Immune Clearance of Phosphatidylserine-expressing Cells by Phagocytes

Abstract

The function of β2-glycoprotein I (β2GPI), a 50-kDa serum glycoprotein, is not completely understood but has been suggested to be involved in the regulation of thrombosis (Brighton, T. A., Hogg, P. J., Dai, Y.-P., Murray, B. H., Choing, B. H., and Chesterman, C. N. (1996) Br. J. Haematol. 93, 185–194) and the clearance of phosphatidylserine (PS)-expressing cells (Chonn, A., Semple S. C., and Cullis P. R. (1995)J. Biol. Chem. 270, 25845–25849). To further understand the role of this protein, we characterized the ability of β2GPI to interact with PS vesicles and influence their uptake by macrophages in vitro. β2GPI bound to and precipitated vesicles containing anionic but not zwitterionic phospholipids in a gel diffusion assay. β2GPI also inhibited the procoagulant activity of PS liposomes. In vitro phagocytosis studies showed 20-fold greater uptake of PS liposomes over phosphatidylcholine liposomes. This enhanced uptake was maintained even after PS was “shielded” with β2GPI and further increased upon the addition of β2GPI antibodies. Similar to liposomes, PS-expressing apoptotic thymocytes and lipid symmetric red blood cell ghosts bound β2GPI. Macrophage uptake of these cells was also maintained or enhanced in the presence of β2GPI and further increased upon the addition of β2GPI antibodies. It is concluded that β2GPI can play a critical role in hemostasis by influencing both thrombosis and the clearance of PS-expressing cells. The function of β2-glycoprotein I (β2GPI), a 50-kDa serum glycoprotein, is not completely understood but has been suggested to be involved in the regulation of thrombosis (Brighton, T. A., Hogg, P. J., Dai, Y.-P., Murray, B. H., Choing, B. H., and Chesterman, C. N. (1996) Br. J. Haematol. 93, 185–194) and the clearance of phosphatidylserine (PS)-expressing cells (Chonn, A., Semple S. C., and Cullis P. R. (1995)J. Biol. Chem. 270, 25845–25849). To further understand the role of this protein, we characterized the ability of β2GPI to interact with PS vesicles and influence their uptake by macrophages in vitro. β2GPI bound to and precipitated vesicles containing anionic but not zwitterionic phospholipids in a gel diffusion assay. β2GPI also inhibited the procoagulant activity of PS liposomes. In vitro phagocytosis studies showed 20-fold greater uptake of PS liposomes over phosphatidylcholine liposomes. This enhanced uptake was maintained even after PS was “shielded” with β2GPI and further increased upon the addition of β2GPI antibodies. Similar to liposomes, PS-expressing apoptotic thymocytes and lipid symmetric red blood cell ghosts bound β2GPI. Macrophage uptake of these cells was also maintained or enhanced in the presence of β2GPI and further increased upon the addition of β2GPI antibodies. It is concluded that β2GPI can play a critical role in hemostasis by influencing both thrombosis and the clearance of PS-expressing cells. The outer leaflet of eukaryotic cell membranes contains most of the cholinephospholipids, whereas the aminophospholipids are mainly present in the inner leaflet of the cell (1Verkleij A.J. Zwaal R.F.A. Roelofsen B. Comfurius P. Kastelijn D. van Deenen L.L.M. Biochim. Biophys. Acta. 1973; 323: 178-193Crossref PubMed Scopus (807) Google Scholar). While asymmetry seems to be the rule for normal cells, loss of membrane lipid sidedness, in particular the emergence of phosphatidylserine (PS) 1The abbreviations used are: PS, phosphatidylserine; β2GPI, β2-glycoprotein I; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; MLV, multilamellar vesicles; RVV, Russel's viper venom; PA, phosphatidic acid; PC, phosphatidylcholine; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; ELISA, enzyme-linked immunosorbent assay. 1The abbreviations used are: PS, phosphatidylserine; β2GPI, β2-glycoprotein I; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; MLV, multilamellar vesicles; RVV, Russel's viper venom; PA, phosphatidic acid; PC, phosphatidylcholine; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; ELISA, enzyme-linked immunosorbent assay. at the cell surface, results in the expression of altered surface properties that modulate cell function and influence the interaction of the cell with its environment (2Zwaal R.F.A. Schroit A.J. Blood. 