Vol.
177,
No.
June
14,
1991
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
610-618
SlGNALTRANSDUCTIONPATHWAYSINTHEINDUCTIONOFHLACLASSIANTIGEN EXPRESSION ON HUH 6 CELLS BY INTERFERON-GAMMA
Takahiro
Towata,
Norio
Hayashi*
, Kazuhiro
Katayama,
Yutaka
Sasaki,
.4kinori
Kasahara,
Hideyuki
and
First
Department
Takenobu
of Medicine,
Received
April
30,
Takehara,
Fusamoto
Kamada
Osaka
Osaka
Tetsuo
University
Medical
School,
553, Japan
1991
This study investigated the intracellular signal transduction regulating the appearance of HLA class I antigens on Huh 6 cells induced by interferon-gamma. The expression was blocked by a protein kinase C inhibitor, H-7, but not by a calmodulin antagonist, W-7, nor by a protein kinase A inhibitor, H-8, at low dose. The antigen expression was induced by a direct activator of protein kinase C, phorbol myristate acetate, but not by calcium ionophore A23187 nor an analog of CAMP, dbcAMP. Therefore, we concluded that protein kinase C is involved in the expression of HL&+ class I antigens on Huh 6 cells induced by interferon-gamma but Ca -calmodulin and cAMP are not. 0 1991 Academic Press. Inc. Interferons properties class
(l),
I
recognize
hepatitis (HBV)-DNA
signal *
and
in various
various
have
has been expression
them.
liver
should
Inc. reserved.
(2-l).
this
be little induces
be addressed.
of is
cells on to
be
as autoimmune
hepatitis
one
T
thought,
IFN-gamma-induced
However,
610
such
HL,.4
expressed
are
example,
may
IFN-gamma
Cytotoxic
mechanisms
For
induces
I antigens
diseases,
to suppress
by which
0006-291X/91 $1.50 Copyright 0 1991 by Academic Prexr. All rights of reproduction in any form
These
shown
and
cells
immunomodulatory
strongly
HL.4 class
(6).
B hepatitis.
To whom correspondence
of
and
of
and
(IFN-gamma)
hepatitis
(7),
type
transduction
antigens lyse
antiviral
kinds
kinds
viral
of
to
interferon-gamma
specific
(5),
or
known
on
both
concerned
chronicity
and
antigens
hepatocytes
antigen
are
B virus HL.4 class
I
the
causes
of
known
about
the
HL.4 class
I antigens
on
Vol.
177,
No.
2, 1991
hepatocytes, of other
BIOCHEMICAL
though cells
much
has
transduction.
receptors, and
of
l-,2-diacylglycerol DG activat.es
response.
On
the
from
concentration
an
kinase
COMMUNICATIONS
signal
transduction
an
post
receptor
intracellular
signal
cell
serves probably
activates
response.
It
adenosine signal
to occurs
in
and
leads
the
of
cells
Ca2+
compartment
also
known
(14)
and
that
the
phosphate by
of Ca2+
calmodulin is
are
to a cell
intracellular
3’:5’-cyclic into
(IP3)
a mediator
and
turn
extracellular
(11) as
(12,13),
intracellular
transduced
1,5-bisphosphate
C (PKC)
store,
in
is
inositol-1,3,5-t,riphosphate
IP3
internal
a
about
kinase
hand,
This
to
of
activating
(CAMP) protein
A.
In the to examine antigens
and
reticulum
leads
transduces
on the
extracellular
protein
rises.
accumulation
RESEARCH
phosphatidylinositol
other
‘endoplasmic
otherwise
reports
known
an
(DG)
produced.
the
some
become
When
hydrolysis
mobilization
are
BIOPHYSICAL
(8-10).
Recently, signal
there
AND
present signal
induced
work,
we used
transduction
the for
hepatoblastoma the
expression
cell of
line, HLA
Huh
6,
class
I
by IFN-gamma.
