Content uploaded by Mourad Errasfa
Author content
All content in this area was uploaded by Mourad Errasfa on Oct 23, 2015
Content may be subject to copyright.
Inhibition
of
02
Generation
by
Dexamethasone
Is
Mimicked
by
Lipocortin
I
in
Alveolar
Macrophages
Isabelle
Maridonneau-Parini,
Mourad
Errasfa,
and
Franmoise
Russo-Marie
Unite
de
Pharmacologie
Cellulaire
associee
Unite"
Institut
Nationale
de
la
Sante
et
de
la
Recherche
Medicale
U285,
Institut
Pasteur,
75015
Paris,
France
Abstract
Glucocorticoids
inhibit
superoxide
(O°)
generation
by
phago-
cytes
through
a
mechanism
that
remains
unclear.
We
investi-
gated
this
effect
by
using
dexamethasone
on
guinea
pig
alveo-
lar
macrophages.
O2
generation
was
induced
either
by
the
calcium
ionophore
A23187,
a
potent
stimulus
of
phospholipase
A2,
or
by
the
protein
kinase
C
activator,
phorbol
myristate
acetate
(PMA).
Dexamethasone
inhibited
O2
generation
initi-
ated
by
A23187
by
50-55%.
This
inhibition
required:
(a)
more
than
45
min
incubation
and
was
maximal
after
2
h;
(b)
gluco-
corticoid
receptor
occupancy;
and
(c)
protein
synthesis.
The
inhibitory
effect
of
dexamethasone
could
not
be
explained
by
an
interaction
with
the
respiratory
burst
enzyme
NADPH
oxi-
dase
since
O2
generation
was
only
weakly
affected
upon
PMA
stimulation.
Lipocortin
I,
a
glucocorticoid
inducible
and
phos-
pholipase
A2
inhibitory
protein,
inhibited
O2
generation
initi-
ated
by
A23187
but
failed
to
modulate
the
respiratory
burst
activated
by
PMA.
These
results
were
obtained
with
lipocortin
I
purified
from
mouse
lungs,
human
blood
mononuclear
cells,
and
with
human
recombinant
lipocortin
I.
We
propose
that
lipocortin
I
is
capable
of
inhibiting
the
activation
of
NADPH
oxidase
only
when
membrane
signal
transduction
involves
phospholipase
A2.
By
mimicking
the
effect
of
dexamethasone,
lipocortin
I
may
extend
its
potential
anti-inflammatory
action
to
the
partial
control
of
the
formation
of
oxygen
reactive
spe-
cies
by
phagocytes.
Introduction
Activation
of
the
superoxide-generating
enzyme
NADPH
oxi-
dase
in
phagocytes
can
be
initiated
either
by
stimuli
involving
binding
to
specific
membrane
receptors
or
by
substances
by-
passing
receptor
interaction
such
as
phorbol
esters
or
calcium
ionophores.
Transmission
of
activation
signals
to
the
NADPH
oxidase
involves
at
least
two
distinct
pathways
(1,
2),
implying
either
phospholipase
A2
(PLA2)'
or
protein
kinase
C
activa-
tion
(3-5).
Generation
of
superoxide
(O°)
by
phagocytes
can
Address
correspondence
to
Dr.
I.
Maridonneau-Parini,
Centre
de
G6netique
Moleculaire,
CNRS,
%
M.
Bornens,
2
avenue
de
la
Ter-
rasse,
91190
Gif
sur
Yvette,
France.
Received
for
publication
4
October
1988
and
in
revised
form
13
February
1989.
1.
Abbreviations
used
in
this
paper:
AM,
alveolar
macrophages;
PAF,
platelet
activating
factor;
PLA2,
phospholipase
A2;
PMA,
phorbol
myristate
acetate.
be
inhibited
by
in
vivo
or
in
vitro
treatment
by
glucocorticoids,
but
their
mechanism
of
action
has
not
been
clarified
(6,
7).
Glucocorticoids
also
affect
other
phagocyte
functions
such
as
AA
hydrolysis
and,
consequently,
the
synthesis
of
eicosanoids
(7-9).
This
particular
effect
of
glucocorticoids
was
first
attrib-
uted
to
the
induction
of
a
PLA2-inhibitory
protein
termed
lipocortin
I
(8-10).
Other
PLA2-inhibitory
proteins
with
se-
quence
homologies
were
then
described
and
included
in
the
lipocortin
family
although
their
induction
by
glucocorticoids
remains
to
be
established.
Recently
it
has
been
reported
that
lipocortin
I
can
inhibit
cellular
PLA2
of
both
guinea
pig
per-
fused
lungs
(1
1)
and
guinea
pig
alveolar
macrophages
(12).
We
report
here
that
in
vitro
treatment
of
guinea
pig
alveolar
mac-
rophages
(AM)
with
dexamethasone
inhibits
O°
generation.
Since
one
of
the
NADPH
oxidase
activation
pathways
has
been
shown
to
involve
PLA2,
we
investigated
the
possibility
that
lipocortin
I
could
mimic
the
glucocorticoid
inhibitory
process
using
human
recombinant
lipocortin
I
or
lipocortin
I
purified
from
either
human
blood
mononuclear
cells
or
mouse
lungs.
Methods
Preparation
of
AM
and
incubation
with
dexamethasone.
