Macrophage Recognition Research Articles

Selective Peroxidation and Externalization of Phosphatidylserine in Normal Human Epidermal Keratinocytes During Oxidative Stress Induced by Cumene Hydroperoxide

Reactive oxygen species not only modulate important signal transduction pathways, but also induce DNA damage and cytotoxicity in keratinocytes. Hydrogen peroxide and organic peroxides are particularly important as these chemicals are widely used in dermally applied cosmetics and pharmaceuticals, and also represent endogenous metabolic intermediates. Lipid peroxidation is of fundamental interest in the cellular response to peroxides, as lipids are extremely sensitive to oxidation and lipid-based signaling systems have been implicated in a number of cellular processes, including apoptosis. Oxidation of specific phospholipid classes was measured in normal human epidermal keratinocytes exposed to cumene hydroperoxide after metabolic incorporation of the fluorescent oxidation-sensitive fatty acid, cis-parinaric acid, using a fluorescence high-performance liquid chromatography assay. In addition, lipid oxidation was correlated with changes in membrane phospholipid asymmetry and other markers of apoptosis. Although cumene hydroperoxide produced significant oxidation of cis-parinaric acid in all phospholipid classes, one phospholipid, phosphatidylserine, appeared to be preferentially oxidized above all other species. Using fluorescamine derivatization and annexin V binding it was observed that specific oxidation of phosphatidylserine was accompanied by phosphatidylserine translocation from the inner to the outer plasma membrane surface where it may serve as a recognition signal for interaction with phagocytic macrophages. These effects occurred much earlier than any detectable changes in other apoptotic markers such as caspase-3 activation, DNA fragmentation, or changes in nuclear morphology. Thus, normal human epidermal keratinocytes undergo profound lipid oxidation with preference for phosphatidylserine followed by phosphatidylserine externalization upon exposure to cumene hydroperoxide. It is therefore likely that normal human epidermal keratinocytes exposed to similar oxidative stress in vivo would under go phosphatidylserine oxidation/translocation. This would make them targets for macrophage recognition and phagocytosis, and thus limit their potential to invoke inflammation or give rise to neoplastic transformations.

Correlation of Antiphospholipid Antibody Recognition with the Structure of Synthetic Oxidized Phospholipids: IMPORTANCE OF SCHIFF BASE FORMATION AND ALDOL CONDENSATION

The oxidation of low density lipoproteins (LDL) has been correlated with atherogenesis through a variety of pathways. The process involves nonspecific fragmentation, oxidative breakdown, and modification of the lipids and protein of LDL. The process yields a variety of bioactive products, including aldehyde-containing phospholipids, which can cross-react with primary amines (i.e. peptides or phospholipid head groups) to yield Schiff base products. We also demonstrate that such oxidized phospholipid products may further react through a post-oxidation chemical pathway involving aldol condensation. EO6, an IgM monoclonal autoantibody to oxidized phospholipids, blocks the uptake of oxidized LDL (OxLDL) by macrophages. Because the epitope(s) of EO6 also blocks the uptake of OxLDL, a series of oxidized phospholipids, their peptide complexes, and their aldol condensates have been synthesized and characterized, and their antigenicity has been determined. This study defines structural motifs of oxidized phospholipids responsible for antigenicity for EO6. Certain monomeric phospholipids containing short chain fatty acids were antigenic whether oxidized or not in the sn-2 position. However, oxidized phospholipids containing sn-1 long chain fatty acids were not antigenic unless the sn-2 oxidized fatty acid contained an aldehyde that first reacted with a peptide yielding a Schiff base or the sn-2 oxidized fatty acid underwent an aldol type self-condensation. Our data indicate that the phosphorylcholine head group is essential for antigenicity, but its availability depends on the oxidized phospholipid conformation. We suggest that upon oxidation, similar reactions occur in phospholipids on the surface of LDL, generating ligands for macrophage recognition. Synthetic imine adducts of oxidized phospholipids of this type are capable of blocking the uptake of OxLDL.