MATERIALS
AND METHODS
Cells Huh 6 cells were rultured in Dulbecco’s (GIBCO, Grand Island, NY) supplemented with (FBS) and penicillin (100 U/ml).
modified 10% fetal
Eagle bovine
medium serum
Reagents l-(5-Isoquinolinesulfonyl)-Z-methylpiperazine dihydrochloride (Hand N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide 7) dihydrochloride (H-8) were purchased from Seikagaku Kogyo Co. (Tokyo, Japan) and dissolved in dimethyl sulfoxide (20 mM). N-(6-Aminohexyl)5-chloro-1-napht alensulfonamide (W-7), phorbol 12-myristate 13acetate (PMA) C>+ lonophore . A23187 and N602-dibutyl-3:5-cyclic monophosphatd (dbcAMP) were obtained from Sigma Chemical Co.(St. Louis, MO). Recombinant IFN-gamma was kindly provided by Shionogi Co.,Ltd. (Osaka, Japan). Anti-HLA class I antibody was purchased from Chemicon International, Inc. (USA). Fluorescein-conjugated goat antimouse immunoglobulin antibody was obtained from Becton Dickinson (Mountain View, CA). Induction of HLA class I antigens Rub 6 cells were cultured in tissue culture dishes (FALCON Becton Dickinson) at 37°C for 48 hr in a humidified atmosphere CO2 in air with or without IFN-gamma. After incubation, cells 611
3002; of 5% were
Vol.
177,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
washed three times with phosphate-buffered saline (PBS) suspension was prepared by 1 hr incubation with 0.05% Suspended cells were analyzed for HLA class I antigens surface.
and a cell collagenase. on their
Flow
cytometry Suspended cells (1.~10~) were washed with PBS containing 2% bovine serum albumin and 0.1% sodium azide (PBS-BSA), and incubated with an appropriate dilution of anti-HLX class I antibody for 30 min at 4°C. After washing twice with PBS-BSA, 4 ~1 of fluorescein isothiocyanate (FITC)-labeled goat anti--mouse immunoglobulin antibody was added, and the cells were incubated for an additional 30 min at 1°C. After washing, the cells were fixed in 0.5% paraformaldehyde solution and analyzed by flow cytometry with the FACScan system (Becton Dickinson). Data analysis was based on reading 10,000 cells per sample. These series of experiments have been done three times. As we observed the same tendency at each time, we presented the following representative data.
RESULTS HLA
class
I antigens
on
incubation
with
IFN-gamma
in a dose-dependent
maximum
expression
antigens
gradually
72 hr
increased
incubation Figure
IFN-gamma suppression
obtained
was observed
2 shows was
that
6 cells
dose-dependent
were
expressed manner
after (Figure
48 hr l),
with
at lo3
U/ml.
The
expression
of HLA plass
until
72 hr
and
80% of the
expression
after
expression
suppressed
was
Huh
48 hr incubation of HLA class
by H-7, an inhibitor
(data
I antigens
not
at
shown).
induced
by
of PKC, at 10 ,Y.Y. The
up to 30 ,Y M.
350
Figure 1. IFN-gamma-induced HLA class I antigens on Huh 6 cells. Huh 6 cells were cultured with or without various concentrations of IFNgamma for 48 hr. Cells were then treated with the anti-HLA class I antibody and analyzed by flow cytometry. The data are presented as histograms, loglO relative fluorescence intensity (x axis, arbitrary units) versus number of cells (y axis).
612
I
Vol.
400
177,
No.
2, 1991
BIOCHEMICAL
AND
7
BlOPHYSlCAL
400
j (, ,$Z’...
..-
:
CONTROL
RESEARCH
: :./:-.; ii
A
-.
COMMUNICATIONS
CONTROL
6
~/ 1. ii
: :: IFN-y +H-7
103 U/ml lOuI.
IFN-y
103U/ml
+H-7
20uM
103U/ml
1
FL
IFN-y +H-7
103U/ml 30pM
IFN-y
0
IFN-y
do
10 I
103lJ/ml
1 FL 1
Figure 2. Effect of H-7 on HLA class I antigen expression induced by IFh-gamma. Cells were cultured with IFN-gamma (10 U/ml) for 48 hr in the presence of the indicated concentration of H-l (A, 10uuM; B, 20 /.LMK;C, 30bM) and then analyzed for HLA class I antigen expression by FACScan.
Huh
6 cells
incubation at the
with
also PMA
(100
concentrations
enhanced, There
induced
was
expression
the
alone
with
particularly no
at 10~ M, 20 /IM,
induce
rig/ml)
together
effect
by IFN-gamma
A23187,
HLA
of 50 ngjml
When PMA was added greatly
expressed
or
which
30bM
of HLA
100 rig/ml,
IFN-gamma,
the
This
but
(Figure of
was
18
hr
observed
not at 200 rig/ml. expression
was
3B). HLA
class
of calmodulin,
I antigens was added
3). Ca 2+ concentrat.ion,
intracellular class
I antigens
after
the antigen
expression
(Figure
of HLA class
and
antigens 3A).
W-7, an antagonist
elevates
expression
I
(Figure
at 100 rig/ml on
when
class
I antigens induced
613
nor by
did
IFN-gamma
it
did
not
change
the
(Figure
5).
