Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3248-3254
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3248-3254.)
© 1997 American Heart Association, Inc.
Evidence That Human Fc
Receptor IIA (CD32) Subtypes Are Not Receptors for Oxidized LDL
Peter M. Morganelli;
Debra S. Groveman;
;
Jason R. Pfeiffer
From the Veterans Administration Hospital in White River Junction, Vt,
and the Department of Microbiology at Dartmouth Medical School, Lebanon, NH.
Correspondence to Peter Morganelli, PhD, Veterans Administration Hospital, Research 151, White River Junction, VT 05009. E-mail Peter.Morganelli{at}Dartmouth.edu
 |
Abstract
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Abstract Several lines of evidence suggest that clearance of
oxidized
LDL (oxLDL) immune complexes by macrophage IgG Fc
receptors
(Fc

Rs) plays a role in atherogenesis. OxLDL may also be
cleared
directly by Fc

Rs, as shown for murine Fc

RII-B2. In
humans, the
homologous Fc

R is Fc

RIIA (CD32), which is abundantly
expressed
on monocytes and macrophages and shares 60% sequence
identity
with murine Fc

RII-B2. As murine Fc

RII-B2 and human
Fc

RIIA also
share similar IgG ligand-binding properties, the purpose
of
this study was to test the hypothesis that human CD32 is a receptor
for
oxLDL. For these studies we used transfected Chinese hamster
ovary
(CHO) cells, monocytes, and cell lines that functionally
express either
of two Fc

RIIA subtypes (R131 or H131) and assayed
binding or
degradation of several preparations of oxLDL. The
integrity of all
oxLDL preparations was checked by studying
their ability to react with
CHO cells expressing human type
I scavenger receptors and by other
characteristics of lipoprotein
oxidation. Although we showed that each
preparation of oxLDL
could recognize class A or class B scavenger
receptors, we did
not detect any differences in the binding or
degradation of
any type of oxLDL preparation among control versus CHO
cell
transfectants. Using monocytes that express Fc

RIIA and CD36,
we
showed that the binding of oxLDL was inhibited by antibodies
to CD36,
but not by Fc

RIIA antibodies. Thus, the data do not
support the
hypothesis that human Fc

RIIA is by itself a receptor
for oxLDL. We
conclude that human CD32 can mediate uptake of
lipoprotein immune
complexes, but does not mediate uptake of
oxLDL in the absence of
anti-oxLDL antibodies. OxLDL may interact
with human mononuclear
phagocytes directly via other types of
receptors, such as class A and
class B scavenger receptors or
CD68.
Key Words: Fc receptors scavenger receptors CD36 oxidized LDL
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Introduction
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Much
evidence suggests that the recognition of modified LDLs
by
macrophage scavenger receptors is an important event in foam
cell
development and other aspects of
atherogenesis.
1,2 To date,
at least three classes
of scavenger receptors have been described.
310
The
type I and type II scavenger receptors (SR-AI and SR-AII) cloned
by
Kodama and associates
3,4 mediate the endocytic
uptake and
degradation of LDL made more electronegative by
acetylation
or malondialdehyde conjugation or by oxidation
in the presence
of copper or iron.
1,2,11 In
addition to class A scavenger receptors,
class B scavenger receptors
have been described (CD36 and SR-BI)
with ligand-binding properties
that are quite distinct from
those of class A
receptors.
5,6 For example, CD36 may bind either
copper-oxidized
LDL,
57
acLDL,
6 or polyanionic
phospholipids.
8 In the case
of human CD36, this
binding is not inhibitable by class A polyanionic
ligands such as
fucoidin.
6 In addition to these newly defined
classes
of scavenger receptors, other molecules capable of binding
oxLDL
have also been identified, such as murine
macrosialin
12 and
its human homolog
CD68,
13 as well as
Fc

Rs.
14 In the case of
Fc

Rs, Stanton et
al
14 provided evidence that oxLDL is recognized
by
murine Fc

RII-B2. Using transfected cells, they demonstrated
that
Fc

RII-B2 bound copper-oxidized LDL in a saturable manner
and that
uptake was inhibited by mAb 2.4G2, which blocks the
IgG ligand-binding
site of this receptor. However, the physiological
relevance
of the recognition of oxLDL by Fc