Identification of a functional role for lipid asymmetry in biological membranes: Phosphatidylserine-skeletal protein interactions modulate membrane stability.

Asymmetric distribution of phospholipids is ubiquitous in the plasma membranes of many eukaryotic cells. The majority of the aminophospholipids are located in the inner leaflet whereas the cholinephospholipids are localized predominantly in the outer leaflet. Several functional roles for asymmetric phospholipid distribution in plasma membranes have been suggested. Disruption of lipid asymmetry creates a procoagulant surface on platelets and serves as a trigger for macrophage recognition of apoptotic cells. Furthermore, the dynamic process of phospholipid translocation regulates important cellular events such as membrane budding and endocytosis. In the present study, we used the red cell membrane as the model system to explore the contribution of phospholipid asymmetry to the maintenance of membrane mechanical properties. We prepared two different types of membranes in terms of their phospholipid distribution, one in which phospholipids were scrambled and the other in which the asymmetric distribution of phospholipids was maintained and quantitated their mechanical properties. We documented that maintenance of asymmetric distribution of phospholipids resulted in improved membrane mechanical stability. The greater difficulty in extracting the spectrin-actin complex at low-ionic strength from the membranes with asymmetric phospholipid distribution further suggested the involvement of interactions between aminophospholipids in the inner leaflet and skeletal proteins in modulating mechanical stability of the red cell membrane. These findings have enabled us to document a functional role of lipid asymmetry in regulating membrane material properties.

Difference in the way of macrophage recognition of target cells depending on their apoptotic states

Dying cells are selectively eliminated from the organism by phagocytosis. Previous studies suggested the existence of some other phagocytosis marker(s) that function together with phosphatidylserine, the best-characterized phagocytosis marker. We obtained here a monoclonal antibody named PH2 that inhibited macrophage phagocytosis of late apoptotic or necrotic cells, but not of early apoptotic cells. On the other hand, phagocytosis of cells at any time during the process of apoptosis was inhibitable by phosphatidylserine-containing liposomes. Inhibition occurred even when target cells were preincubated with PH2 and separated from unbound antibodies. Moreover, PH2 bound to apoptotic cells at late stages more efficiently than to those at early stages, and it did not bind to normal cells unless their plasma membrane was permeabilized. These results suggest that the putative PH2 antigen is a novel phagocytosis marker that translocates to the cell surface at late stages of apoptosis, resulting in maximal recognition and engulfment by macrophages.

Distinct modes of macrophage recognition for apoptotic and necrotic cells are not specified exclusively by phosphatidylserine exposure.

The distinction between physiological (apoptotic) and pathological (necrotic) cell deaths reflects mechanistic differences in cellular disintegration and is of functional significance with respect to the outcomes that are triggered by the cell corpses. Mechanistically, apoptotic cells die via an active and ordered pathway; necrotic deaths, conversely, are chaotic and passive. Macrophages and other phagocytic cells recognize and engulf these dead cells. This clearance is believed to reveal an innate immunity, associated with inflammation in cases of pathological but not physiological cell deaths. Using objective and quantitative measures to assess these processes, we find that macrophages bind and engulf native apoptotic and necrotic cells to similar extents and with similar kinetics. However, recognition of these two classes of dying cells occurs via distinct and noncompeting mechanisms. Phosphatidylserine, which is externalized on both apoptotic and necrotic cells, is not a specific ligand for the recognition of either one. The distinct modes of recognition for these different corpses are linked to opposing responses from engulfing macrophages. Necrotic cells, when recognized, enhance proinflammatory responses of activated macrophages, although they are not sufficient to trigger macrophage activation. In marked contrast, apoptotic cells profoundly inhibit phlogistic macrophage responses; this represents a cell-associated, dominant-acting anti-inflammatory signaling activity acquired posttranslationally during the process of physiological cell death.