Hartley
guinea
pigs
were
anesthetized
by
intraperitoneal
injection
of
sodium
pentobarbitone
(30
mg/kg).
The
trachea
was
cannulated
and
AM
were
obtained
by
repeated
lung
lavages
with
5
ml
sterile
PBS,
pH
7.4,
at
37°C
as
previously
described
(13),
except
that
no
lidocaine
was
added
to
the
medium.
Cells
were
washed
and
pelleted.
Viability
(>
90%)
was
assessed
by
trypan
blue
exclusion
and
the
cell
number
was
adjusted
to
2
X
106
viable
cells/ml
of
MEM
(Eurobio,
Paris,
France)
without
phenol
red
supplemented
with
0.5
mM
CaC12;
0.25
mM
MgCl2;
20
mM
Hepes,
and
0.5
g/liter
glucose,
pH
7.4,
at
370C.
AM
were
incubated
for
2
h
in
the
presence
of
5
,M
dexamethasone
and
stimulated.
In
some
experiments
cycloheximide
(Sigma
Chemical
Co.,
St.
Louis,
MO)
or
RU486
(Roussel-Uclaf,
Paris,
France)
was
added
to
the
cell
suspension
just
before
dexamethasone
(Sigma
Chemi-
cal
Co.).
Superoxide
measurement.
Superoxide
generation
was
assessed
by
the
SOD-inhibiting
reduction
of
cytochrome
c,
continuously
moni-
tored
for
4
min
at
550
nm
with
a
spectrophotometer
(Uvikon
860;
Kontron
Analytical,
Redwood
City,
CA)
thermostated
at
37°C
as
pre-
viously
described
(3).
Phorbol
myristate
acetate
(PMA)
and
A23187
were
obtained
from
Sigma
Chemical
Co.
Preparation
of
lipocortin
I.
Lipocortin
I
was
purified
from
human
blood
mononuclear
cells
or
from
mouse
lungs.
The
procedure
for
isolation
of
human
mononuclear
cells
(70%
lymphocytes,
30%
mono-
cytes)
is
reported
in
reference
14.
Mouse
lungs
were
isolated
from
thoracic
cages,
homogenized
using
a
teflon
potter,
and
sonicated.
Pro-
teins
were
extracted
(14)
and
purified
as
previously
described
for
human
blood
mononuclear
cells
(15)
or
mouse
lungs
(12).
The
peak
corresponding
to
lipocortin
I
was
selected
and
analyzed
in
SDS-PAGE
(14).
A
single
protein
band
at
40
kD
was
obtained
with
the
preparation
from
mouse
lungs
and
a
single
band
at
35-38
kD
with
the
protein
from
mononuclear
cells.
The
two
proteins
exhibited
PLA2
inhibitory
activ-
1936
I.
Maridonneau-Parini,
M.
Errasfa,
and
F.
Russo-Marie
J.
Clin.
Invest.
©
The
American
Society
for
Clinical
Investigation,
Inc.
0021-9738/89/06/1936/05
$2.00
Volume
83,
June
1989,
1936-1940
ity
as
assessed
on
porcine
pancreatic
PLA2
using
membranes
of
Esche-
richia
coli
labeled
with
[3H]oleic
acid
(16).
Western
blot
analyses
were
performed
as
described
(14,
17).
The two
proteins
were
recognized
by
the
lipocortin
I
antiserum
(15,
18)
raised
against
the
recombinant
protein
(19)
kindly
provided
by
Biogen
Corp.
(Cambridge,
MA).
Anti-
sera
directed
against
lipocortin
II,
III,
and
32
kD
(now
referred
to
as
lipocortin
V;
Rothhut,
B.,
C.
Comera,
S.
Cortial,
P.
Y.
Haumont,
K.
H.
Diep-le,
J.
C.
Cavadore,
J.
Connard,
F.
Russo-Marie,
and
F.
Lederer,
manuscript
submitted
for
publication)
were
also
tested;
the
32-kD
and
lipocortin
III
antisera
were
shown
to
be
monospecific
(20),
and
the
lipocortin
II
antiserum
(provided
by
Biogen
Corp.)
has
a
low
titer
of
antibodies
that
recognize
lipocortin
I
(18).
The
lipocortin
II
and
the
32-kD
antisera
(14)
did
not
immunoreact
with
recombinant-,
mouse
lung-
(12),
or
human
blood
mononuclear
cell-lipocortin
I
(20).
The
lipocortin
III
antiserum
tested
on
recombinant
lipocortin
I
and
lipocortin
I
from
human
blood
mononuclear
cells
did
not
immunore-
act
with
these
proteins
(20).
Finally,
our
purified
proteins
cannot
be
similar
to
lipocortin
IV
since
the
lipocortin
I
antiserum
raised
against
the
recombinant
protein
does
not
immunoreact
with
lipocortin
IV
(R.
B.
Pepinsky,
personal
communication).
Before
the
use
of
the
two
purified
lipocortin
I's
in
biological
exper-
iments,
the
chromatographic
buffer
was
removed
by
sequential
micro-
concentrations
on
Centrcon
10
(Amicon
Corp.,
Danvers,
MA).
The
human
recombinant
lipocortin
I
(19)
was
kindly
provided
by
Biogen
Corp.
RIA
of
eicosanoids.
Measurement
of
PGE2
and
TXB2
were
per-
formed
as
previously
described
(21).
Statistical
analysis.