Vol.
177,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
400
1
+ PMA
100 rig/ml
0 1 FL
1
FL1
Figure 3. Effect of PMA on the induction of HLA class I antigen expression. Cells were cultured with or without PMA (100 n /ml) for 48 hr and then analyzed for class I antigen expression (A). Huh 6 cells were cultured with IFN-gamma (lo3 U/ml) in the presence or absence of PMA (100 rig/ml) for 48 hr and analyzed by FACScan (B).
These
experiments
were
performed
with
100 nM, 200 n!l,
1 fi Y, and
5~ M
of .423187. F’igure cells yM
when did
pressed
6 shows H-8,
the
an inhibitor
no1 influence
the
it at 20 fl ?I and
0
expression
of HLA
of protein expression
kinasr induced
class
I antigens
4, was
added.
by IFN-gamma,
on
Huh
H-8 at 10 but. sup-
30 by?.
1 FL
1
Figure 4. Effect of W-7 on HLA class I antigen expression induced by IFN-gamma. Cells were cultured with IFN-gamma (10 U/ml) for 48 hr in the presence of W-7 (30 ,YM) and then analyzed for class I antigen expression by FACScan. 614
6
Vol.
177,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
B L_
i
CONTROL
... IFN-y
103U/ml
IFN-y 103U/ml +A23187 1OOnM
0 FL
1
FL
1
Figure 5. Effect of A23187 on the induction of HLA class I antigen expression. Huh 6 cells were cultured with or without A23187 (100 nM) for 48 hr and then analyzed for class I antigen expression (A). Cells were cultured with IFN-gamma (lo3 U/ml) in the presence or absence of A23187 (100 nM) for 48 hr. HLA class I antigen expression was analyzed by FACScan (B).
300
300
IFN-y
I
103U/mI -lFN-y
103U/ml
+H-8
20uM
t
I
m
L
I
CONTROL
Figure 6. Effect of H-8 on HLA class I antigen expressiop induced by IFN-gamma. Cells were cultured with interferon-gamma (10 U/ml) for 48 hr in the presence of the indicated concentration of H-8 (A, 10fiM; B, 20,uM; C, 30~M) and then analyzed for HLA class I antigen expression by FACScan.
61.5
Vol.
177,
No.
BIOCHEMICAL
2, 1991
FL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
I
induction of HLA class I antigen Figure 7. Effect of dbcAMP on the expression. Cells were cultured with or without dbcAMP (100 ~(1) for 48 hr and then analyzed for HLA class I 3ntigen expression (A). Huh 6 cells were cultured with IFN-gamma (10 U/ml) in the presence or absence of dbcAMP (100,uM) for 48 hr and analyzed by FACScan (B).
dbcAMP Huh
could
6 cells
enhance
not
at 1 fiI\I,
or inhibit
induce
t.he expression
of HLA class
10 y X, 100 ;r Y, or
t.he expression
1 mY (Figure
induced
I antigens
7.4), nor
by IF!+gamma
on
did
(Figure
it 7B).
DISCUSSION
.4t
least
three
transduction: which
the
is
H-7,
A with
protein
by IF!%-gamma.
But
kinase
I antigen
expression
(16).
D-M,
a Ki value
showed it
H-8 616
HL.4
also
did
no
class
at 30 fiCLM. H-8 inhibits not
inhibit
I
protein
we
These antigen kinase
a Ki value
be involved
effect
find
inhibited
manner.
by IFN-gamma,
of 1.2 y ?I and of
of If-7
PKC with
A might
To
I antigens,
H-7 also inhibited
inhibiting
inhibited
.4s 10 yM
the
signal
pathways.
HL.4 class
in
induced H-8
but
cAMP
addition
involved
as protein
.4 with
15 fiu”l
is
Thus,
10
intracellular
in a dose-dependent
while
at
and
Simultaneous
PKC
A inhibitor.
knoti n for
induces
of 3.0 DLL,
kinase
expression
kinase
that
value
6.0 DLIM (16). HLA class
IFN-gamma
expression
induced a Iii
now PKC,
a PRC inhibitor.
suggested
expression
of
when
IFN-gamma-induced
results
are
Cazt-calmodulin,
involved
employed the
pathways
we tested on
the
inhibits
PKC with the
of
in the H-8,
a
antigen protein
a Ki value HL.4
class
I
Vol.
177,
No.
2, 1991
BIOCHEMICAL
antigen
expression,
c4MP
antigen
expression
induced
antigen
expression
possibly
confirmed
class
was
dbcAMP.
