of label into phosphatidylcholine. (PC) (90% increase compared to approximately. 50% in the other phospholipids). In macrophages labeled with [3H]-choline,.
Journal
of Leukocyte
Biology
48:38-42
(1990)
Lipopolysaccharide (LPS) Alters Phosphatidylcholine Metabolism in Elicited Peritoneal Macrophages Robert
I. Grove,
Nick
J. Allegretto,
Bristol-Myers
Peter PROD,
Company,
A. Kiener,
Wallingford,
and
Glenn
A. Warr
Connecticut
We investigated the effects of LPS on mouse peritoneal macrophage phospholipids using radiolabeled precursors. LPS (200 ng/mI) stimulated incorporation of [32P] into all classes of phospholipids within 0.5 hr, and after 2 hr the increase was 60% greater than controls. Separation of the phospholipid classes by thin-layer chromatography revealed a selective increase in incorporation of label into phosphatidylcholine (PC) (90% increase compared to approximately 50% in the other phospholipids). In macrophages labeled with [3H]-choline, LPS stimulated both the incorporation of label into PC and the release of incorporated label into the medium. The time dependencies of stimulated [3HJ release and [32P] incorporation were similar. These data are consistent with the hypothesis that LPS activates macrophages via a PC-specific phospholipase-dependent mechanism. Key words:
macrophage,
activation,
endotoxin
INTRODUCTION In response phages undergo free radicals,
to
bacterial endotoxins phagocytosis, produce synthesize arachidonic
and secrete a variety growth factors) [ 1 51 necrosis has
factor
been
has
induces
endotoxic other
C-catalyzed stimulated related
by which
long
been
reported
shock
[4,5]
of intense
diacylglycerol
inositide-
and
inositol
trisphosphate, in activating protein kinase C and mobilizing intracellular Ca2 are well known [2]. However, inositide breakdown lasted for only 15 mm, while the macrophage for
up
(PLA,)
(and
protein
to 6 hr
[16,18].
activity
and
kinase In
prostanoid
macrophages treated gests, however, that
with LPS
C)
synthesis
activation
are
yates
in cell of
activation
mouse
LPS on peritoneal
© 1990
Wiley-Liss,
fects
in
explain the mechanism these cells. Since other
volved
[8,9],
phospholipid macrophages. Inc.
A,
is increased
in sugPLA,
,
rather than directly activates some of the well-known important
activated
phospholipase
LPS [ 1 1 7] . The evidence primes macrophages for
activation Although cannot
remained
addition,
the enzyme [1]. pathways for cell
[ 1 16]
macrophages by which phospholipases we
,
endotoxin may
investigated
metabolism Our evidence
Lipopolysaccharide
(Escherichia
was purchased The culture media
(FCS) Island,
were NY). (Boston,
from and
co/i, Sigma, certified
0 1 1 1 :84 phenol Inc. fetal
(St. calf
Louis, serum
purchased from Gibco Laboratories (Grand Radioisotopes were from New England NuMA).
Brewer’s
modified
medium (4%) came from BBL (Cockeysville, MD). All other Sigma, Inc. (St. Louis, MO).
thioglycollate
Microbiology reagents
System from
were
Mice Outbred female Swiss CD- I mice (20-25 g) Charles River Breeding Laboratories (Rockville, were the source for peritoneal macrophages. Four cent ml)
sterile Brewer’s modified thioglycollate was injected into the peritoneal cavity;
macrophages
were
collected
by peritoneal
from MD) per-
medium (2 5 days later lavage
with
8
they
,
actibe inthe
in
METHODS
extract) MO).
clear is
,
messengers,
It
phospholipase
breakdown of polyphosphoinositides by LPS [ 1 1 6] . The effects of the second
and
investigation.
inositide-specific
AND
of phosphatiSome of the [ 10].
