Vol. 172,
No.
November
BIOCHEMICAL
3, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
15, 1990
Pages
GLUCOCORTICOIDS OF NITRIC M.
Di Rosa,*
1NHlBlT
OXIDE M.
SYNTHASE
Radomski,
R.
INDUCTION
IN MACROPHAGES
Carnuccio*
Wellcome Research Court, Beckenham,
Langley
THE
1246-1252
and
Laboratories, Kent, U.K.
S. Moncada
BR3
3BS
*Dept. of Experimental Pharmacology, University of Naples “Federico II”, via Domenico Montesano, 80131 Naples, italy Received
September
24,
1990
The effect of glucocorticoids on the production of NO2- and NO by the macrophage cell line J774 was investigated. Stimulation of the cells with lipopolysaccharide (LPS) resulted in a time-dependent accumulation of NO in the medium, reaching a plateau after 48h. Concomitant incubation of t&e cells for 24h with dexamethasone (0.001-1.0 pM) or hydrocortisone (0.01-10.0 pM) caused a concentration-dependent inhibition of NO formation. The cytosol of J774 cells stimulated LPS and with ?FN-y produced a time-dependent increase in the release of NO. This was blocked in a concentration-dependent manner by dexamethasone and hydrocortisone, but not progesterone, administered concomitantly with the immunological stimulus. None of these compounds had any effect on the release of NO once the enzyme had been induced. The inhibitory effect of hydrocortisone on NO formation was blocked by cortexolone. These data suggest that part of the anti-inflammatory and immunosuppressive actions of glucocorticoids is due to their inhibition of the induction of the NO synthase. 0 1990 Academic Press, Inc.
Macrophages When
play
activated
they
microorganisms action
(2-4).
nitrite This
macrophages common
been
(4,5)
.
7-9)
(NO;
L-arginine been
inducible
by
and
vascular
and
both
metabolite endothelial one
cells
constitutive
inc. reserved.
of tumour
of
cytostaticlcytotoxic
The
I246
the
and
of
amino
and
the
been
are
2L
acid
generation
the
L-arginine of activated
formed
identified
Two types -dependent
Ca
(LPS)
mechanism N03-
has
(10,ll). latter
the
cells
lipopolysaccharide
cytolytic
and
mechanism.
understood.
This
discovery
defence
variety
from
NO2-
(6).
( 12) .
Press, in an? form
fully
host
bacterial (N03-)
Furthermore,
Ca’+-independent.
0006-291x/90 $1.50 Copyrighr 0 1990 bv Arudemic
of reproduction
is not
to be a major
the
the
mechanism
nitrate
shown
in
of a wide
to
exposed
following
identified,
macrophages
rights
cells
L-arginine-derived
oxide have
on target ( N02-)
has
growth the
macrophages
synthesize
the
However,
(1) .
Mouse
All
inhibit
of macrophages
role
a significant
only
of
from
a
as nitric NO
from
of NO synthase and the other one
found
in
Vol.
BIOCHEMICAL
172, No. 3, 1990
Since a fully
studies
tumouricidal
is known including effect
several
that
state
these
their
have
shown
is inhibited
compounds
anti-microbial
of glucocorticoids
AND BIOPHYSICAL
on the
that
the
RESEARCH COMMUNICATIONS
activation
by glucocorticoids
inhibit
other
activity
(16),
production
of macrophages (13-15)
and
since
to it
cytotoxic actions of macrophages we have now investigated the of NO by
the
macrophage
cell
line
J774.