I antigens
but
I antigen
results
supported
the
via
A23187
alone
did
not
did
show
any
effect
our
is
antigens
by IFhi-gamma.
.!E HLA
induced we
stated
class it
has
diseases.
Now,
expression
integration
of HBV
reason
we have conclusion,
gamma-induced CAMP are
by
in
on
integration
the
cells
Huh
our HL.4 class
and
of
of HLA
IFN-gammaThese
HL4
effect
Huh
class
I
expression.
HLA
v-cry
indicated
that
expression
class
I
important
kinds
were
of
hepatic
HL.4 class
established
order
by
to elucidate
transduction.
as a model
that
IFN-gamma-induced is
in
nor
indicated of
various
6 cells,
HL.4
I antigens
A23187
the
which
t.he
ionophore
IFh-gamma-induced
signal
I antigen
on
Calcium
expression
of
(17),
6 cells
results
further
activator
ant.igen
hepatocytes
g the
to the
chosen
no
of HLR class
wit.h
investigatin
into
was
induced
IFN-gamma.
W-7
relationship
genome
had
investigation
on HE611
of HBV
effect
In
we are
IFN-gamma
with
expression
a close
the
had no effect.
IFN-gamma-induced
involved
previously,
I antigen
becaulae
antigen
no1
dbc.iMP
I
a CAMP pathway.
obtained
Ca2+ -calmodulin
This
enhanced
expression
the
inhibited
PMA greatly
that
the
on
HLA class
to be a direct
but
induced
induce
results
H-8 at 30 UY
antagonist,
expression
in the
the expression
not.
not
COMMUNICATIONS
PM.4 induced
expression
calmodulin
I antigen
Therefore,
did
hypothesis
c!ass
involved
PM.4, known
a PKC pathway,
W--7, a potent
not
RESEARCH
to PKC involvement.
alone,
dbc.4MP
HL.4 class
it
due
added
induced
antigens
probably
with
When
BIOPHYSICAL
by IFN-gamma.
by experiments
PKC, and
AND
And
this
I the the
is
the
of hepatocytes. PKC is but
involved
in
Ca2+-calmodulin
IFNand
not.
REFERENCES 1. Trincherei, 135. 2. Durandy, Immunol.
G. and A., Virelizier, 52. 173-178.
Perussia, J.L.
B. (1985) and
617
Immunology
Griscelli,
C.
Today (1983)
6, 131Clin.
Exp.
Vol.
177,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
3. Pignatelli, M., Waters, J., Brown, D., Lever, A., Iwarson, S., Schaff, Z., Gerety, R. and Thomas, H.C. (1986) Hepatology 6, 319-353. 1. Ikeda, T., Pignatelli, ?I., Lever, A.M.L. and Thomas, H.C. (1986) Gut 27, 1198-1501. 5. Chu, C.?iI., Shyu, W.C., Kuo, R.W. and Liaw, Y.F. (1987) Hepatology 7, 1311-1316. 6. Yeo, L.A., Senaldi, G., Portmann, B., Mowat, A.P., Vergani, GM. and Vergani, D. (1990) Hepatology 12, 22$-232. 7. Onji, M., Lever, A.M.L., Saito, I. and Thomas, H.C. (1989) Hepatology 9, 92-968. 8. Erusalimsky, J.D., Kefford, R.F., Gilmore, D.J. and Milstein, C. (1989) Proc. Natl. Acad. Sci. US.4. 86, 1973-1976. 9. Ina, Y., Koide, Y., Nezu, N. and Yoshida, T.O. (1987) J. Immunol. 139, 1711-1717. 10. Mattila, P., Hayry, P. and Renkonen, R. (1989) FEBS Lett. 250, 362-366. 11. Nishizuka, Y. (1984) Nature 308, 693-697. 12. Berridge, M.J. and Irvine, R.F. (198-l) Nature 312, 315-321. 13. Berridge, M.J. and Irvine, R.F. (1989) Nature 331, 197-205. 15. Cheung, W.Y. (1980) Science 207, 19-27. 15. Nakabayashi, H., Taketa, Ii., Miyano, Ii., Yamane, T. and Sato, J. (1982) Cancer Res. 12, 3858-3863. 16. Hidaka, H., Inagaki, Y., Kawamoto, S. and Sasaki, Y. (1984) Biochemistry 23, 5036-50.41. 17. Tsurimoto, T., Fujiyama, :4. and Matsubara, K. (1987) Proc. Natl. Acad. Sci. VS.4 81, U-118.
