Vol. 182, No. 3, 1992 February 14, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1100-1107
INDUCTION OF Mn-SUPEROXIDE DISMUTASE BY TUMOR NECROSIS FACTOR, INTERLEUKIN-1 AND INTERLEUKIN-6 IN HUMAN HEPATOMA CELLS Minoru
Ono'r*,
Hironobu
Chihiro
Kohda',
Sekiya',
Tetsuo
Masayoshi and Naoyuki
'Department
of Internal 4-5 Nishikagura,
Namiki',
Akira .3 Taniguchi
Motoyuki
Ube Industries,
Ube 755,
Osaka University Osaka, Japan
of Biochemistry,
Ohhiral,
Takeyasu'
Medicine (III), Asahikawa Asahikawa 078, Hokkaido,
'Ube Laboratory, 3Department
Kawaguchi2,
Medical Japan
College,
Japan
School
of Medicine,
Received December 20, 1991 Effects of Tumor Necrosis Factor (TNF), Interleukin-1 (IL(IL-6) and Interferon-gamma (IFN-gamma) on the 11, Interleukin-6 expression of Mn-superoxide dismutase (Mn-SOD) protein were investigated in human hepatoma cells, Hu-HI, which revealed resistance to the cytotoxicity of TNF and IL-l. Both TNF and IL1 enhanced the Mn-SOD production to the level of 30- to IO-fold. IL-6 also increased the enzyme protein to 2- to 3-fold of the basal level without any cell proliferative effect. A specific antibody against IL-6 almost completely inhibited the induction of Mn-SOD. IL-6, as well as TNF and IL-l, appears to play some role in the Mn-SOD protein expression in human hepatoma cells. 0 1992Academic Press.Inc.
Superoxide cells
against
radiation of
reactive
or through
superoxide
(Cu,Zn-SOD), *
dismutases
(EC
free other
dismutase,
are
1.15.1.1)
radicals
produced
mechanisms designated
Mn-superoxide
known to
(I).
on ionization There
Cu-Zn-superoxide
dismutase
protect
(Mn-SOD)
are
three
types
dismutase
and extra
cellular
To whomcorrespondence should be addressed.
The abbreviations used are: Mn-SOD, Mn-Superoxide dismutase; TNF, tumor necrosis factor; IL-l, interleukin-1; IL-6, interleukin-6; IFN-gamma, interferon-gamma; PBC, primary biliary cirrhosis; ELISA, enzyme-linked immunoadsorbent assay. 0006-291X/92$1.50 Copyright All rights
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
1100
Vol.
182,
SOD
No.
3, 1992
BIOCHEMICAL
Mn-SOD
(2-4).
whereas
Cu,Zn-SOD
SOD in
extra-cellular
striking
effects
induction
were
(8)
and
such
we examined Mn-SOD first and
the
protein report
IL-l
Materials
protein
in
TNF and/or
of
in
IL-l
the
Mn-SOD (LPSl(9)
effects human
effect
matrix, and extra-cellular
recently sera
which
we found
from
a
patients
macrophage
on the
Mn-SOD
of
in
Mn-SOD
protein
hepatoma
of
TNF,
induction in
hepatoma
expression
human
in
COMMUNICATIONS
with
or
T cell-
protein
(5).
biological in
cytosol
Mn-SOD
showed
investigated
the
Very
(PBC),
RESEARCH
mitochondrial
(2-4).
suggested
works
the
in
lipopolysaccharide
However, been
of
BIOPHYSICAL
fluid
cirrhosis
mediated
at
localized
of
biliary
Recent
located
is
increase
primary
is
AND
several
cell
TNF and
IL-l
cells.
In
IL-l, human
by TNF
IL-6
and
hepatoma
induction
(6,7), lines.
have the
not
by IL-6
as
yet
present
IFN-gamma
cells.