1997; 89: 1121-1132Crossref PubMed Google Scholar). For example, the exposure of PS promotes coagulation and thrombosis by platelets (3Zwaal R.F.A. Comfurius P. Bevers E.M. Biochem. Soc. Trans. 1993; 21: 248-253Crossref PubMed Scopus (106) Google Scholar) and seems to be involved in the recognition and elimination of apoptotic (4Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216PubMed Google Scholar) and senescent cells (5Connor J. Pak C.C. Schroit A.J. J. Biol. Chem. 1994; 269: 2399-2404Abstract Full Text PDF PubMed Google Scholar). Several mechanisms might be responsible for the clearance of PS-expressing cells by phagocytes. These include direct recognition via specific PS receptors (4Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216PubMed Google Scholar, 6Ramprasad M.P. Fischer W. Witztum J.L. Sambrano G.R. Quehenberger O. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9580-9584Crossref PubMed Scopus (296) Google Scholar, 7Sambrano G.R. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1396-1400Crossref PubMed Scopus (271) Google Scholar) and class B scavenger receptors (8Rigotti A. Acton S.L Krieger M. J. Biol. Chem. 1995; 270: 16221-16224Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 9Ren Y. Silverstein R.L. Allen J. Savill J. J. Exp. Med. 1995; 181: 1857-1862Crossref PubMed Scopus (346) Google Scholar, 10Fukasawa M. Adachi H. Hirota K. Tsujimoto M. Arai H. Inoue K. Exp. Cell Res. 1996; 222: 246-250Crossref PubMed Scopus (136) Google Scholar) or indirect mechanisms that might include thrombospondin-dependent vitronectin receptors (11Savill J. Fadok V. Henson P. Haslett C. Immunol. Today. 1993; 14: 131-136Abstract Full Text PDF PubMed Scopus (981) Google Scholar) and antibody-dependent recognition via Fc receptors. Recent studies have raised the possibility that autologous serum proteins might bind PS-expressing cells. Indeed, evidence has been presented suggesting that at least one of these proteins, β2-glycoprotein I (β2GPI), could play a critical role in regulating platelet-dependent thrombosis (12Brighton T.A. Hogg P.J. Dai Y.-P. Murray B.H. Choing B.H. Chesterman C.N. Br. J. Haematol. 1996; 93: 185-194Crossref PubMed Scopus (89) Google Scholar) and clearance of liposomes containing negatively charged lipids (13Chonn A. Semple S.C. Cullis P.R. J. Biol Chem. 1995; 270: 25845-25849Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Evidence is accumulating which suggests that the majority of so-called antiphospholipid antibodies present in the serum of patients with a variety of autoimmune disorders are in fact directed to plasma proteins (β2GPI, prothrombin, and protein C) that bind lipids (14Galli M. Comfurius P. Maassen C. Hemker H.C. De Baets M.H. van Breda-Vriesman Barbui T. Zwaal R.F.A. Bevers E.M. Lancet. 1990; 335: 1544-1547Abstract PubMed Scopus (1285) Google Scholar, 15McNeil H.P. Simpson R.J. Chesterman C.N. Krilis S.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4120-4124Crossref PubMed Scopus (1622) Google Scholar, 16OoSting J.D. Derksen R.H.W.M. Bobbink I.W.G. Hackeng T.M. Bouma B.N. de Groot P.G. Blood. 1993; 81: 2618-2625Crossref PubMed Google Scholar, 17Roubey R.A.S. Blood. 1994; 84: 2854-2867Crossref PubMed Google Scholar, 18Cines D.B. McCrae K.R. J. Clin. Immunol. 1995; 15: 86-100Crossref Scopus (41) Google Scholar). Interestingly, while deep venous thrombosis is characteristic of antiphospholipid syndrome, in vitro diagnostic tests of the serum of the patient exhibit prolonged clotting times. This apparent paradox suggests that the interaction of these complexesin vivo are complicated and may be mediated via Fc receptor-dependent binding to circulating cells. This report focuses on the potential role of β2GPI in the recognition and clearance of antibody-β2GPI-cell complexes using PS-containing liposomes, red blood cell ghosts, and apoptotic thymocytes as models. Our findings support the notion that β2GPI can regulate the interaction of PS-expressing cells with macrophages. Phospholipids were purchased from Avanti Polar Lipids (Birmingham, AL). Methylprednisolone and fluorescein isothiocyanate (FITC) were from Sigma. Carrier-free125I-labeled PE was synthesized as described previously (19Schroit A.J. Madsen J. Biochemistry. 1983; 22: 3617-3623Crossref PubMed Scopus (16) Google Scholar). β2GPI was purified from pooled human plasma (Gulf Coast Regional Blood Center, Houston, TX) by perchloric acid precipitation, ion exchange chromatograhy, and heparin affinity chromatography as described previously (20Poltz E. Kostner G.M. FEBS Lett. 1979; 102: 183-186Crossref PubMed Scopus (191) Google Scholar, 21Wurm H.J. Int. J. Biochem. 