AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 359-365

BIOCHEMICAL

Vol. 179, No. 1, 1991 August 30, 1991

THE CONTRIBUTION

OF THE MACROPHAGE TO ITS CELLULAR Michael

Lipid Family

Research Institute

RECEPTOR

FOR

OXIDIZED

LDL

UPTAKE

Aviram

Laboratory, Rambam Medical Center, Rappaport for Research in the Medical Sciences,Technion Faculty of Medicine, Haifa, Israel

Received June 28, 1991 SUMMARY: Oxidized LDL (Ox-LDL) was shown to be taken up by macrophages via several receptors including the acetyl-LDL(Acthe LDL,and the Ox-LDL receptors. Cellular uptake and LDL), degradation of Ox-LDL could be dissociated from that of LDL and AC-LDL as demonstrated by usingmacrophages that lack the LDL or the AC-LDL receptors.In J-774 A.1 macrophage-like cell line unlabeled Ox-LDL reduced the '"I-Ox-LDL by up to degradation of 91% whereas unlabeled AC-LDL and native LDL reduced'"I-Ox-LDLdegradation by only 51% and 23%, respectively. Analysis of macrophage degradation of'"I-Ox-LDLin the presence of 30-fold excess concentration of LDL + AC-LDL(to block uptake of'261-Ox-LDLvia the LDL and the AC-LDL receptors) revealed that cellular degradation via the Ox-LDL receptor could account for 45% of the macrophage uptake of Ox-LDL. a 1991Academicmess, Inc. Oxidized

low density

was shown

to

foam

formation

to

cell

the

different

Ox-LDL

bind

The present

to the

of

with

similar

study the

cellular

LDL,

AC-LDL

uptake

Ox-LDL

as well

as with

we have

LDL receptor

was undertaken

addition

(1,4).

recently

to the

LDL and

in

receptors

Furthermore,

can also

contribution

on macrophages

interact

native

accumulation possesses,

scavenger

(5,6).

unlike

cholesterol

Macrophage

(I-3).

receptors that

(Ox-LDL),

macrophage

(AC-LDL)

macrophages(7). relative

induce

LDL receptor,several

and acetyl-LDL

shown

lipoprotein

on to assess

and Ox-LDL

and degradation

the

receptors of

Ox-LDL. Abbreviations: LDL, low density Oxidized-LDL; AC-LDL,acetyl-LDL; Eagle's Medium.

lipoproteins(s); DMEM, Dulbecco's

Ox-LDL, Modified 0006-291X/91 $1.50

359

Copyright 0 1991 by Academic Press, Inc. AlI rights of reproduction in any form reserved.

Vol.

179, No. 1, 1991

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

METHODS Cells. J-774 A.1 murine macrophage-like cell line and U-937 macrophage histiocytes were purchased from American Tissue Rockville, MD. Cells were plated at Culture Collection (ATTC), 2.5X10 cells/l6-mm dish in DMEM supplemented with 10% fetal serum (FCS). The cells were fed every 3 days and were calf used for experiments within 7 days of plating. Human monocytederived macrophages (HMDM) were prepared as previously described (8), from healthy volunteers as well as from one patient with homozygous familial hypercholesterolemia (HFH). Lipoproteins. LDL was prepared from human plasma derived from fasted normolipidemic volunteers. LDL was prepared by discontinuous density gradient ultracentrifugation as described previously (9). The lipoprotein was washed at d = 1.063 g/ml and dialyzed against 150 mM NaCl, ImM EDTA (pH 7.4) under nitrogen in the dark at 4C. LDL was then sterillized by filtration (0.22 nm) and used within 2 weeks.LDL was iodinated by the method of McFarlane as modified for lipoproteins (IO). in the presence of 10pM CuSO4 (5). LDL was LDL was oxidized acetylated by repeated additions of acetic anhydride to 4mg/ml LDL diluted I:1 (v:v) with saturated ammonium acetate (11). Cellular degradation of lipoproteins. LDL degradation was measured following incubation of the iodinated lipoprotein with cells for 5h at 37?Z. The hydrolysis of LDL protein was assayed in the incubation medium by measurment of tricholoroacetic acid soluble, non-iodide radioactivity(l2). LDL degradation was minimal and was subtracted from Cell-free times with total degradation. Thecell layer was washed three PBS and extracted by a l-h incubation at room temperature with 0.5 ml of O.lN NaOH for measurement of cell-associated radioactivity and of cellular protein content by the method of Lowry et al. (13). Competition studies were performed using a constant amount of'"I-Ox-LDL (5pg of protein/ml) and increasing concentration of unlabeled lipoproteins (LDL, AC-LDL, Ox-LDL).