618
177,
No.
June
14,
1991
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
610-618
SlGNALTRANSDUCTIONPATHWAYSINTHEINDUCTIONOFHLACLASSIANTIGEN EXPRESSION ON HUH 6 CELLS BY INTERFERON-GAMMA
Takahiro
Towata,
Norio
Hayashi*
, Kazuhiro
Katayama,
Yutaka
Sasaki,
.4kinori
Kasahara,
Hideyuki
and
First
Department
Takenobu
of Medicine,
Received
April
30,
Takehara,
Fusamoto
Kamada
Osaka
Osaka
Tetsuo
University
Medical
School,
553, Japan
1991
This study investigated the intracellular signal transduction regulating the appearance of HLA class I antigens on Huh 6 cells induced by interferon-gamma. The expression was blocked by a protein kinase C inhibitor, H-7, but not by a calmodulin antagonist, W-7, nor by a protein kinase A inhibitor, H-8, at low dose. The antigen expression was induced by a direct activator of protein kinase C, phorbol myristate acetate, but not by calcium ionophore A23187 nor an analog of CAMP, dbcAMP. Therefore, we concluded that protein kinase C is involved in the expression of HL&+ class I antigens on Huh 6 cells induced by interferon-gamma but Ca -calmodulin and cAMP are not. 0 1991 Academic Press. Inc. Interferons properties class
(l),
I
recognize
hepatitis (HBV)-DNA
signal *
and
in various
various
have
has been expression
them.
liver
should
Inc. reserved.
(2-l).
this
be little induces
be addressed.
of is
cells on to
be
as autoimmune
hepatitis
one
T
thought,
IFN-gamma-induced
However,
610
such
HL,.4
expressed
are
example,
may
IFN-gamma
Cytotoxic
mechanisms
For
induces
I antigens
diseases,
to suppress
by which
0006-291X/91 $1.50 Copyright 0 1991 by Academic Prexr. All rights of reproduction in any form
These
shown
and
cells
immunomodulatory
strongly
HL.4 class
(6).
B hepatitis.
To whom correspondence
of
and
of
and
(IFN-gamma)
hepatitis
(7),
type
transduction
antigens lyse
antiviral
kinds
kinds
viral
of
to
interferon-gamma
specific
(5),
or
known
on
both
concerned
chronicity
and
antigens
hepatocytes
antigen
are
B virus HL.4 class
I
the
causes
of
known
about
the
HL.4 class
I antigens
on
Vol.
177,
No.
2, 1991
hepatocytes, of other
BIOCHEMICAL
though cells
much
has
transduction.
receptors, and
of
l-,2-diacylglycerol DG activat.es
response.
On
the
from
concentration
an
kinase
COMMUNICATIONS
signal
transduction
an
post
receptor
intracellular
signal
cell
serves probably
activates
response.
It
adenosine signal
to occurs
in
and
leads
the
of
cells
Ca2+
compartment
also
known
(14)
and
that
the
phosphate by
of Ca2+
calmodulin is
are
to a cell
intracellular
3’:5’-cyclic into
(IP3)
a mediator
and
turn
extracellular
(11) as
(12,13),
intracellular
transduced
1,5-bisphosphate
C (PKC)
store,
in
is
inositol-1,3,5-t,riphosphate
IP3
internal
a
about
kinase
hand,
This
to
of
activating
(CAMP) protein
A.
In the to examine antigens
and
reticulum
leads
transduces
on the
extracellular
protein
rises.
accumulation
RESEARCH
phosphatidylinositol
other
‘endoplasmic
otherwise
reports
known
an
(DG)
produced.
the
some
become
When
hydrolysis
mobilization
are
BIOPHYSICAL
(8-10).
Recently, signal
there
AND
present signal
induced
work,
we used
transduction
the for
hepatoblastoma the
expression
cell of
line, HLA
Huh
6,
class
I
by IFN-gamma.
