N.J.. Farr, A. and Randall, R. (1951) J. Biol. Chem. 193:265-275. Laemmli, U. K. (1970) Nature 227:680-685. Bjerrum. 0. J. and Schafer-Nielsen, C. (1988) Analytical Electrophoresis (M.J. Dunn, Ed.) pp.315-350 Verlag Chcmie, Wein heim. Loeper, J., Descatoire, V.. Maurice, M., Beaune. P., Feldmann, G.. Larrey, D. and Pessayre, D. (1990) Hepatology 11:850-858. Kenna, J. G., Neuberger, J. and Williams, R. (1987) J. Pharmacol. Exp. Ther. 242~733-740. Kenna, J. G., Satoh, H., Christ, D. and Pohl, L. R. (1988) J. PhcmnacoZ. i&p. Ther. 245:1103-l 109. Vergani. D.. Miely-Vergani, G.. Alberti. A., Neuberger, J., Eddleston. A., Davis, M. and Williams, R. (1980) N. Engl. J. Med. 303:66-71.
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