Involvement of calcium signaling in the fibronectin-stimulated macrophage recognition of oxidatively damaged erythrocytes

Macrophages recognize oxidatively damaged autologous erythrocytes, and cell surface fibronectin of macrophages enhances the recognition (Beppu et al., FEBS Lett. 295 (1991) 135–140). In the present study, mechanisms of enhanced macrophage recognition of oxidatively damaged erythrocytes by fibronectin were investigated. Monolayers of thioglycollate-induced mouse peritoneal macrophages with cell surface fibronectin recognized autologous erythrocytes oxidized with an iron catalyst ADP/Fe 3+. The macrophage recognition of the oxidized erythrocytes was inhibited partially by pretreatment of the macrophage monolayers with a Ca 2+ channel blocker (diltiazem), calmodulin inhibitors (W-7, trifluoperazine, chlorpromazine and dibucaine), an inhibitor of myosin light chain kinase (ML-9), a microfilament formation inhibitor (cytochalasin B), phospholipase A 2 inhibitors (4-bromophenacyl bromide, mepacrine) and cyclooxygenase inhibitors (indomethacin and aspirin). Monolayers of macrophages depleted of fibronectin by trypsinization lost the ability of recognizing oxidized erythrocytes, but acquired the ability when stimulated with a fibronectin-coated coverslip. The recognition of fibronectin-stimulated trypsinized macrophages was also inhibited by the above inhibitors. On treatment with Ca ionophore A23187, trypsinized macrophages acquired the ability to recognize oxidized erythrocytes. The recognition of Ca ionophore-stimulated trypsinized macrophages was inhibited by the above inhibitors except the Ca 2+ channel blocker. These results indicate that the Ca 2+ signaling including Ca 2+ influx, calmodulin activation and myosin light chain phosphorylation are involved in the fibronectin stimulation of the recognition of macrophages for oxidized erythrocytes. Involvement of microfilament formation and arachidonate cascade in the fibronectin stimulation was also suggested.

Involvement of phosphatidylserine exposure in the recognition and phagocytosis of uremic erythrocytes

Cell surface-exposed phosphatidylserine (PS) represents a signal for macrophage recognition and cell phagocytosis. This study examines PS exposure and susceptibility to erythrocyte phagocytosis in patients with chronic uremia in an attempt to assess the possible pathogenic mechanism behind cell removal in a condition associated with shortened erythrocyte life. Both PS-expressing erythrocytes and erythrophagocytosis (human monocyte-derived macrophages ingesting one or more erythrocytes) were significantly increased in uremic patients compared with healthy controls. Phagocytosed uremic erythrocytes appeared intact, suggesting they were identified before lysis through some surface change recognized by the macrophages. The degree of phagocytosis was markedly greater for PS-positive than PS-negative fluorescence-activated cell sorter (FACS)-sorted uremic erythrocytes. A significant correlation (r = 0.655) was found between the percentage of PS-expressing red blood cells (RBCs) and the percentage of phagocytosing macrophages in uremic patients. Reconstitution experiments showed the ability of uremic plasma to promote both PS exposure and erythrophagocytosis, the latter without direct interaction with the macrophage population. Phagocytosis of uremic erythrocytes was strongly inhibited when the macrophages were preincubated with glycerophosphorylserine (GPS), a structural derivative of PS, but this was not the case with the equivalent derivative of phosphatidylethanolamine, glycerophosphorylethanolamine. This inhibition appeared to be specific because GPS failed to inhibit the phagocytosis of opsonized uremic erythrocytes that occurs through an Fc receptor-mediated pathway. These findings suggest that a PS-recognition mechanism may promote the susceptibility of uremic RBCs to phagocytosis and thus be involved in the shortened erythrocyte life span of uremia.