Reagents
pathophysiological [12]. activates macro-
endotoxin
an area
that
MATERIALS
,
atherosclerosis
mechanism
been
(LPS), macrooxygen-derived acid metabolites,
proteins (cytokines, enzymes, of the secreted proteins tumor
in several
including
phages has
(TNF)
implicated
conditions,
The
of One
.
that LPS stimulates a sustained degradation dylcholine via a specific phospholipase. results have been reported in abstract form
elicited suggests
ef-
Received Reprint istry, 06492.
August requests: Bristol-Myers
24.
1989:
Robert
accepted
I. Grove,
Company,
October Dept.
5 Research
26,
1989.
of Cardiovascular Parkway,
BiochemWallingford,
CT
in Macrophages
39
180
A p E
R
C
iso
E
iso
N T
i40
C 0 N T R 0 L
iso
i20
iio iOO
so
,
,
,
I
0
30
50
90
-
INCUBATION
TIME
iso
PHO8PHOLIPID
Fig. 1 . Effect of LPS on (32PJ incorporation into MO phospholipids. The data represent the means (± SEM) of three different experiments, each done in duplicate, except for the I .5 hr point in A, which comes from one experiment. A: Total phospholipids from cells treated for the indicated times and labeled with [32P1 (25-50 pCi/mb) were analyzed as described in Materials and Methods. B: Phospholipids from cells stimulated with LPS for 2
ml phosphate (pH
buffered
saline
#{149} P1
Pa
(Mm)
containining
3 mM
CLA8S
hr were analyzed for incorporated label after separation by thinlayer chromotography. PC = phosphatidylcholine; Pb = phosphatidylinositol; PS = phosphatidylserine; PE = phosphatidylethanolamine; PA = phosphatidic acid. For one experiment in which the cells were labeled with 25 Ci/ml, control total incorporation was 9,125 cpm. Control incorporation for PC, Pb + PS, PA + PE was 6,141 , 2,016, and 968 cpm, respectively.
EDTA
7.4).
Macrophage Freshly trifugation
moved
Culture collected (400g
for
by hypotonic
macrophages 5 mm).
lysis
addition of 10-fold excess The cells were collected pended
in Dulbecco’s
glucose; DMEM) 95% as determined phages
were
were pelleted blood cells
Red
(water
for 30 sec followed
modified
into
by
phosphate buffered saline). by centrifugation and resusEagle’s
with 10% fetal by differential
plated
by cenwere re-
12 well
bridge, MA) at 1 x 106 ml/well for 2 hr in a 37#{176}C humidified
medium
calf serum. staining. dishes
(high
[3H]-choline
chloride
pCi/ml [32P]-orthophosphoric well. In some studies the
with
LPS
for various
times
removed DMEM
0 0 0
(Costar,
Cam-
allowed to adhere CO, incubator. Non-
CONTROL
and 5%
(60
Ci/mmol)
or
10-25
acid in 0.5 ml DMEM per macrophages were challenged
and the radiolabel
for the last hour of the incubation period. ies, the macrophages were prelabeled. label was equilibrated
x
Purity was The macro-
adherent cells were removed with 2 washes of DMEM. Macrophage monolayers were incubated with 0.25-2.0
pCi/ml
C P M
and replaced with for 0.5 hr. The
Fig. 2. Effect of LPS on [3H]-choline incorporation into phospholipids. Cells were treated with LPS for 2 hr and radiolabebed (5 pCi/mb) the last hour of incubation. Phospholipids were analyzed as in Figure 1A. The data represent the means (± SEM) for four different experiments, each done in triplicate.
was added
In other studAfter 2 hr the
fresh temperature monolayers were
washed 4 times with 0.5 ml aliquots of DMEM and the macrophages were incubated for an additional 2 hr in 0.5
phospholipids and either counted
were subjected
Thin-Layer The
dried
method
with
phases
of the
extracted
down in a nitrogen evaporator thin-layer chromatography
phospholipids spotted
(TLC) were
on Silica in a solvent
Authentic the
labeled
phospholipid phospholipids
solubilized
Gel 60 system
ACETIC ACID:WATER::50:30:8: rate phosphatidylcholine from [7].
or
counter.
