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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3442-3448.)
© 1997 American Heart Association, Inc.

Articles

Independent Mechanisms for Macrophage Binding and Macrophage Phagocytosis of Damaged Erythrocytes

Evidence of Receptor Cooperativity

Gilberto R. Sambrano; Valeska Terpstra; ; Daniel Steinberg

From the Department of Medicine, University of California San Diego, La Jolla, Calif.

	Abstract

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Abstract The binding and phagocytosis of oxidatively damaged red blood cells (OxRBCs) by mouse peritoneal macrophages can be inhibited by oxidatively modified LDL (OxLDL), implying some commonality at their receptor-binding domains. Studies from many different laboratories support the view that OxRBC binding is due to the disruption of plasma membrane phospholipid asymmetry and the subsequent exposure of phosphatidylserine (PS) on the outer membrane leaflet. Presumably, oxidation of LDL creates a surface structure on it in some way homologous to the PS-rich domain on OxRBCs. Apoptotic cells in some instances are also recognized because of PS exposure on the outer leaflet of the membrane, and apoptotic cells are a common feature of atherosclerotic lesions. In the present studies, the mechanisms of binding and internalization of cells recognized by virtue of their membrane PS were studied using OxRBCs or vanadate-treated erythrocytes (VaRBCs) as models. Disruption of phospholipid asymmetry with vanadate produced cells that were bound by macrophages in the same divalent cation–dependent manner as OxRBCs. However, whereas OxRBCs were rapidly phagocytosed, VaRBCs were not. Stimulation of mouse macrophages with phorbol myristate acetate resulted in a concentration-dependent induction of phagocytosis of bound VaRBCs, an effect that could be prevented by the protein kinase C inhibitor staurosporine. Because phagocytosis of OxRBCs occurred unassisted, we speculated that there must be additional membrane changes induced by oxidation (over and above the disruption of phospholipid asymmetry) that contribute to phagocytosis of OxRBCs, possibly resulting in the ligation of a distinct receptor that does not necessarily contribute to adherence. This proposal is supported by the finding that ligation of macrophage Fc receptors by the anti-FcRII/RIII antibody 2.4G2 triggers the phagocytosis of bound VaRBCs. Phagocytosis is also triggered by subthreshold opsonization of VaRBC, ie, by antibody concentrations that do not by themselves cause binding and phagocytosis of native RBCs. Finally, treatment with low concentrations of glutaraldehyde, which causes membrane protein cross-linking, promotes the phagocytosis of VaRBCs, but, at the low concentration used, has little or no effect on binding and phagocytosis of native RBCs. We suggest that the internalization of damaged cells, bound because of PS exposure, requires the cooperation of a PS-binding receptor with at least one additional receptor to trigger an intracellular signaling pathway to initiate phagocytosis.

Key Words: macrophages • apoptosis • scavenger receptors

	Introduction

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The removal of damaged or dying cells is fundamental to the developmental process, tissue repair, and homeostasis.¹ Macrophages play a central role in these processes by recognizing, internalizing, and degrading dying cells before they can necrose and damage surrounding tissues. It is now clear that atherosclerotic lesions include large numbers of apoptotic cells.^{2 3 4} Membrane PS exposed on the surface of dying cells is one of the key markers for macrophage recognition.^{5 6 7} Mammalian cells are believed to maintain an asymmetrical distribution of their plasma membrane phospholipids by actively transporting anionic phospholipids such as PS from the outer membrane leaflet to the inner membrane leaflet by an ATP/Mg²⁺-dependent aminophospholipid translocase.^{8 9} Disruption of this asymmetry and the concomitant exposure of PS on the outer membrane leaflet has been shown to accompany apoptosis of various cell types, including lymphocytes and neutrophils, contributing to their phagocytosis by macrophages.^{10 11} A loss of membrane phospholipid asymmetry has also been shown to occur in oxidized or sickled erythrocytes.^{12 13 14} Recognition of cells via exposed PS residues has already been described by several investigators and is attributed to an as yet uncharacterized PS receptor.^{11 14 15}

