Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3248-3254

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Morganelli, P. M.
Right arrow Articles by Pfeiffer, J. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Morganelli, P. M.
Right arrow Articles by Pfeiffer, J. R.
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3248-3254.)
© 1997 American Heart Association, Inc.

Articles

Evidence That Human Fc{gamma} 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
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract Several lines of evidence suggest that clearance of oxidized LDL (oxLDL) immune complexes by macrophage IgG Fc receptors (Fc{gamma}Rs) plays a role in atherogenesis. OxLDL may also be cleared directly by Fc{gamma}Rs, as shown for murine Fc{gamma}RII-B2. In humans, the homologous Fc{gamma}R is Fc{gamma}RIIA (CD32), which is abundantly expressed on monocytes and macrophages and shares 60% sequence identity with murine Fc{gamma}RII-B2. As murine Fc{gamma}RII-B2 and human Fc{gamma}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{gamma}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{gamma}RIIA and CD36, we showed that the binding of oxLDL was inhibited by antibodies to CD36, but not by Fc{gamma}RIIA antibodies. Thus, the data do not support the hypothesis that human Fc{gamma}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


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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{gamma}Rs.14 In the case of Fc{gamma}Rs, Stanton et al14 provided evidence that oxLDL is recognized by murine Fc{gamma}RII-B2. Using transfected cells, they demonstrated that Fc{gamma}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{gamma}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{gamma}RII-B2 is expressed predominantly on macrophages and binds monomeric IgG poorly and polyvalent IgG immune complexes with high affinity.17 Fc{gamma}RII-B2 shares approximately 60% amino acid sequence homology with its human counterpart, Fc{gamma}RIIA (CD32). Fc{gamma}RIIA is also expressed on mononuclear phagocytes and like Fc{gamma}RII-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 Fc{gamma}Rs in general, and human Fc{gamma}Rs in particular, can function as oxLDL receptors. We therefore wanted to test the hypothesis that human Fc{gamma}RIIA is a receptor for oxLDL. There are two allelic subtypes of Fc{gamma}RIIA 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 Fc{gamma}RIIA.18,19 To determine whether Fc{gamma}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.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 Fc{gamma}RIIA.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Materials
Anti-Fc{gamma}RII mAb IV.3 (murine IgG2b) was obtained from Medarex, Inc. Anti-Fc{gamma}RII 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 Fc{gamma}RIIA 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; Fc{gamma}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{gamma}RIIA transfectants, cells were stained as described28 with anti-Fc{gamma}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{gamma}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
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Expression of Fc{gamma}RIIA Subtypes on CHO Cells
CHO cell transfectants expressing either the R131 or H131 subtype of human Fc{gamma}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{gamma}RIIA subtype on stable CHO cell transfectants is shown in Fig 1Down. Cell surface expression was measured by quantitative flow cytometry using anti-Fc{gamma}RIIA mAbs IV.3 and 41H16. Monoclonal antibody IV.3 recognizes both the R131 and H131 subtypes of Fc{gamma}RIIA while mAb 41H16 recognizes only the R131 subtype.21 As shown in Fig 1ADown, each Fc{gamma}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{gamma}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{gamma}RIIA subtype on stable CHO cell transfectants.



View larger version (41K):
[in this window]
[in a new window]
  		Figure 1. Histograms of cell surface expression (A) and functional activity (B) of human Fc{gamma}RIIA subtypes in CHO cell transfectants. A, CHO cells expressing the R131 or H131 subtype of Fc{gamma}RIIA were stained with mAbs IV.3 or 41H16. Binding was detected by flow cytometry with fluorescein isothiocyanate-conjugated goat anti-mouse Fab'2 as outlined in "Methods." Shown are the mean±SD of triplicate measurements. B, CHO cell transfectants were treated with 50 µg/ml of 125I-nLDL for 4 hours at 37°C in the presence or absence of an anti-Fc{gamma}RIIA x anti-nLDL bispecific antibody, followed by assay of LDL degradation as outlined in "Methods."

To confirm functional Fc receptor activity, CHO cell transfectants were treated with 125I-nLDL in the presence of bispecific antibodies consisting of anti-Fc{gamma}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{gamma}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{gamma}Rs. Fig 1BUp shows the results of targeting 125I-nLDL to control transfectants as well as to transfectants expressing the R131 or H131 subtype of Fc{gamma}RIIA. In the absence of bispecific antibodies, the degradation of 125I-nLDL was minimal and the same for both control and Fc{gamma}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{gamma}RIIA transfectants expressing the R131 or H131 subtype of Fc{gamma}RIIA was enhanced >6-fold in the presence of the Fc{gamma}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{gamma}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 TableDown. 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.


