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

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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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McCord,

J.M.,

and

Fridovich,

I.

J.

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6049-6055.

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Weisiger, 3582-3592. Weisiger, 4793-4796. Marklund,

R.A.,

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I.

(1973)

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Ono, M., Sekiya, C., Ohhira, M-O., Ohhira, M.A., Namiki, M., Endo, Y., Suzuki, K., Matsuda, Y., and Taniguchi, N. (1991) J. Lab. Clin. Med. 118, 476-483. 6. Wong, G.H.W., and Goeddel, D.V. (1988) Science 242, 941-944. 7. Kawaguchi, T., Takeyasu, A., Matsunobu, K., Uda, T., Ishizawa, M., Suzuki, K., Nishiura, T., Ishikawa, M., and Biochem. Biophys. Res. Commun. 171, 1378Taniguchi, T. (1990)

5.

1386. 8.

Matsuda, A., Longo, D.L., Kobayashi, Oppenheim, J.J., and Matsushima, K.

Y., (1988)

Appela, E., FASEB. J.

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3091.

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Visner, G.A., Dougall, W.C., Wilson, J.M., Burr, I.A., and Nick, H.S. (1990) J. Biol. Chem. 265, 2856-2864. Kohda, H., Uede, T., Yuasa, H., Yamaki, T., Osawa, H., Diamantstein, T., Yodoi, J., and Kikuchi, K. (1986) 137, 1557-1563. J. Immunol. Yamaszaki, S., Onishi, E., Enami, K., Natori, K., Kohase, M., Skamoto, H., Tanouchi, M., and Hayashi, H. (1986) Japan. J. Med. Sci. Biol. 39, 105-118. Lowry, O-H., Rosebrough, N-J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Vilcek, J., and Lee, T.H. (1991) J. Biol. Chem. 266, 73137316.

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16. 17. 18. 19.

20. 21. 22.

Kakumu, S., Yoshioka, K., Tsutsumi, Y., Wakita, T., and Arao, M. (1990) Clin. Immunol. Immunopathol. 56, 54-65. Wong, G.H.W., Elwell, J.H., Oberley, L-W., and Goeddel, D.V. (1990) Cell 58, 923-931. Scheuer, P.J. (1980) In the Liver biopsy interpretation (Scheuer P.J., Ed.), 3rd edition, pp. 47-56. Bailliere Tindall, London, U.K. Dinarello, C. (1991) Blood 177, 1627-1652. Osborn, L., Kunkel, S., and Nabel, G-J. (1989) Pro. Natl. Acad. Sci. U.S.A. 86, 2336-2340. Brenner, D.A., O'Hara, M., Angel, P., Chojikier, M., and Karin, M. (1989) Nature 337, 661-663. Akira S., Isshiki, H., Sugita, T., Tanabe, O., Kinoshita, S ., Nlshio, Y., Nakajima, T., Hirano, T., and Kishimoto, T. (1990) EMBO J. 9, 1897-1906. Gauldie, J., Richards, C., Haarnish, D., Lansdorp, P., and Baumann, H. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7251-7255. Fujii, J., and Taniguchi, N. (1991) J. Biol. Chem., in press.

1107

Induction of Mn-superoxide dismutase by tumor necrosis factor, interleukin-1 and interleukin-6 in human hepatoma cells.

Effects of Tumor Necrosis Factor (TNF), Interleukin-1 (IL-1), Interleukin-6 (IL-6) and Interferon-gamma (IFN-gamma) on the expression of Mn-superoxide...
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