MATERIALS
AND
METHODS
Materials All reagents for cell culture except foetal calf serum (Flow Labs.) were from Gibco. Salmonella typhosa LPS (Difco), IFN- 5 , (Genzyme) , L-arginine, dithiothreitol, hydrocortisone, dexamethasone, progesterone and cortexolone (all Sigma) and NADPH (Boehringer) were obtained as indicated. Human haemoglobin was prepared as described (17). Cell
culture
The experiments were carried out as a collabo_ration between two laboratories, one in Naples where the production of NO by intact J774 cells was studied and the other in Beckenham where the pro 4 uction of NO by J774 cell cytosol was investigated. In Naples, the murine monocyte/macrophage cell line J774 (American Tissue Culture Catalogue TlB 67 page 231) was grown on 100 mm plastic culture dishes in Dulbecco’s modified Eagles medium supplemented with 2 mM glutamine, 25 mM HEPES, penicillin (100 units/ml), streptomycin (100 ug/ml) and 10% foetal calf serum. Cells were passaged every 3-6 days by diluting a suspension of the cells 1 :lO in fresh medium. These cells were used to determine N02- production in response to LPS. In Beckenham, the cells were grown in suspension culture in Techne stirrer bottles, spun at 25 rpm and incubated at 37OC in RPM1 1640 medium containing 25 mM HEPES supplemented with 10% foetal calf serum, 2 mM glutamine, penicillin (100 units/ml) and streptomycin (100 ug/ml). The cells were subcultured by removing 95% of the cell suspension and replacing it with fresh growth medium. The cell viability was shown by the trypan blue exclusion test to be >99%. The production of NO by the cytosol of these cells in response to LPS and IFN-\( was determined. Measurement
of NO,-
and
NO production
by J774 cells
and
their
cytosols
J774 cells were removed from culture dishes by vigorous pipetting and e centrifuged and resuspended in the medium to a concentration of 1 x YF cells/ml . Cells were plated in 24 well culture plates (Falcon] and allowed to adhere for 2h. Thereafter the medium was replaced with fresh medium and cells were activated by LPS (0.1 pglml). They were incubated at 37OC for up to 72h in the presence or absence of steroids. After the appropriate incubation time the supernatants from the plated cells (300 pl) were mixed with an equal volume of Griess reagent (1% sulphanilamide / 0.1% naphthylethylenediamine dihydrochloride / 2.5% H P04; 18) and incubated at room temperature for 10 min to form a chromop #Iore. The absorbance was read at 550 nm using a Beckman DU40 spectrophotometer and NO2 was measured using NaN02 as a standard. In order to induce substantial amounts of NO synthase J774 cells cultured in stirrer bottles were activated by LPS (10 pg/ml) and IFN-x(150 U/ml) for up to 72 h in the presence or absence of steroids. The cells were harvested by centrifugation, washed twice with HEPES buffer (0.1 M, pH 1247
Vol.
172,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
7.4) and finally resuspended in the same buffer (O.lM, pH 7.4) containing 100 pM dithiothreitol. They were homogenized by sonicating twice for 10 sec. The homogenate was then centrifuged at 105,000 x g for 30 min at 4°C and the supernatant was incubated with AC50-X8 (100 mg/ml of supernatant) for 5 min at 4OC to deplete endogenous L-arginine (19). The formation of NO by cytosol was measured by a spectrophotometric method of Feelisch and Noack (20). This method is based on the rapid oxidation of oxyhaemoglobin to methaemoglobin by NO. The synthesis of NO was initiated by addition of a submaximally effective concentration of L-arginine (30 pM) in the presence of oxyhaemoglobin (5 pM) and NADPH (100 FM). The rate of NO production was determined as the difference in absorbance between 401 and 411 nm in a dual wavelength spectrophotometer (Shimadzu). Statistics All ANOVA
by
values and
are means +- S .E .M. p < 0.05 was considered
of
n experiments. as statistically
They significant.
NO
by
were
analysed
RESULTS Time-course their
of
induction
of
NO,-
N02-
by
generation
J774
cells
and
by
cytosols The
1
production
nmol
of
N02-/lo6
cells;
concentration-dependent cells in
and
with the
release
LPS
(0.1
medium
pg/ml)
which
nmol/106
cells,
n=2
nmol/106
cells,
n=2).
The (< IFN-
pmol r
NO/mg in
following
challenge
experiment
(72h,
Inhibition
of
the
Fig.
NO
obtained
at
reached
was
the
N02-
and
74.9
72h
(73.2
undetectable by
the
LPS
cytosol
and which
maximum
at
24h
duration
of
the
cells
by
a
for
24h
cells
in
of of
at
J774
synthase
3h,
stimulation
at
cytosol
a
1). of
NO,-
and
NO
in
generation
J774
steroids J774
of (0.01-10.0
1)
cells pM)
with or
as
well
as
an
inhibition
1 & Fig. cells The
concentrations cells
did
was Longer
2). not
increase
inhibitory of was
of
(up
to
actions
hydrocortisone
(Fig. affected
less
2). by 1248
progesterone
NO
potent 72h) (Fig. was
The
pM)
formation of
x
inhibitory of
NO*-
generation 10
steroids
(0.001-1.0 of
the
approx.
their
cortexolone not
dexamethasone
incubations
effect
anti-inflammatory
the
inhibition
Hydrocortisone
2).
(Table
n=4
was
of
unchanged
a concentration-dependent
(Table
cells,
cell
NO
the
(
No.