酸化LDL中の酸化リン脂質とその意義

Oxidatively modified low density lipoprotein (OxLDL), which is recognized by scavenger receptors, is now thought to be an important factor in atherogenesis. Since modifications occuring in OxLDL are complex and heterogenous, it is difficult to clarify the precise structures, metabolism, and localization of OxLDL in vivo. We have utilized a monoclonal antibody against OxLDL, which recognizes oxidized phosphatidylcholine (OxPC) molecules as its epitope, to study the feature of OxLDL present in vivo. So far we found from a number of co-laborating works that: (i) OxPC-apolipoprotein B complex occurs in OxLDL, (ii) OxLDL detected as LDL particles complexed with OxPC is present in human circulating plasma, (iii) the plasma OxLDL level is high in patients with certain diseases including heart diseases, (iv) OxPC-apolipoprotein B complex which corresponds to partially degraded OxLDL is present in atherosclerotic lesions in the human carotid artery and in foamy shaped macrophages in culture, and finally (v) OxPC is likely to be involved in macrophage recognition of OxLDL particles. Recently several studies showed OxPC can stimulate vessel wall cells including endothelial cells and macrophages. OxPC is thought to be an important molecule as a marker for OxLDL in vivo, as well as a pathological ustimulant involved in atherogenesis.

Capture of Stealth Nanoparticles by the Body’s Defences

This article highlights and discusses our experience in the design, handling, and biological evaluation of long-circulating nanoparticles based on polyoxypropylene/polyoxyethylene copolymer nonionic surfactants. A frequently observed but ignored phenomenon following intravenous injection of such polymer-modified long-circulating colloidal systems is their eventual recognition and clearance by macrophages of the reticuloendothelial system. The pharmacokinetics as well as the tissue distribution of such so-called 'stealth" nanoparticles are also altered after repeated intravenous injection in a time-dependent manner. An understanding of immunological and pathological factors that control the pharmacokinetic and biological behavior of long-circulating particles after single or repeated administration is therefore crucial for the design of a system with an optimal diagnostic and/or therapeutic performance. Therefore, we have also examined and discussed the concept of macrophage recognition of a 'stealth-like nature" and factors that initiate this cascade. A critical discussion of the future of this interesting area of nano-biotechnology/engineering is also provided.

Loss of Phospholipid Asymmetry and Surface Exposure of Phosphatidylserine Is Required for Phagocytosis of Apoptotic Cells by Macrophages and Fibroblasts

Removal of apoptotic cells during tissue remodeling or resolution of inflammation is critical to the restoration of normal tissue structure and function. During apoptosis, early surface changes occur, which trigger recognition and removal by macrophages and other phagocytes. Loss of phospholipid asymmetry results in exposure of phosphatidylserine (PS), one of the surface markers recognized by macrophages. However, a number of receptors have been reported to mediate macrophage recognition of apoptotic cells, not all of which bind to phosphatidylserine. We therefore examined the role of membrane phospholipid symmetrization and PS externalization in uptake of apoptotic cells by mouse macrophages and human HT-1080 fibrosarcoma cells by exposing them to cells that had undergone apoptosis without loss of phospholipid asymmetry. Neither mouse macrophages nor HT-1080 cells recognized or engulfed apoptotic targets that failed to express PS, in comparison to PS-expressing apoptotic cells. If, however, their outer leaflets were repleted with the l-, but not the d-, stereoisomer of sn-1,2-PS by liposome transfer, engulfment by both phagocytes was restored. These observations directly demonstrate that loss of phospholipid asymmetry and PS expression is required for phagocyte engulfment of apoptotic cells and imply a critical, if not obligatory, role for PS recognition in the uptake process.