We have previously demonstrated that recognition of OxRBCs by mouse peritoneal macrophages is mediated by a receptor with affinity for both membrane PS and OxLDL.¹⁴ We also showed that recognition of OxRBCs is inhibited by polyanionic molecules such as fucoidan, polyinosinic acid, and malondialdehyde-modified albumin,¹⁶ all characteristic inhibitors of binding to scavenger receptors (reviewed in Reference 17¹⁷ ). However, AcLDL did not interfere with OxRBC binding. This observation raised the possibility that alternative receptors recognizing OxLDL might be involved. Recently, macrosialin and the scavenger receptor class B members (SRBI and CD36) have been shown also to bind PS-containing liposomes.^{18 19} However, a functional role for these proteins in mediating recognition of cell membranes expressing surface PS has not yet been demonstrated.

While there is considerable evidence that an increase in PS content of the external leaflet of cell membranes, particularly membranes of aging or oxidatively damaged RBCs, is a basis for their recognition by macrophages, the properties and nature of the receptor(s) involved remain unknown. Identification of the specific ligand domains recognized by scavenger receptors is difficult because of the structural complexity of the damaged membrane on which the ligand resides. Sodium vanadate has proven useful in generating a ligand with less complexity as a result of its ability to specifically inhibit the RBC aminophospholipid translocase and thereby create cells expressing PS on their membrane surface but avoiding additional changes resulting from oxidation.²⁰ We previously demonstrated that treatment of RBCs with vanadate produced RBCs that were comparable to OxRBCs in the levels of exposed surface PS and their propensity to be bound by macrophages.¹⁴ Vanadate treatment thus seemed to provide a simplified model for studying PS receptor activity in macrophages. Interestingly, VaRBCs, unlike OxRBCs, were not readily phagocytosed by macrophages despite avid binding.

The present studies were undertaken to further characterize the interactions between macrophages, OxRBCs, and VaRBCs, taking advantage of the dissociation between binding and phagocytosis, which we have previously reported.¹⁴

	Methods

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Materials
PMA, staurosporine, RGDS peptide, trypsin, pronase, collagenase, CuSO₄, ascorbate, and sodium orthovanadate, were obtained from Sigma Chemical Company; phospholipids from Avanti Polar Lipids; glutaraldehyde 25% in water from Fisher Scientific; and rat anti-mouse FcRII/RIII mAb (2.4G2)²¹ from Pharmingen. Mouse monoclonal IgG₁ anti-human glycophorin A (10F7) was a generous gift of Dr A. Rearden (University of California, San Diego)²² ; rat mAb 2F8, directed against the mouse AcLDL receptor was a generous gift from Dr I. Fraser, D.A. Hughes, and Dr S. Gordon (University of Oxford, UK).²³

Lipoproteins
Human LDL (d=1.019 to 1.063) was isolated in EDTA (1 mg/mL) from fresh plasma by preparative ultracentrifugation as previously described.²⁴ Acetylation of LDL with acetic anhydride was as described by Basu et al.²⁵

Cells
Human RBCs were isolated as previously described.¹⁶ OxRBCs (4% hematocrit in PBS were prepared by incubating at 37°C for 90 minutes in the presence of 0.2 mmol/L CuSO₄ plus 5 mmol/L ascorbate as previously described.¹⁶ Membrane phospholipid asymmetry was disrupted by treatment with 100 µmol/L sodium orthovanadate (10% hematocrit in PBS) for 18 to 20 hours. In some experiments, RBCs were treated with glutaraldehyde or with an antibody against glycophorin A by incubating for 45 minutes at 37° on a shaker (concentrations shown with data below).

Resident mouse peritoneal macrophages were isolated by peritoneal lavage as previously described¹⁶ and plated in RPMI 1640 supplemented with 10% fetal bovine serum and gentamycin. After 4 hours, nonadherent cells were removed by washing three times with PBS. Macrophages were used immediately after the washing step because overnight incubation results in an acquired ability to bind even normal human RBCs via a sialic acid–dependent mechanism. The adherent macrophages were kept in Dulbecco's modified Eagle's medium for binding and phagocytosis experiments.