November
BIOCHEMICAL
3, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
15, 1990
Pages
GLUCOCORTICOIDS OF NITRIC M.
Di Rosa,*
1NHlBlT
OXIDE M.
SYNTHASE
Radomski,
R.
INDUCTION
IN MACROPHAGES
Carnuccio*
Wellcome Research Court, Beckenham,
Langley
THE
1246-1252
and
Laboratories, Kent, U.K.
S. Moncada
BR3
3BS
*Dept. of Experimental Pharmacology, University of Naples “Federico II”, via Domenico Montesano, 80131 Naples, italy Received
September
24,
1990
The effect of glucocorticoids on the production of NO2- and NO by the macrophage cell line J774 was investigated. Stimulation of the cells with lipopolysaccharide (LPS) resulted in a time-dependent accumulation of NO in the medium, reaching a plateau after 48h. Concomitant incubation of t&e cells for 24h with dexamethasone (0.001-1.0 pM) or hydrocortisone (0.01-10.0 pM) caused a concentration-dependent inhibition of NO formation. The cytosol of J774 cells stimulated LPS and with ?FN-y produced a time-dependent increase in the release of NO. This was blocked in a concentration-dependent manner by dexamethasone and hydrocortisone, but not progesterone, administered concomitantly with the immunological stimulus. None of these compounds had any effect on the release of NO once the enzyme had been induced. The inhibitory effect of hydrocortisone on NO formation was blocked by cortexolone. These data suggest that part of the anti-inflammatory and immunosuppressive actions of glucocorticoids is due to their inhibition of the induction of the NO synthase. 0 1990 Academic Press, Inc.
Macrophages When
play
activated
they
microorganisms action
(2-4).
nitrite This
macrophages common
been
(4,5)
.
7-9)
(NO;
L-arginine been
inducible
by
and
vascular
and
both
metabolite endothelial one
cells
constitutive
inc. reserved.
of tumour
of
cytostaticlcytotoxic
The
I246
the
and
of
amino
and
the
been
are
2L
acid
generation
the
L-arginine of activated
formed
identified
Two types -dependent
Ca
(LPS)
mechanism N03-
has
(10,ll). latter
the
cells
lipopolysaccharide
cytolytic
and
mechanism.
understood.
This
discovery
defence
variety
from
NO2-
(6).
( 12) .
Press, in an? form
fully
host
bacterial (N03-)
Furthermore,
Ca’+-independent.
0006-291x/90 $1.50 Copyrighr 0 1990 bv Arudemic
of reproduction
is not
to be a major
the
the
mechanism
nitrate
shown
in
of a wide
to
exposed
following
identified,
macrophages
rights
cells
L-arginine-derived
oxide have
on target ( N02-)
has
growth the
macrophages
synthesize
the
However,
(1) .
Mouse
All
inhibit
of macrophages
role
a significant
only
of
from
a
as nitric NO
from
of NO synthase and the other one
found
in
Vol.
BIOCHEMICAL
172, No. 3, 1990
Since a fully
studies
tumouricidal
is known including effect
several
that
state
these
their
have
shown
is inhibited
compounds
anti-microbial
of glucocorticoids
AND BIOPHYSICAL
on the
that
the
RESEARCH COMMUNICATIONS
activation
by glucocorticoids
inhibit
other
activity
(16),
production
of macrophages (13-15)
and
since
to it
cytotoxic actions of macrophages we have now investigated the of NO by
the
macrophage
cell
line
J774.