Chromatography
chloroform, chromatographed
chloroform
dried to
in a scintillation
ml DMEM to which LPS was added. At the end of 2 hr. the medium was removed and counted on a scintillation counter to assay for released label. Macrophage monolayers were solubilized in 0. 1% SDS and the phospholipids extracted using a modification of the Bligh-Dyer
[6, 1 1] . The
LPS
the
standards or
in
100
TLC plates, (CHCL3:MEOH:
i.l and
1) designed to sepaother phospholipids were
either
chromatographed
mixed on
Grove
40
et al.
B
P E R C E N T
P E R C E N I
180
C 0 N I R 0 L
C 0 N I R 0 L
110
i20
100
t
90
so
0
so
INCUBATION
eo TIME
I
I
11111$ 100
10
(Mba)
I
LP8 CONCENTRATION
:c
1000
tItlist
I
120
I
111(111 1000
(nglml)
D 1000
T N F
T N F
U N
U N 100
8 I
8 /
M L
M L I
so
so
0
INCUBATION
so TIME
i20
1
10
(Mln)
100
1000
LP8 CONCENTRATION
(ag/mI)
Fig. 3. Effect of LPS on phosphatidylcholine turnover and tumor necrosis factor (TNF) secretion. MO monolayers were prelabeled with [3H]-chobine, washed, and treated with LPS at the concentrations and times indicated. The media were analyzed for released label (A,B) and TNF (C,D) as described in Materials
and Methods. Control values for released radiolabeb in both A and B were approximately 9,000 cpm. The data represent means (± SEM) for triplicate (A,B) or duplicate (C,D) experiments. A,C: LPS concentrations were 200 ng/ml. B,D: Stimulation time was 2 hr.
separate lanes. The with iodine vapors,
(50%;
scintillation
resolved scraped
phospholipids into vials,
were located assayed in a
and
counter.
from
Fig. lB). Atlhough phosphatidylethanolamine,
label
was
To
Tumor
Necrosis
Biologically measured using previously
Factor
(TNF)
Assay
active TNF secreted the L929 fibroblast
described
RESULTS
from
The
of effect
approximately 1A). Separation thin-layer lectively dylcholine
LPS
Greater
[32P] elicited occurred
stimulated
an
orthophosphoric mouse peritoneal within
60% greater of individual
0.5
increase
acid into phosmacrophages.
hr and
by 2 hr was
than control values classes of phospholipids
chromatography (TLC) indicated increased incorporation of label (90%) compared to the other
in
(Fig. by
that LPS sein phosphatiphospholipids
with
effect
metabolism,
with
[3H]-choline than
90%
with authentic and LPS-treated in a less
of PC
acid
the
grate
not
resolved remaining
(Fig.
1B).
specifically
on phosphati-
LPS-treated
macrophages
[3H]-choline.
After
2 hr, (Fig.
by 60%
incorporated
label
comi-
standard on TLC plates in both M#{216}.The remaining 10% was
lipophilic
or sphingomyelin
The effect by measuring
entirely of the
incorporation
dylcholine (data
always most
phosphatidic
of LPS
pulse-labeled
increased
recovered
incorporation pholipids LPS
(PC)
(M#{248})were 2).
[13].