RII-B2 is uncertain as
mAb
2.4G2 failed to inhibit binding or uptake of oxLDL or oxidized
red
blood cells by murine
macrophages.
5,15,16
Fc
RII-B2 is expressed predominantly on macrophages and binds
monomeric IgG poorly and polyvalent IgG immune complexes with high
affinity.17 Fc
RII-B2 shares approximately 60%
amino acid sequence homology with its human counterpart, Fc
RIIA
(CD32). Fc
RIIA is also expressed on mononuclear phagocytes and like
Fc
RII-B2 recognizes IgG immune complexes with high affinity while
reacting poorly with monomeric IgG.1719 Because
of these similarities, there may be a tendency to presume that Fc
Rs
in general, and human Fc
Rs in particular, can function as oxLDL
receptors. We therefore wanted to test the hypothesis that human
Fc
RIIA is a receptor for oxLDL. There are two allelic subtypes of
Fc
RIIA that differ by amino acid substitutions at positions 27 and
131.1921 Receptors that contain histidine at
position 131 are able to bind human IgG2 immune
complexes, while those that possess arginine at that position are
not.20 The important point is that the
ligand-binding properties of these subtypes are clearly unique, at
least with respect to the binding of IgG immune complexes.
Human mononuclear phagocytes express at least three molecules that may
bind oxLDL4,5,6,13 as well as significant amounts
of one or both subtypes of Fc
RIIA.18,19 To
determine whether Fc
RIIA does indeed possess the ability to react to
oxLDL, we studied the interactions of several different forms of oxLDL
with CHO cells transfected with R131 or H131 subtypes, as well as with
normal cells and cell lines expressing different subtypes. In previous
studies, similarly prepared oxLDLs were shown to react strongly to type
I scavenger receptors22, as well as to the newer
classes discussed above.57,13,14 Although the
ligands that we prepared were able to react to human class A scavenger
receptors also expressed in CHO cells or to CD36, we did not obtain any
evidence that these ligands could react with either subtype of
Fc
RIIA.
 |
Methods
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Materials
Anti-Fc

RII mAb IV.3 (murine IgG
2b) was
obtained from Medarex,
Inc. Anti-Fc

RII mAb 41H16
(IgG
2a) was obtained from Dr Paul
Guyre,
Dartmouth Medical School, Lebanon, NH. Anti-CD36 mAbs
clone 89
(IgG
2b) and OKM5 (IgG
1)
were obtained from Serotec
and Ortho Diagnostics,
respectively. Human SR-AI cDNA in vector
CMV
4 was
obtained from Dr Todd Kodama, University of Tokyo,
Tokyo, Japan. Human
Fc