MATERIALS
AND METHODS
Cells Huh 6 cells were rultured in Dulbecco’s (GIBCO, Grand Island, NY) supplemented with (FBS) and penicillin (100 U/ml).
modified 10% fetal
Eagle bovine
medium serum
Reagents l-(5-Isoquinolinesulfonyl)-Z-methylpiperazine dihydrochloride (Hand N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide 7) dihydrochloride (H-8) were purchased from Seikagaku Kogyo Co. (Tokyo, Japan) and dissolved in dimethyl sulfoxide (20 mM). N-(6-Aminohexyl)5-chloro-1-napht alensulfonamide (W-7), phorbol 12-myristate 13acetate (PMA) C>+ lonophore . A23187 and N602-dibutyl-3:5-cyclic monophosphatd (dbcAMP) were obtained from Sigma Chemical Co.(St. Louis, MO). Recombinant IFN-gamma was kindly provided by Shionogi Co.,Ltd. (Osaka, Japan). Anti-HLA class I antibody was purchased from Chemicon International, Inc. (USA). Fluorescein-conjugated goat antimouse immunoglobulin antibody was obtained from Becton Dickinson (Mountain View, CA). Induction of HLA class I antigens Rub 6 cells were cultured in tissue culture dishes (FALCON Becton Dickinson) at 37°C for 48 hr in a humidified atmosphere CO2 in air with or without IFN-gamma. After incubation, cells 611
3002; of 5% were
Vol.
177,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
washed three times with phosphate-buffered saline (PBS) suspension was prepared by 1 hr incubation with 0.05% Suspended cells were analyzed for HLA class I antigens surface.
and a cell collagenase. on their
Flow
cytometry Suspended cells (1.~10~) were washed with PBS containing 2% bovine serum albumin and 0.1% sodium azide (PBS-BSA), and incubated with an appropriate dilution of anti-HLX class I antibody for 30 min at 4°C. After washing twice with PBS-BSA, 4 ~1 of fluorescein isothiocyanate (FITC)-labeled goat anti--mouse immunoglobulin antibody was added, and the cells were incubated for an additional 30 min at 1°C. After washing, the cells were fixed in 0.5% paraformaldehyde solution and analyzed by flow cytometry with the FACScan system (Becton Dickinson). Data analysis was based on reading 10,000 cells per sample. These series of experiments have been done three times. As we observed the same tendency at each time, we presented the following representative data.
RESULTS HLA
class
I antigens
on
incubation
with
IFN-gamma
in a dose-dependent
maximum
expression
antigens
gradually
72 hr
increased
incubation Figure
IFN-gamma suppression
obtained
was observed
2 shows was
that
6 cells
dose-dependent
were
expressed manner
after (Figure
48 hr l),
with
at lo3
U/ml.
The
expression
of HLA plass
until
72 hr
and
80% of the
expression
after
expression
suppressed
was
Huh
48 hr incubation of HLA class
by H-7, an inhibitor
(data
I antigens
not
at
shown).
induced
by
of PKC, at 10 ,Y.Y. The
up to 30 ,Y M.
350
Figure 1. IFN-gamma-induced HLA class I antigens on Huh 6 cells. Huh 6 cells were cultured with or without various concentrations of IFNgamma for 48 hr. Cells were then treated with the anti-HLA class I antibody and analyzed by flow cytometry. The data are presented as histograms, loglO relative fluorescence intensity (x axis, arbitrary units) versus number of cells (y axis).
612
I
Vol.
400
177,
No.
2, 1991
BIOCHEMICAL
AND
7
BlOPHYSlCAL
400
j (, ,$Z’...
..-
:
CONTROL
RESEARCH
: :./:-.; ii
A
-.
COMMUNICATIONS
CONTROL
6
~/ 1. ii
: :: IFN-y +H-7
103 U/ml lOuI.
IFN-y
103U/ml
+H-7
20uM
103U/ml
1
FL
IFN-y +H-7
103U/ml 30pM
IFN-y
0
IFN-y
do
10 I
103lJ/ml
1 FL 1
Figure 2. Effect of H-7 on HLA class I antigen expression induced by IFh-gamma. Cells were cultured with IFN-gamma (10 U/ml) for 48 hr in the presence of the indicated concentration of H-l (A, 10uuM; B, 20 /.LMK;C, 30bM) and then analyzed for HLA class I antigen expression by FACScan.
Huh
6 cells
incubation at the
with
also PMA
(100
concentrations
enhanced, There
induced
was
expression
the
alone
with
particularly no
at 10~ M, 20 /IM,
induce
rig/ml)
together
effect
by IFN-gamma
A23187,
HLA
of 50 ngjml
When PMA was added greatly
expressed
or
which
30bM
of HLA
100 rig/ml,
IFN-gamma,
the
This
but
(Figure of
was
18
hr
observed
not at 200 rig/ml. expression
was
3B). HLA
class
of calmodulin,
I antigens was added
3). Ca 2+ concentrat.ion,
intracellular class
I antigens
after
the antigen
expression
(Figure
of HLA class
and
antigens 3A).
W-7, an antagonist
elevates
expression
I
(Figure
at 100 rig/ml on
when
class
I antigens induced
613
nor by
did
IFN-gamma
it
did
not
change
the
(Figure
5).