Binding and Phagocytosis Assays
RBCs (hematocrit 0.1%) were incubated with macrophages at 37°C for 1 hour. After washing to remove unbound RBCs, the percentage of macrophages binding (and/or phagocytosing) one or more RBCs was determined as previously described.¹⁶ Macrophage-bound RBCs were removed by hypotonic lysis with 5 mmol/L phosphate buffer and macrophages were fixed with methanol before determination of RBC phagocytosis.

	Results

Top Abstract Introduction Methods Results Discussion References

 
We previously suggested that macrophages recognize VaRBCs and OxRBCs via the same surface receptor(s) because binding of both is dependent on expression of membrane PS and is inhibited almost completely by the same scavenger receptor ligands.¹⁴ Further studies in this laboratory continue to support this view. We find that binding is not dependent on the presence of sialic acid residues or proteins on the RBC surface because treatment of RBCs with neuraminidase, trypsin, or pronase after vanadate treatment has no effect on their recognition by macrophages (data not shown). Macrophage recognition of both OxRBCs and VaRBCs is dependent on divalent cations and is largely unaffected by temperature (Fig 1). Binding of OxRBCs requires macrophage membrane surface proteins, since treatment of macrophages with either trypsin or pronase eliminates the binding activity (Fig 2); results with VaRBCs were very similar (data not shown). We also find that recognition of both OxRBCs and VaRBCs is most evident in resident mouse peritoneal macrophages, whereas thioglycollate-elicited macrophages¹⁸ and several macrophage-like cell lines tested were deficient in this activity (including RAW 264.7, P388D1, J774.1, and PMA-stimulated THP-1 cells). The Table presents a summary of the characteristics of OxRBC and VaRBC binding by mouse peritoneal macrophages.

View larger version (29K): [in this window] [in a new window]   Figure 1. A, Divalent cation dependency of OxRBC (solid bars) or VaRBC (hatched bars) binding to mouse peritoneal macrophages. RBCs were incubated with macrophages in HBSS with Ca2+ and Mg2+ or in HBSS with 5 mmol/L EDTA for 1 hour. Macrophages binding one or more RBCs were considered positive. B, Temperature dependency of RBC binding to macrophages. OxRBCs (solid bars) or VaRBCs (hatched bars) were incubated with macrophages at 4°C, 22°C, and 37°C for 1 hour. Values in both A and B are the mean±SE of four determinations from a representative experiment. Two other experiments gave essentially the same results.

View larger version (14K): [in this window] [in a new window]   Figure 2. Effect of protease treatment of macrophages on their ability to bind OxRBCs. Mouse peritoneal macrophages were treated with 1 mg/mL trypsin or pronase for 1 hour or with purified collagenase before incubation with RBCs. Data represent the mean±SE of four determinations of a representative experiment. Two additional experiments with pronase and trypsin gave similar results. Collagenase was studied only once.

View this table: [in this window] [in a new window]   Table 1. Comparison of Copper-Oxidized and Vanadium-Treated RBCs With Respect to Membrane Changes and Characteristics of Their Binding to Macrophages

The failure of VaRBCs to undergo phagocytosis even when they expressed an amount of PS on the external leaflet comparable to that found in OxRBCs implied that oxidation must alter the RBC in an additional way or ways that account for the triggering of their phagocytosis. It has been previously shown that the complement receptor CR3 (iC3b receptor) on neutrophils and monocytes will bind iC3b-coated particles but will not phagocytose them unless the cells are first activated with PMA. On the other hand, when ß-glucan particles bind CR3, they are phagocytosed without any need to add PMA, presumably because ß-glucan can itself promote phagocytosis, either through a distinct receptor or specific binding to a distinct region of CR3.^{24 27} Because of the parallelism between these findings and our findings with VaRBCs versus OxRBCs, we tested whether the addition of PMA would trigger phagocytosis of VaRBCs. As shown in Fig 3 pretreatment of macrophages with PMA for 10 minutes led to a significant induction of VaRBC phagocytosis, increasing to a level comparable to that seen with OxRBCs,^{14 16} but with no apparent change in the overall binding. This effect was prevented by preincubating the macrophages for 15 minutes with 1 µmol/L staurosporine, suggesting a role for protein kinase C in the induction of phagocytosis. To rule out a nonspecific cytotoxic effect of staurosporine, we did a duplicate study using normal RBCs opsonized with a mAb against human glycophorin A. Phagocytosis was not inhibited.