IL-l
study on the
This
is
the
well
as
TNF
cells.
and Methods
Chem$cals: Human recombinant TNF-alpha (TNF), specific activity 2 x 10 unit/mg protein; human recombinant IL-l-alpha (IL-l), specific activity, 8 x lo6 units/mg prqtein; human recombinant IL-6 (IL-6), specific activity, 1 x 10 units/mg protein; human reco binant interferon-gamma (IFN-gamma), specific activity, 2.5 protein and polyclonal rabbit anti-human IL-6 were x 10 5 units/mg obtained from Genzyme Corp., Cambridge, USA. a human hepatocellular carcinoma Cells and cell culture: Hu-HI, ___-cell line, was kindly supplied by Dr. Naraki, Eisai Laboratory. Cells were grown in Eagle's Minimum Essential Medium (MEM) containing 10% fetal calf serum (Gibco), and 3% glutamine, 100 and 100 ug/ml streptomycin. units/ml penicillin, The cells were plated in culture flasks (Costar) and routinely maintained in a CO? incubator at 37OC. HU-~1 cells (1 x 10' /well) were plated in Cyeokine treatment: 96-well elates and incubated for 24 to 72 h with or without an addition-of TNF, IL-l, IL-6 and IFN-gamma in the culture medium. After incubation, the medium was removed and each well was washed 1 mM EDTA in PBS was added, with PBS. Solution of 0.01% trypsin, The enzyme reaction was and the plates were incubated for 5 min. stopped by adding PBS. All cells were removed by centrifugation at 1500 rpm for 10 min. Finally to each tube was added 0.5 ml PBS, and the mixture was frozen at -8OOC until used. Neutralization of IL-6 activity by anti-IL-6 antibody: A polyclonal rabbz x-human IL-6 was diluted by culture medium to various concentrations, and added to the IL-6 solution (100 U/ 1) followed incubation at 4OC over night. The cells (1 x 10 '5 /well) plated in 96 well plates were incubated for 24 h in the 1101
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
pre-incubated culture medium containing IL-6 and anti-IL-6 antibody. After incubation, the cells were harvested in the same way described above and stored at -8OOC. available Assay of Mn-SOD: Mn-SOD was measured with commercially ELISA kit (Mn-SOD ELISA kit. Ube Industries, Jaoan) as we previously‘described (5,7).' Each sample was thawed at room temperature, followed sonication. After centrifugation at 3,000 rpm for 15 min, the supernatant was diluted 10 to 100 fold with 10 mM phosphate buffer, pH 7.4 containing 0.5 M NaCl, 0.1% bovine serum albumin and 0.09% Kathon CG (w/v) (Rohm and Haas, Japan). Effect of IL-6 on Hu-Hl cell proliferation: An eff ct of IL-6 on toEferati= of-HI cells was assessed by [ f Hlthymidine ( 9 H-TdR) incorporation as we previously described (10). The cells were cultured for 24 and 48 h in the presence or absence of various concentra ions of recombinant human IL-6 at a cell density of 1 x 10 fi /ml in 96 wells microculture plates, then cells were pulsed for 4 h with 1 uCi/well 3H-TdR (New England Nyclear). After incubation, the cells were harvested and uptake of H-TdR was measured by liquid scintillation counter. Assay for cytotoxicity of TNF and IL-l: Cells (1 x lo5 /well) were plated in 96-well plates and incubated for 48 h with the addition of TNF or IL-l. Viability of the cells were examined by morphological observation (11). Protein concentration was determined according to Protein assay: the procedure by Lowry -et al. (12) using bovine serum albumin as the standard.
Results Effects
-of TNF,
expression
in
Hu-Ml
When Hu-Hl protein
level
protein
in
IL-l,
IFN-gamma
(Fiq.
and ---~
with
no addition
was 99 ng/mg protein 118
on
Mn-SOD
protein
1,2).
was cultured
48 h, and
IL-6
of
in 24 h culture;
ng/mg protein
in
cytokine, 111
72 h culture.
Mn-SOD ng/mg Addition
5ooor
5OOOr
TNF
(nglml)
IL- 1 W/ml)
Fiq. 1. Effect of TNF and IL-l on Mn-SOD protein induction in Hu-Hl cells. Hu-HI cells were treated with TNF and IL-l in the indicated concentrations for 24, 48 and 72 h, Mn-SOD protein levels were determined by the ELISA as described in the "Materials and Methods". Each value was obtained from duplicate samples, in which the coefficient of variation was within 5%. Open circle, 24 h culture; closed circle, 48 h culture; open triangle, 72 h culture.
1102
Vol.
