Macrophage cholesterol removal by triglyceride-phospholipid emulsions

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 155, No. 2, 1988

Pages 709-713

September 15, 1988

MACROPHAGE CHOLESTEROL REMOVAL BY TRICLYCERIDE-PHOSPHOLIPID EMULSIONS Michael Aviram~ Kevin Jon Williams, and Richard J. Deckelbaum Columbia University, College of Physicians & Surgeons New York, New York 10032 Received May 25, 1988

SUMMARY: Phospholipid liposomes were previously shown to mobilize cholesterol from cultured macrophage foam cells. Because Intralipid, a clinically available triglyceride-phospholipid emulsion, contains both phospholipid liposomes and triglyceride-emulsion particles, we sought to study its effect on macrophage clholesterol mobilization. Following an 18h incubation of J774 macrophages in serum-free medium supplemented with Intralipid, cholesteryl ester content decreased by up to 50% in previously cholesterol-loaded cells, and by 25% in non-loaded cells. Both components of Intralipid, liposomes and emulsion particles, independently caused reductions in cellular cholesteryl ester. We conclude that clinically available triglyceride-phospholipid emulsions can mobilize macrophage cholesterol in vitro. © 1988 AcademicPress, Inc.

Removal of "accepter"

cholesterol from

particles,

cultured cells

including

HDL

is mediated by

(i),

red

a variety

blood

cells

apoprotein-phospholipid disks (3), and cholesterol-poor phospholipid (4).

of cholesteryl

ester

membrane (2,3).

Of

the

phospholipid liposomes

the cells, uptake of the freely

the accepter, and to make

up for

loss of

cholesterol at

particles studied

definitively shown

the

floating

hydrolysis of intracellular

the

various accepter

have been

the cell

diffusion of the accepter particle into

unstirred water layer that surrounds cholesterol molecule by

(2),

liposomes

The process involves spontaneous desorption of cholesterol from

membrane into the aqueous medium,

of

stores

the

cell

in vitro,

only

to mobilize

cholesterol

from endogenous stores in the whole animal (5,6). In

the

current

study,

we

sought

to

determine

if

Intralipid,

triglyceride-phospholipid emulsion that is commonly administered to humans, macrophages. component is

can

mobilize

cholesterol

Intralipid

contains

cholesteryl

phospholipid

triglyceride-emulsion particles

size (approximately 500nm into the unstirred cholesterol.

and

water layer, and

However,

liposomes,

(5-7).

diameter), emulsion

Because

from but

cultured its

of their

particles would diffuse

hence have poor

emulsion particles

ester

have

access to cell

a large

a

intravenously

capacity

major large poorly

membrane to

carry

*Dr. Aviram was a visiting scientist from the Lipid Research Laboratory, Rambam Medical Center, Haifa, Israel, where reprint requests should be sent. Abbreviations: DMEM, Dulbeceo's Modified Eagle's lipoprotein; HDL, high-density lipoprotein.

709

Medium;

LDL,

low-density

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 155, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

unesterified cholesterol, which can distribute to the phospholipid surface

and

to the triglyceride core (8). MATERIALS & METHODS Egg phospholipid liposomes were prepared by prolonged sonication (9). 10% and 20% Intralipid were purchased from KabiVitrum (Alameda, CA). Each deciliter of 10% Intralipid contains 10g triglyceride emulsified with 1.2g of phospholipid; 20% Intralipid contains twice the triglyceride (20g/dl) but the same amount of phospholipid (l.2g/dl). Triglyceride emulsion particles were purified from 20% Intralipid by separation from the liposome-rich fraction by ultracentrifugal floatation at d=l.OO6g/ml, to yield "washed" Intralipid (7). Murine J774 macrophage-like cells were maintained in DMEM supplemented with 10% fetal calf serum. Cholesterol-loaded foam cells were prepared by an 18~ incubation with LDL (0.6mg proteig/ml); to cholesterol-label cells, [(-H)-cholesteryl ester]-LDL was added (I0 cpm/ml) (i0,ii). All experiments involved 18h incubations at 37°C in DMEM supplemented with 0.5% bovine serum albumin and the various lipid dispersions described above. To avoid confounding effects from lipoproteins and apolipoproteins, the experimental media contained no serum. Following the experimental incubations, the cells were washed, then analyzed for protein (12), unesterified cholesterol, and cholesteryl ester mass (13). Radioactivity r~tained by the choles~erol-labeled macrophages was determined. Cellular [~H]-cholesterol and [~H]-cholesteryl ester were separated by thin-layer chromatography (14) .±~., Cellular cholesterol synthesis [W~c]aSsessed by the conversion of [1,2- C]-sodium acetate (i0 Ci/ml) into -cholesterol and cholesteryl ester. All data are displayed as means ± standard error of the mean, n=6. RESULTS An 18h

incubation

with 10%

Intralipid

cellular cholesteryl ester mass, in

A.

o5

IOO

Fc

reductions At

B. CHOLESTEROLLOADED CELLS

NONLOADED CELLS

o.'i

caused pronounced

a dose-dependent fashon (fig. i).