RIIA R131 and H131 cDNAs in vector pcDX
20
were
obtained from Dr Jan van de Winkel, University of
Utrecht, the
Netherlands. Defined FBS was obtained from
Hyclone. Na
125I was
obtained from Amersham. All
chemicals used were of standard
reagent grade and obtained from Fisher
Scientific unless otherwise
indicated.
Lipoproteins
nLDL (density 1.019 to 1.063 g/ml) was prepared and
labeled with 125I by the McFarlane method as
described23 and used within 2 weeks. The specific
activity of five preparations averaged 422±141 cpm/ng of protein and
had undetectable TBARS; <2% was trichloroacetic acid-soluble for each
preparation. To prepare oxLDL, the LDL was first passed through a
Bio-Rad P6-DG column to remove EDTA and adjusted to 0.2 mg/ml
protein in Ca2+- and
Mg2+-free PBS. Copper-oxidized LDL was incubated
at 37°C for the times indicated with 5 µmol/L copper
sulfate; after the oxidation period EDTA and butylated hydroxytoluene
were added to achieve final concentrations of 0.01 mol/L and
20 µmol/L, respectively. Preparations of copper-oxidized
LDL were either dialyzed into 0.15 mol/L NaCl containing
0.01 mmol/L EDTA, pH 7.4, before use or in some cases were
used without dialysis. All preparations of oxLDL were assayed for the
presence of TBARS before dialysis according to a standard fluorimetric
method24 using malonaldehyde bis(dimethyl acetal)
(Aldrich) as the standard. Cell-modified LDL was prepared by
incubating 10 mL of 0.1 mg/ml LDL protein in Ham's F10 medium
in 100-mm tissue culture dishes containing confluent monolayers of
14-day cultured human monocyte-derived macrophages (see below)
for 20 hours at 37°C, conditions similar to the production of
endothelial cell-modified LDL.24
Peroxide/peroxidase-modified LDL was prepared by incubating LDL with
H2O2 and horseradish
peroxidase as described.25 For both
peroxide/peroxidase-modified LDL and cell-modified LDL, 0.01
mmol/L EDTA and 20 µmol/L butylated hydroxytoluene
were added after the oxidation period, and each preparation was used
without dialysis. acLDL was prepared by treatment with acetic anhydride
(Sigma) as described.26 For cold competition
experiments, unlabeled preparations were concentrated with
Centriprep-50 concentrators (Amicon). All lipoprotein preparations were
filter-sterilized through 0.2-µm filter units, stored at 4°C, and
used within 1 week of oxidation. REM of lipoproteins was determined by
running 1 to 5 µg of protein per lane in 1% agarose gels prepared in
50 mmol/L barbital buffer, pH 8.6, for 1 hour at 90 V.
Bands were visualized by staining with Sudan black. Protein
concentrations were determined by modified Lowry assay as
described.27 For the characteristics of all
lipoprotein preparations used in these studies, two preparations of
4-hour 125I-copper-oxidized LDL had 67±18 nmol
malondialdehyde equivalents per mg of protein (mean±range) and a REM
of 1.4; six preparations of 20-hour
125I-copper-oxidized LDL had 75±22 nmol
malondialdehyde equivalents per mg of protein (mean±SD) and a REM of
2.2±0.1; three preparations of cell-modified LDL had 70±14 nmol
malondialdehyde equivalents per mg of protein and a REM of 2.3±0.5;
five preparations of acLDL had a REM of 2.9±0.2; and one preparation
of 125I-peroxide/peroxidase-modified LDL had a
REM of 2.6.
Production of CHO Cell Transfectants
CHO-K1 cells (American Type Culture Collection) were grown in
Ham's F12 medium plus 10% FBS. SR-AI cDNA4 in
vector CMV was transfected into subconfluent CHO cells grown in 100-mm
tissue culture dishes with Lipofectin (Gibco) according to the
manufacturer's instructions; Fc
RIIA R131 and H131 cDNAs in vector
pcDX20 were cotransfected with vector
pcDNA3 (Invitrogen). For controls, CHO cells were
also transfected with CMV or pcDNA3 empty
vectors. In the case of SR-AI transfectants, cells that arose after 2