The
results
were
statistically
analyzed
using
a
paired
t
test.
Results
It
has
been
reported
that
glucocorticoid
treatment
may
modify
the
number
of
various
membrane
receptors
(22-24).
There-
fore,
we
decided
to
stimulate
dexamethasone-treated
AM
with
substances
bypassing
membrane
receptors,
the
calcium
iono-
phore
A23187
and
the
protein
kinase
C
activator
PMA.
Generation
of
O2
induced
by
A23187
in
AM
was
inhibited
by
dexamethasone
(Table
I).
The
inhibitory
effect
was
detected
45
min
after
dexamethasone
addition
and
was
maximal
2
h
later.
The
dexamethasone
effect
was
abolished
when
the
pro-
tein
synthesis
inhibitor,
cycloheximide,
or
the
glucocorticoid
receptor
antagonist,
RU486,
was
present
(25;
Table
I).
RU486
or
cycloheximide
did
not
affect
the
generation
of
O2
per
se
and
did
not
enhance
it
when
AM
were
stimulated
with
A23187
in
the
absence
of
dexamethasone
(Table
I).
At
10
,sg/ml
cyclo-
Table
I.
Effect
of
Cycloheximide
and
RU486
on
Dexamethasone-induced
Inhibition
of
°2
Generation
by
Guinea
Pig
Alveolar
Macrophages
Control
Dexamethasone
nmol
Oi/min
per
106
cells
None
1.92±0.35
0.99±0.22
RU486
1.51±0.12
2.43±0.41
Cycloheximide
1.96±0.45
2.11±0.33
AM
were
preincubated
for
2
h
with
5
,uM
dexamethasone,
stimulated
with
8
,uM
A23
187,
and
generation
of
O2
was
measured.
5
uM
RU486
or
10
ug/ml
cycloheximide
were
added
just
before
dexameth-
asone.
The
results
are
expressed
as
mean±SD
of
two
experiments
performed
in
triplicate.
O2
production
before
A23
187
was
not
de-
tectable.
Table
II.
Effect
of
Dexamethasone
Removal
on
°2
Generation
Control
Dexamethasone
P
nmol
Ojymin
per
106
cells
8
,M
A23187
1.89±0.21
0.87±0.11
<0.02
10
AM
A23187
2.63±0.23
1.09±0.17
<0.02
50
ng/ml
PMA
1.81±0.24
1.65±0.26
<0.05
AM
were
incubated
for
2
h
with
5
qM
dexamethasone,
washed,
and
incubated
for
an
additional
2-h
period
without
dexamethasone.
The
cells
were
stimulated
with
A23187
or
PMA,
and
O2
generation
was
recorded.
The
results
expressed
as
mean±SD
of
four
experiments
were
statistically
analyzed
using
a
paired
t
test.
heximide
abolished
the
protein
synthesis
both
in
control
and
dexamethasone-treated
AM
as
examined
by
autoradiography
of
[355]methionine-labeled
proteins
separated
by
SDS-PAGE
(21;
data
not
shown).
To
assess
whether
inhibition
of
O°
generation
persists
even
after
removal
of
glucocorticoids,
cells
were
exposed
to
dexa-
methasone,
then
washed
and
incubated
for
an
additional
2
h
in
the
absence
of
dexamethasone
before
stimulation.
During
this
particular
procedure,
which
has
been
previously
used
for
the
study
of
the
inhibitory
effect
of
glucocorticoids
on
PLA2,
the
cells
were
still
synthesizing
proteins
induced
by
glucocorticoids
for
3-5
h
(25).
Under
these
conditions,
generation
of
O2
by
AM
stimulated
with
A23187
was
inhibited
(Table
II)
to
a
simi-
lar
extent
as
in
the
presence
of
dexamethasone
(Table
I),
indi-
cating
that
preincubation
with
dexamethasone
is
sufficient
to
induce
the
inhibitory
process.
Stimulation
of
AM
with
PMA
was
only
inhibited
9%
by
dexamethasone,
whereas
50
ng/ml
PMA
or
8
AM
A23187
provoked
cell
responses
of
similar
magnitude
(Table
II).
This
indicates
that
NADPH
oxidase
was
probably
not
directly
inhibited
since
O°
generation
was
weakly
modified
upon
PMA
stimulation.
Therefore,
the
inhibitory
factor(s)
seems
most
likely
to
interfere
with
the
NADPH
oxi-
dase
activation
pathway
initiated
by
A23187.
The
calcium
ionophore
A23187
initiates
AA
hydrolysis
which
mainly
results
from
PLA2
activation
(26,
27).
Synthesis
of
a
derivative
of
AA,
PGE2,
was
measured
after
incubation
of
AM
for
2
h
with
or
without
dexamethasone,
and
with
or
with-
out
stimulation
with
A23187
for
15
min
(picograms/106
cells
in
1
ml,
mean±SD,
n
=
4):
(a)
unstimulated
cells:
control,
671.7±119.5;
dexamethasone,
344.2+27.2,
P
<
0.03;
(b)
A23187
stimulated
cells:
control,
1038.2±53.1;
dexametha-
sone,
774.2+66.7,
P
<
0.02.