2OO

i

~=E so

Ioo

..I .J i,i 0

O TRIG .YCERIDE

I

I

I

I

I00

200

I00

200

PHO~;PHOLIPID

12

24

12

24

0

INTRALIP1D CONCENTRATION (mg/dl)

Figure !. Effect of a triglyceride-phospholipid emulsion on the cholesterol content of macrophages. J774 cells were incubated for 18h with 10% Intralipid at the indicated concentrations in serum-free media. Cells were then analyzed for unesterified (e) and esterified (0) cholesterol contents. A: Cells not pre-leaded; B: cells pre-loaded with cholesterol.

710

in the

Vol. 155, NO. 2, 1988

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

g.

A.

CHOLESTEROL LOADED

NONLOADED CELLS

pz ~ I.-

z o ,£,)~ n-

~ .~ELLS

IO0

IOO

FCj,

o

p-,._ if) o

~

s0

50

7"-O _.1 --I U.I rO I

'

0

12

I

24

I

12

0

24

PHOSPI-IOIIPID CONCENTRATION (mg/dl)

EMULSION

Figure 2. Effect of emulsion phospholipid concentrations on maorophage cholesterol. J774 cells were incubated for 18h in media containing 200mg of emulsion triglyceride/dl and the indicated concentrations of emulsion phospholipid. Cells were then analyzed for unesterified (e) and esterified (0) cholesterol contents. A: Cells not pre-loaded; B: cells pre-loaded with cholesterol. Washed, 20%, and 10% Intralipid were used to achieve the phospholipid concentrations of 6, 12, and 24mg/dl of medium, respectively.

highest

concentration

of

Intralipid

tested,

J774

cells

ester and cells

preloaded

with

that had not

been

cholesterol lost: 55%

of their cholesteryl

pre-loaded lost 28%.

Free cholesterol content also decreased (fig. i).

Triglyeeride-emulsions particles, without liposomes, reducing cellular cholesteryl

ester mass

were also capable

(fig. 2A and

2B, leftmost

At the same triglyceride concentration, 20% and 10% Intralipid (24 phospholipid/dl of

medium,

cellular cholesterol phospholipid/dl),

respectively)

and cholesteryl

indicating

were

ester

more

than was

additive effects

effective

and 12mg of at

removing

washed Intralipid

from the

of

points).

content of

(6mg

liposomes

(fig. 2). Similarly,

in

comparing

lipid

concentration, those with higher

dispersions

of

constant

phospholipid

triglyceride concentrations effected

losses of cellular cholesterol and cholesteryl ester (fig. 3). pre-loaded cells,

media containing

media, 12mg phospholipid/dl) mass

than

did

phospholipid/dl) Reduction in

media

containing

liposomes

macrophage cholesterol (fig.

cellular

losses.

cholesterol

4A).

cholesterol mobilization by lipid from the cells (fig. 4B). the

(200mg triglyceride/dl

lower cellular cholesteryl (no

of

ester

triglyceride,

12mg

(fig. 3B).

cholesterol synthesis

paralleled

20% Intralipid

produced a 30%

greater

In cholesterol

patterns

phospholipid concentration

content was not

Synthesis The

mechanism

caused by

increased

to

responsible

compensate for

macrophage

(12mg/dl

cholesterol

of medium),

dispersions with higher triglyceride contents. 711

more

mass: loss was

for

macrophage

dispersions was enhanced cholesterol

The patterns for retained cholesterol for

decreased

efflux

radioactivity at

a

fixed

produced

by

Vol. 155, No. 2, 1988

A.

l,.-

z bJ l-Z 0

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

NONLOADEDCELLS

B. CHOLESTEROL LOADED

I00

IOO

50

50

J~

w

F-,,.

mo -r .-1 -J

0

I

I

0

I

I00

200

I

I

0

I

IOO

200

EMULSION TRIGLYCERIDE CONCENTRATION (mg/dl) Figure 3. Effect of triglyceride concentrations on macrophage cholesterol. Cells (A: not pre-loaded; B: pre-loaded with cholesterol) were incubated in media containing 12mg of phospholipid/dl and the indicated concentrations of triglyceride. Cells were analyzed for unesterified cholesterol (I) and esterified cholesterol (0). Pure phospholipid liposomes, 10% Intralipid, and 20% Intralipid were used to achieve the triglyceride concentrations of 0, i00, and 200mg/dl, respectively.

