BIOCHEMICAL
Vol. 168, No. 3, 1990 May 16, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1217-1222
EFFECT OF INTERLEUKIN 1 (IL-l) ON THE LEVELS OF CYTOCHROME P-450 INVOLVING IL-l RECEPTOR ON THE ISOLATED HEPATOCYTES OF RAT Kazufumi
Sujita.
Yoshiaki
Hirano,
The First Department Labora tory, University of Medicine, l-l Received
April
Fumio
Okuno.
Yoshiya
Yoshito Inamoto, and Masao Arai*
Tanaka,
Sumiya
Eto,
of Internal Medicine, and *The of Occupational and Environmental Iseigaoka Yahatanishiku Kitakyushu
Central Clinical Health, School 807, Japan
9, 1990
In the present study, the expression of IL- 1 receptor (IL-1 R) on the primary cultured hepatocytes was determined and the effect of IL-1 on the intracellular amount of cytochrome P-450 was investigated. The kinetics of IL-1 R on the hepatocytes was studied by C’2511-IL-la and revealed that 5,000 molecules of IL-l bound to a hepatocyte with a Kd of 4.0 x lo-’ M. Incubation of the hepatocytes with IL- 1 for 18hrs decreased the contents of cytochrome P-450 in a dose-dependent manner. These findings suggest that IL-1 potentially depresses the drug metabolism involving IL-1 R which is expressed on hepatccytes. 01990 Academic Press,
Inc.
Interleukin and
1 (IL-l)
is a potent
regulator
effects of IL-l
located drug
and
Kupffer
anatomically
closely
metabolism.
enzyme
activities
Previously
we
metabolism
in
whether The
of albumin
since
this
purpose
hepatocytes using primary
Recent in endotoxin reported
of IL-1
of the present and the in vitro cultured
include
product
cells,
(1 ) (2)
it
to hepatocytes, showed
sensitive
(5 ) .
effect
Since
considered
IL- 1
that
monccytes inflammatory
but not in
it
still
hepatocytes
is to examine
reactions.
of macrophage that
IL-1
the
is
The
synthesis
IL- 1 affects hepatic
protein
origin,
are
can affect hepatic
decreased
drug-metabolising
resistant
mice (3 ) (4 1 .
effect of in vivo administration However,
or macrophages
of acute phase protein
is possible
mice,
on the rat study
.
are
by
and
stimulation
which
works
released
responses
the
synthesis
the inhibitory
the rat liver action
soluble
of immunological
on the liver
and the inhibition synthesis
is a
of IL-1
remains
to
be
receptor - mediated
existence
of
IL-1R
of IL- 1 on the levels of cytochrome
on drug
investigated or
not.
on
rat
P-450
by
hepatocytes.
interleukin 1; rIL-1, recombinant Abbreviations used in this naner: IL-l, interleukin 1; IL-1R. interleukin 1 receptor; FCS, fetal calf serum; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; PBS, phosphate buffered saline; RES, reticuloendothelial system; LPS. lipopolysaccharide. ooo6-291xBo 1217
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
BIOCHEMICAL
166, No. 3, 1990
AND BiOPHYSlCAL
RESEARCH COMMUNICATIONS
METHODS Isolation of rat hepatocytes. Hepatocytes were isolated from male Wistar rats perfusion technique as described by Seglen (250 - 300 g) by the two step-liver were suspended (6) and modified by Williams et al. (7) . The isolated hepatocytes in Williams medium E containing 10 % FCS, 100 U/ml penicillin, 100 pg/ml streptomycin, and 0.25jfg/ml fungizone at a concentration of 1 x 10’ cells/ml. The cells were plated in plastic culture dishes (Falcon 3002 ;Falcon Plastics, Oxnard, CA) and cultured at 37’C in 5% COZ and 95% air. IL-l was labeled by the method of Dower Radiolabeling of human rIL-1 . was et al. (8) . Briefly, 1 pg of IL-la in 50 ~1 of O.lM borate buffer (pH 8.5) added to 1 mCi [ iz5 11 -labeled Bolton Hunter reagant (2200 Ci/mM; New England Nuclear, Boston, MA) that had been evaporated by a gentle stream of dry nitrogen. The reaction was allowed to proceed on ice for 1 hr and was terminated by the of 5/11 of 1M