The expression of CD10 by apoptotic lymphocytes is preceded by a pronounced externalization of phosphatidylserine

Cutrona et al[1][1] demonstrated the expression of CD10 on human postthymic T cells when undergoing apoptosis and discussed how, in vivo, this effect may act to limit potential inflammation at the site of the apoptotic cell or aid macrophage recognition. Identification of apoptosis and CD10 in many

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Phosphatidylserine expression on apoptotic lymphocytes of Xenopus laevis, the South African clawed toad, as a signal for macrophage recognition

Inflammation is avoided in apoptosis by early removal of dying cells by macrophages (MØs). In mammalian cells, an early aspect of apoptosis is the translocation of phosphatidylserine (PS) from the inner leaflet of the cell membrane to the surface. PS recognition can serve as a signal for triggering removal of dying cells. PS expression on splenocytes and thymocytes of Xenopus laevis was quantified using FITC-Annexin and flow cytometry following exposure in vitro to several known apoptogens for this species. All apoptogens used induced PS expression. Dose dependency and the kinetics of PS expression following exposure to the calcium ionophore, A23187, were also examined. Peritoneal exudate cells (PEC’s) were cultured with A23187-treated thymocytes to test MØ capacity for recognition of PS. MØ binding to apoptotic thymocytes was reduced following exposure of PEC’s to a water soluble analogue of PS, phospho- L-serine. The presence of a phagocytic PS-dependent recognition system in amphibia is supportive of the evolutionary conservation of this function in mammals that is crucial in limiting inflammation induced by dying cells.

Oxidation and erythrocyte senescence.

Direct macrophage recognition of an externalized phosphatidylserine signal on senescent erythrocytes is a process of erythrophagocytic clearance that is in line with the general clearance process of all other circulating cells that become apoptotic. Advances in deciphering this process suggest that oxidation of the erythrocyte's hemoglobin, the salient target of the free radicals encountered in the circulatory environment, may drive subsequent steps. The progressive accumulation of oxidized hemoglobin covalently bound to the membrane skeleton not only disrupts membrane organization but also threatens eventual phospholipid oxidation via a calcium-promoted quasi-lipoxygenase activity. The emergence on the cell surface of a threshold concentration of oxidized phospholipids, principally phosphatidylserine, signals recognition by the CD36 macrophage receptor.

The porin OmpC ofSalmonella typhimuriummediates adherence to macrophages

Macrophages recognize, adhere to, and phagocytose Salmonella typhimurium. The major outer membrane protein OmpC is a candidate ligand for macrophage recognition. To confirm this we used transposon mutagenesis to develop an ompC-deficient mutant in a known virulent strain of S. typhimurium; mutant and wild type were compared in macrophage adherence and association assays. Radiolabeled wild type S. typhimurium bound to macrophages at five-fold higher levels than did the ompC mutant. In association assays, macrophages in monolayers bound and internalized three-fold more wild type than mutant, while macrophages in suspension bound and internalized 40-fold more wild type than mutant. The ompC gene of our test strain of S. typhimurium contains several discrete differences compared with the ompC genes of Salmonella typhi and Escherichia coli. The deduced OmpC amino acid sequence of S. typhimurium shares 77 and 98% identity with OmpC amino acid sequence of E. coli and S. typhi, respectively. Evidence from this study supports a role for the OmpC protein in initial recognition by macrophages and distinguishes regions of this protein that potentially participate in host-cell recognition of bacteria by phagocytic cells.

Macrophage Recognition of ICAM-3 on Apoptotic Leukocytes

Cells undergoing apoptosis are cleared rapidly by phagocytes, thus preventing tissue damage caused by loss of plasma membrane integrity. In this study, we show that the surface of leukocytes is altered during apoptosis such that the first Ig-like domain of ICAM-3 (CD50) can participate in the recognition and phagocytosis of the apoptotic cells by macrophages. Macrophage recognition of apoptotic cell-associated ICAM-3 was demonstrated both on leukocytes and, following transfection of exogenous ICAM-3, on nonleukocytes. The change in ICAM-3 was a consistent consequence of apoptosis triggered by various stimuli, suggesting that it occurs as part of a final common pathway of apoptosis. Alteration of ICAM-3 on apoptotic cells permitting recognition by macrophages resulted in a switch in ICAM-3-binding preference from the prototypic ICAM-3 counterreceptor, LFA-1, to an alternative macrophage receptor. Using mAbs to block macrophage/apoptotic cell interactions, we were unable to obtain evidence that either the alternative ICAM-3 counterreceptor alpha d beta 2 or the apoptotic cell receptor alpha v beta 3 was involved in the recognition of ICAM-3. By contrast, mAb blockade of macrophage CD14 inhibited ICAM-3-dependent recognition of apoptotic cells. These results show that ICAM-3 can function as a phagocytic marker of apoptotic leukocytes on which it acquires altered macrophage receptor-binding activity.