DISCUSSION We

have

emulsion,

shown

Intralipid,

cultured macrophages. which were

that can

a

clinically

available

substantially

triglyceride-phospholipid

reduce the

cholesterol

The effect was m o s t p r o n o u n c e d

pre-loaded with

m e d i a t e d b y an e n h a n c e m e n t

cholesterol. of

Cellular

cholesterol

in our

cholesterol

reduction

e f f l u x b y the emulsion.

g. ..J 0

~

content

of

m o d e l foam cells,

Both

was types

CHOLESTEROL EFFLUX

I0,-

w I-

A.

w _J

=

z

7

CHOLESTEROL SYNTHESIS

Q.

w"

'

~u m o-r

~

2

~ Control 0

Z

-1

/ ufl t.9

-x

IOO

20

3OO

~ EMULSION TRIGLYCERIDE CONCENTRATION (rng/dl)

o i

Oontrol 0

i

I00

i

i

200

300

EMULSION TRIGLYCERIDE CONCENTRATION (rag/all)

Figure 4. Effect of lipid dispersions on m~crophage cholesterol synthesis and efflux.- J774 cells were pre-loaded with [ H]-cholesterol, then incubated for 18h in lipid-free medium (control) or in media supplemented with 12mg of phospholipid/dl and the indicated concentrations of triglyceride. Cells were then analyzed f o r cholesterol 3synthesis rate (A) and retained radioactivity (B). [ H]-cholesterol, |; [ H]-cholesteryl ester, 0. Pure phospholipid liposomes, 10% Intralipid, 20% Intralipid, and washed Intralipid were used to achieve the triglyceride concentrations of 0, i00, 200, and 250mg/dl, respectively.

712

Vol. 155, No. 2, 1988

of

particles

particles,

in

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Intralipid,

liposomes

and

large

triglyeeride-emulsion

contributed to the effect.

Our results with pure

triglyeeride-emulsion particles show that, in

spite

of their large size, they were extremely effective cholesterol accepters.

Per

unit mass of phospholipid, macrophages.

It

has

they are more effective than liposomes

been

shown

elsewhere

partitions into the

triglyceride core

large

core

triglyceride

triglyceride-emulsion

can

act

that

unesterified

of emulsion particles

as

a

cholesterol

at unloading cholesterol

( 8 ) . Thus,

sink.

the

Efficacy

particles as cholesterol accepters in vivo, however,

be limited by t]heir short

residence time in the circulation (15), which

of may

would

limit the time to equilibrate with endogenous cholesterol stores (3,5,6). Overall,

we

have

shown

that

intravenously into humans, can vitro. particles,

Recent and

studies

have

Intralipid

atherogenie lipoproteins

Intralipid,

which

is

promote cholesterol efflux shown

can

that

also

liposomes,

block

influx

into model foam cells (11,16).

commonly

infused

from foam cells

i_nn

triglyceride-emulsion of

cholesterol

These results

from suggest

a possible anti.-atherogenic role for emulsions. ACKNOWLEDGEMENTS This work was supported by grants HL38956, HL21006, and HL40404 from the National Institutes of Health. K.J. Williams is a recipient of a Clinician-Scientist Award from the American Heart Association, with funds contributed in part by the AHA-New York City Affiliate.

REFERENCES i. Baily, J.M. (1965) Exp. Cell Res. 37,175-182. 2, He, Y.K., Brown, M.S., and Goldstein, J.L. (1980) J. Lipid Res. 21,391-398. 3. Phillips, M.C., Johnson, W.J., and Rothblat, G.H. (1987) Biochim. Biophys. Aeta 906,223-276. 4. Yau-Young, A.O., Rothblat, G.H., and Small, D.M. (1982) Biochim. Biophys. Acta 710,181-187. 5. Williams, K.J., and Scanu, A.M. (1986) Biochim. Biopbys. Aeta 875,183-194. 6. Williams, K.J., Vallabhajosula, S., Rahman, I.U. Dcnnelly, T.M., Parker, T.S., Weinraueh, M., and Goldsmith, S.J. (1987) P~oc. Natl. Acad. Sci. USA 85,242-246. 7. Granot, E., Deckelbaum, R.J., Eisenberg, S., Oschry, Y., and Olivecrona, G.B. (1985) Biochim. Biophys. Acta 833,308.~;[5 8. Miller, K.W., and Small, D.M. (1982) J. Ce!!~id. Inter. Sci. 89,466-478. 9. Huang, C., and Thompson, T.E. (1974) Methods Enzymol. 32,485-489. I0. Tabas, I., Weiland, D.A., and Tall. A.R. (1985) Prec. Natl. Acad. Sci. USA 82,416-420. ii. Williams, K.J., Tall, A.R., Bisgaier, C., and Broeia, R. (1987) J. Clin. Invest. 79,1466-1472. 12. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall~, R.J. (1951) J. Biol. Chem. 193,265-275. 13. Ishikawa, T.T., MacGee, J., Morrison, J.A., and Glueck, C.J. (1974) J. Lipid Res. 15,286-291. 14. Mangold, H.K. (1969) In Thin-layer Chromatography. A Laboratory Handbook. (E. Stahl, Ed.), pp. 363-421. Springer-Verlag, New York. 15. Untraeht, S.H. (1982) Bioehim. Biophys. Acta 711,176-192. 16. Aviram, M., Williams, K.J., MeIntosh, R.A., Carpentier, Y., Tall, A., and Deckelbaum, R.J. (1988) Arteriosclerosis, in press.

713