glycine ethyl ester. Then 30~1 of 2% gelatin in PBS (0.05M addition (pH 7.4) were added as a carrier, and labeled phosphate, 0.15M buffered saline) chromatography on a l-ml column of Bio-Gel P-10 IL-la was separated by (Bio-Rad Laboratories, Richmond, CA) . Aliquots (100,nl) were collected, and The specific activity fractions containing protein- binding radioactivity were pooled. of C I 25 11 -labeled IL- la was 7.6 x lo6 cpm/pg protein. [12511-labeled IL- la band on SDS polyacrylamide gels. Although the migrated as a single 17-kDa radiolabeled IL-la lost about 80% of their activity when assayed by murine thymocyte proliferative responses, they retained .a binding activity to rabbit anti - IL- la antibody. Binding of C 12511-labeled IL- la to rat hepatocvtes. After 16 hrs of culture, hepatocytes were harvested by pipetting. The’ cells were washed three times with PBS containing 1 mg/ml BSA (Sigma Chemical Co., St. Louis, MO) , and the cell was adjusted to 5x106 cells/O.2 ml PBS. Different concentrations of number C 1 25 13 -IL-la were added to cell suspensions in Eppendorf tubes at 4’C . After the incubation at 4°C for 1 hr. the cell suspensions were layered over ZOO@ of a the method of mixture of 20% olive oil and 80% di-N-butyl phthalate by Uchiyama et al. (9) , and were centrifuged at 10,000 x g for 1 min (4’C ) to separate free and bound IL-l. The nonspecific binding of CiZ511 -IL-la was determined by incubating cells with labeled IL-l in the presence of a loo-fold excess of cold IL- la. In the competitive binding assay study, we had confirmed that IL-l R is specific for IL-l, and IL-la and IL-la share identical IL-1R (10) . The results were expressed as the mean cpm (specific binding) in which the nonspecific binding was subtracted and as the standard error (SE) of triplicate cultures. Effect of IL-l on cytochrome P- 450 in cultured hepatccytes. After placing hepatocytes in 5% COZ and 95% air for 3 hrs, various amounts of human rIL-la (4eU/mi, 20U/ml, lOOU/ml, 500U/ml) (supplied by Dr. M. Yamada, Dainippon Pharmaceutical Co., Osaka, Japan) were added to culture dishes. Eighteen hours later, cultured hepatocytes were washed with cold PBS and scraped off the dishes using a u rubber policeman” . The cell suspensions were washed by cold PBS three times, and were homogenized using a Teflon -glass Potter -Elvehjem type homogenizer in cold To measure cytochrome P- 450, homogenates were prepared in cold 1 .15% KCl. 1.15% KC1 at a concentration of 1 x lo7 cells/ml. contents Cytochrome P- 450 were determined by the method of Omura and Sato (11 ) . The results were expressed as mean nmol/mg protein and SE of triplicate cultures. The method of Lowry et al. (12) was used to determine the protein contents of homogenates.
RESULTS Binding C lZ5 I] -IL-h were
incubated
dose response
assav to
of
C12511-IL-1~
hepatocytes for 1 hr
in
reached
on
of
hepatocvtes.
the maximum
the following
curve of the binding
rat
binding
C 12511-IL-la 1218
levels within assay study. to
As the binding 1 hr Fig.2A
rat hepatocytes.
(Fig. 1),
of cells
shows Scatchard
a
Vol.
166, No. 3, 1990
BIOCHEMICAL
010
30
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
60
160 Incubation
Fig. 1:
plot
analysis
of
these data
Fig.3,
of
by
were
nmol/ mg
protein.
cytochrome
500 U/ml release
for 18 hrs with
P-450
P-450
The
addition
levels
in
0.084kO.008 IL-la,
that
a
in
The
rat
IL-la
cultured
IL-la
the
incubation
manner, protein
blue exclusion
test
in were
hepatocytes
were
is
with and
was not affected even with
of The
0.118f0.005
medium
that
nmol/mg
shown
of IL-la.
without into
estimated
hepatocytes
concentrations
dose-dependent
the cell viability
As
rat
only
M.
hepatccytes.