Vol.
177,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
400
1
+ PMA
100 rig/ml
0 1 FL
1
FL1
Figure 3. Effect of PMA on the induction of HLA class I antigen expression. Cells were cultured with or without PMA (100 n /ml) for 48 hr and then analyzed for class I antigen expression (A). Huh 6 cells were cultured with IFN-gamma (lo3 U/ml) in the presence or absence of PMA (100 rig/ml) for 48 hr and analyzed by FACScan (B).
These
experiments
were
performed
with
100 nM, 200 n!l,
1 fi Y, and
5~ M
of .423187. F’igure cells yM
when did
pressed
6 shows H-8,
the
an inhibitor
no1 influence
the
it at 20 fl ?I and
0
expression
of HLA
of protein expression
kinasr induced
class
I antigens
4, was
added.
by IFN-gamma,
on
Huh
H-8 at 10 but. sup-
30 by?.
1 FL
1
Figure 4. Effect of W-7 on HLA class I antigen expression induced by IFN-gamma. Cells were cultured with IFN-gamma (10 U/ml) for 48 hr in the presence of W-7 (30 ,YM) and then analyzed for class I antigen expression by FACScan. 614
6
Vol.
177,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
B L_
i
CONTROL
... IFN-y
103U/ml
IFN-y 103U/ml +A23187 1OOnM
0 FL
1
FL
1
Figure 5. Effect of A23187 on the induction of HLA class I antigen expression. Huh 6 cells were cultured with or without A23187 (100 nM) for 48 hr and then analyzed for class I antigen expression (A). Cells were cultured with IFN-gamma (lo3 U/ml) in the presence or absence of A23187 (100 nM) for 48 hr. HLA class I antigen expression was analyzed by FACScan (B).
300
300
IFN-y
I
103U/mI -lFN-y
103U/ml
+H-8
20uM
t
I
m
L
I
CONTROL
Figure 6. Effect of H-8 on HLA class I antigen expressiop induced by IFN-gamma. Cells were cultured with interferon-gamma (10 U/ml) for 48 hr in the presence of the indicated concentration of H-8 (A, 10fiM; B, 20,uM; C, 30~M) and then analyzed for HLA class I antigen expression by FACScan.
61.5
Vol.
177,
No.
BIOCHEMICAL
2, 1991
FL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
I
induction of HLA class I antigen Figure 7. Effect of dbcAMP on the expression. Cells were cultured with or without dbcAMP (100 ~(1) for 48 hr and then analyzed for HLA class I 3ntigen expression (A). Huh 6 cells were cultured with IFN-gamma (10 U/ml) in the presence or absence of dbcAMP (100,uM) for 48 hr and analyzed by FACScan (B).
dbcAMP Huh
could
6 cells
enhance
not
at 1 fiI\I,
or inhibit
induce
t.he expression
of HLA class
10 y X, 100 ;r Y, or
t.he expression
1 mY (Figure
induced
I antigens
7.4), nor
by IF!+gamma
on
did
(Figure
it 7B).
DISCUSSION
.4t
least
three
transduction: which
the
is
H-7,
A with
protein
by IF!%-gamma.
But
kinase
I antigen
expression
(16).
D-M,
a Ki value
showed it
H-8 616
HL.4
also
did
no
class
at 30 fiCLM. H-8 inhibits not
inhibit
I
protein
we
These antigen kinase
a Ki value
be involved
effect
find
inhibited
manner.
by IFN-gamma,
of 1.2 y ?I and of
of If-7
PKC with
A might
To
I antigens,
H-7 also inhibited
inhibiting
inhibited
.4s 10 yM
the
signal
pathways.
HL.4 class
in
induced H-8
but
cAMP
addition
involved
as protein
.4 with
15 fiu”l
is
Thus,
10
intracellular
in a dose-dependent
while
at
and
Simultaneous
PKC
A inhibitor.
knoti n for
induces
of 3.0 DLL,
kinase
expression
kinase
that
value
6.0 DLIM (16). HLA class
IFN-gamma
expression
induced a Iii
now PKC,
a PRC inhibitor.
suggested
expression
of
when
IFN-gamma-induced
results
are
Cazt-calmodulin,
involved
employed the
pathways
we tested on
the
inhibits
PKC with the
of
in the H-8,
a
antigen protein
a Ki value HL.4
class
I
Vol.
177,
No.
2, 1991
BIOCHEMICAL
antigen
expression,
c4MP
antigen
expression
induced
antigen
expression
possibly
confirmed
class
was
dbcAMP.