182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
500-
-
500
O 010 IL-0
100
o-
1000
RESEARCH COMMUNICATIONS
’
010
w/ml)
loo IFN-T
1000 (U/ml)
2. Effect of IFN-gamma and IL-6 on Mn-SOD protein induction in Hu-Hl cells. Hu-Hl cells were treated with IFN-gamma and IL-6 and then Mn-SOD protein levels were determined by the ELISA as described in Fig. 1. Open circle, 24 h culture; closed circle, 48 h culture; open triangle, 72 h culture. Fig.
of
TNF resulted
dependent
manner.
addition 2405, fold
of
TNF to concentrations
the
expression
basal
after
of
ability
to
contrast,
in Mn-SOD levels
of
1,
IL-l
also
and
induce
with 100
and
10
induced
U/ml.
IL-l
Mn-SOD protein
with
was
were 20- to
40-
Mn-SOD protein increase
IL-l
was
to
seemed to
in Hu-HI
effect
rig/ml
100
the
of
in a dose-
48 culture
30-fold
addition
had little
after
which
approximately
10
IFN-gamma
level
respectively,
48 h culture
concentrations the
level.
significantly;
observed
increase
The Mn-SOD protein
and 4481 rig/ml,
4191
of
in a marked
share
cells.
with
TNF
In
on Mn-SOD protein
expression. The cytotoxicity the
Materials
and Methods.
inhibitory
effect
that
is
Hu-Hl
Effect
of ---~ IL-6 Since
biologic both
IL-l
of TNF or IL-l
on cell
one of
the
viability
(data
revealed not
shown), cells.
on Mn-SOD protein
expression
in Hu-HI --
TNF and IL-6 and also
and TNF (13), expression. induction
have
been known to
IL-6
As shown in Fig.
an effect 2, IL-6
in a dose dependent 1103
share
gene expression
we investigated
in
no
IL-l-resistant
activities,
Mn-SOD protein
These cytokines
as described
TNF-or
IL-l,
SOD protein
was examined
is of
indicating
several induced IL-6
on Mn-
stimulated
manner,
by
although
the the
Vol.
182,
No.
BIOCHEMICAL
3, 1992
AND
OL 0 ;
BIOPHYSICAL
10
60 IL-6
RESEARCH
loo
6601000
COMMUNICATIONS
.J
W/ml)
Fiq. & L3H]thymidine incorporation of Hu-HI after IL-6 treatment. An ef ect of IL-6 o the proliferation of Hu-Hl cells was assessed by [ 5 Hlthymidine ( 9 H-TdR). The cells were cultured for 24 and 48 h in the presence of indicated concentgations of recombinant human IL-6, then cells were pulsed with H-TdR. Each value was obtained from 5 samples. Bar indicates the standard deviation. Open circle, 24 h culture; closed circle, 48 h culture.
effect
was
not
so
as in the
marked
Mn-SOD protein
level
after
concentrations
of
ng/mg protein,
respectively,
10,
cases
48 culture and
100
of
with
TNF and IL-l. addition
U/ml was 237,
1000
which
were 2- to
of
The IL-6
to
288 and 354
3-fold
of
the
basal
level. Effect ___-----
of
IL-6
on Hu-Hl
cell
whether
the
To examine was merely
a result
influence
of
investigated. to
1000
U/ml,
incorporation, culture
of
IL-6
to
Mn-SOD protein
cell
on the
resulted
have
of
IL-6
no significant
that cell
of
of Hu-Hl
an was
concentrations,
difference
in the
was observed
24 h culture.
proliferative
by IL-6
or not,
to various
some decrease
with
little
effect
3H-TdR incorporation
in
although
induction
proliferative
The addition
as compared
appeared
proliferation
3H-TdR
in 48 h
Therefore,
effect
1
IL-6
on Hu-Hl
(Fig.
3). Neutralization IL-6
of --
IL-6
induced
Mn-SOD p rotein
expression
by anti-
antibody To verify
an inhibition
the
potency
by specific
of
IL-6
antibody 1104
on Mn-SOD protein against
IL-6
expression,
was examined.
As
Vol.
182,
No.
BIOCHEMICAL
3, 1992
Table
I.
