BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 185, No. 1,1992
Pages 465-472
May 29,1992
P H O S P H O L I P A S E A 2 - M O D I F I E D LDL IS TAKEN UP AT E N H A N C E D RATE BY MACROPHAGES
Michael Aviram and Irit Maor
Lipid R e s e a r c h Laboratory, Rambam Medical Center, Rappaport Family Institute for R e s e a r c h in the Medical Sciences, Technion Faculty of Medicine, Haifa, Israel Received April 18, 1992 SUMMARY:
M o d i f i c a t i o n of the low density lipoprotein
(LDL) core
or surface lipids were shown to affect the cellular uptake of the
lipoproteins
and
hence
the
formation
of
macrophages. In the present study phospholipase of
LDL
with
was
increased
of
lysolechitine.
This
taken
up
and
by
effect
in
modified J-774
(up to 97% when
comparison
to
control
revealed that
the
uptake
of
PLase
the m a c r o p h a g e s was specific and was m e d i a t e d via
the LDL receptor.
Since
tissues,
PLase
thus
certain p a t h o l o g i c a l
A2
was
found
to
exist
in
the production of PLase A2-LDL under
conditions can potentially contribute
foam cell formation and accelerated atherosclerosis. Press,
A. I
of PLase A2 was enzyme dose dependent.
Competition experiments
various
degraded
cell line at enhanced rate
10 units/ml of PLase A2 was used)
by
treatment
content
was
macrophage-like
A2-LDL
A2
cell
shown to produce negatively charged l i p o p r o t e i n
lipoprotein
LDL. This
foam
to
®1992Ac~de=io
Inc.
P e r t u r b a t i o n of the core of low density lipoprotein triglyceride
hydrolysis
(1,2) or cholesteryl esterase
(3),
significantly
LDL,
by
lipases
ester hydrolysis with cholesterol
the m o d i f i e d l i p o p r o t e i n s Abbreviations:
(LDL)
with lipoprotein or hepatic
increased or decreased the uptake of
by the macrophages respectively.
low density lipoprotein(s);
p h o s p h o l i p a s e A2; DMEM,
PLase A=
Dulbecco's Modified Eagles Medium. 0006-291X/92 $4.00 465
Copyright © 1992 by Academic Press, lnc. All rights of reproduction in any form reserved.
Vol. 185, No. 1, 1992
Similarly,
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
changes
in the surface unesterified cholesterol by
LDL treatment with cholesterol oxidase the LDL coat phospholipids, phospholipases
(5-8)
resulted
PLase
and was taken phagocytic
its
incubation
with
in the formation of m o d i f i e d
lipoproteins which d e m o n s t r a t e d macrophages.
(4) or p e r t u r b a t i o n of
following
increased
uptake
by
C-modified LDL was shown to be a g g r e g a t e d
up
by
process
macrophages that
was
at
enhanced
rate
by
a
mediated via the LDL receptor
(5,6). PLase D - m o d i f i e d LDL was not aggregated and demonstrated
increased uptake
by
macrophages
receptor m e d i a t e d endocytosis
(8).
PLase
and
A2,
unlike
phosphoryl group, position cause
of
the
in
LDL
lipoprotein
nonspecifically
physicochemical
the
LDL
the
cells
lipoproteins
also
other
and
since
(9-11)
is distinct
fibroblasts
(12),
we
the
LDL
A2
was
(7). shown
In to
d e m o n s t r a t e d very (7).
Since
possess in addition to
even
the
from the classical in
sn-2
characterization
receptors
analyzed
the
was shown to
fibroblasts
the
cellular uptake of PLase A2-modified LDL by macrophages,
at
PLase
and
unlike human fibroblasts,
receptor,
macrophages
with
PLase A2-modified LDL
limited cellular d e g r a d a t i o n by macrophages,
acids
phospholipid,
incubation
to
an
PLase D, do not attack the fatty
diacylglycerol
human skin fibroblasts, bind
C
but rather the
changes
following
PLase
via
for LDL LDL
modified receptor on
receptor
present J-774
on
study the A.I
a cell line which was shown to possess all these
various receptors
for lipoproteins
(11,12).
METHODS Cells. J-774 A.I murine macrophage-like
cell line was p u r c h a s e d
from the A m e r i c a n Tissue Culture Collection plated
at
(ATCC). Cells were
2.5xi0 s cells/16mm dish in DMEM supplemented with
10% fetal calf serum
(FCS). The cells were fed
every
3
days
and were used for experiments within 7 days of plating. Lipoproteins.
LDL
was prepared from human plasma derived from
fasted n o r m o l i p i d e m i c
volunteers.
LDL was prepared by density
gradient u l t r a c e n t r i f u g a t i o n
as described previously(13).
LDL
was iodinated by the method
of
for
lipopoproteins of
Lowry
et
(14). LDL al.