RESULTS The ability

of

unlabeled

incuced-oxidized its

uptake

line of'"

LDL (Ox-LDL) and degradation

was tested I-Ox-LDL

of

in

of

unlabeled

30 fold

AC-LDL

or native

reduced

by 91%,

results

were

shown).

To assess

for

Ox-LDL

to

excess

LDL,

to

LDL (AC-LDL)

compete

5 hours

protein/ml the

acetyl

by J-774

following

concentrations presence

LDL,

the

of cell

presence

the

the

(Fig of

degradation

contribution

360

of

with

1).

5pg

In

1).

macrophage

Ox-LDL,

cells

the

Ox-LDL,

of'261-Ox-LDL

cell-association the

cell

unlabeled

(Fig

of

uptake

for

increasing

lipoproteins

for'% I-Ox-LDL

cellular

incubation of

51% and 23%,respectively

obtained

I-Ox-LDL

A.1 macrophages-like

concentration

cellular

with'=

and copper

was

Similar

(data receptor were

not

Vol.

179, No. 1, 1991

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

251-Ox-LDL

f, t(u)LtAc-LDL)

75 W

Competitor

Concentration

Qg of protein/ml)

Lipoprotein

Concentration

(pg of protein/ml)

Fig 1. Competition for macrophage degradation of '251-0x-LDL by unlabeled LDL, AC-LDL and Ox-LDL. Competition studies were performed using 5pg ofi2%-Ox-LDL protein/ml that was incubated with J-774 A.1 macrophages in the presence of the indicated concentrations of unlabeled lipoproteins for 5 hours at 37oC prior to analysis of lipoprotein degradation. Each point is the mean of duplicate determinations (each performed in triplicates). Fig 2. Saturation curves for degradation of'251-Ox-LDL by J-114 A.1 macrophages. Macrophage degradation of12%-Ox-LDL was determined using increasing concentrations of1251-Ox-LDL in the absence or presence of 30 fold excess concentrations of unlabeled LDL + AC-LDL. Each point is the mean of duplicate determination (each performed in triplicates).

incubated

increasingconcentrationsof

with

absence

(control)

or presence

of

unlabeled

LDL and AC-LDL

both

the

macrophage

Ox-LDL

was about

absence that

of

were

via

receptor

the

were

excess used

Fucoidin

for

to the

macrophages

was inhibited

of

inhibited

out

by fucoidin,

30 fold

total

of'"

concentration the

degradation

effect

of

was tested of

361

via

the

the

results not

shown). The

by J-774

A.1

presences

of

50pg/ml

Ox-LDL

receptor

is

also

this

compound

in

on cellular

the absenceor Fucoidin

in both

the

LDL and

on macrophages(4).

LDL + AC-LDL.

of?-Ox-LDL

in

LDL+Ac-LDL,

(data

of protein/ml)

the

of

Similar

of'= I-Ox-LDL

(5pg

of

(obtained

respectively).

whether

I-Ox-LDL

degradation

unlabeled

receptor

the

The contribution

of Ox-LDL

by 81% in

the

2).

degradation

uptake

in

concentration

to the

of

AC-LDL

of'% I-AC-LDL

degradation

the

the

degradation

blocked

(Fig

Ox-LDL

cell-association

To find

30 fold

for

LDL receptors

binds

fucoidin.

of

concentrations

to block

acetyl

found

45% of

'2~ I-Ox-LDL

cases

presence (50pg/ml)

by

Vol.

BIOCHEMICAL

179, No. 1, 1991

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3 ~‘251-Ox-LDL + kDL+Ac-LDI Lm+hJc I

Fiq 3. The effect of fucoidin on macrophage degradation of Ox-LDL. J-774 A.1 macrophages were incubated with 1Opg of 1251-Ox-LDL/ml in the absence or presence of 50pg/ml fucoidin(A). Similar study was carried out using 101.19 of 1251-0x-LDL/m1+300pg of protein/ml of LDL and AC-LDL (B). Lipoprotein degradation rates were studied as described under Methods.

(Fig

77%

31,

LDL receptor

inhibited cells,

in

as well

as the

by fucoidin.

U-937

were

receptors

degraded

from

healthy

fold

higher

degradation

subjects than of

In

that

of

AC-LDL

native

(Table

Lipoprotein type

u-937

HMDM-Normal HMDM-HFH

study

Ox-LDL

1).