Neutralization
AND
of IL-6
BIOPHYSICAL
RESEARCH
activity
COMMUNICATIONS
by anti-IL-6
antibody
Mn-SOD protein no addition IL-6 IL-6 plus Ab ( 1 pglml) Ab (100 pg/ml) Ab 1 pglml 100
(%)
100
237 177 106 105
pg/ml
119
Inhibition of anti-human IL-6-antibody on the Mn-SOD protein induction by IL-6 (100 U/ml) was examined. The basal level (no addition) of Mn-SOD protein in Hu-Hl cells is expressed as 100%. IL-6, human recombinant IL-6; Ab, rabbit polyclonal anti-human IL-6-antibody. Each value was obtained from triplicate samples.
shown in Table induced to
I,
a polyclonal
Mn-SOD protein
concentration
inhibited,
of
whereas
expression. ug/ml
100
the
Mn-SOD induction
antibody
(Table
With
the
antibody
neutralized an addition
induction
alone
the
of antibody
was almost
had little
IL-6
completely
effect
on the
I).
Discussion Mn-SOD is (9) is
in
several
cell
known about
In the
the
present
stimulated
not
Very protein
study,
of
level
in
an increased lymphocytes
liver
production
potent
expression
that
of
found
both
(8)
however,
and LPS little
on Mn-SOD induction.
TNF and IL-l
and also
a marked with
IL-6
markedly
had similar,
elevation
investigators
(5).
Some
of
IL-l,
IFN-gamma,
of PBC patients of
in human hepatocytes. 1105
demonstrated
immunohistologically
tissues
inducibility
of Mn-SOD
PBC, and also
Mn-SOD protein
TNF,
IL-l
effect.
patients
and macrophages the
cytokines
stimulatory
sera of
(6,7),
human hepatocytes,
induction
we have
expression
by TNF
these
we found
so marked,
corresponding
protein
In
effect
recently,
an enhanced
speculated
lines.
Mn-SOD protein
although
the
known to be induced
these
reported
and IL-6 (14),
so that
cytokines
Wong et --
al.
in
in we have
on Mn-SOD showed that
Vol.
182,
cell
No.
killing
RNAs,
by TNF could
and
indicated
resistance
to
induction In
in
PBC,
of
it
TNF or
lymphocytes
for
influence
of
TNF, to
IL-l
initiate
AP-1
and
(19),
are
has
been
encoding
to
shown
of
understood. ester
also
mRNA
cell
the
affected
Mn-SOD
cellular
Mn-SOD
been
activated
present
the
reaction.
and
IL-6
have
expression
lines
shown
(6).
(16). hepatocytes
the
cytotoxicity
macrophages
of
or
to
Mn-SOD Very
there
pathways,
protein
dependent
ones
signaling
pathways
induction,
and
EL-6
share
the
(17).
expression
(17).
in
the
--et
are
least
two
C dependent
al.
and
1106
not
in
still phorbol
cells
and
transduction kinase
C non-
intracellular in the
the IL-
A precise
that
protein
factors
be involved
in
genes
TNF-resistant signal
(la),
itself,
(21). is
reported
may be several
transcription
several
IL-1
ability
TNF and
IL-6,
liver
the
(NF-kB)
both
of
have
Several
Again,
in
might
almost
(13).
IL-l
Mn-SOD
and
was
be involved
mRNA of
There
little
to
Fujii
kinase
with
factor-kB
by TNF and
at
to
verified
proteins
indicated
proteins
induction
(22).
to
transcription
recently
induced
which
nuclear
gene
activate
effect
reported
several
are
revealed
expression
antibody,
TNF and IL-6
IL-6
This
been
(20),
induce
that
protein
including
NF-IL-6
suggested
study,
for
human acute-phase
mechanism
the
against
by anti-IL-6
actions known
of
has
proliferation.
factors,
biological 1
cell
gene
transcription
of
transformed
that
Mn-SOD
inhibited
specificity
as
immunity
the
stimulate
on the
completely
found
cytoprotection
in
to
COMMUNICATIONS
by expression
a determinant
also
by the
RESEARCH
PBC.
Interestingly, an ability
is
be possible
produced
in
BIOPHYSICAL
as well
cellular
Mn-SOD IL-1
They
cells
might
produce
Mn-SOD
TNF (15). normal
AND
be modulated
that
an altered
Therefore, also
BIOCHEMICAL
3, 1992
the
process(es).
Mn-SOD
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Acknowledgments We would like to thank Miss Izumi Okumura for her excellent technical assistance. This work was partially supported by grants from the Ministries of Education, Science and Culture, Japan.
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