weeks in the presence of 0.5 mg/ml Geneticin (Gibco) were
harvested by treatment with Hanks' balanced salt solution plus 1
mmol/L EDTA for 20 minutes at 37°C, then passed and allowed to
grow to confluency in 75-mm2 culture flasks. To
obtain transfectants expressing scavenger receptor activity,
transfected cells were treated for 24 hours at 37°C with 5
µg/ml of DiI-labeled acLDL (Molecular Probes, Inc). After 24
hours, the cells were washed, harvested as above, and then resuspended
in complete medium. Cell-associated red fluorescence (emission
wavelength 630 nm) was analyzed by a FACStar Plus flow
cytometer (Becton Dickinson). The brightest 0.1% were sorted into a
culture dish and grown to confluency. The sorting step was then
repeated, and cells were grown to confluency a second time before being
subcloned by limiting dilution. Resulting subclones were checked for
scavenger receptor activity as described below. In the case of
Fc
RIIA transfectants, cells were stained as
described28 with anti-Fc
RIIA mAb IV.3 (which
recognizes equally both the R131 and H131 subtypes). The brightest
0.1% were sorted, grown to confluency, and then subcloned by limiting
dilution. Resulting subclones were checked for expression of each
subtype by staining with anti-Fc
RIIA mAbs IV.3 and 41H16.
Quantitation of mAb binding was determined as
described28 and is expressed as the amount of
second antibody molecules bound per cell.
Cells and Cell Lines
Suspensions of human monocytes of >90% purity were prepared
from peripheral blood drawn from healthy donors as
described.28 Cells were seeded in 100-mm Corning
tissue culture dishes (15 mL at 2x106/ml) in
serum-free DMEM containing 5x10-5 mol/L
ß-mercaptoethanol, 15 mmol/L HEPES buffer, pH 7.4, and 50
µg/ml gentamicin. After 1 hour of culture at 37°C and 10%
CO2, 1.5 mL of heat-inactivated
pooled human serum (prepared locally) was added per dish. The medium
was replaced after 1 week, and cells were used after 14 days of
culture. For lipoprotein-binding assays (see below), cells were washed
in PBS and then detached by treatment with a freshly-prepared solution
of PBS containing 15 mmol/L lidocaine-HCl (Sigma) plus
5 mmol/L EDTA, pH 7.4, for 20 minutes at
37°C.29 The cells were washed and then
resuspended in DMEM containing 10% FBS or 5 mg/ml
lipoprotein-deficient FBS. Viability as assessed by Trypan blue
exclusion was always >90%. K562 and U937 cells were obtained from
American Type Culture Collection and maintained in suspension culture
in RPMI-1640 supplemented with 10% FBS and gentamicin.
Lipoprotein Binding and Degradation Assays
All binding assays with transfected cells or macrophages
were done by incubating 1 to 1.5 million cells in DMEM containing 10%
FBS or 5 mg/ml lipoprotein-deficient FBS with the indicated
concentrations of ligand in Eppendorf tubes or 96-well plates with
gentle shaking for 4 hours at 4°C. After 4 hours, the suspensions
were immediately layered on top of 200 µL of 9.4% dextran sulfate
prepared in PBS and spun in a microfuge for 1 minute. Cell pellets were
removed and cell-bound radioactivity was quantitated by gamma
spectroscopy. For lipoprotein degradation studies, ligands were
prepared in DMEM containing 10% FBS and 15 mmol/L HEPES,
pH 7.4, and added to adherent cells in 12- or 24-well plates or to
cells in suspension in Eppendorf tubes. After 4 hours at 37°C,
supernatants were assayed for the presence of acid-soluble products
that did not contain free iodide.30 All
treatments were corrected for background by subtracting the amount of
degradation obtained in the absence of cells. Bispecific antibody
targeting experiments were done as
described.31
 |
Results
|
|---|
Expression of Fc
RIIA Subtypes on CHO Cells
CHO cell transfectants expressing either the R131 or H131 subtype
of
human Fc