MATERIALS
AND
METHODS
Materials All reagents for cell culture except foetal calf serum (Flow Labs.) were from Gibco. Salmonella typhosa LPS (Difco), IFN- 5 , (Genzyme) , L-arginine, dithiothreitol, hydrocortisone, dexamethasone, progesterone and cortexolone (all Sigma) and NADPH (Boehringer) were obtained as indicated. Human haemoglobin was prepared as described (17). Cell
culture
The experiments were carried out as a collabo_ration between two laboratories, one in Naples where the production of NO by intact J774 cells was studied and the other in Beckenham where the pro 4 uction of NO by J774 cell cytosol was investigated. In Naples, the murine monocyte/macrophage cell line J774 (American Tissue Culture Catalogue TlB 67 page 231) was grown on 100 mm plastic culture dishes in Dulbecco’s modified Eagles medium supplemented with 2 mM glutamine, 25 mM HEPES, penicillin (100 units/ml), streptomycin (100 ug/ml) and 10% foetal calf serum. Cells were passaged every 3-6 days by diluting a suspension of the cells 1 :lO in fresh medium. These cells were used to determine N02- production in response to LPS. In Beckenham, the cells were grown in suspension culture in Techne stirrer bottles, spun at 25 rpm and incubated at 37OC in RPM1 1640 medium containing 25 mM HEPES supplemented with 10% foetal calf serum, 2 mM glutamine, penicillin (100 units/ml) and streptomycin (100 ug/ml). The cells were subcultured by removing 95% of the cell suspension and replacing it with fresh growth medium. The cell viability was shown by the trypan blue exclusion test to be >99%. The production of NO by the cytosol of these cells in response to LPS and IFN-\( was determined. Measurement
of NO,-
and
NO production
by J774 cells
and
their
cytosols
J774 cells were removed from culture dishes by vigorous pipetting and e centrifuged and resuspended in the medium to a concentration of 1 x YF cells/ml . Cells were plated in 24 well culture plates (Falcon] and allowed to adhere for 2h. Thereafter the medium was replaced with fresh medium and cells were activated by LPS (0.1 pglml). They were incubated at 37OC for up to 72h in the presence or absence of steroids. After the appropriate incubation time the supernatants from the plated cells (300 pl) were mixed with an equal volume of Griess reagent (1% sulphanilamide / 0.1% naphthylethylenediamine dihydrochloride / 2.5% H P04; 18) and incubated at room temperature for 10 min to form a chromop #Iore. The absorbance was read at 550 nm using a Beckman DU40 spectrophotometer and NO2 was measured using NaN02 as a standard. In order to induce substantial amounts of NO synthase J774 cells cultured in stirrer bottles were activated by LPS (10 pg/ml) and IFN-x(150 U/ml) for up to 72 h in the presence or absence of steroids. The cells were harvested by centrifugation, washed twice with HEPES buffer (0.1 M, pH 1247
Vol.
172,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
7.4) and finally resuspended in the same buffer (O.lM, pH 7.4) containing 100 pM dithiothreitol. They were homogenized by sonicating twice for 10 sec. The homogenate was then centrifuged at 105,000 x g for 30 min at 4°C and the supernatant was incubated with AC50-X8 (100 mg/ml of supernatant) for 5 min at 4OC to deplete endogenous L-arginine (19). The formation of NO by cytosol was measured by a spectrophotometric method of Feelisch and Noack (20). This method is based on the rapid oxidation of oxyhaemoglobin to methaemoglobin by NO. The synthesis of NO was initiated by addition of a submaximally effective concentration of L-arginine (30 pM) in the presence of oxyhaemoglobin (5 pM) and NADPH (100 FM). The rate of NO production was determined as the difference in absorbance between 401 and 411 nm in a dual wavelength spectrophotometer (Shimadzu). Statistics All ANOVA
by
values and
are means +- S .E .M. p < 0.05 was considered
of
n experiments. as statistically
They significant.
NO
by
were
analysed
RESULTS Time-course their
of
induction
of
NO,-
N02-
by
generation
J774
cells
and
by
cytosols The
1
production
nmol
of
N02-/lo6
cells;
concentration-dependent cells in
and
with the
release
LPS
(0.1
medium
pg/ml)
which
nmol/106
cells,
n=2
nmol/106
cells,
n=2).
The (< IFN-
pmol r
NO/mg in
following
challenge
experiment
(72h,
Inhibition
of
the
Fig.
NO
obtained
at
reached
was
the
N02-
and
74.9
72h
(73.2
undetectable by
the
LPS
cytosol
and which
maximum
at
24h
duration
of
the
cells
by
a
for
24h
cells
in
of of
at
J774
synthase
3h,
stimulation
at
cytosol
a
1). of
NO,-
and
NO
in
generation
J774
steroids J774
of (0.01-10.0
1)
cells pM)
with or
as
well
as
an
inhibition
1 & Fig. cells The
concentrations cells
did
was Longer
2). not
increase
inhibitory of was
of
(up
to
actions
hydrocortisone
(Fig. affected
less
2). by 1248
progesterone
NO
potent 72h) (Fig. was
The
pM)
formation of
x
inhibitory of
NO*-
generation 10
steroids
(0.001-1.0 of
the
approx.
their
cortexolone not
dexamethasone
incubations
effect
anti-inflammatory
the
inhibition
Hydrocortisone
2).
(Table
n=4
was
of
unchanged
a concentration-dependent
(Table
cells,
cell
NO
the
(
