© 1997 American Heart Association, Inc.
Articles |
Evidence That Human Fc
Receptor IIA (CD32) Subtypes Are Not Receptors for Oxidized LDL
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
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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 FcRs, as shown for murine FcRII-B2. In
humans, the homologous FcR is FcRIIA (CD32), which is abundantly
expressed on monocytes and macrophages and shares 60% sequence
identity with murine FcRII-B2. As murine FcRII-B2 and human
FcRIIA 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 FcRIIA 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 FcRIIA and CD36, we
showed that the binding of oxLDL was inhibited by antibodies to CD36,
but not by FcRIIA antibodies. Thus, the data do not support the
hypothesis that human FcRIIA 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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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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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.3–10 The type I and type II scavenger receptors (SR-AI and SR-AII) cloned by Kodama and associates3,4 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,5–7 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 macrosialin12 and its human homolog CD68,13 as well as Fc
Rs.14 In the case of FcRs, Stanton et
al14 provided evidence that oxLDL is recognized
by murine FcRII-B2. Using transfected cells, they demonstrated that
FcRII-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 FcRII-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
FcRII-B2 is expressed predominantly on macrophages and binds
monomeric IgG poorly and polyvalent IgG immune complexes with high
affinity.17 FcRII-B2 shares approximately 60%
amino acid sequence homology with its human counterpart, FcRIIA
(CD32). FcRIIA is also expressed on mononuclear phagocytes and like
FcRII-B2 recognizes IgG immune complexes with high affinity while
reacting poorly with monomeric IgG.17–19 Because
of these similarities, there may be a tendency to presume that FcRs
in general, and human FcRs in particular, can function as oxLDL
receptors. We therefore wanted to test the hypothesis that human
FcRIIA is a receptor for oxLDL. There are two allelic subtypes of
FcRIIA that differ by amino acid substitutions at positions 27 and
131.19–21 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 FcRIIA.18,19 To
determine whether FcRIIA 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.5–7,13,14 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
FcRIIA.
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Methods |
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Materials
Anti-Fc
RII mAb IV.3 (murine IgG2b) was
obtained from Medarex, Inc. Anti-FcRII mAb 41H16
(IgG2a) was obtained from Dr Paul Guyre,
Dartmouth Medical School, Lebanon, NH. Anti-CD36 mAbs clone 89
(IgG2b) and OKM5 (IgG1)
were obtained from Serotec and Ortho Diagnostics,
respectively. Human SR-AI cDNA in vector CMV4 was
obtained from Dr Todd Kodama, University of Tokyo, Tokyo, Japan. Human
FcRIIA R131 and H131 cDNAs in vector pcDX20
were obtained from Dr Jan van de Winkel, University of
Utrecht, the Netherlands. Defined FBS was obtained from
Hyclone. Na125I 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; FcRIIA 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
FcRIIA transfectants, cells were stained as
described28 with anti-FcRIIA 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-FcRIIA 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
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Results |
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Expression of Fc
RIIA Subtypes on CHO CellsCHO 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
FcRIIA subtype on stable CHO cell transfectants is shown in Fig 1
. Cell surface expression was measured
by quantitative flow cytometry using anti-FcRIIA mAbs IV.3 and
41H16. Monoclonal antibody IV.3 recognizes both the R131 and H131
subtypes of FcRIIA while mAb 41H16 recognizes only the R131
subtype.21 As shown in Fig 1A, each FcRIIA
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 FcRIIA, 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 FcRIIA subtype on stable CHO cell transfectants.
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To confirm functional Fc receptor activity, CHO cell transfectants were
treated with 125I-nLDL in the presence of
bispecific antibodies consisting of anti-FcRIIA mAb IV.3 conjugated
to an anti-nLDL mAb. In previous studies we used bispecific antibodies
to study the effects of targeting LDL to FcRs 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 FcRs. Fig 1B
shows the results of targeting
125I-nLDL to control transfectants as well as to
transfectants expressing the R131 or H131 subtype of FcRIIA. In the
absence of bispecific antibodies, the degradation of
125I-nLDL was minimal and the same for both
control and FcRIIA 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 FcRIIA
transfectants expressing the R131 or H131 subtype of FcRIIA was
enhanced >6-fold in the presence of the FcRIIA 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 FcRIIA
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.
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Reactivity of Copper-Oxidized LDL to Human FcRIIA
Subtypes
To test the hypothesis that human FcRIIA 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 FcRIIA 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 FcRIIA 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 FcRIIA
transfectants did not exceed that of the control
(pcDNA3 empty vector) transfectants.