In
contrast,
PMA,
which
is
a
potent
activator
of
the
respiratory
burst,
is
unable,
at
the
con-
centration
and
the
stimulation
length
we
used,
to
initiate
the
release
of
AA
from
membrane
phospholipids
(3,
27,
data
not
shown)
and,
therefore,
to
modify
the
basal
synthesis
of
PGE2
(control,
684±125;
PMA,
709±86
pg/106
cells
in
1
ml;
mean±SD,
n
=
3)
or
TXA2
(measurement
of
its
stable
metabo-
lite
TXB2,
control,
19.8+4.3;
PMA,
17.1±3.9
ng/106
cells
in
1
ml)
as
measured
by
RIA
15
min
after
stimulation
of
AM
with
50
ng/ml
PMA.
These
results
agree
with
previous
reports
on
the
absence
of
PLA2
involvement
in
the
transmission
of
acti-
vation
signals
to
NADPH
oxidase
upon
PMA
stimulation
(3,
28).
Therefore,
we
examined
whether
the
effect
of
dexametha-
sone
observed
upon
A23187
stimulation
could
be
mimicked
by
the
PLA2
inhibitory-
and
glucocorticoid-induced
protein,
lipocortin
I.
Inhibition
of
Superoxide
Generation
by
Dexamethasone
or
Lipocortin
I
1937
co
2,0
-
Figure
1.
Effect
of
in-
creasing
concentrations
1,5A-
of
lipocortin
I
on
O°
oj
\generation.
Lipocortin
I
0e
1,0-
T
purified
from
mouse
x
lungs
was
added
at
the
o
0,5
-
indicated
concentration
to
the
cell
suspension
0,0-
--------
and
incubated
at
370C
1
1
o
1
o0
1,000
1
choo
LIPOCORTIN
1
(ng/ml)
for
30
min.
Macro-
phages
were
stimulated
with
8
X
10-6
M
A23187
and
O2
generation
was
recorded.
Shown
are
the
means±SD
of
three
experiments.
The
statistical
analysis
was
performed
using
a
paired
t
test.
From
10
ng/ml
to
5
Ag/ml
lipocortin
I,
the
differences
in
superoxide
production
were
significative
when
compared
with
control
cells
with
P
<
0.04.
As
shown
in
Fig.
1,
O2
generation
by
AM
stimulated
with
A23187
was
inhibited
by
lipocortin
I
purified
from
mouse
lungs
in
a
concentration-dependent
manner
from
10
ng/ml
(2.5
X
10-10
M)
to
1
,ug/ml,
which
provided
the
maximal
effect.
The
inhibitory
effect
was
detected
15
min
after
addition
of
lipocortin
I,
was
maximal
at
30
min,
and
was
not
changed
by
the
concomitant
addition
of
10,gg/ml
cycloheximide
or
by
supplementing
the
calcium-containing
incubation
medium
with
1
mM
CaCl2.
When
1
gg/ml
of
the
protein
was
boiled
for
10
min,
its
inhibitory
effect
was
abolished
(53.6±15.5%
inhibi-
tion
vs.
7.8±8.3%).
Control
proteins,
BSA
and
ovalbumin
(10
tig/ml),
tested
in
the
same
conditions
as
lipocortin
I,
did
not
modify
O-
generation.
The
inhibitory
effect
of
dexamethasone
(53.8±10.5%,
n
=
3)
was
not
potentiated
by
addition
of
1
jig/ml
lipocortin
I
to
the
cell
suspension
30
min
before
stimu-
lation
(47.7±12.3%,
n
=
3).
Inhibition
of
PLA2
by
extracellular
lipocortin
I
was
exam-
ined.
Since
inhibition
of
the
release
of
AA
by
AM
has
been
published
elsewhere
using
the
same
preparations
of
lipocortin
I
as
in
the
present
work
(see
reference
12),
here
we
only
report
the
effect
of
1
gg/ml
lipocortin
I
on
PGE2
synthesis
(pg/106
cells
in
1
ml,
mean±SD,
n
=
3):
(a)
unstimulated
cells:
control,
345.3±35;
lipocortin
I,
222.3±23.6,
P
<
0.03;
(b)
A23187-
stimulated
cells:
control,
865.3±79;
lipocortin
I,
611±45.6,
P
<
0.01).
The
effects
of
three
lipocortin
I's
purified
from
different
sources
were
then
compared.
The
human
recombinant-,
the
mouse
lung-,
and
the
human
blood
mononuclear
cell
lipocor-
tin
I
exhibited
similar
inhibitory
ranges
on
the
O2
generation
initiated
by
A23187
(Fig.
2).
Upon
PMA
stimulation,
none
of
the
tested
lipocortins
inhibited
O2
generation,
suggesting
that
lipocortins
did
not
interact
with
NADPH
oxidase.
The
effect
of
the
mouse
lung
lipocortin
I
was
investigated
in
the
presence
of
another
agonist
that
stimulates
PLA2
(12,
29),
platelet-activating
factor
(PAF),
a
stimulus
binding
spe-
cific
membrane
receptors.
As
observed
with
A23187,
lipocor-
tin
I
inhibited
°2
generation
initiated
by
5
X
l0-7
M
PAF
(0.71
vs.
0.37
nmol
0i/min
per
106
cells,
mean
of
duplicate
determinations).
Discussion
In
the
present
paper
we
report
that
in
vitro
treatment
of
AM
with
dexamethasone
is
able
to
inhibit
O-
generation
through
a
mechanism
involving
glucocorticoid
receptor
occupancy
and
8
-
Figure
2.