Monoclonal antibodies against oxidized low-density lipoprotein bind to apoptotic cells and inhibit their phagocytosis by elicited macrophages: evidence that oxidation-specific epitopes mediate macrophage recognition.

Apoptosis is recognized as important for normal cellular homeostasis in multicellular organisms. Although there have been great advances in our knowledge of the molecular events regulating apoptosis, much less is known about the receptors on phagocytes responsible for apoptotic cell recognition and phagocytosis or the ligands on apoptotic cells mediating such recognition. The observations that apoptotic cells are under increased oxidative stress and that oxidized low-density lipoprotein (OxLDL) competes with apoptotic cells for macrophage binding suggested the hypothesis that both OxLDL and apoptotic cells share oxidatively modified moieties on their surfaces that serve as ligands for macrophage recognition. To test this hypothesis, we used murine monoclonal autoantibodies that bind to oxidation-specific epitopes on OxLDL. In particular, antibodies EO6 and EO3 recognize oxidized phospholipids, including 1-palmitoyl 2-(5-oxovaleroyl) phosphatidylcholine (POVPC), and antibodies EO12 and EO14 recognize malondialdehyde-lysine, as in malondialdehyde-LDL. Using FACS analysis, we demonstrated that each of these EO antibodies bound to apoptotic cells but not to normal cells, whereas control IgM antibodies did not. Confocal microscopy demonstrated cell-surface expression of the oxidation-specific epitopes on apoptotic cells. Furthermore, each of these antibodies inhibited the phagocytosis of apoptotic cells by elicited peritoneal macrophages, as did OxLDL. In addition, an adduct of POVPC with BSA also effectively prevented phagocytosis. These data demonstrate that apoptotic cells express oxidation-specific epitopes-including oxidized phospholipids-on their cell surface, and that these serve as ligands for recognition and phagocytosis by elicited macrophages.

Receptors for oxidized low-density lipoprotein on elicited mouse peritoneal macrophages can recognize both the modified lipid moieties and the modified protein moieties: implications with respect to macrophage recognition of apoptotic cells.

It has been shown previously that the binding of oxidized low-density lipoprotein (OxLDL) to resident mouse peritoneal macrophages can be inhibited (up to 70%) by the apoprotein B (apoB) isolated from OxLDL, suggesting that macrophage recognition of OxLDL is primarily dependent on its modified protein moiety. However, recent experiments have demonstrated that the lipids isolated from OxLDL and reconstituted into a microemulsion can also strongly inhibit uptake of OxLDL (up to 80%). The present studies show that lipid microemulsions prepared from OxLDL bind to thioglycollate-elicited macrophages at 4 degrees C in a saturable fashion and inhibit the binding of intact OxLDL and also of the apoB from OxLDL. Reciprocally, the binding of the OxLDL-lipid microemulsions was strongly inhibited by intact OxLDL. A conjugate of synthetic 1-palmitoyl 2(5-oxovaleroyl) phosphatidylcholine (an oxidation product of 1-palmitoyl 2-arachidonoyl phosphatidylcholine) with serum albumin, shown previously to inhibit macrophage binding of intact OxLDL, also inhibited the binding of both the apoprotein and the lipid microemulsions prepared from OxLDL. Finally, a monoclonal antibody against oxidized phospholipids, one that inhibits binding of intact OxLDL to macrophages, also inhibited the binding of both the resolubilized apoB and the lipid microemulsions prepared from OxLDL. These studies support the conclusions that: (i) at least some of the macrophage receptors for oxidized LDL can recognize both the lipid and the protein moieties; and (ii) oxidized phospholipids, in the lipid phase of the lipoprotein and/or covalently linked to the apoB of OxLDL, likely play a role in that recognition.