RIIA were prepared to study the ability of these
receptors
to interact with oxLDL independently of other
macrophage cell
surface molecules. The expression of each
Fc

RIIA subtype on
stable CHO cell transfectants is shown in Fig 1

. Cell surface
expression was measured
by quantitative flow cytometry using
anti-Fc

RIIA mAbs IV.3 and
41H16. Monoclonal antibody IV.3 recognizes
both the R131 and H131
subtypes of Fc

RIIA while mAb 41H16 recognizes
only the R131
subtype.
21 As shown in Fig 1A

, each Fc

RIIA
transfectant
expressed >500 000 antibody binding sites per cell,
which
is on average greater than 10 times normal expression of these
receptors
on monocytes.
18,19 As expected, there
was essentially no reactivity
of mAb 41H16 to CHO cells transfected
with the H131 subtype
of Fc

RIIA, while the reactivity to cells
expressing the R131
subtype was the same as that of mAb IV.3. There was
no reactivity
of both mAbs to CHO cells transfected with the empty
vector
(not shown). Thus, these data demonstrate the unique expression
of
each Fc

RIIA subtype on stable CHO cell transfectants.
To confirm functional Fc receptor activity, CHO cell transfectants were
treated with 125I-nLDL in the presence of
bispecific antibodies consisting of anti-Fc
RIIA mAb IV.3 conjugated
to an anti-nLDL mAb. In previous studies we used bispecific antibodies
to study the effects of targeting LDL to Fc
Rs on human
monocytes.31 Those studies revealed that
bispecific antibodies were effective in triggering
metabolic uptake and degradation of LDL in the context of
specific Fc
Rs. Fig 1B
shows the results of targeting
125I-nLDL to control transfectants as well as to
transfectants expressing the R131 or H131 subtype of Fc
RIIA. In the
absence of bispecific antibodies, the degradation of
125I-nLDL was minimal and the same for both
control and Fc
RIIA transfectants. The same amount of degradation was
seen with control transfectants in the presence of the bispecific
antibody. However, the amount of nLDL degradation for Fc
RIIA
transfectants expressing the R131 or H131 subtype of Fc
RIIA was
enhanced >6-fold in the presence of the Fc
RIIA bispecific antibody.
These results demonstrate that the transfected receptors, like their
macrophage counterparts, can trigger LDL uptake and degradation
in response to receptor cross-linking, ie, that the Fc
RIIA
transfectants express functional Fc receptors.
SR-AI Activity in CHO Cell Transfectants
As a model cell line expressing class A scavenger receptor
activity, we prepared CHO cell transfectants expressing human SR-AI.
The binding and degradation of 125I-acLDL or
125I-oxidized LDL for SR-AI transfectants
demonstrated saturation kinetics, where maximum binding and degradation
occurred by 20 µg/ml for each lipoprotein (not shown). Another
important feature of ligand reactivity to SR-AI is the phenomenon of
nonreciprocal cross competition.1,22 This feature
of our SR-AI transfectants is shown in the
Table
. The results demonstrate the same
type of nonreciprocal cross competition observed for SR-AI by
others.22 Thus, the degradation of
125I-acLDL was completely inhibited by excess
unlabeled acLDL, but only partially inhibited by an 80-fold excess of
unlabeled oxLDL. Conversely, the degradation of
125I-oxidized LDL was completely inhibited by
each unlabeled ligand. As expected, there was no effect of excess nLDL
on the degradation of either 125I-oxLDL or
125I-acLDL. These transfectants were used
throughout the course of this study as an additional measure of LDL
modification for each radiolabeled lipoprotein preparation. Each
125I-labeled preparation of oxLDL used was shown
to undergo scavenger receptor activity in these transfectants.
Reactivity of Copper-Oxidized LDL to Human Fc
RIIA
Subtypes
To test the hypothesis that human Fc
RIIA is a receptor for
oxLDL, several experiments were performed where the interactions of
oxLDL prepared by a variety of methods were studied with CHO cell
transfectants at both 4 and 37°C. Fig 2A
shows a typical experiment where
control, SR-AI, and Fc
RIIA transfectants were treated with varying
amounts of 20-hour 125I-copper-oxidized LDL for 4
hours at 37°C followed by assay of oxLDL degradation. In the case of