Effect
of
lipocor-
tin
I
on
generation
of
O°
6
by
AM
stimulated
with
A23187
(top)
or
PMA
(bot-
tom).
Alveolar
macro-
mi
4
-
t
phages
were
incubated
for
cJ
*
_
>>/>30
min
with
each
lipocor-
W
2
-
tin
I(1
jg/ml)
before
stim-
0
//2
ulation
with
8
jiM
A23187
Xj
Sor
50
ng/ml
PMA,
and
O2-
o
0
-
generation
was
recorded
for
z
200
s.
Shown
are
the
w
means±SD
of
triplicate
de-
a
X
8
terminations
from
one
ex-
O
periment
representative
of
w
two
to
seven
separate
ex-
g
6
periments.
This
particular
experiment
is
shown
be-
4
-
cause
the
three
lipocortin
I's
were
tested
in
parallel
on
the
same
cells.
a,
con-
2
-
trol;
*,
mouse
lung;
o,
re-
combinant;
m,
mononu-
0
clear
cells.
Seven
experi-
ments
performed
with
mouse
lung
lipocortin
I
were
statistically
analyzed
using
a
paired
t
test.
The
results
are
expressed
in
nmol
0O/I06
cells
per
200
s
(mean±SD);
A23
187
stimulation:
control,
5.54±2.51;
lipocortin
I,
2.52±1.35
(P
<
0.02).
PMA
stimulation:
control,
5.26±2.43;
lipocor-
tin
I,
5.59±3.71
(NS).
protein
synthesis.
The
PLA2-inhibitory
protein
lipocortin
I
mimicked
the
inhibitory
effect
of
dexamethasone.
Like
dexa-
methasone,
extracellular
application
of
lipocortin
I
resulted
in
a
decrease
in
both
O2-
generation
and
PGE2
synthesis
in
re-
sponse
to
A23187.
In
contrast,
the
calcium-
and
PLA2-inde-
pendent
NADPH
oxidase
activation
pathway
initiated
by
PMA
(1,
3,
4)
was
not
modified.
This
indicates
that
NADPH
oxidase
was
not
directly
inhibited.
More
likely,
activation
of
NADPH
oxidase
appeared
to
be
affected
only
when
mem-
brane
signal
transduction
involved
PLA2.
This
was
also
sup-
ported
by
the
results
obtained
with
PAF,
which
stimulates
PLA2
(12,
29)
and
NADPH
oxidase
(13);
both
enzyme
activi-
ties
were
inhibited
by
lipocortin
1(12;
this
paper).
Moreover,
dexamethasone
and
lipocortin
I
inhibitory
effects
were
not
additive,
suggesting
that
they
may
affect
the
same
step
of
the
activation
pathway
of
NADPH
oxidase.
The
differences
ob-
served
between
the
mechanisms
of
action
of
lipocortin
I
and
dexamethasone
strengthened
the
hypothesis
that
lipocortin
I
could
account
for
the
dexamethasone
effect;
i.e.,
lipocortin
I
inhibitory
effect
occurred
more
rapidly
than
dexamethasone
and
it
did
not
require
protein
synthesis.
Nevertheless,
it
cannot
be
excluded
that
other
glucocorticoid-inducible
proteins
may
operate
in
that
process.
Although
this
study
further
supports
the
involvement
of
PLA2
in
the
activation
pathway
of
NADPH
oxidase
(3,
5,
30),
the
relationship
between
these
two
enzymes
was
not
investi-
gated.
It
has
been
previously
reported
that
AA
and
eicosanoids
play
a
role
in
the
activation
of
NADPH
oxidase
(3,
5,
28,
30,
31),
but
their
mechanisms
of
action
are
still
unclear.
As
previously
described
(8-12),
neither
lipocortin
I
nor
dexamethasone
provided
a
total
inhibition
of
O°
generation
or
AA
hydrolysis.
This
may
be
explained
either
by
the
existence
1938
L
Maridonneau-Parini,
M.
Errasfa,
and
F.
Russo-Marie
of
a
pool
of
lipocortin
I-
and
dexamethasone-insensitive
PLA2,
or
by
the
activation
of
several
distinct
phospholipid-de-
grading
processes
in
macrophages
stimulated
with
A23
187
or
PAF
(26,
29).
Therefore,
it
is
possible that
the
remaining
frac-
tion
of
O2
formation
results
from
the
hydrolysis
of
AA
by
enzymatic
processes
insensitive
to
lipocortin
I
and/or
from
the
liberation
of
diacylglycerol
by
phospholipase
C
(although
it
is
weakly
stimulated
by
A23187
[26])
which
is
involved
in
NADPH
oxidase
activation
through
protein
kinase
C
stimula-
tion
(1,
4,
32).
It
has
been
shown
that
treatment
of
macrophages
with
glucocorticoids
induces
the
release
of
lipocortin
(8,
33).
How-
ever,
induction
of
its
synthesis
by
glucocorticoids
is
still
con-
troversial
although
an
increase
in
lipocortin
I
mRNA
was
re-
ported
(19).
Since
lipocortin
I
is
also
a
constitutive
protein,
another
possible
effect
of
glucocorticoids
would
be
to
provoke
its
secretion
through
a
mechanism
involving
protein
synthesis.
When
purified
lipocortin
I
is
applied
extracellularly
it
may,
therefore,
mimic
the
effect
of
glucocorticoids.