I antigens
but
I antigen
results
supported
the
via
A23187
alone
did
not
did
show
any
effect
our
is
antigens
by IFhi-gamma.
.!E HLA
induced we
stated
class it
has
diseases.
Now,
expression
integration
of HBV
reason
we have conclusion,
gamma-induced CAMP are
by
in
on
integration
the
cells
Huh
our HL.4 class
and
of
of HLA
IFN-gammaThese
HL4
effect
Huh
class
I
expression.
HLA
v-cry
indicated
that
expression
class
I
important
kinds
were
of
hepatic
HL.4 class
established
order
by
to elucidate
transduction.
as a model
that
IFN-gamma-induced is
in
nor
indicated of
various
6 cells,
HL.4
I antigens
A23187
the
which
t.he
ionophore
IFh-gamma-induced
signal
I antigen
on
Calcium
expression
of
(17),
6 cells
results
further
activator
ant.igen
hepatocytes
g the
to the
chosen
no
of HLR class
wit.h
investigatin
into
was
induced
IFN-gamma.
W-7
relationship
genome
had
investigation
on HE611
of HBV
effect
In
we are
IFN-gamma
with
expression
a close
the
had no effect.
IFN-gamma-induced
involved
previously,
I antigen
becaulae
antigen
no1
dbc.iMP
I
a CAMP pathway.
obtained
Ca2+ -calmodulin
This
enhanced
expression
the
inhibited
PMA greatly
that
the
on
HLA class
to be a direct
but
induced
induce
results
H-8 at 30 UY
antagonist,
expression
in the
the expression
not.
not
COMMUNICATIONS
PM.4 induced
expression
calmodulin
I antigen
Therefore,
did
hypothesis
c!ass
involved
PM.4, known
a PKC pathway,
W--7, a potent
not
RESEARCH
to PKC involvement.
alone,
dbc.4MP
HL.4 class
it
due
added
induced
antigens
probably
with
When
BIOPHYSICAL
by IFN-gamma.
by experiments
PKC, and
AND
And
this
I the the
is
the
of hepatocytes. PKC is but
involved
in
Ca2+-calmodulin
IFNand
not.
REFERENCES 1. Trincherei, 135. 2. Durandy, Immunol.
G. and A., Virelizier, 52. 173-178.
Perussia, J.L.
B. (1985) and
617
Immunology
Griscelli,
C.
Today (1983)
6, 131Clin.
Exp.
Vol.
177,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
3. Pignatelli, M., Waters, J., Brown, D., Lever, A., Iwarson, S., Schaff, Z., Gerety, R. and Thomas, H.C. (1986) Hepatology 6, 319-353. 1. Ikeda, T., Pignatelli, ?I., Lever, A.M.L. and Thomas, H.C. (1986) Gut 27, 1198-1501. 5. Chu, C.?iI., Shyu, W.C., Kuo, R.W. and Liaw, Y.F. (1987) Hepatology 7, 1311-1316. 6. Yeo, L.A., Senaldi, G., Portmann, B., Mowat, A.P., Vergani, GM. and Vergani, D. (1990) Hepatology 12, 22$-232. 7. Onji, M., Lever, A.M.L., Saito, I. and Thomas, H.C. (1989) Hepatology 9, 92-968. 8. Erusalimsky, J.D., Kefford, R.F., Gilmore, D.J. and Milstein, C. (1989) Proc. Natl. Acad. Sci. US.4. 86, 1973-1976. 9. Ina, Y., Koide, Y., Nezu, N. and Yoshida, T.O. (1987) J. Immunol. 139, 1711-1717. 10. Mattila, P., Hayry, P. and Renkonen, R. (1989) FEBS Lett. 250, 362-366. 11. Nishizuka, Y. (1984) Nature 308, 693-697. 12. Berridge, M.J. and Irvine, R.F. (198-l) Nature 312, 315-321. 13. Berridge, M.J. and Irvine, R.F. (1989) Nature 331, 197-205. 15. Cheung, W.Y. (1980) Science 207, 19-27. 15. Nakabayashi, H., Taketa, Ii., Miyano, Ii., Yamane, T. and Sato, J. (1982) Cancer Res. 12, 3858-3863. 16. Hidaka, H., Inagaki, Y., Kawamoto, S. and Sasaki, Y. (1984) Biochemistry 23, 5036-50.41. 17. Tsurimoto, T., Fujiyama, :4. and Matsubara, K. (1987) Proc. Natl. Acad. Sci. VS.4 81, U-118.
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