the SR-AI transfectant, there was a significant amount of lipoprotein
degradation that tended to reach a plateau by 20 µg/ml of
ligand. However, for each of the other transfectants, the amount of
lipoprotein degradation was severalfold less than that seen for the
SR-AI transfectant and was linear up to 40 µg/ml of ligand. As
shown in Fig 2
, there was no difference in degradation between the
control and Fc
RIIA transfectants. Similar results were also obtained
with 4-hour 125I-copper-oxidized LDL, as well as
in 4°C binding assays with the same preparations of ligands (not
shown). The important point is that in each of these experiments the
amount of oxLDL binding or degradation associated with the Fc
RIIA
transfectants did not exceed that of the control
(pcDNA3 empty vector) transfectants.
Reactivity of Other OxLDLs to Human Fc
RIIA Subtypes
To determine if other oxLDL preparations could react to Fc
RIIA,
both macrophage-modified LDL and peroxide/peroxidase-modified
LDL preparations were also studied. Macrophage-modified LDL was
prepared by incubating 125I-nLDL in serum-free
Ham's F10 medium with confluent monolayers of human monocyte-derived
macrophages as outlined in "Methods." The ability of this
preparation to undergo degradation via SR-AI is shown in Fig 2B
. As
shown in the figure, 125I-nLDL incubated in
Ham's F10 medium in the absence of cells did not undergo degradation
by SR-AI transfectants, while 125I-nLDL incubated
in Ham's F10 medium in the presence of cells underwent significant
degradation. The amount of degradation seen exceeded that of 20-hour
copper-oxidized LDL, which as shown also reacted significantly. The
same result was obtained with peroxide/peroxidase-modified LDL. In this
case, the amount of degradation seen with SR-AI transfectants also
exceeded that of 20-hour copper-oxidized LDL (not shown). The results,
however, were different in the case of the Fc
RIIA transfectants. As
was seen with copper-oxidized LDL, these different LDL modifications
also failed to show more than minimal reactivity to each subtype of
Fc
RIIA, and in no case was reactivity greater than that seen with
the control transfectant. The same results were obtained in two other
similar experiments. Thus, these different preparations of oxLDL, each
of which displayed SR-AI activity, also failed to react to R131 and
H131 Fc
RIIA subtypes.
Reactivity of OxLDL to CD36
All preparations of ligands used to this point demonstrated SR-AI
reactivity. Based on the results of others,57
these preparations would also be expected to react to type B scavenger
receptors. To show this with our ligands, we studied the 4°C binding
of 20-hour 125I-copper-oxidized LDL to freshly
prepared human monocytes in the presence and absence of anti-Fc
RIIA
mAb IV.3 or anti-CD36 mAb OKM5 (Fig 3
).
As determined by flow cytometric analysis, the monocytes from
this particular donor expressed both the R131 and H131 subtypes of
Fc
RIIA (for a total of >50 000 antibody-binding sites per cell)
and >100 000 binding sites per cell of CD36. As shown in Fig 3
, the
binding of 125I oxLDL was inhibited approximately
60% in the presence of 25-fold excess unlabeled oxLDL. This level of
binding (
20 ng/mg protein bound in the presence of unlabeled
ligand) represents a minimal amount due mostly to nonspecific
binding, as even greater amounts of unlabeled ligand (up to 400
µg/ml) typically did not decrease binding any further in these
cells and CHO cell transfectants as well (not shown). There was no
inhibition of binding in the presence of excess acLDL (which has been
reported to bind to CD36 in transfected cells).6
125I-oxLDL binding was also inhibited by 60% in
the presence of anti-CD36 mAb OKM5, but not with saturating amounts of
mAb IV.3, which blocks the IgG ligand-binding site of
Fc
RIIA.32 The same results were obtained in
another experiment with cells from a different donor; again, mAb IV.3
had no effect on 125I-oxLDL binding while mAb
OKM5 inhibited binding by 60% (not shown). These data are
consistent with the interpretation that a preparation of oxLDL
with class B scavenger receptor reactivity did not react to Fc
RIIA
subtypes.