Primary
hepatocytes
trypan
as 4 .O x lo-’
manner. different
expressed
la on the cells were
cultured
four
of
hepatocytes
in primary
and 0.067+0.007
respectively.
showed
in
rat
was calculated
P-450
P-450
that
C1z511 -IL-
values
in a dose-dependent
cytochrome
0.095+0.001,
the Kd
on cvtochrome
incubated of
revealed
sites of
of cytochrome
IL-la
contents
(Fig.2 B)
binding
and
IL-la
the contents
decreased rats
The
molecules/cell
Effect
(min)
Kinetic study of the binding of ClZ5 11 -IL-la to rat hepatocytes. Rat hepatocytes were cultured for 16 hrs and were harvested by pipetting. The harvested cells were washed with PBS and incubated with 0.27 nmol C12511-IL-la at 4’C for the period indicated. After incubation, the cells were centrifuged on 20% olive oil and 80% di-N- butyl phthalate, and the radioactivities of cell- bound C12513- IL- la were counted by a gamma counter. The results were expressed as the mean specific binding (cpm) f SE of triplicate cultures.
one type of IL-1R. as 5.000
360 time
decreased
0.093iO.005. 4, 20, 100 and
cytoplasmic
enzyme
the
dose of
highest
IL- la. DISCUSSION It
is now
(RES)
activity
the liver. zymosan variety
a
well
affects drug
An earlier prolonged of drugs
established
study
barbiturate which
phenomenon
metabolism,
that
which
is
the
one of the important
by Wooles et al. demonstrated anesthesia
(13)
modify RES activity
.
Di Carlo
also prolonged
1219
reticuloendothelial
stimulation et al. sleeping
system
functions of the RES
showed time
of by
that a wide caused
by
Vol.
BIOCHEMICAL
168, No. 3, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Added1251-ILl= O.OB-
(nM)
(B) t?
Ol 0
Fig. 2:
wall hepatic the
gram
negative
decrease
consequence
in
be
responsible
for
immunoactive metabolism
organisms,
LPS- treated indicate
other
hepatic
drug
been
hepatic
drug
metabolism
the chemical
the
Renton
depression (16)
LPS
.
of However,
is a weaker is stable
mediator(s)
the
system, products
than interferon
loss of the
It has been suggested agents
perhaps
1220
on
that
unlike
be an
that
(sl
by
is some
depress drug
the humoral
LPS-induced
been
interferon
caused
LPS can markedly that
and/or
these cells have
metabolism
and
that
indirect
monocytes
proposed
may be a candidate
metabolism.
may
released from
drug
the facts
58%
to cause a marked
Mannering
hepatic
of
of the cell
these
interferon-inducer,
to heat
component
(15 ) .
by
and
(LPS)
shown
system
effects on the immune
responsible.
serum
has oxidase
agents while
lipopolysaccharide
function
In particular,
to
the
mixed
of their
macrophages.
20
cells)
hepatocytes.
Endotoxin,
microsomal
thought
to rat
(14 ) .
of
‘*6I-ILlo:
Binding of C’2511 -IL-la to rat hepatocytes and Scatchard plot analysis. Panel A shows the dose-response curve of C’z511IL-la binding to rat hepatocytes. Rat hepatocytes were treated with various doses of C’2511-IL-la at 4’C for 1 hr. After washing, cell- bound radioactivities were counted. The results are (cpm) of [lz511 -IL-la. expressed as the specific binding Panel B shows the Scatchard plot analysis of C12511-IL-la bound
barbiturates
16
12 (fmol/5x106
a
ILd
interferon
factor (17)
for a depressant
in , of
Vol.
BIOCHEMICAL
168, No. 3, 1990
IL-1
logx)[
Fig.