In

both

degradation

only

protein/

2135+158 5478t239 305223 95*-17

HMDM, Human monocyte-derived macrophages Results are the mean + S.D. of 3 experiments. 362

AC-LDL

of

I/B

was 3

the

a patient types

with of

(25ug of protein/ml)

LDL

4635t224 1418t61 1003*57 911+42

that

macrophages

degradation in various macrophages

(ng/mg cell

of

degradation

HMDM from

Degradation

A.1

we found

cellular

LDL, but

Ox-LDL J-774 A.1

J-774

monocyte-derived

(HMDM-Normaol),

Ac-

similary

A.1 macrophages, the

(the

number

small

present

of

human

TABLE 1. Lipoprotein

Cell

l/4

are

unlike

relatively the

receptors

receptor)

macrophages,

J-774

to only 1).

scavenger

Ox-LDL

In

(14).

asopposedto

LDL (Table

both

to possess

AC-LDL

cells

and Ox-LDL native

shown

for

U-937

that

suggesting

5h) AC-LDL 70392339 1407+58 3015?166 3068i195

Vol.

BIOCHEMICAL

179, No. 1, 1991

homogygous lack

familial

the

hypercholesterolemia

LDL receptor,

degradation

of

AND BIOPHYSICAL

Ox-LDL

AC-LDL

(HMDM-HFH)

which

was also

30% the

degradation

(Table

RESEARCH COMMUNICATIONS

1).

DISCUSSION Oxidized

LDL can lead

following

its

Unlike

the

cellular

are

not

Ox-LDL. on Ox-LDL which

is

(5). not

only

A.1

'261-0x-LDL affect

suggesting by the

cells of

of

degradation

that

Ox-LDL

might

various

shown that

cellular

degradation

from

that

existence Macrophage half

the

lipoprotein different

LDL and AC-LDL

of

specific

of

degradation uptake intracellular

of

of AC-LDL. via

the

up in AC-LDL

further

Ox-LDL

pathway 363

(18)

might

receptor

is

that

of

did

only

LDL

it

could

be

be dissociate

for

it

than

with

cells

support

was shown Thus,

unlike

shown),

or the

could

in

cellular

not

U-937

receptor(s)

Ox-LDL

the

LDL+Ac-LDL

(data

Ox-LDL

and thus

was shown

incubated

of macrophages

macrophage

degradation

to cells

by the

Ox-LDL

macrophages,

'2~ I-Ox-LDL

types

of

to

on

epitopes

for

of unlabeled

and not

By using

various

LDL (5,6)

In U-937

be taken

receptors.

the

receptor

addition

of

formation

epitopes

contribute

Ox-LDL.

can cause

populations,

different of

concentrations

receptor

cell

or native

and HMDM, the

the

Ox-LDL

and foam heterogeneous

to substantially

excess

AC-LDL

macrophage

by AC-LDL

receptors

and thus

a fraction

(1).

by the AC-LDL

may recognize

and degradation

J-774

receptors

several

the

of

The specific

study

via

(15),

consists

shared

accumulation

regulated

accumulation

mimics

cholesterol

is

regulated

receptors

present

uptake

content

LDL

AC-LDL

cells

which

cholesterol

different

the

similarly

Oxidized

(l-4).

not

into

LDL receptor

macrophage

the

uptake

cholesterol

(16,17)

thus

to macrophage

the

Ox-LDL. to

be about

be that followed AC-LDL.

by Ox-LDL

is

Vol.

179, No. 1, 1991

differently than It

BIOCHEMICAL

targeting

not

cholesterol

than

the

the

uptake

about

cellular

of AC-LDL

half

of

receptor

the

(which

is

In

U-937

receptors

to

both

to

share

similar

blocks

the

cellular that Ox-LDL

vesicles

is

of

cell

that

If

that

up via

the

found

to

these

purified

AC-LDL

number

of

be low

in

yet

to allow

to macrophage

a better

cholesterol

may

which inhibited receptor

macrophage

(16,17),the

AC-LDL

receptors

fucoidin

the

to

only

the

AC-LDL by macrophages,similarly Unlike

so,

cholesterol

line,

were

by

comparison

fact

by cellular

Furthermore,

Ox-LDL.

purified

in

taken

suggesting

regulated

LDL receptor.

to the

and Ox-LDL

LDL,

is

of Ox-LDL

regulated

AC-LDL

of

contribution

the

macrophage-like

uptake

was not

like

Ox-LDL

properties.

was recently

receptor

be related

may

not

native

uptake

Ox-LDL

uptake

degraded

content).

comparison

the

content

reduced

or peripheral

(19,20).

known whether

cellular

its

to perinuclear

LDL or B-VLDL is

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

however

receptor

for

understanding

of

metabolism.

ACKNOWLEDGMENTS The author wishes to thank expert technical assistance this manuscript.

Mira Rosenblat and to Illana

and Judith Cohen for

Oiknine typing

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