Indeed,
some
recent
reports
demonstrate
an
inhibition
of
PLA2
by
extracel-
lular
lipocortin
I
(1
1,
12).
These
and
our
results
are,
however,
in
disagreement
with
the
data
of
Northup
and
co-workers
(34),
who
reported
that
extracellular
lipocortin
I
does
not
inhibit
PLA2
of
mouse
peritoneal
macrophages,
in
contrast
to
what
they
observe
by
treating
the
cells
with
dexamethasone.
In
their
experiments,
lipocortin
I
was
applied
to
the
cells
for
only
15
min;
therefore,
we
can
question
whether
longer
incubation
time
would
have
favored
the
action
of
lipocortin
I.
The
mechanism
of
action
of
extracellular
lipocortin
I
has
not
yet
been
defined.
It
seems
unlikely
that
a
strongly
polar
40-kD
protein
could
enter
the
plasma
membrane.
The
sim-
plest
model
would
be
that
lipocortin
I
interacts
with
a
putative
membrane
receptor
which
could
be
PLA2
itself,
assuming
that
PLA2
is
a
transmembrane
protein.
However,
it
has
been
pro-
posed
that
the
inhibition
of
PLA2
is
dependent
on
the
ability
of
lipocortin
I
to
bind
negatively
charged
phospholipids
(14,
35).
Whether
the
low
concentration
of
lipocortin
I
providing
a
biological
effect
(11,
12,
this
report)
is
compatible
with
this
hypothesis
remains
to
be
elucidated.
Albumin,
which
has
also
the
ability
to
bind
phospholipids,
did,
however,
not
modify
the
generation
of
O2,
further
suggesting
a
specific
effect
of
lipo-
cortin
I.
Glucocorticoids
are
by
far
the
most
potent
anti-inflamma-
tory
molecules
active
against
virtually
every
type
of
inflamma-
tory
disease.
Lipocortin,
which
has
been
recently
defined
as
a
"second
messenger"
of
glucocorticoids
(33),
can
account
in
part
for their
anti-inflammatory
action
by
controlling
the
syn-
thesis
of
eicosanoids
and
PAF
(33).
From
the
present
study,
the
action
of
lipocortin
I
can
be
extended
to
the
partial
control
of
proinflammatory
and
tissue
damaging
oxygen
reactive
spe-
cies.
In
conclusion,
we
report
that
lipocortin
I
mimics
the
effect
of
dexamethasone
and
we
propose
that
it
could
account
for
part
of
the
action
of
glucocorticoids.
We
also
confirm
that
lipocortin
I
is
biologically
active
when
applied
extracellularly
(1
1,
12).
In
addition,
our
data
further
support
the
previously
proposed
critical
role
of
PLA2
in
the
transduction
of
activa-
tion
signals
to
NADPH
oxidase
(3,
5,
30)
together
with
the
involvement
of
a
PLA2-independent
activation
pathway
of
the
oxidase
(1-3,
32).
We
gratefully
acknowledge
Dr.
B.
Rothhut
and
Dr.
C.
Comera
for
the
preparation
of
lipocortin
I
from
human
blood
mononuclear
cells
and
Dr.
J.
F.
Browning
for
the
gift
of
human
recombinant
lipocortin
I,
and
lipocortin
I
and
II
antisera.
References
1.
Craig,
G.,
L.
C.
McPhail,
A.
Marfat,
N.
P.
Stimler-Gerard,
D.
A.
Bass,
and
C.
E.
McCall.
1986.
Role
for
protein
kinases
in
stimulation
of
human
polymorphonuclear
leukocyte
oxidative
metabolism
by
various
agonists.
J.
Clin.
Invest.
77:61-65.
2.
Serhan,
C.
N.,
M.
J.
Broekman,
H.
M.
Korchak,
J.
E.
Smolen,
A.
J.
Marcus,
and
G.
Weissmann.
1983.
Changes
in
phosphatidylino-
sitol
and
phosphatidic
acid
in
stimulated
human
neutrophils:
relation-
ship
to
calcium
mobilization,
aggregation
and
superoxide
radical
gen-
eration.
Biochim.
Biophys.
Acta.
762:420-428.
3.
Maridonneau-Parini,
I.,
S.
M.
Tringale,
and
A.
I.
Tauber.
1986.
Identification
of
distinct
activation
pathways
of
the
human
neutrophil
NADPH
oxidase.
J.
Immunol.
137:2925-2929.
4.
Tauber,
A.
I.
1987.
Protein
kinase
C
and
the
activation
of
human
neutrophil
NADPH
oxidase.
Blood.
69:711-720.
5.
Sakata,
A.,
E.
Ida,
M.
Tominaga,
and
K.
Onoue.
1987.
Arachi-
donic
acid
acts
as
an
intracellular
activator
of
NADPH
oxidase
in
Fc
receptor-mediated
superoxide
generation
in
macrophages.
J.
Im-
munol.
138:4353-4359.
6.
Peters-Golden,
M.,
and
P.
Thebert.
1987.
Inhibition
by
methyl-
prednisolone
of
zymosan-induced
leukotriene
synthesis
in
alveolar
macrophages.
Am.
Rev.
Respir.
Dis.
135:1020-1026.
7.
Webb,
D.
S.
A.,
and
J.
A.
Roth.
1987.