1984; 16: 511-515Crossref PubMed Scopus (216) Google Scholar). Human serum albumin (25% USP) was from Alpha Therapeutics (Los Angeles, CA). Antibodies were produced in rabbits by injecting ∼0.5 mg of purified β2GPI in complete Freund's adjuvant at multiple intradermal sites, followed by two boosters (0.25 mg of protein) at 2-week intervals in incomplete Freund's adjuvant. The rabbits were bled 2 weeks after the last injection. IgG was purified from the immune serum by protein A affinity chromatography. Purified anti-β2GPI IgG was labeled with FITC using standard procedures (protein/FITC:1/4). Macrophages were obtained from the peritoneal cavity of female C3H mice 4 days after an intraperitoneal injection of 2 ml of thioglycollate broth. The cells were washed, resuspended in minimal essential medium containing 5% fetal bovine serum, and plated in 24-well tissue culture plates (Costar) (0.5 × 106 cells/well). Resealed, lipid symmetric red blood cell ghosts were prepared from 125I-labeled red blood cells (IODO-BEADs, Pierce) as described previously (22Connor J. Gillum K. Schroit A.J. Biochim. Biophys. Acta. 1990; 1025: 82-86Crossref PubMed Scopus (73) Google Scholar). Thymocytes were obtained from BALB/c mice. Multilamellar vesicles (MLV) and small lipid vesicles containing 125I-PE (1 μCi/ml) were prepared from the indicated lipids (1 mg/ml) by vortexing and sonication, respectively. Flow cytometry was done with a Coulter Epics Profile flow cytometer employing EPICS elite software. The binding specificity of β2GPI to phospholipids was monitored by ELISA using lipid-coated polystyrene plates and by gel diffusion using small sonicated vesicles. Polystyrene ELISA plates were coated overnight with 6 μg of the indicated phospholipids (60 μg/ml in ethanol). The plates were then blocked with 1% ovalbumin in Tris-buffered saline (TBS; 20 mm Tris, 150 mm sodium chloride, pH 7.4) for 60 min and incubated for 60 min (20 °C) with serial dilutions of β2GPI. After removing unbound β2GPI, the plates were incubated for an additional 60 min with 100 μl of anti-β2GPI (0.5 μg of IgG/well). The plates were then washed, and bound antibody was quantified with horseradish peroxidase-conjugated goat anti-rabbit IgG. TMB-ELISA (3,3′-5,5′-tetramethylbenzidine base, Life Technologies, Inc.) was used as the substrate. In the gel diffusion, microscope slides were coated with 0.9% agarose in 10 mm Tris-HCl, pH 7.4, to yield a punch hole volume of 20 μl. The center holes were filled with β2GPI (300 μg/ml) and the surrounding wells with small unilamellar vesicles of the indicated lipid composition. The plates were developed for 24 h, and unbound protein and lipid was removed by washing for 24 h in the same buffer. The gels were then dried. Precipitates containing protein and lipid were detected by staining with Coomassie Blue and autoradiography, respectively. Platelet-poor plasma was prepared by high speed centrifugation of human blood (collected from normal volunteers in acid citrate dextrose). Plasma was pretreated with Russel's viper venom (RVV) (50 μg/ml) for 2 h at 20 °C (Sigma). PC/PS (1/1) MLV (1 mg lipid/ml) were incubated with the indicated amounts of β2GPI or human serum albumin overnight. Clotting time was determined by continuously mixing MLV (20 μl), activated plasma (25 μl), and 25 mmCaCl2 (15 μl) with a 50-μl Eppendorf pipette until the suspension could no longer be aspirated. Control clotting times using protein-free PC/PS MLV was 23 s. PC MLV did not promote clotting. Red blood cells, red blood cell ghosts, or thymocytes were resuspended in TBS containing 1 mm EDTA (TBS-EDTA) and incubated with β2GPI (16 μg/ml) and fluorescent rabbit anti-β2GPI IgG (50 μg/ml) for 30 min at 20 °C. Unbound β2GPI and antibody were removed by washing. Labeled ghosts and cells were photographed usingepi-fluorescence illumination with appropriate excitation and emission filters. Thymocytes (5 × 107 cells/ml) in RPMI 1640 medium were incubated with 10 μm methylprednisolone (diluted from a 10 mm stock in Me2SO) at 37 °C. Control thymocytes were incubated with identical concentrations of Me2SO alone. At the indicated times, aliquots were stained with FITC-anti-β2GPI as described above and analyzed by flow cytometry. Forward and side angle light scatter were set to eliminate dead cells. Fluorescence channels were set logarithmically. 