McFarlane
as
modified
protein was measured by the method
(15). Vitamin
466
E
and
carotenoids
were
Vol. 185, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
determined as p r e v i o u s l y described
(16) and the
fatty
acids
d i s t r i b u t i o n was analyzed by gas liquid chromatography Phospholipolysis. venom
was used
of protein/ml)
Phospholipase
150mM NaCI,pH=8)
prepared
by
the
10
min
at
for
lh
at
37°C. The
from excess enzyme by
minicolumn
Iodine
methanol
(4). For
solution
(2:1,v:v).
TLC plates.
on
subclasses
(TLC) on silica
chloroform:
(60:35:8,v:v:v).
were used to visualize the lipid spots on the
The
appropriate
spots
ananlyzed for their phosphourous
were
content
C u l l u l a r uptake of lipoproteins.
J-774A.I
macrophages
then
scraped
and
(18).
Degradation of 12sI-
LDL or PLase A - 1 2 S I - L D L was measured with
Phospholipid
using a d e v e l o p i n g solution of
a m m o n i u m hydroxide
The
phospholipid
by thin layer chromatography
vapors
was
passage
lipid extracts of LDL were prepared using
separated
PLase
reaction
analysis,
methanol:
(Img
addition of ImM EDTA and refrigeration.
G-100
gel plates,
LDL
37°C.
Sephadex
were
bee
by incubation of the LDL solution with
modified LDL was separated
chloroform:
(17).
from
in the presence of 3mM CaCI2
free albumin,for
5 units/ml of PLase A2 stopped
A2)
was incubated with an equal volume of a buffer
(180 mM Tris,
was
(PLase
(Boehringer Mannheim GmBH, Germany).
and 0.6% fatty-acid A2-LDL
A2
following their incubation
for 5h at 37°C. The hydrolysis of
the LDL protein was assayed in the incubation medium Cellular c h o l e s t e r o l
esterification
previously d e s c r i b e d
(4).
rates
were
(19)o
measured
as
RESULTS Upon
incubation
units/ml) the
of LDL
(Img of protein/ml)
for I hours at 37°C,
modified
lipoportein
followed
(by
by
with PLase A2 reseparation
ultracentrifugation),
lysolechitine
content
protein
The electrophoretic mobility of
on
(n=3).
cellulose
acetate
native
LDL
PLase
Am-LDL
was increased and it migrated
14±3 mm
and
6±2%
to
32±4%
to migration of 9±2 mm o b t a i n e d
(p
Vol. 185, No. 1,1992
Pages 465-472
May 29,1992
P H O S P H O L I P A S E A 2 - M O D I F I E D LDL IS TAKEN UP AT E N H A N C E D RATE BY MACROPHAGES
Michael Aviram and Irit Maor
Lipid R e s e a r c h Laboratory, Rambam Medical Center, Rappaport Family Institute for R e s e a r c h in the Medical Sciences, Technion Faculty of Medicine, Haifa, Israel Received April 18, 1992 SUMMARY:
M o d i f i c a t i o n of the low density lipoprotein
(LDL) core
or surface lipids were shown to affect the cellular uptake of the
lipoproteins
and
hence
the
formation
of
macrophages. In the present study phospholipase of
LDL
with
was
increased
of
lysolechitine.
This
taken
up
and
by
effect
in
modified J-774
(up to 97% when
comparison
to
control
revealed that
the
uptake
of
PLase
the m a c r o p h a g e s was specific and was m e d i a t e d via
the LDL receptor.
Since
tissues,
PLase
thus
certain p a t h o l o g i c a l
A2
was
found
to
exist
in
the production of PLase A2-LDL under
conditions can potentially contribute
foam cell formation and accelerated atherosclerosis. Press,
A. I
of PLase A2 was enzyme dose dependent.
Competition experiments
various
degraded
cell line at enhanced rate
10 units/ml of PLase A2 was used)
by
treatment
content
was
macrophage-like
A2-LDL
A2
cell
shown to produce negatively charged l i p o p r o t e i n
lipoprotein
LDL. This
foam
to
®1992Ac~de=io
Inc.
P e r t u r b a t i o n of the core of low density lipoprotein triglyceride
hydrolysis
(1,2) or cholesteryl esterase
(3),
significantly
LDL,
by
lipases
ester hydrolysis with cholesterol
the m o d i f i e d l i p o p r o t e i n s Abbreviations:
(LDL)
with lipoprotein or hepatic
increased or decreased the uptake of
by the macrophages respectively.
low density lipoprotein(s);
p h o s p h o l i p a s e A2; DMEM,
PLase A=
Dulbecco's Modified Eagles Medium. 0006-291X/92 $4.00 465
Copyright © 1992 by Academic Press, lnc. All rights of reproduction in any form reserved.