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, SR-AI transfectants;
, control pcDNA3 transfectants; 
, R131
transfectants; 
, H131 transfectants. B, Histogram of the
degradation of different preparations of 125I-oxLDL by CHO
cell transfectants. Cells were treated for 4 hours at 37°C with 30
µg/ml of each preparation followed by assay of oxLDL degradation as
outlined in "Methods." Shown are the mean±SD of triplicate
measurements.
Reactivity of Other OxLDLs to Human FcRIIA Subtypes
To determine if other oxLDL preparations could react to FcRIIA,
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 FcRIIA transfectants. As
was seen with copper-oxidized LDL, these different LDL modifications
also failed to show more than minimal reactivity to each subtype of
FcRIIA, 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 FcRIIA subtypes.
Reactivity of OxLDL to CD36
All preparations of ligands used to this point demonstrated SR-AI
reactivity. Based on the results of others,5–7
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-FcRIIA
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
FcRIIA (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
FcRIIA.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 FcRIIA
subtypes.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Recent evidence suggests that the clearance of lipoprotein immune complexes through macrophage Fc
Rs plays a role in
atherogenesis.33–37 Human mononuclear phagocytes
may express three functionally distinct classes of human
FcRs,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-FcR x anti-LDL antibodies to study lipoprotein
metabolism in the context of each specific type of
FcR.31,38 As we have begun to address the
question of oxLDL immune complex metabolism, the
possibility that macrophage FcRs 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 FcRIIA subtypes are low-affinity IgG receptors
that share significant amino acid sequence homology with murine
FcRII-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 FcRIIA (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 FcRIIA 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 FcRIIA was significantly
inhibited by anti-CD36 mAb (Fig 3), but not by an anti-FcRIIA mAb
that blocks the IgG ligand-binding site, also consistent with a
lack of FcRIIA reactivity.
Taken together, the data do not support the hypothesis that human
FcRIIA is a receptor for oxLDL. This contrasts with the results of
Stanton et al,14 who reported that murine
FcRII-B2 is a receptor for copper-oxidized LDL. As discussed above,
murine FcRII-B2 and human FcRIIA share 60% amino acid sequence
homology in the extracellular domain.17 We
therefore expected that human FcRIIA 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 FcRIIA 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 FcRIIA to bind oxLDL (in the case of CHO
cell transfectants).
To address the first point, although it is not known which sequences of
murine FcRII-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 FcRII-B2, blocks oxLDL uptake
in transfected cells expressing FcRII-B2.14
Whereas the extracellular domains of murine FcRII-B2 and human
FcRIIA 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 FcRII-B2 and human FcRIIA. 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 FcRIIA 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-FcRIIA 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-FcRI mAb
2240 or human IgG (consistent with lack
of interaction with FcRI), as well as anti-FcRIIA mAb IV.3. K562
cells (that expressed no CD36 but both FcRIIA subtypes) also showed
minimal reactivity to copper-oxidized LDL that was unaffected by
anti-FcRIIA 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 FcRIIA. 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 FcRIIA
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
FcRIIA 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 FcRIIA because binding of oxLDL to
monocytes that expressed both high amounts of CD36 and FcRIIA 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 FcRIIA 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 FcRIIA. 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, FcRIIA appears not to be a major factor in
oxLDL uptake by human mononuclear phagocytes.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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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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1. Krieger Acton A, Ashkenas J, Pearson A, Penman M, Resnick D. Molecular flypaper, host defense, and atherosclerosis: structures, binding properties, and functions of macrophage scavenger receptors. J Biol Chem. 1993;268:4569–4572.
2. Brown MS, Goldstein JL. Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Annu Rev Biochem. 1983;52:223–261.[Medline] [Order article via Infotrieve]
3.
Kodama T, Freeman M, Rohrer L, Zabrecky J, Matsudaira
P, Krieger M. Macrophage scavenger receptor contains
-helical and collagen like coiled coils. Nature. 1990;343:531–535.[Medline]
[Order article via Infotrieve]
4.
Matsumoto A, Naito M, Itakura H, Ikemoto S, Asaoka H,
Hayakawa I, Kanamori H, Aburatani H, Takaku F, Suzuki H, Kobari Y,
Miyai T, Takahashi K, Cohen EH, Wydro R, Housman DE, Kodama T. Human
macrophage scavenger receptors: primary structure, expression,
and localization in atherosclerotic lesions. Proc Natl Acad Sci
U S A.. 1990;87:9133–9137.