125I-Labeled liposomes, -red blood cells, -red blood cell ghosts, and 51Cr-labeled thymocytes were incubated with β2GPI and anti-β2GPI as described above. The macrophages were washed, resuspended in TBS, and incubated with the targets (vesicles or cells) for the indicated time. The cultures were then washed and uptake was determined by scintillation counting. Western blot analysis with antibodies produced against β2GPI reacted specifically with the antigen when tested against whole human serum (Fig. 1, lanes 3 and4). The high molecular weight band stained in whole serum (lane 3) is multimeric β2GPI because it did not stain with the anti-rabbit IgG alone and resolved to a 50-kDa band upon reduction with 2-mercaptoethanol (not shown). Several assays were developed to test the reactivity of the protein to phospholipids. Fig.2 shows the binding of soluble β2GPI to lipid-coated ELISA plates. It can be seen that the binding of β2GPI to negatively charged lipids could be detected at protein concentrations <10 ng/ml. Binding to PC and PE was weak.Figure 2Binding of β2GPI to phospholipids in a modified ELISA. Serial dilutions of β2GPI (from 500 ng/ml) were incubated with the indicated lipid-coated plates as described under “Experimental Procedures.” β2GPI not bound to the wells was removed by washing, and bound protein was quantitated with rabbit anti-β2GPI IgG. ▴, PC; ▾, PE; •, PS; ○, PG; ▪, CL; □, PA.View Large Image analysis of β2GPI has that the protein contains a binding A. C. A.J. Biochem. J. PubMed Scopus (173) Google Scholar, J. Krilis S. J. Immunol. 1994; Google suggesting that are studies have that β2GPI suggesting a interaction Biochim. Biophys. Acta. 1979; PubMed Scopus Google Scholar). To test the ability of β2GPI to lipid vesicles in a gel diffusion was shows of vesicles containing amounts of PS in PC (Fig. A and liposomes containing PS, PG, PA, and PE in PC (Fig. and of the by protein staining and showed that the were of 125I-labeled lipids and protein, that the were the of anionic The of β2GPI on the procoagulant activity of PS-containing vesicles was determined by the shows that β2GPI concentrations in in clotting times. Human serum albumin as a even at a β2GPI was the binding of β2GPI to PS seems to for the of the plasma coagulation on the These results that β2GPI can the lipids from β2GPI PS-containing vesicles in H.J. Int. J. Biochem. 1984; 16: 511-515Crossref PubMed Scopus (216) Google Scholar, Y. Y. H. T. J. Biochem. 1995; PubMed Scopus Google Scholar) and in vivo (13Chonn A. Semple S.C. Cullis P.R. J. Biol Chem. 1995; 270: 25845-25849Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar) and their clearance from the (13Chonn A. Semple S.C. Cullis P.R. J. Biol Chem. 1995; 270: 25845-25849Abstract Full Text Full Text PDF PubMed Scopus (173) Google it seems to that this protein might as a specific for the recognition of PS-expressing cells. To the influence of β2GPI on the uptake of PS-containing liposomes and PS-expressing lipid symmetric red blood cell ghosts was The presented in that uptake of and PS liposomes were the the addition of β2GPI antibodies increased the uptake of PC liposomes with β2GPI and was these to or no influence of β2GPI on the recognition of PS by it is that the protein and recognition Indeed, showed increased uptake of lipid symmetric red blood cell ghosts (Fig. but not red blood cells suggesting that β2GPI in the interaction of PS-expressing targets with of lipid symmetric red blood cell ghosts by macrophages. 125I-labeled red blood cell ghosts were incubated with for the indicated time at 37 °C. The plates were then washed, and uptake was quantified by scintillation counting. of and red blood cell and fluorescent of a blood cell the specificity of β2GPI binding to PS-expressing red blood cell that red blood cells are not fluorescent of FITC Large Image of 2 and 2 antibodies on the uptake of lipid symmetric red blood cell ghosts and red blood are as uptake of blood rabbit IgG symmetric 125I-labeled red blood cell ghosts and lipid 125I-labeled red blood cells were incubated with β2GPI, or both as described under “Experimental Procedures.” was after 1 h of with at 37 are as uptake of cells. in a symmetric 125I-labeled red blood cell ghosts and lipid 125I-labeled red blood cells were incubated with β2GPI, or both as described under “Experimental Procedures.” was after 1 h of with at 37 °C. studies have that the exposure of PS to the outer leaflet is one of the characteristic of cells Blood. 