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Figure 3. Histogram of the effects of unlabeled lipoproteins
or monoclonal antibodies on the binding of copper-oxidized LDL to
freshly prepared human monocytes. Human monocytes were treated with 20
µg/ml of 20-hour 125I-copper-oxidized LDL for 2 hours at
4°C followed by assay of oxLDL binding as outlined in "Methods,"
in the presence of 250 µg/ml of unlabeled oxLDL, 500 µg/ml of
unlabeled acLDL, 5 µg/ml of anti-CD36 mAb OKM5, or 5 µg/ml of
anti-Fc RIIA mAb IV.3. Shown are the mean±SD of quintuplicate
measurements.
|
|
 |
Discussion
|
|---|
Recent evidence suggests that the clearance of lipoprotein immune
complexes
through macrophage Fc

Rs plays a role in
atherogenesis.
3337 Human mononuclear phagocytes
may express three functionally
distinct classes of human
Fc

Rs,
18,19 making it difficult to
understand
the extent to which each class (and their isoforms
and subtypes)
participates in immune complex clearance. In previous
experiments we
prepared native or aggregated LDL immune complexes
with bispecific
anti-Fc

R x anti-LDL antibodies to study lipoprotein
metabolism
in the context of each specific type of
Fc

R.
31,38 As we have
begun to address the
question of oxLDL immune complex metabolism,
the
possibility that macrophage Fc

Rs could react directly with
oxLDL
in the absence of antibodies posed an additional variable
that
needed to be addressed. Such a result would also have implications
for
antigen presentation and the immune response to oxLDL
and whether
this response is proatherogenic or antiatherogenic. As
discussed
above, the Fc

RIIA subtypes are low-affinity IgG receptors
that
share significant amino acid sequence homology with murine
Fc

RII-B2,
17 a receptor shown to recognize
copper-oxidized LDL when expressed
on transfected
cells.
14 Several different preparations of oxLDL
were
tested in 4 and 37°C binding or degradation assays,
respectively,
using CHO cells expressing high amounts of either the
R131 or
H131 subtype of human Fc

RIIA (Fig 1

). To ensure ligand
integrity,
each preparation of ligand was characterized before use for
the
presence of TBARS, for REM (see "Methods"), and for the ability
to
undergo degradation in CHO cells expressing human SR-AI. The
types
of preparations used included 4- and 20-hour copper-oxidized
LDL, human
macrophage-modified LDL, and peroxide/peroxidase-modified
LDL.
These preparations were similar with respect to TBARS,
but less so with
respect to REM and (as expected) ability to
react to SR-AI. Despite the
fact that all preparations were
significantly oxidized and reacted
specifically to SR-AI, their
reactivity to CHO cells expressing either
of the Fc

RIIA subtypes
was not different from that of the control
CHO cell transfectants
(Fig 2

). Also, the binding of copper-oxidized
LDL to cells expressing
both CD36 and Fc

RIIA was significantly
inhibited by anti-CD36
mAb (Fig 3

), but not by an anti-Fc

RIIA mAb
that blocks the IgG
ligand-binding site, also consistent with a
lack of Fc