Vol. 185, No. 1, 1992
Similarly,
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
changes
in the surface unesterified cholesterol by
LDL treatment with cholesterol oxidase the LDL coat phospholipids, phospholipases
(5-8)
resulted
PLase
and was taken phagocytic
its
incubation
with
in the formation of m o d i f i e d
lipoproteins which d e m o n s t r a t e d macrophages.
(4) or p e r t u r b a t i o n of
following
increased
uptake
by
C-modified LDL was shown to be a g g r e g a t e d
up
by
process
macrophages that
was
at
enhanced
rate
by
a
mediated via the LDL receptor
(5,6). PLase D - m o d i f i e d LDL was not aggregated and demonstrated
increased uptake
by
macrophages
receptor m e d i a t e d endocytosis
(8).
PLase
and
A2,
unlike
phosphoryl group, position cause
of
the
in
LDL
lipoprotein
nonspecifically
physicochemical
the
LDL
the
cells
lipoproteins
also
other
and
since
(9-11)
is distinct
fibroblasts
(12),
we
the
LDL
A2
was
(7). shown
In to
d e m o n s t r a t e d very (7).
Since
possess in addition to
even
the
from the classical in
sn-2
characterization
receptors
analyzed
the
was shown to
fibroblasts
the
cellular uptake of PLase A2-modified LDL by macrophages,
at
PLase
and
unlike human fibroblasts,
receptor,
macrophages
with
PLase A2-modified LDL
limited cellular d e g r a d a t i o n by macrophages,
acids
phospholipid,
incubation
to
an
PLase D, do not attack the fatty
diacylglycerol
human skin fibroblasts, bind
C
but rather the
changes
following
PLase
via
for LDL LDL
modified receptor on
receptor
present J-774
on
study the A.I
a cell line which was shown to possess all these
various receptors
for lipoproteins
(11,12).
METHODS Cells. J-774 A.I murine macrophage-like
cell line was p u r c h a s e d
from the A m e r i c a n Tissue Culture Collection plated
at
(ATCC). Cells were
2.5xi0 s cells/16mm dish in DMEM supplemented with
10% fetal calf serum
(FCS). The cells were fed
every
3
days
and were used for experiments within 7 days of plating. Lipoproteins.
LDL
was prepared from human plasma derived from
fasted n o r m o l i p i d e m i c
volunteers.
LDL was prepared by density
gradient u l t r a c e n t r i f u g a t i o n
as described previously(13).
LDL
was iodinated by the method
of
for
lipopoproteins of
Lowry
et
(14). LDL al.
McFarlane
as
modified
protein was measured by the method
(15). Vitamin
466
E
and
carotenoids
were
Vol. 185, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
determined as p r e v i o u s l y described
(16) and the
fatty
acids
d i s t r i b u t i o n was analyzed by gas liquid chromatography Phospholipolysis. venom
was used
of protein/ml)
Phospholipase
150mM NaCI,pH=8)
prepared
by
the
10
min
at
for
lh
at
37°C. The
from excess enzyme by
minicolumn
Iodine
methanol
(4). For
solution
(2:1,v:v).
TLC plates.
on
subclasses
(TLC) on silica
chloroform:
(60:35:8,v:v:v).
were used to visualize the lipid spots on the
The
appropriate
spots
ananlyzed for their phosphourous
were
content
C u l l u l a r uptake of lipoproteins.
J-774A.I
macrophages
then
scraped
and
(18).
Degradation of 12sI-
LDL or PLase A - 1 2 S I - L D L was measured with
Phospholipid
using a d e v e l o p i n g solution of
a m m o n i u m hydroxide
The
phospholipid
by thin layer chromatography
vapors
was
passage
lipid extracts of LDL were prepared using
separated
PLase
reaction
analysis,
methanol:
(Img
addition of ImM EDTA and refrigeration.
G-100
gel plates,
LDL
37°C.
Sephadex
were
bee
by incubation of the LDL solution with
modified LDL was separated
chloroform:
(17).
from
in the presence of 3mM CaCI2
free albumin,for
5 units/ml of PLase A2 stopped
A2)
was incubated with an equal volume of a buffer
(180 mM Tris,
was
(PLase
(Boehringer Mannheim GmBH, Germany).
and 0.6% fatty-acid A2-LDL
A2
following their incubation
for 5h at 37°C. The hydrolysis of
the LDL protein was assayed in the incubation medium Cellular c h o l e s t e r o l
esterification
previously d e s c r i b e d
(4).
rates
were
(19)o
measured
as
RESULTS Upon
incubation
units/ml) the
of LDL
(Img of protein/ml)
for I hours at 37°C,
modified
lipoportein
followed
(by
by
with PLase A2 reseparation
ultracentrifugation),
lysolechitine
content
protein
The electrophoretic mobility of
on
(n=3).
cellulose
acetate
native
LDL
PLase
Am-LDL
was increased and it migrated
14±3 mm
and
6±2%
to
32±4%
to migration of 9±2 mm o b t a i n e d
(p