3:
a
study,
considerable
levels of cytochrome al.
found
that
hepatocytes. significant
suggest
(U/ml)1
amount
in
system. a
hepatic
demonstrated
that
primary
of
and
found
P- 450 contents IL-l
decreases
that
depress
we
lowered
Subsequently,
monooxygenase
concentration
RESEARCH COMMUNICATIONS
Effect of IL- la on the contents of cvtochrome P-450 in cultured rat hepatocytes. Isolated hepatocyteswere cultured with various amounts of IL-la (4U/ml. 20U/ml. lOOU/ml. 500U/mll for 18 hrs. The cultured hepatocytes were washed with PBS and scraped off the dishes. Cytochrome P-450 and protein contents of cultured hepatocytes were assayed as described in the Method section. The results were expressed as means (nmol/mg protein) + SE of triplicate cultures.
In the present express
AND BIOPHYSICAL
Kupffer drug
in
activity
Shedlofsky
et al.
cytochrome Our
metabolism are clinically
depressed
when
levels
are
of
chemical is IL-l,
and
and
(20 1 , and alcoholic
which
that
IL-l
into
mice
liver disease
example,
Gezzi
et
in
rat
and
found
of the microsomal authors
has a potent action
because the hepatic for
decreases the
diethylase
in the activities
mediator
hepatocytes
In 1986,
as those of the above
important increased,
ethoxycoumarin IL-l
rat
IL-1
manner.
injected
levels
results as well
These findings
( 19 ) .endotoxemia
P-450
cell-derived
that
a dose-dependent
the
IL- 1R.
IL-1
IL-1 R
cultured
strongly activity
is mediated
drug metabolism
in trauma
to via is
(18 ) , burns
(21 ) .
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Bauer, J., Weber, W., and Tran-Thi. TA (1985) FEBS 190, 271-274. C.A. (1985 ) J. Exp. Med. 162.930-942 Ramadori, G., Sipe, J.D., and Dinarello. and Robinson, J.M. (1987) Life Science 40, Shedlofsky, S.I.. Swim, A.T., 2331-2336. Infec. Immunol., 837-840. Gezzi, P., Beatrice, S., and Pia, V. (1986) Sujita, K., Okuno, F.. and Hirano, Y. (1988) IASL Abstracts 145. Seglen, P-0. (1976) Meth. Cell. Biol. 13, 29-83. In Vitro 13, Williams, G.M.. Bermudez, E., and Scaramuzzino, D. (1978) 809-817. 1221
Vol. 168, No. 3, 1990 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Dower, SK., S.R. Kronheim, and C.J. March. (1985) J. Exp. Med. 162, 501-515. Uchiyama, T., Hori, T.. Tsudo, M., Wano, Y., Umadome, H., Tamori, S., (1985) J. Clin. Invest. 76, Yodoi. J., Maeda, M., Sawai, H.. and Uchino, H. 446-453. Tanaka, Y ., Shirakawa, F., and Oda, S. ( 1989) J. Immunol., 167- 172. Gmura, T., Sato, R. (1964) J. Biol. Chem. 239, 2379-2385. Lowry, O.H., Rosenbrough, N.J., and Farr, A.L. (1951) J. Biol. Chem.193, 265 -275 Wooles, W.R., Borzelleca, J.F. (1966) J. Reticuloendothel. Sot. 3, 41-47. DiCarlo, F.J., Haynes, L.J., Coutinho, C.B., and Phillips, G.E. (1965) J. Reticuloendothel. Sot. 2, 360-361. Gorodischer, R., Krasner. J., McDevitt. J. J., Nolan, J.P., and Yaffe, S.J. ( 1976) Biochem. Pharmacol. 25, 351-353. Renton, K.W.. Mannering, G.J. (1976) Biochem. Biophys. Res. Commun. 73, 343-348. Egawa, K., Yoshida, M., and Kasai, N. (1981) Microbial. Immun. 25. 1091-1096. Cohen, D.A., Ott, L., Young, B., Kaplan, A.M., and McClain, C.J. (1986 ) Fed. Proc. 45, 263, Boosalis, M., Solem, L.. McCall, J., and McClain, C.J. (1984 ) JPEN 8,102. Wolff, S.M. Dinarello, C.A., Clowes, G.H.A.. Gordon, A.H.. Saravis, C.A., and (1984 ) J. Immunol. 133,1332- 1338. McClain, C.J., Cohen, D.A., Dinarello, C.A., Cannon, J.G., Shedlofsky, S.I., and Kaplan, A.M. (1986) Life Sciences 39, 1479-1485.