Relationship
of
glucocor-
ticoid
suppression
of
arachidonic
acid
metabolism
to
alteration
of
neutrophil
function.
J.
Leukocyte
Biol.
41:156-164.
8.
Blackwell,
G.
J.,
R.
Carnuccio,
M.
DiRosa,
R.
J.
Flower,
L.
Parente,
and
P.
Persico.
1980.
Macrocortin:
a
polypeptide
causing
the
antiphopholipase
effect
of
glucocorticoids.
Nature
(Lond.).
287:147-
149.
9.
Hirata,
F.,
E.
Schiffmann,
K.
Venkatsubramanian,
D.
Salomon,
and
J.
A.
Axelrod.
1980.
Phospholipase
A2
inhibitory
protein
in
rabbit
neutrophils
induced
by
glucocorticoids.
Proc.
Natl.
Acad.
Sci.
USA.
77:2533-2536.
10.
Cloix,
J.
F.,
0.
Colard,
B.
Rothhut,
and
F.
Russo-Marie.
1983.
Characterization
and
partial
purification
of'renocortins':
two
polypep-
tides
formed
in
renal
cells
causing
the
anti-phospholipase-like
action
of
glucocorticoids.
Br.
J.
Pharmacol.
7:313-321.
11.
Cirino,
G.,
R.
J.
Flower,
J.
L.
Browning,
L.
K.
Sinclair,
and
R.
B.
Pepinsky.
1987.
Recombinant
human
lipocortin
1
inhibits
thromboxane
release
from
guinea-pig
isolated
perfused
lung.
Nature
(Lond.).
328:270-272.
12.
Errasfa,
M.,
M.
Bachelet,
and
F.
Russo-Marie.
1988.
Inhibition
of
phospholipase
A2
activity
of
guinea-pig
alveolar
macrophages
by
lipocortin-like
molecules
purified
from
mice
lung.
Biochem.
Biophys.
Res.
Commun.
153:1267-1270.
13.
Maridonneau-Parini,
I.,
V.
Lagente,
J.
Lefort,
J.
Randon,
F.
Russo-Marie,
and
B.
B.
Vargaftig.
1985.
Desensitization
to
PAF-in-
duced
bronchoconstriction
and
to
activation
of
alveolar
macrophages
by
repeated
inhalations
of
PAF
in
the
guinea-pig.
Biochem.
Biophys.
Res.
Commun.
131:42-49.
14.
Rothhut,
B.,
C.
Comera,
B.
Prieur,
M.
Errasfa,
G.
Minassian,
and
F.
Russo-Marie.
1987.
Purification
and
characterization
of
a
32kD
phospholipase
A2
inhibitory
protein
(lipocortin)
from
human
periph-
eral
blood
mononuclear
cells.
FEBS
(Fed.
Eur.
Biochem.
Soc.)
Lett.
219:169-175.
15.
Huang,
K.
S.,
B.
P.
Wallner,
R.
J.
Mattaliano,
R.
Tizard,
C.
Burne,
A.
Frey,
C.
Hession,
P.
McGray,
L.
K.
Sinclair,
E.
P.
Chow,
J.
L.
Browning,
K.
L.
Ramachandran,
J.
Tang,
J.
E.
Smart,
and
R.
B.
Inhibition
of
Superoxide
Generation
by
Dexamethasone
or
Lipocortin
I
1939
Pepinsky.
1986.
Two
human
35
kD
inhibitors
of
phospholipase
are
related
to
substrate
of
pp6O
v-src
and
of
epidermal
growth
factor
re-
ceptor/kinase.
Cell.
46:191-199.
16.
Rothhut,
B.,
F.
Russo-Marie,
J.
Wood,
M.
DiRosa,
and
R.
J.
Flower.
1983.
Further
characterization
of
the
glucocorticoid-induced
anti-phospholipase
protein
"renocortin".
Biochem.
Biophys.
Res.
Commun.
117:878-884.
17.
Towbin,
H.,
T.
Staehelin,
and
J.
Gordon.
1979.
Electrophoretic
transfer
of
proteins
from
polyacrylamide
gels
to
nitrocellulose
sheets:
procedure
and
some
applications.
Proc.
Nail.
Acad.
Sci.
USA.
76:4350-4354.
18.
Pepinsky,
R.
B.,
R.
Tizard,
R.
J.
Mattaliano,
L.
K.
Sinclair,
G.
T.
Miller,
J.
L.
Browning,
E.
P.
Chow,
C.
Burne,
K.
S.
Huang,
D.
Pratt,
L.
Watcher,
C.
Hession,
A.
Z.
Frey,
and
B.
P.
Wallner.
1988.
Five
distinct
calcium
and
phospholipid
binding
proteins
share
homol-
ogy
with
lipocortin
I.
J.
Biol.
Chem.
263:10799-10811.
19.
Wallner,
B.
P.,
R.
J.
Mattaliano,
C.
Hession,
R.
L.
Cate,
R.
Tizard,
L.
K.
Sinclair,
C.
Foeller,
E.
Pingchang
Chow,
J.
L.
Browning,
K.
L.
Ramachandran,
and
R.
B.
Pepinsky.
1986.
Cloning
and
expres-
sion
of
human
lipocortin,
a
phospholipase
A2
inhibitor
with
potential
anti-inflammatory
activity.
Nature
(Lond.).
320:77-80.
20.
Comera,
C.,
B.