View larger version (29K): [in this window] [in a new window]   Figure 3. Phagocytosis (solid bars) and binding (hatched bars) of VaRBCs by macrophages pretreated with 200 nmol/L PMA for 10 minutes and/or 1 µmol/L staurosporine (stauro) for 15 minutes. The values presented are the mean±SE of four determinations from a representative experiment. Two additional experiments gave similar results.

The nature of the additional changes induced by oxidation that promote OxRBC phagocytosis is not known. Possibilities include structural changes, such as membrane protein cross-linking or proteolytic degradation, generating new ligands recognized by a signaling receptor.²⁸ Phagocytosis of apoptotic cells by CD36 requires that it interact with the vitronectin receptor (_vß₃), and RGDS peptide has been shown to inhibit interaction of that complex with thrombospondin and to inhibit also the phagocytosis of apoptotic cells.²⁹ We tested for any potential involvement of this system in the phagocytosis of VaRBCs but, as shown in Fig 4, RGDS neither inhibited nor significantly enhanced phagocytosis.

View larger version (13K): [in this window] [in a new window]   Figure 4. Effect of RGDS peptide (1 mmol/L) and anti-FcRII/RIII antibody (4 µg/mL) on phagocytosis of VaRBCs by mouse peritoneal macrophages. Ligands were present during incubation of RBCs with macrophages. Effect of PMA pretreatment of macrophages is shown for comparison. The values, shown as a percentage of control, are the mean±SE of four determinations from a representative experiment. The PMA effect and the effect of the antibody against Fc receptor were similar in two additional experiments; the negative result with RGDS was demonstrated only once.

Cross-linking of Fc receptors due to binding of immune complexes or of antibodies against the Fc receptor causes protein tyrosine phosphorylation³⁰ and can trigger phagocytosis. We therefore tried adding an antibody to the FcRII/RIII receptor during the binding of VaRBCs to macrophages. As shown in Fig 4, this significantly increased phagocytosis of VaRBCs.

Although AcLDL does not compete for binding of VaRBCs to macrophages, it is the most characteristic ligand for some scavenger receptors¹⁷ and could in principle interact with one or more of them to trigger phagocytosis. However, addition of neither AcLDL nor 2F8 (a mAb against scavenger receptor A) had any effect (data not shown).

To extend our investigation and draw a closer analogy to what might be happening with OxRBCs, we tested several modifications of VaRBCs to see if they could trigger their phagocytosis. A critical requirement was that the modification would not by itself mediate significant binding to the macrophage. It is known that senescent RBCs isolated from healthy volunteers contain associated autoantibodies, but in numbers too few to promote binding or phagocytosis.³¹ However, if these senescent RBCs are additionally opsonized with complement component C3b, efficient phagocytosis is observed.^{31 32 33} By analogy, we asked whether binding of a small number of IgG antibodies to a VaRBC might be sufficient to stimulate subsequent phagocytosis without contributing significantly to adherence. Both native and VaRBCs were opsonized with an IgG₁ antibody directed against glycophorin A (10F7) using several dilutions of antibody-containing hybridoma medium to find concentrations that did not result in significant binding or phagocytosis of native RBCs. As shown in Fig 5, mild opsonization at the concentrations shown had little or no effect on binding but increased VaRBC phagocytosis significantly. There was no comparable increase in phagocytosis of native RBCs.

View larger version (30K): [in this window] [in a new window]   Figure 5. Binding (A) and phagocytosis (B) of normal RBCs (solid bars) and VaRBCs (hatched bars) opsonized with an anti-glycophorin A mAb (10F7). RBCs were treated with several dilutions of the hybridoma culture supernatant at 37°C for 45 minutes. Macrophages having engulfed one or more RBCs were considered positive. Data represent the mean±SE of three determinations in each of six separate experiments (total n=18).