1222
Vol. 168, No. 3, 1990 May 16, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1217-1222
EFFECT OF INTERLEUKIN 1 (IL-l) ON THE LEVELS OF CYTOCHROME P-450 INVOLVING IL-l RECEPTOR ON THE ISOLATED HEPATOCYTES OF RAT Kazufumi
Sujita.
Yoshiaki
Hirano,
The First Department Labora tory, University of Medicine, l-l Received
April
Fumio
Okuno.
Yoshiya
Yoshito Inamoto, and Masao Arai*
Tanaka,
Sumiya
Eto,
of Internal Medicine, and *The of Occupational and Environmental Iseigaoka Yahatanishiku Kitakyushu
Central Clinical Health, School 807, Japan
9, 1990
In the present study, the expression of IL- 1 receptor (IL-1 R) on the primary cultured hepatocytes was determined and the effect of IL-1 on the intracellular amount of cytochrome P-450 was investigated. The kinetics of IL-1 R on the hepatocytes was studied by C’2511-IL-la and revealed that 5,000 molecules of IL-l bound to a hepatocyte with a Kd of 4.0 x lo-’ M. Incubation of the hepatocytes with IL- 1 for 18hrs decreased the contents of cytochrome P-450 in a dose-dependent manner. These findings suggest that IL-1 potentially depresses the drug metabolism involving IL-1 R which is expressed on hepatccytes. 01990 Academic Press,
Inc.
Interleukin and
1 (IL-l)
is a potent
regulator
effects of IL-l
located drug
and
Kupffer
anatomically
closely
metabolism.
enzyme
activities
Previously
we
metabolism
in
whether The
of albumin
since
this
purpose
hepatocytes using primary
Recent in endotoxin reported
of IL-1
of the present and the in vitro cultured
include
product
cells,
(1 ) (2)
it
to hepatocytes, showed
sensitive
(5 ) .
effect
Since
considered
IL- 1
that
monccytes inflammatory
but not in
it
still
hepatocytes
is to examine
reactions.
of macrophage that
IL-1
the
is
The
synthesis
IL- 1 affects hepatic
protein
origin,
are
can affect hepatic
decreased
drug-metabolising
resistant
mice (3 ) (4 1 .
effect of in vivo administration However,
or macrophages
of acute phase protein
is possible
mice,
on the rat study
.
are
by
and
stimulation
which
works
released
responses
the
synthesis
the inhibitory
the rat liver action
soluble
of immunological
on the liver
and the inhibition synthesis
is a
of IL-1
remains
to
be
receptor - mediated
existence
of
IL-1R
of IL- 1 on the levels of cytochrome
on drug
investigated or
not.
on
rat
P-450
by
hepatocytes.
interleukin 1; rIL-1, recombinant Abbreviations used in this naner: IL-l, interleukin 1; IL-1R. interleukin 1 receptor; FCS, fetal calf serum; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; PBS, phosphate buffered saline; RES, reticuloendothelial system; LPS. lipopolysaccharide. ooo6-291xBo 1217
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
BIOCHEMICAL
166, No. 3, 1990
AND BiOPHYSlCAL
RESEARCH COMMUNICATIONS
METHODS Isolation of rat hepatocytes. Hepatocytes were isolated from male Wistar rats perfusion technique as described by Seglen (250 - 300 g) by the two step-liver were suspended (6) and modified by Williams et al. (7) . The isolated hepatocytes in Williams medium E containing 10 % FCS, 100 U/ml penicillin, 100 pg/ml streptomycin, and 0.25jfg/ml fungizone at a concentration of 1 x 10’ cells/ml. The cells were plated in plastic culture dishes (Falcon 3002 ;Falcon Plastics, Oxnard, CA) and cultured at 37’C in 5% COZ and 95% air. IL-l was labeled by the method of Dower Radiolabeling of human rIL-1 . was et al. (8) . Briefly, 1 pg of IL-la in 50 ~1 of O.lM borate buffer (pH 8.5) added to 1 mCi [ iz5 11 -labeled Bolton Hunter reagant (2200 Ci/mM; New England Nuclear, Boston, MA) that had been evaporated by a gentle stream of dry nitrogen. The reaction was allowed to proceed on ice for 1 hr and was terminated by the of 5/11 of 1M glycine