Rothhut,
J.
C.
Cavadore,
I.
Vilgrain,
C.
Cochet,
E.
Chambaz,
and
F.
Russo-Marie.
Further
characterization
of
four
lipocortins
from
human
peripheral
blood
mononuclear
cells.
J.
Cell.
Biochem.
In
press.
21.
Maridonneau-Parini,
I.,
J.
Clerc,
and
B.
S.
Polla.
1988.
Heat
shock
inhibits
NADPH
oxidase
in
human
neutrophils.
Biochem.
Biophys.
Res.
Commun.
54:
i
79-186.
22.
Davies,
A.
O.,
and
R.
J.
Lefkowitz.
1980.
Corticosteroid-in-
duced
differential
regulation
of
beta-adrenegic
receptors
in
circulating
human
polymorphonuclear
leukocytes
and
mononuclear
leukocytes.
J.
Clin.
Endocrinol.
Metab.
51:599-605.
23.
Yodoi,
J.,
M.
Hirashima,
and
K.
Ishizaka.
1981.
Lymphocytes
bearing
Fc
receptors
for
IgE:
suppressive
effect
of
glucocorticoids
on
the
expression
of
Fc
receptors
and
glycosylation
of
IgE-binding
factors.
J.
Immunol.
127:471-482.
24.
Petroni,
K.
C.,
L.
Shen,
and
P.
M.
Guyre.
1988.
Modulation
of
human
polymorphonuclear
leukocyte
IgG
Fc
receptor-mediated
func-
tions
by
IFN-'y
and
glucocorticoids.
J.
Immunol.
140:3467-3472.
25.
Russo-Marie,
F.,
and
D.
Duval.
1982.
Dexamethasone
induced
inhibition
of
prostaglandin
production
does
not
result
from
a
direct
inhibition
of
phospholipase
activities
but
is
mediated
through
a
ste-
roid-inducible
factor.
Biochim.
Biophys.
Acta.
712:177-185.
26.,Emilsson,
A.,
and
R.
Sundler.
1985.
Studies
of
the
enzymatic
pathways
of
calcium
ionophore
induced
phospholipid
degradation
and
arachidonic
acid
mobilization
in
peritoneal
macrophages.
Biochim.
Biophys.
Acta.
816:265-274.
27.
Walsh,
C.
E.,
B.
M.
Waite,
M.
J.
Thomas,
and
L.
R.
DeChate-
let.
1981.
Release
and
metabolism
of
arachidonic
acid
in
human
neu-
trophils.
J.
Bid.
Chem.
256:7228-7234.
28.
Smolen,
J.
E.,
and
G.
Weissmann.
1980.
Effect
of
indometha-
cin,
5,8,11,14-eicosatetraenoic
acid
and
p-bromophenacylbromide
on
lysosomal
enzyme
release
and
superoxide
generation
by
human
poly-
morphonuclear
leukocytes.
Biochem.
PharmacoL
29:533-538.
29.
Shaw,
J.
O.,
S.
J.
Klusick,
and
D.
J.
Hanahan.
1981.
Activation
of
rabbit
platelet
phospholipase
and
thromboxane
synthesis
by
1-0-
hexadecyl/octadecyl-2-acetyl-sn-glyceiyl-3-phosphorylcholine
(plate-
let
activating
factor).
Biochim.
Biophys.
Acta.
663:222-229.
30.
Maridonneau-Parini,
I.,
and
A.
I.
Tauber.
1986.
Activation
of
NADPH
oxidase
by
arachidonic
acid
involves
phospholipase
A2
in
intact
neutrophils
but
not
in
the
cell
free
system.
Biochem.
Biophys.
Res.
Commun.
138:1099-1105.
31.
Bromberg,
Y.,
and
E.
Pick.
1983.
Unsaturated
fatty
acids
as
second
messengers
of
superoxide
generation
by
macrophages.
Cell.
Immunol.
79:240-252.
32.
Grzeskowiak,
M.,
V.
DellaBianca,
P.
De
Togni,
E.
Papini,
and
F.
Rossi.
1985.
Independence
with
respect
to
calcium
changes
of
the
neutrophil
respiratory
and
secretory
response
to
exogenous
phospholi-
pase
C
and
possible
involvement
of
diacylglycerol
and
protein
kinase
C.
Biochim.
Biophys.
Acta.
844:81-90.
33.
Flower,
R.
J.
1988.
Lipocortin
and
the
mechanism
of
action
of
the
glucocorticoids.
Br.
J.
Pharmacol.
94:987-1015.
34.
Northup,
J.
K.,
K.
A.
Valentine-Braun,
L.
K.
Johnson,
D.
L.
Severson,
and
M.
D.
Hollenberg.
1988.
Evaluation
of
the
antimiflam-
matory
and
phospholipase-inhibitory
activity
of
calpactin
II/lipocortin
I.
J.
Clin.
Invest.
82:1347-1352.
35.
Davidson,
F.
F.,
E.
A.
Dennis,
M.
Powell,
and
J.
R.
Glenney,
Jr.
1987.
Inhibition
of
phospholipase
A2
by
lipocortins
and
calpactins:
an
effect
of
binding
to
substrate
phospholipids.
J.
Biol.
Chem.
262:1698-
1705.
1940
I.
Maridonneau-Parini,
M.
Errasfa,
and
F.
Russo-Marie