It has been shown previously that treatment of RBCs with aldehydes can increase their binding and phagocytosis by macrophages much as treatment with oxygen radicals does.^{16 34} We previously showed that glutaraldehyde-treated RBCs were recognized by a macrophage scavenger receptor similar or identical to that which recognizes OxRBCs.¹⁶ Currently it is not clear whether recognition of these RBCs is mediated by aldehyde-modified membrane components per se or by exposed membrane PS. In any case, it is clear that aldehyde treatment produces RBCs that are readily phagocytosed. Conditions were found under which glutaraldehyde treatment of native RBCs resulted in only minimal binding and phagocytosis by macrophages yet generated membrane protein cross-linking detectable by SDS–polyacrylamide gel electrophoresis (data not shown). We tested whether similar glutaraldehyde treatment of VaRBCs would increase their phagocytosis. As shown in Fig 6, binding and phagocytosis of native RBCs was not significantly affected by the glutaraldehyde concentrations used, whereas phagocytosis of VaRBCs was increased at least threefold. These results demonstrate that changes produced by glutaraldehyde that are by themselves unable to mediate significant macrophage adherence can stimulate phagocytosis of bound VaRBCs.

View larger version (33K): [in this window] [in a new window]   Figure 6. Effect of treatment with glutaraldehyde on binding (A) and phagocytosis (B) of normal RBCs (solid bars) and VaRBCs (hatched bars) by macrophages. RBCs were treated with glutaraldehyde at different concentrations at 37°C for 45 minutes. Data represent the mean±SE of three determinations in each of four separate experiments (total n=12).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The receptor mechanism used by macrophages to recognize and subsequently internalize damaged or apoptotic cells via membrane PS has not yet been clearly defined. We have previously suggested that a yet undefined scavenger receptor can recognize damaged cells expressing surface membrane PS¹⁴ , and more recently the membrane proteins CD36, SRBI, and macrosialin have been cited as possible receptor candidates owing to their ability to bind PS liposomes.^{18 19} However, it is still unclear which, if any, of the now known scavenger receptors actually participate in the recognition of damaged or apoptotic cells via membrane PS. Scavenger receptors, specifically those that mediate macrophage uptake of OxLDL, almost certainly play a role in the development of foam cells and of the early atherosclerotic lesion.³⁵ The finding that OxLDL can compete for the binding of oxidatively damaged RBCs or apoptotic cells to macrophages strongly implies a role for these receptors also in the clearance of nonviable cells. Because apoptosis is a prominent feature of atherosclerotic lesions,^{36 37} this more recently recognized function of "OxLDL receptors" requires elucidation. Whether the presence of scavenger receptors in atherosclerotic lesions is damaging (eg, by generating lipid-filled foam cells) or beneficial (eg, by clearing out damaged cells) is unclear. It is interesting to note, however, that more than one known scavenger receptor has been suggested to have such a dual role. CD36 has already been shown to cooperate with the vitronectin receptor and thrombospondin in the recognition of apoptotic cells.²⁹ In addition, a recent study comparing monocyte-derived macrophages from normal and CD36-deficient patients has shown that CD36 makes a significant contribution to the uptake of OxLDL by these cells.³⁸ Recent evidence suggests that the extensively studied modified lipoprotein receptors, SRAI and II, are also capable of recognizing apoptotic thymocytes.³⁹ Ryfom et al⁴⁰ have shown that CD36 on rat retinal epithelium plays a role in phagocytosis of photoreceptor outer segments and does so by recognizing PS.