(LPS)
the
dylcholine
into the medium was cytotoxicity assay as
grated control
Lipopolysaccharide
associated
study
not
area
would
where
lysophosphati-
be expected
to mi-
shown).
of LPS on PC degradation was investigated the release into the medium of previously
incorporated [3H]-chobine. LPS apparently increased
These
studies
revealed
that
the rate of degradation of PC. Stimulated release was detected within 0.5 hr and reached 30% above control values after 2 hr (Fig. 3A). Concentrations of LPS as low as 10-50 ng/ml induced
Phosphatidybcholine significant
increases
while
maximal
induction
at 100 ng/ml and higher (Fig. 3B). In order to compare the relevance
of the
to M#{248}activation,
dose
the
LPS
time
and
PC
occurred
binds
response
A third possibility over are two separate
degradation
(Fig.
secretion that are
is stimulated very similar
a in
with time and to those for PC
has
generated
of LPS
action
considerable
in macrophages
interest.
activation of polyphosphoinositide within 10 sec, peaked at approx levels
however,
These
by
findings by
Protein
elevated
suggest
from
that
protein
inositide-related
account
for
nase. Recently
a
the
In one
LPS
occurred declined to
kinase
30 mm
to 6 hr [1,16].
kinase
C is activated but
activation
state
phosphatidylcholine-specific
represents a possible vation of the C kinase results
we
mechanism [9].
present
alterations
the
here
they
do ki-
suggest
that
produces
and diacylglycerol.
degradation. and PC turnof LPS on the
indicate that PC turnover plays More work is needed before the
degradation
is involved
in TNF
se-
Alternative data are also
The curves
correlation for both PC
of
the
released
label
J.M.
time and
and tumor
necrosis
factor:
the
alternative
which Our
possibility
the
ofendotoxin.
Bligh.
E.G.
traction
and
Med.
164.165-
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266,695-707.
Mechanisms
Some
G.
brain
-dependent
A23l87.
Exton,
Gombos,
.
adult
to phosphatide
growth
TNF
Exp.
phosphatidylcholine
Production,
effects
noylphorbol
dose-response necrosis factor
secreted
,
cachectinltumor
I 1 . Grove,
appears to be important data, however, do not that
second
against
elicited
(TNF) secretion suggests a common mechanism. One possibility is that LPS activates diacylglycerol formation via a PC-PLC in order to maintain the activated state of protein kinase C, for TNF secretion [14].
a
V. from
Milsark,
B.
lethality
between metabolism
Duronio,
Lipopolysaccharide
in
trisphosphate
83,6785-6789,
,
Beutler,
ionophore
meis
J.
J. 220,345-350.
,
of
2676.
are possible, an LPS stim-
metabolites.
of diarylglycerol
nists:
of a phospholipase D (PLD) which degrades PC choline and phosphatidic acid [9,20] or a phos-
Characterization
Inositol Biochem.
Besterman,
tol
9.
the
A2 which causes the release of lysophosphati(or platelet activating factor) into the
acid
and Cohn, Z.A. for enhanced re-
1959.
8.
into phosphaactivation which
with
Mi.
U.S.A. 4.
synthesis from PC
PC synthesis. however. The
mechanisms consistent
arachidonic
2. Berridge,
6.
more
Both
of
D.S., Wright, S.D., prime macrophages
179, 1986.
caused
and
A.A.. Cohen, lipopolysaccharide
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of PC from the membrane and the increase levels could signal the M#{248} to initiate
out
PC
vivo.
disappearance in diacylglycerol
dium. progress.
lease
acti-
LPS
metabolism
both the degradation and stimulated release of label
phosphorylcholine
I. Aderem, Bacterial
7.
incorporation a PC-PLC
rule
PC
be determined.
generation
of the
sustained
and the stimulation of [32P] tidic acid are consistent with
pholipase dylcholine
can
mation
,
for
in phospholipid
specifically increased of PC in M#{248}.The
ulation to free
to which
cretion
3.
phospholi-
,
The
extent
messengers.
pase C (PC-PLC) has been reported and can be activated by phorbol esters to degrade PC and produce phosphorylcholine and diacylglycerol [3 1 1 1 9] . The formation of diacylglycerol from phosphatidylcholine breakdown
general
Clearly, these findings a role in M#{216} activation.
C activity,
diacylglycerol
continued
(M#{248}) study,
breakdown 60 sec, and
mm.
5-15
remained
initially not
induces
is that TNF secretion and unrelated effects
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