ethyl ester. Then 30~1 of 2% gelatin in PBS (0.05M addition (pH 7.4) were added as a carrier, and labeled phosphate, 0.15M buffered saline) chromatography on a l-ml column of Bio-Gel P-10 IL-la was separated by (Bio-Rad Laboratories, Richmond, CA) . Aliquots (100,nl) were collected, and The specific activity fractions containing protein- binding radioactivity were pooled. of C I 25 11 -labeled IL- la was 7.6 x lo6 cpm/pg protein. [12511-labeled IL- la band on SDS polyacrylamide gels. Although the migrated as a single 17-kDa radiolabeled IL-la lost about 80% of their activity when assayed by murine thymocyte proliferative responses, they retained .a binding activity to rabbit anti - IL- la antibody. Binding of C 12511-labeled IL- la to rat hepatocvtes. After 16 hrs of culture, hepatocytes were harvested by pipetting. The’ cells were washed three times with PBS containing 1 mg/ml BSA (Sigma Chemical Co., St. Louis, MO) , and the cell was adjusted to 5x106 cells/O.2 ml PBS. Different concentrations of number C 1 25 13 -IL-la were added to cell suspensions in Eppendorf tubes at 4’C . After the incubation at 4°C for 1 hr. the cell suspensions were layered over ZOO@ of a the method of mixture of 20% olive oil and 80% di-N-butyl phthalate by Uchiyama et al. (9) , and were centrifuged at 10,000 x g for 1 min (4’C ) to separate free and bound IL-l. The nonspecific binding of CiZ511 -IL-la was determined by incubating cells with labeled IL-l in the presence of a loo-fold excess of cold IL- la. In the competitive binding assay study, we had confirmed that IL-l R is specific for IL-l, and IL-la and IL-la share identical IL-1R (10) . The results were expressed as the mean cpm (specific binding) in which the nonspecific binding was subtracted and as the standard error (SE) of triplicate cultures. Effect of IL-l on cytochrome P- 450 in cultured hepatccytes. After placing hepatocytes in 5% COZ and 95% air for 3 hrs, various amounts of human rIL-la (4eU/mi, 20U/ml, lOOU/ml, 500U/ml) (supplied by Dr. M. Yamada, Dainippon Pharmaceutical Co., Osaka, Japan) were added to culture dishes. Eighteen hours later, cultured hepatocytes were washed with cold PBS and scraped off the dishes using a u rubber policeman” . The cell suspensions were washed by cold PBS three times, and were homogenized using a Teflon -glass Potter -Elvehjem type homogenizer in cold To measure cytochrome P- 450, homogenates were prepared in cold 1 .15% KCl. 1.15% KC1 at a concentration of 1 x lo7 cells/ml. contents Cytochrome P- 450 were determined by the method of Omura and Sato (11 ) . The results were expressed as mean nmol/mg protein and SE of triplicate cultures. The method of Lowry et al. (12) was used to determine the protein contents of homogenates.
RESULTS Binding C lZ5 I] -IL-h were
incubated
dose response
assav to
of
C12511-IL-1~
hepatocytes for 1 hr
in
reached
on
of
hepatocvtes.
the maximum
the following
curve of the binding
rat
binding
C 12511-IL-la 1218
levels within assay study. to
As the binding 1 hr Fig.2A
rat hepatocytes.
(Fig. 1),
of cells
shows Scatchard
a
Vol.
166, No. 3, 1990
BIOCHEMICAL
010
30
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
60
160 Incubation
Fig. 1:
plot
analysis
of
these data
Fig.3,
of
by
were
nmol/ mg
protein.
cytochrome
500 U/ml release
for 18 hrs with
P-450
P-450
The
addition
levels
in
0.084kO.008 IL-la,
that
a
in
The
rat
IL-la
cultured
IL-la
the
incubation
manner, protein
blue exclusion
test
in were
hepatocytes
were
is
with and
was not affected even with
of The
0.118f0.005
medium
that
nmol/mg
shown
of IL-la.
without into
estimated
hepatocytes
concentrations
dose-dependent
the cell viability
As
rat
only
M.
hepatccytes.