View this table:
[in this window]
[in a new window]
  		Table 1. Human SR-AI Activity in Transfected CHO Cells

Reactivity of Copper-Oxidized LDL to Human Fc{gamma}RIIA Subtypes
To test the hypothesis that human Fc{gamma}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 2ADown shows a typical experiment where control, SR-AI, and Fc{gamma}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 2Down, there was no difference in degradation between the control and Fc{gamma}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{gamma}RIIA transfectants did not exceed that of the control (pcDNA3 empty vector) transfectants.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 2. A, Line plots of 125I-copper-oxidized LDL degradation in CHO cell transfectants. Each transfectant was treated with varying amounts of 20-hour 125I-copper-oxidized LDL for 4 hours at 37°C followed by assay of oxLDL degradation as outlined in "Methods." Shown are the mean±SD of triplicate measurements. {blacksquare}, SR-AI transfectants; {diamond}, control pcDNA3 transfectants; {bullet}, R131 transfectants; {bigtriangleup}, 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 Fc{gamma}RIIA Subtypes
To determine if other oxLDL preparations could react to Fc{gamma}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 2BUp. 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{gamma}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{gamma}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{gamma}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,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-Fc{gamma}RIIA mAb IV.3 or anti-CD36 mAb OKM5 (Fig 3Down). As determined by flow cytometric analysis, the monocytes from this particular donor expressed both the R131 and H131 subtypes of Fc{gamma}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 3Down, the binding of 125I oxLDL was inhibited approximately 60% in the presence of 25-fold excess unlabeled oxLDL. This level of binding ({approx}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{gamma}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{gamma}RIIA subtypes.



View larger version (71K):
[in this window]
[in a new window]
  		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{gamma}RIIA mAb IV.3. Shown are the mean±SD of quintuplicate measurements.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Recent evidence suggests that the clearance of lipoprotein immune complexes through macrophage Fc{gamma}Rs plays a role in atherogenesis.33–37 Human mononuclear phagocytes may express three functionally distinct classes of human Fc{gamma}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{gamma}R x anti-LDL antibodies to study lipoprotein metabolism in the context of each specific type of Fc{gamma}R.31,38 As we have begun to address the question of oxLDL immune complex metabolism, the possibility that macrophage Fc{gamma}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{gamma}RIIA subtypes are low-affinity IgG receptors that share significant amino acid sequence homology with murine Fc{gamma}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{gamma}RIIA (Fig 1Up). 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{gamma}RIIA subtypes was not different from that of the control CHO cell transfectants (Fig 2Up). Also, the binding of copper-oxidized LDL to cells expressing both CD36 and Fc{gamma}RIIA was significantly inhibited by anti-CD36 mAb (Fig 3Up), but not by an anti-Fc{gamma}RIIA mAb that blocks the IgG ligand-binding site, also consistent with a lack of Fc{gamma}RIIA reactivity.

Taken together, the data do not support the hypothesis that human Fc{gamma}RIIA is a receptor for oxLDL. This contrasts with the results of Stanton et al,14 who reported that murine Fc{gamma}RII-B2 is a receptor for copper-oxidized LDL. As discussed above, murine Fc{gamma}RII-B2 and human Fc{gamma}RIIA share 60% amino acid sequence homology in the extracellular domain.17 We therefore expected that human Fc{gamma}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{gamma}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{gamma}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{gamma}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{gamma}RII-B2, blocks oxLDL uptake in transfected cells expressing Fc{gamma}RII-B2.14 Whereas the extracellular domains of murine Fc{gamma}RII-B2 and human Fc{gamma}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{gamma}RII-B2 and human Fc{gamma}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{gamma}RIIA expressed on CHO cells or human monocytes (Figs 2Up and 3Up). In the case of monocytes, oxLDL binding was significantly inhibited by anti-CD36 mAb OKM5, but not by anti-Fc{gamma}RIIA mAb IV.3 (Fig 3Up). 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{gamma}RI mAb 2240 or human IgG (consistent with lack of interaction with Fc{gamma}RI), as well as anti-Fc{gamma}RIIA mAb IV.3. K562 cells (that expressed no CD36 but both Fc{gamma}RIIA subtypes) also showed minimal reactivity to copper-oxidized LDL that was unaffected by anti-Fc{gamma}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{gamma}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{gamma}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{gamma}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{gamma}RIIA because binding of oxLDL to monocytes that expressed both high amounts of CD36 and Fc{gamma}RIIA was completely accounted for in terms of reactivity to CD36 (Fig 3Up).

In summary, the evidence obtained in this study did not support the hypothesis that human Fc{gamma}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{gamma}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{gamma}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{gamma}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.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  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.[Free Full Text]
  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 {alpha}-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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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{gamma} receptor II (CD32). J Exp Med. 1990;172:19–25.[Abstract/Free Full Text]
  21. Gosselin EJ, Brown MF, Anderson CL, Zipf TF, Guyre PM. The monoclonal antibody 41H16 detects the LEU 4 responder form of Fc{gamma}RII. 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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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-{gamma} 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{gamma} 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.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  35. Haberland ME, Fong D, Cheng L. Malondialdehyde-altered protein occurs in atheroma of Watanabe heritable hyperlipidemic rabbits. Science. 1988;241:215–218.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  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 Fc{gamma}RI are able to trigger receptor function. J Immunol. 1989;143:1650–1655.[Abstract]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
A. Munteanu, M. Taddei, I. Tamburini, E. Bergamini, A. Azzi, and J.-M. Zingg
Antagonistic Effects of Oxidized Low Density Lipoprotein and {alpha}-Tocopherol on CD36 Scavenger Receptor Expression in Monocytes: INVOLVEMENT OF PROTEIN KINASE B AND PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-{gamma}
J. Biol. Chem., March 10, 2006; 281(10): 6489 - 6497.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Morganelli, P. M.
Right arrow Articles by Pfeiffer, J. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Morganelli, P. M.
Right arrow Articles by Pfeiffer, J. R.

ATVB Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 1997 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.