S-nitrosoglutathione enhances neutrophil DNA fragmentation and cell death.

Enhancing the clearance of neutrophils by enhancing apoptotic cell death and macrophage recognition may be beneficial in acute lung injury. Exogenous nitric oxide gas depresses neutrophil oxidative functions and accelerates cell death (A. H. Daher, J. D. Fortenberry, M. L. Owens, and L. A. Brown. Am. J. Respir. Cell Mol. Biol. 16: 407-412, 1997). We hypothesized that S-nitrosoglutathione (GSNO), a physiologically relevant nitric oxide donor, could also enhance neutrophil DNA fragmentation. Neutrophils were incubated for 2-24 h in the absence and presence of GSNO (dose range 0.1-5 mM) and evaluated for cell death by a fluorescent viability/cytotoxicity assay. Neutrophil DNA fragmentation was assessed by cell death detection ELISA and by terminal deoxynucleotidyltransferase-mediated fluorescence-labeled dUTP nick end labeling assay. Neutrophil oxidative function was also determined. Incubation with GSNO increased cell death at 2, 4, and 24 h. GSNO incubation for 24 h significantly increased DNA fragmentation in a dose-dependent fashion at 0.5 (median 126% of control value; P = 0.002) and 5 mM (185% of control value; P = 0.002) by terminal deoxynucleotidyltransferase-mediated fluorescence-labeled dUTP nick end labeling and at 0.5 mM by ELISA (164% of control value; P = 0.03). The apoptosis-to-total cell death ratio increased with increasing GSNO concentration (P < 0.05). Effects were mitigated by coincubation with superoxide dismutase. Five millimolar GSNO decreased overall superoxide generation and O2 consumption but not when adjusted for dead neutrophils. GSNO significantly enhances cell death and neutrophil DNA fragmentation in a dose-dependent fashion.

Characterization of Phosphatidylserine-dependent β2-Glycoprotein I Macrophage Interactions: IMPLICATIONS FOR APOPTOTIC CELL CLEARANCE BY PHAGOCYTES

The binding and uptake of phosphatidylserine (PS)-expressing cells appears to involve multiple receptor-mediated systems that recognize the lipid either directly or indirectly through intermediate proteins that form a molecular bridge between the cells. Here we show that beta2-glycoprotein I (beta2GPI), a 50-kDa serum glycoprotein, binds PS-containing vesicles and serves as an intermediate for the interaction of these vesicles with macrophages. Chemical modification of lysines and cysteines abolished beta2GPI-dependent PS uptake by inhibiting the binding of PS to beta2GPI and the binding of PS.beta2GPI complex to macrophages, respectively. Recognition was mediated by beta2GPI and not by the lipid because antibodies to beta2GPI inhibited binding of the complex to macrophages. These results indicate that human (THP-1-derived) macrophages bind beta2GPI only after it is bound to its lipid ligand. Competition experiments with monosaccharides that inhibit lectin-dependent interactions, and PS.beta2GPI binding experiments using deglycosylated beta2GPI, suggested that carbohydrate residues were not required for macrophage recognition of the complex. Antibodies to putative macrophage PS receptors (CD36, CD68, and CD14) did not inhibit uptake of the complex. These data suggest that beta2GPI can bind cells that fail to maintain membrane lipid asymmetry and generate a specific bridging moiety that is recognized for clearance by a phagocyte receptor that is distinct from CD36, CD68, and CD14.