Primary
hepatocytes
trypan
as 4 .O x lo-’
manner. different
expressed
la on the cells were
cultured
four
of
hepatocytes
in primary
and 0.067+0.007
respectively.
showed
in
rat
was calculated
P-450
P-450
that
C1z511 -IL-
values
in a dose-dependent
cytochrome
0.095+0.001,
the Kd
on cvtochrome
incubated of
revealed
sites of
of cytochrome
IL-la
contents
(Fig.2 B)
binding
and
IL-la
the contents
decreased rats
The
molecules/cell
Effect
(min)
Kinetic study of the binding of ClZ5 11 -IL-la to rat hepatocytes. Rat hepatocytes were cultured for 16 hrs and were harvested by pipetting. The harvested cells were washed with PBS and incubated with 0.27 nmol C12511-IL-la at 4’C for the period indicated. After incubation, the cells were centrifuged on 20% olive oil and 80% di-N- butyl phthalate, and the radioactivities of cell- bound C12513- IL- la were counted by a gamma counter. The results were expressed as the mean specific binding (cpm) f SE of triplicate cultures.
one type of IL-1R. as 5.000
360 time
decreased
0.093iO.005. 4, 20, 100 and
cytoplasmic
enzyme
the
dose of
highest
IL- la. DISCUSSION It
is now
(RES)
activity
the liver. zymosan variety
a
well
affects drug
An earlier prolonged of drugs
established
study
barbiturate which
phenomenon
metabolism,
that
which
is
the
one of the important
by Wooles et al. demonstrated anesthesia
(13)
modify RES activity
.
Di Carlo
also prolonged
1219
reticuloendothelial
stimulation et al. sleeping
system
functions of the RES
showed time
of by
that a wide caused
by
Vol.
BIOCHEMICAL
168, No. 3, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Added1251-ILl= O.OB-
(nM)
(B) t?
Ol 0
Fig. 2:
wall hepatic the
gram
negative
decrease
consequence
in
be
responsible
for
immunoactive metabolism
organisms,
LPS- treated indicate
other
hepatic
drug
been
hepatic
drug
metabolism
the chemical
the
Renton
depression (16)
LPS
.
of However,
is a weaker is stable
mediator(s)
the
system, products
than interferon
loss of the
It has been suggested agents
perhaps
1220
on
that
unlike
be an
that
(sl
by
is some
depress drug
the humoral
LPS-induced
been
interferon
caused
LPS can markedly that
and/or
these cells have
metabolism
and
that
indirect
monocytes
proposed
may be a candidate
metabolism.
may
released from
drug
the facts
58%
to cause a marked
Mannering
hepatic
of
of the cell
these
interferon-inducer,
to heat
component
(15 ) .
by
and
(LPS)
shown
system
effects on the immune
responsible.
serum
has oxidase
agents while
lipopolysaccharide
function
In particular,
to
the
mixed
of their
macrophages.
20
cells)
hepatocytes.
Endotoxin,
microsomal
thought
to rat
(14 ) .
of
‘*6I-ILlo:
Binding of C’2511 -IL-la to rat hepatocytes and Scatchard plot analysis. Panel A shows the dose-response curve of C’z511IL-la binding to rat hepatocytes. Rat hepatocytes were treated with various doses of C’2511-IL-la at 4’C for 1 hr. After washing, cell- bound radioactivities were counted. The results are (cpm) of [lz511 -IL-la. expressed as the specific binding Panel B shows the Scatchard plot analysis of C12511-IL-la bound
barbiturates
16
12 (fmol/5x106
a
ILd
interferon
factor (17)
for a depressant
in , of
Vol.
BIOCHEMICAL
168, No. 3, 1990
IL-1
logx)[
Fig.