5.
Endemann G, Stanton LW, Madden KS, Bryant CM, White
RT, Protter AA. CD36 is a receptor for oxidized low density
lipoprotein. J Biol Chem. 1993;268:11811–11816.
6.
Acton SL, Scherer PE, Lodish HF, Krieger MK.
Expression cloning of SR-BI, a CD36-related class B scavenger receptor.
J Biol Chem. 1994;269:21003–21009.
7.
Nicholson AC, Frieda S, Pearce A, Silverstein RL.
Oxidized LDL binds to CD36 on human monocyte-derived
macrophages and transfected cell lines. Arterioscler
Thromb Vasc Biol. 1995;15:269–275.
8.
Ryeom SW, Silverstein RL, Scotto A, Sparrow JR.
Binding of anionic phospholipids to retinal pigment epithelium may be
mediated by the scavenger receptor CD36. J Biol Chem. 1996;271:20536–20539.
9. Acton S, Rigotti R, Landshultz KT, Xu S, Hobbs HH, Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science.. 1996;271:518–520.[Abstract]
10. Pearson A, Lux A, Krieger M. Expression cloning of dSR-CI, a class C macrophage-specific scavenger receptor from Drosophila melanogaster. Proc Natl Acad Sci U S A. 1995;92:4056–4060.
11. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med. 1989;320:915–924.[Medline] [Order article via Infotrieve]
12.
Ramprasad MP, Fischer W, Witztum J, Sambrano G,
Quehenberger O, Steinberg D. The 94- to 97-kDa mouse macrophage
membrane protein that recognizes oxidized low density lipoprotein and
phosphatidyl serine rich-liposomes is identical to macrosialin, the
mouse homolog of human CD68. Proc Natl Acad Sci U S A.. 1995;92:9580–9584.
13.
Ramprasad MP, Terpstra V, Kondratenko N, Quehenberger
O, Steinberg D. Cell surface expression of mouse macrosialin and human
CD68 and their role as macrophage receptors for oxidized low
density lipoprotein. Proc Natl Acad Sci U S A.. 1996;93:14833–14838.
14.
Stanton LW, White RT, Bryant CM, Protter AA, Endemann
G. A macrophage Fc receptor for IgG is also a receptor for
oxidized low density lipoprotein. J Biol Chem. 1992;267:22446–22451.
15.
Ottnad E, Parthasarathy S, Sambrano GR, Ramprasad MP,
Quehenberger O, Kondratenko N, Green S, Steinberg D. A
macrophage receptor for oxidized low density lipoprotein
distinct from the receptor for acetyl low density lipoprotein: partial
purification and role in recognition of oxidatively damaged cells.
Proc Natl Acad Sci U S A. 1995;92:1391–1395.
16.
Sambrano GR, Parthasarathy S, Steinberg D. Recognition
of oxidatively damaged erythrocytes by a macrophage receptor
with specificity for oxidized low density lipoprotein. Proc Natl
Acad Sci U S A.. 1994;91:3265–3269.
17. Hulett MD, Hogarth PM. Molecular basis of Fc receptor function. In: Dixon, FJ, ed. Advances in Immunology. San Diego, Calif: Academic Press, Inc, 1994:1–127.
18. van de Winkel JGJ, Anderson CL. Biology of human immunoglobulin G Fc receptors. J Leukocyte Biol. 1991;49:511–524.[Medline] [Order article via Infotrieve]
19. van de Winkel JGJ, Capel PJA. Human IgG Fc receptor heterogeneity: molecular aspects and clinical implications. Immunol Today. 1993;14:215–221.[Medline] [Order article via Infotrieve]
20.
Warmerdam PAM, van de Winkel JGJ, Gosselin EJ, Capel
PJA. Molecular basis of a polymorphism of human Fc receptor
II (CD32). J Exp Med. 1990;172:19–25.
21.
Gosselin EJ, Brown MF, Anderson CL, Zipf TF, Guyre PM.
The monoclonal antibody 41H16 detects the LEU 4 responder form of
FcRII. J Immunol. 1990;144:1817–1822.[Abstract]
22.
Freeman M, Ekkel Y, Rohrer L, Penman M, Freedman NJ,
Chisolm GM, Krieger M. Expression of type I and type II bovine
scavenger receptors in Chinese hamster ovary cells: lipid droplet
accumulation and nonreciprocal cross competition by acetylated
and oxidized low density lipoprotein. Proc Natl Acad Sci
U S A.. 1991;88:4931–4935.