RIIA reactivity.
Taken together, the data do not support the hypothesis that human
Fc
RIIA is a receptor for oxLDL. This contrasts with the results of
Stanton et al,14 who reported that murine
Fc
RII-B2 is a receptor for copper-oxidized LDL. As discussed above,
murine Fc
RII-B2 and human Fc
RIIA share 60% amino acid sequence
homology in the extracellular domain.17 We
therefore expected that human Fc
RIIA allelic subtypes would also
possess the ability to bind oxLDL and questioned whether there might be
a relationship between the presence of either subtype and
cardiovascular disease in patients. While our results
were unexpected, there are several reasons to explain the discrepancy
with the results reported by Stanton et al.14
These include the facts (1) that the inability of human Fc
RIIA to
bind oxLDL is due to sequence differences between the two receptors,
(2) that there may have been significant differences in the
characteristics of our oxLDL preparations, (3) that differences in the
level of Fc receptor expression may be related to their ability to bind
oxLDL, and (4) that coreceptor molecules not expressed on CHO cells are
needed for the ability of Fc
RIIA to bind oxLDL (in the case of CHO
cell transfectants).
To address the first point, although it is not known which sequences of
murine Fc
RII-B2 are involved in oxLDL binding, they are probably
those that make up the IgG ligand-binding site, since mAb 2.4G2, which
blocks the IgG ligand-binding site of Fc
RII-B2, blocks oxLDL uptake
in transfected cells expressing Fc
RII-B2.14
Whereas the extracellular domains of murine Fc
RII-B2 and human
Fc
RIIA are highly conserved, the ligand-binding sites are probably
different, as reflected by differences in human and murine mAb
cross-reactivity between both receptors.17 Thus,
amino acid sequence differences in the IgG ligand-binding sites between
both receptors could explain the discrepancy in oxLDL binding between
murine Fc
RII-B2 and human Fc
RIIA. Regarding the second point, a
difficulty in comparing results between different laboratories lies in
differences between lipoprotein preparations and cell models.
Peroxidative modifications of LDL result in several structural
alterations including apoprotein B fragmentation and derivatization of
lysine residues by reactive aldehydic
groups.11,39 Because there may be some
differences in the composition of oxLDL among different laboratories
(and in sequential preparations within a given laboratory), our
conclusions were based on studies using several different oxLDL
preparations. These included two or more preparations of mildly and
extensively oxLDL labeled with 125I as well as
two preparations of 20-hour copper-oxidized LDL labeled with
fluorescein isothiocyanate (used for assay of
cell-associated oxLDL by flow cytometry, not shown). While these types
of oxidative modifications result in the generation of unique
epitopes,25,39 none of these ligands showed
evidence of specific reactivity to Fc
RIIA expressed on CHO cells or
human monocytes (Figs 2
and 3
). In the case of monocytes, oxLDL binding
was significantly inhibited by anti-CD36 mAb OKM5, but not by
anti-Fc
RIIA mAb IV.3 (Fig 3
). We also studied K562 cells, and U937
cells that were either high or low expressors of CD36 (data not shown).
In the case of U937 cells, the binding of 4- and 20-hour
copper-oxidized LDL paralleled the expression of CD36 and was
unaffected by the presence of anti-Fc
RI mAb
2240 or human IgG (consistent with lack
of interaction with Fc
RI), as well as anti-Fc
RIIA mAb IV.3. K562
cells (that expressed no CD36 but both Fc
RIIA subtypes) also showed
minimal reactivity to copper-oxidized LDL that was unaffected by
anti-Fc
RIIA or anti-CD36 mAbs. Thus, our data confirm that oxLDL is
able to react to CD36, but are not consistent with oxLDL
reactivity to human Fc
RIIA. Nonetheless, although there is an
apparent redundancy in the expression of molecules able to react to
oxLDL ligands prepared in vitro, the possibility remains that Fc
RIIA
could prefer a different oxidative modification of LDL, such as one
that more closely mimics the oxidative modifications that occur
in vivo. With regard to the third point that differences in the level
of Fc receptor expression may be related to their ability to bind
oxLDL, the monocytes and cell lines studied provided a wide range in
Fc
RIIA expression (between 40 000 and >500 000 receptors per
cell). And lastly, our data suggest that coreceptor molecules are not
needed for oxLDL binding to Fc
RIIA because binding of oxLDL to
monocytes that expressed both high amounts of CD36 and Fc
RIIA was
completely accounted for in terms of reactivity to CD36 (Fig 3
).
In summary, the evidence obtained in this study did not support the
hypothesis that human Fc
RIIA is a receptor for oxLDL. These
conclusions were based on the results of studies of the reactivity of
several different types of oxLDL preparations with several different
cell types expressing one or both subtypes of human Fc
RIIA. The
clearance of oxLDL by human mononuclear phagocytes may therefore be
affected by type A and type B scavenger receptors, by CD68, and by Fc
receptors in the context of oxLDL immune complexes. In the absence of
anti-oxLDL antibodies, Fc
RIIA appears not to be a major factor in
oxLDL uptake by human mononuclear phagocytes.
 |
Selected Abbreviations and Acronyms
|
|---|
| acLDL |
= |
acetylated LDL |
| CHO |
= |
Chinese hamster ovary |
| DMEM |
= |
Dulbecco's modified Eagle's medium |
| FBS |
= |
fetal bovine serum |
Fc R |
= |
IgG Fc receptors |
| mAb |
= |
monoclonal antibody |
| nLDL |
= |
native LDL |
| oxLDL |
= |
oxidized LDL |
| PBS |
= |
phosphate-buffered saline |
| REM |
= |
relative electrophoretic mobility |
| TBARS |
= |
thiobarbituric acid-reactive substances |
|
 |
Acknowledgments
|
|---|
This work was supported by a grant from the United States
Department
of Veterans Affairs (Merit Review) and by National
Institutes
of Health Grant AG-14405-01.
Received June 25, 1997;
accepted August 7, 1997.
 |
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