3:
a
study,
considerable
levels of cytochrome al.
found
that
hepatocytes. significant
suggest
(U/ml)1
amount
in
system. a
hepatic
demonstrated
that
primary
of
and
found
P- 450 contents IL-l
decreases
that
depress
we
lowered
Subsequently,
monooxygenase
concentration
RESEARCH COMMUNICATIONS
Effect of IL- la on the contents of cvtochrome P-450 in cultured rat hepatocytes. Isolated hepatocyteswere cultured with various amounts of IL-la (4U/ml. 20U/ml. lOOU/ml. 500U/mll for 18 hrs. The cultured hepatocytes were washed with PBS and scraped off the dishes. Cytochrome P-450 and protein contents of cultured hepatocytes were assayed as described in the Method section. The results were expressed as means (nmol/mg protein) + SE of triplicate cultures.
In the present express
AND BIOPHYSICAL
Kupffer drug
in
activity
Shedlofsky
et al.
cytochrome Our
metabolism are clinically
depressed
when
levels
are
of
chemical is IL-l,
and
and
(20 1 , and alcoholic
which
that
IL-l
into
mice
liver disease
example,
Gezzi
et
in
rat
and
found
of the microsomal authors
has a potent action
because the hepatic for
decreases the
diethylase
in the activities
mediator
hepatocytes
In 1986,
as those of the above
important increased,
ethoxycoumarin IL-l
rat
IL-1
manner.
injected
levels
results as well
These findings
( 19 ) .endotoxemia
P-450
cell-derived
that
a dose-dependent
the
IL- 1R.
IL-1
IL-1 R
cultured
strongly activity
is mediated
drug metabolism
in trauma
to via is
(18 ) , burns
(21 ) .
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Bauer, J., Weber, W., and Tran-Thi. TA (1985) FEBS 190, 271-274. C.A. (1985 ) J. Exp. Med. 162.930-942 Ramadori, G., Sipe, J.D., and Dinarello. and Robinson, J.M. (1987) Life Science 40, Shedlofsky, S.I.. Swim, A.T., 2331-2336. Infec. Immunol., 837-840. Gezzi, P., Beatrice, S., and Pia, V. (1986) Sujita, K., Okuno, F.. and Hirano, Y. (1988) IASL Abstracts 145. Seglen, P-0. (1976) Meth. Cell. Biol. 13, 29-83. In Vitro 13, Williams, G.M.. Bermudez, E., and Scaramuzzino, D. (1978) 809-817. 1221
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Dower, SK., S.R. Kronheim, and C.J. March. (1985) J. Exp. Med. 162, 501-515. Uchiyama, T., Hori, T.. Tsudo, M., Wano, Y., Umadome, H., Tamori, S., (1985) J. Clin. Invest. 76, Yodoi. J., Maeda, M., Sawai, H.. and Uchino, H. 446-453. Tanaka, Y ., Shirakawa, F., and Oda, S. ( 1989) J. Immunol., 167- 172. Gmura, T., Sato, R. (1964) J. Biol. Chem. 239, 2379-2385. Lowry, O.H., Rosenbrough, N.J., and Farr, A.L. (1951) J. Biol. Chem.193, 265 -275 Wooles, W.R., Borzelleca, J.F. (1966) J. Reticuloendothel. Sot. 3, 41-47. DiCarlo, F.J., Haynes, L.J., Coutinho, C.B., and Phillips, G.E. (1965) J. Reticuloendothel. Sot. 2, 360-361. Gorodischer, R., Krasner. J., McDevitt. J. J., Nolan, J.P., and Yaffe, S.J. ( 1976) Biochem. Pharmacol. 25, 351-353. Renton, K.W.. Mannering, G.J. (1976) Biochem. Biophys. Res. Commun. 73, 343-348. Egawa, K., Yoshida, M., and Kasai, N. (1981) Microbial. Immun. 25. 1091-1096. Cohen, D.A., Ott, L., Young, B., Kaplan, A.M., and McClain, C.J. (1986 ) Fed. Proc. 45, 263, Boosalis, M., Solem, L.. McCall, J., and McClain, C.J. (1984 ) JPEN 8,102. Wolff, S.M. Dinarello, C.A., Clowes, G.H.A.. Gordon, A.H.. Saravis, C.A., and (1984 ) J. Immunol. 133,1332- 1338. McClain, C.J., Cohen, D.A., Dinarello, C.A., Cannon, J.G., Shedlofsky, S.I., and Kaplan, A.M. (1986) Life Sciences 39, 1479-1485.
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