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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-

as second

1984.

and Cuatrecasas,

,

P. Rapid

for-

a pathway

Proc.

NatI.

for

Acad.

Sci.

1986.

1W.

and

,

Cerami.

A. Cachectin/Tumor

distribution,

and

135,3972-3977, lW.

and

,

and diarylglycerol

messenger.

and

Cerami, factor

,

Dyer,

purification.

metabolic

fate

in

1985.

necrosis Science

Breckenridge.

W.C.

composition

of

Biocheim.

Biophys.

Cockcroft,

S. Ca’

A. Passive

immunization

protects

229.869-871.

mice

from

lethal

1985.

Wi.

A rapid

Can.

J. Biochem.

10.

rat

J.H.

Acta

method

of total

Physiol.

lipid

37,91

cx-

1-9

17.

RI.

RI.,

and

K.A..

Tumor

PA.

37-46,

or 1984.

of calcium-mobilizing theme.

Hajjar.

FASEB.

agoJ.

2,2670-

PA.

S.D.

R.L.

release

endothelial

GA. in 1988.

12-0-tetradecain

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Silverstein,

cultured

of

metabolism

Acta

D.P.,

l07(6),866a.

Effects

glycerolipid

Warr.

metabolism Biol.

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from

and

.

cultured

1982. , and Nachman. of platelet-derived

cells.

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, Marek. J. Induction

mice

F. , Rodgers, G. , Lin. P.F. . Warr, G. . and of tumor necrosis factor. IFN-b and acute

by

toxic

and

non-toxic

forms

of

lipid

A.

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, Radzioch, D. , Young, HA. , and Varesio. L. Difinhibition of IL- 1 and TNF a mRNA expression by

E.J. which

rophages.

block

second

J. Immunol. CF.

79,319-326,

V. ,

Kiener. J. Cell

Biophys.

141,870-874,

ferential

Prpic, S.L.

fmet-leu-phe

795:

1987.

in

Kovacs,

Nathan,

on

necrosis

factor

Kiener.

.

Schimmel,

I 3-acetate

Immunol.

16.

lipid

phosphatidylinosi-

with

phosphatidylcholine

macrophages.

Biochim.

Hajjar.

agents

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peritoneal

Desiderio,

15.

of Acta

of action a young

The membrane.

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1G.

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Morgan.

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Grove,

R.L.

13.

and

conversion

in neutrophils

variations

myoblasts. 12.

,

synaptosomal

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

J. Immunol. 5. Beutler, B. , Milsark,

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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