The phagocytosis of cells targeted for removal by macrophages is a two-step process initiated by recognition and binding of the target followed by its internalization. The dissociation between binding and internalization of damaged cells described here exemplifies the independence of ligand binding from ligand internalization: Binding is not necessarily followed by internalization; rather, internalization may be dependent on the activation state of the macrophage or the cooperation of additional membrane components. In our model, the PS receptor appears to function primarily as an adhesion receptor for damaged cells because VaRBCs are bound but are not internalized. In the case of OxRBCs, recognition is achieved in the same way, but internalization of the damaged cell is initiated by a signaling event probably resulting from an interaction with a distinct macrophage receptor(s). The membrane topography of OxRBCs is quite complex due to the various structural changes induced by oxidation, and many of these newly formed "epitopes" could potentially stimulate phagocytosis without necessarily contributing to adherence to the macrophage. By analogy, apoptotic cells also recognized by virtue of their membrane PS, may exhibit additional unknown ligands that promote their phagocytosis.

Initiation of phagocytosis requires cytoskeletal rearrangement at the point of particle contact, a process that is triggered by intracellular signals. Phagocytosis-promoting receptors such as the Fc receptor are able to stimulate actin polymerization and internalization through multiple signaling pathways.⁴¹ The signaling event induced by ligation of Fc or fibronectin receptors has been shown to assist phagocytosis via CR3, which is itself able to bind C3bi-coated particles but is inefficient in mediating their internalization.^{26 42} Binding of VaRBCs to the macrophage receptor does not itself stimulate phagocytosis, suggesting that, like CR3, the putative PS receptor may require the cooperation of additional membrane receptors if internalization is to occur. We have shown here that opsonization of VaRBCs with antibody concentrations that are insufficient to mediate binding of normal RBCs can promote the phagocytosis of bound VaRBCs by mouse peritoneal macrophages. Similarly, membrane changes induced by glutaraldehyde treatment that are insufficient to mediate binding of native RBCs once again promote phagocytosis of bound VaRBCs. Therefore, mild opsonization that may occur in vivo or modifications that may be present on OxRBCs can contribute to the internalization of the bound target cell. Experiments showing that a soluble ligand like the 2.4G2 antibody or a trigger of cytoplasmic signaling like PMA can also promote the phagocytosis of VaRBCs suggest that internalization is initiated by a general signaling event that directs internalization of ligands bound to the PS receptor. Thus, we propose that the exposure of membrane PS that occurs as a consequence of apoptosis or cellular damage is sufficient to explain binding by macrophages but is not sufficient to drive phagocytosis.

In summary, we suggest that phagocytosis of damaged RBCs bound by way of exposed PS requires the cooperation of other membrane receptors to generate a signal for phagocytosis. Oxidation of RBCs must involve many changes in addition to the increase in PS expression on the outer leaflet of the membrane. Lipids undergo oxidation, proteins undergo cross-linking, and the topography of the membrane may be altered in major ways. Further studies will be needed to define exactly which features of the OxRBCs are essential. Whether or not similar considerations apply to apoptotic cells remains to be determined. Competition for binding of apoptotic cells by either PS liposomes or OxLDL is only partial, and it seems unlikely that only one mechanism would have evolved by which damaged and dying cells are recognized and phagocytosed. Most likely there is a redundancy of mechanisms available to support such an important function.

	Selected Abbreviations and Acronyms

 

AcLDL = acetylated LDL mAb = monoclonal antibody OxLDL = oxidatively modified LDL OxRBC = oxidatively damaged RBC PMA = phorbol myristate acetate PS = phosphatidylserine RBC = red blood cell VaRBC = vanadate-treated RBC

	Acknowledgments

 
This work was supported by a Specialized Center of Research Grant from the National Heart, Lung, and Blood Institute (HL-14197) and by the Stein Institute for Research on Aging. Valeska Terpstra was supported in part by a fellowship from the Dutch Heart Foundation (NHS) and the Verenigde Spaar Bank (VSB). Mouse monoclonal IgG₁ anti-human glycophorin A (10F7) was a generous gift of Dr A. Rearden, University of California, San Diego, and rat monoclonal antibody 2F8, directed against the mouse AcLDL receptor was a generous gift from Dr I. Fraser, D.A. Hughes, and Dr S. Gordon, University of Oxford, UK. The authors thank Audrey Threlkeld for preparation of the manuscript.

	Footnotes

 
Reprint requests to Dr Daniel Steinberg, Department of Medicine, 0682, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0682.

Received March 17, 1997; accepted May 16, 1997.

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