23. Goldstein JL, Basu SK, Brown MI. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells. Methods Enzymol. 1983;98:241–260.[Medline] [Order article via Infotrieve]
24.
Steinbrecher UP, Parthasarathy S, Leake DS, Witztum JL,
Steinberg D. Modification of low density lipoprotein by
endothelial cells involves lipid peroxidation and
degradation of low density lipoprotein phospholipids. Proc Natl
Acad Sci U S A.. 1984;81:3883–3887.
25.
Seccia M, Albano E, Maggi E, Bellomo G. Circulating
autoantibodies recognizing peroxidase-oxidized low density lipoprotein:
evidence for new antigenic epitopes formed in vivo independently from
lipid peroxidation. Arterioscler Thromb Vasc Biol. 1997;17:134–140.
26.
Basu SK, Goldstein JL, Anderson RGW, Brown MI.
Degradation of cationized low density lipoprotein and regulation of
cholesterol metabolism in homozygous familial
hypercholesterolemia fibroblasts. Proc
Natl Acad Sci U S A.. 1976;73:3178–3182.
27. Markwell MK, Haas SM, Bieber LL, Tolbert NE. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978;87:206–210.[Medline] [Order article via Infotrieve]
28.
Morganelli PM, Guyre PM. IFN- plus
glucocorticoids stimulate the expression of newly identified human
mononuclear phagocyte specific antigen. J Immunol. 1988;140:2296–2304.[Abstract]
29. Fraser I, Doyle A, Hughes D, Gordon S. Use of surface molecules and receptors for studying macrophages and mononuclear phagocytes. J Immunol Methods. 1994;174:95–102.[Medline] [Order article via Infotrieve]
30. Drevon CA, Attie AD, Pangburn SH, Steinberg D. Metabolism of homologous and heterologous lipoproteins by cultured rat and and skin fibroblasts. J Lipid Res. 1981;22:37–46.[Abstract]
31.
Morganelli PM, Kitzmiller TJ, Hemmer R, Fanger MW.
Redirected targeting of LDL to human monocyte Fc receptors with
bispecific antibodies. Arterioscler Thromb. 1992;12:1131–1137.[Abstract]
32. Looney RJ, Abraham GN, Anderson CL. Human monocytes and U937 cells bear two distinct receptors for IgG. J Immunol. 1986;136:1641–1647.[Abstract]
33.
Griffith RL, Virella GT, Stevenson HC, Lopes-Virella,
MI. Low density lipoprotein metabolism by human
macrophages activated with low density lipoprotein
immune complexes. J Exp Med. 1988;168:1041–1059.
34.
Ylä-Herrttuala S, Palinski W, Butler SW, Picard
S, Witztum JL. Rabbit and human atherosclerotic lesions contain IgG
that recognizes epitopes of oxidized LDL. Arterioscler
Thromb. 1994;14:32–40.
35.
Haberland ME, Fong D, Cheng L. Malondialdehyde-altered
protein occurs in atheroma of Watanabe heritable
hyperlipidemic rabbits. Science. 1988;241:215–218.
36.
Rosenfeld ME, Palinski W, Ylä-Herrttuala S,
Butler SW, Witztum JL. Distribution of oxidation specific lipid-protein
adducts and apolipoprotein B in atherosclerotic lesions of varying
severity from WHHL rabbits.
Arteriosclerosis. 1989;10:336–349.
37. Ylä-Herrttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler SW, Witztum JL, Steinberg D. Evidence for the presence of oxidatively modified LDL in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989;84:1086–1097.
38. Morganelli PM, Rogers RA, Kitzmiller TJ, Bergeron A. Enhanced metabolism of LDL aggregates mediated by specific human monocyte IgG Fc receptors. J Lipid Res. 1995;36:714–724.[Abstract]
39. Esterbauer H, Dieber-Rotheneder M, Waeg G, Striegl G, Jurgens G. Biochemical, structural, and functional properties of oxidized low-density lipoprotein. Chem Res Toxicol. 1990;3:77–92.[Medline] [Order article via Infotrieve]
40.
Guyre PM, Graziano RF, Vance BA, Morganelli PM, Fanger
MW. Monoclonal antibodies that bind to distinct epitopes on
FcRI are able to trigger receptor function. J
Immunol. 1989;143:1650–1655.[Abstract]
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