1994; 84: PubMed Google Scholar). β2GPI is an anionic protein in it is that it the recognition and uptake of apoptotic cells. To test thymocytes were to with and the exposure of PS was quantified by analysis of β2GPI and cells. shows that cultures incubated with (Fig. but not cultures developed a of cells that stained with for the fluorescent cell increased with time (Fig. of the cells by showed of fluorescent cells (Fig. and cells were not To β2GPI the binding and uptake of these cells by thymocytes were to with β2GPI alone or β2GPI and and monitored for shows that of apoptotic thymocytes with β2GPI in in uptake Similar to the results obtained with PS liposomes and red cell ghosts, cells with β2GPI and β2GPI antibodies in of cultures incubated with apoptotic thymocytes (Fig. showed macrophages with bound fluorescent of thymocytes by macrophages. 51Cr-labeled thymocytes were incubated with cultures for 1 h at 37 °C. The plates were then washed, and uptake was quantified by scintillation counting. the of apoptotic cells bound by and fluorescent of FITC apoptotic Large Image Our that β2GPI vesicles containing negatively charged phospholipids in a gel diffusion (Fig. 3) the possibility that are binding were not these could be by binding of or by β2GPI The that both I Y. Y. H. T. J. Biochem. 1995; PubMed Scopus Google Scholar) and J. Krilis S. J. Immunol. 1994; Google Scholar) of β2GPI bind of that are the in vivo function of β2GPI has not been in it is to interact with plasma proteins (20Poltz E. Kostner G.M. FEBS Lett. 1979; 102: 183-186Crossref PubMed Scopus (191) Google Scholar) and play a role in regulating coagulation (12Brighton T.A. Hogg P.J. Dai Y.-P. Murray B.H. Choing B.H. Chesterman C.N. Br. J. Haematol. 1996; 93: 185-194Crossref PubMed Scopus (89) Google Scholar) and immune clearance (13Chonn A. Semple S.C. Cullis P.R. J. Biol Chem. 1995; 270: 25845-25849Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Recent studies have that liposomes containing negatively charged phospholipids bind β2GPI, a that with the clearance of the vesicles. (13Chonn A. Semple S.C. Cullis P.R. J. Biol Chem. 1995; 270: 25845-25849Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). In this we the interaction of β2GPI with PS-expressing liposomes, red blood cell ghosts, and cells and determined their to interact with macrophages. β2GPI clotting (Fig. 4 and J. Bevers E.M. U. H. Kostner G.M. Zwaal R.F.A. Biochim. Biophys. Acta. PubMed Scopus Google it is that its binding to anionic the binding of this to be the β2GPI be to the uptake of anionic lipid vesicles by macrophages. The that uptake was or even enhanced in the of PS-expressing red blood cell ghosts, suggests that uptake on direct These results the possibility that the expression of PS on cells to that the cells interaction with its the of lipids in the cells plasma membrane that results in the surface expression of PS Blood. 1994; 84: PubMed Google we in thymocytes and determined β2GPI to PS-expressing apoptotic cells and the influence of bound β2GPI to the recognition of these cells by macrophages. an of the cells bound fluorescent β2GPI antibodies (Fig. increased expression of β2GPI binding at the cell cells with β2GPI and antibody enhanced Similar to PS liposomes, of apoptotic cells with β2GPI alone did not this could also be the of of PS recognition The results presented with on the clearance of negatively charged liposomes (13Chonn A. Semple S.C. Cullis P.R. J. Biol Chem. 1995; 270: 25845-25849Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar) that β2GPI may be one of the for the recognition of PS-expressing cells in this interaction is further mediated by the binding affinity of β2GPI to its lipid G.M. M.P. Comfurius P. M. Zwaal R.F.A. Bevers E.M. Biochemistry. 1996; PubMed Scopus Google Scholar). This suggests that antibody could influence the interaction of PS-expressing cells with the Indeed, is a of evidence that antibodies to β2GPI complexes is the of antiphospholipid (14Galli M. Comfurius P. Maassen C. Hemker H.C. De Baets M.H. van Breda-Vriesman Barbui T. Zwaal R.F.A. Bevers E.M. Lancet. 1990; 335: 1544-1547Abstract PubMed Scopus (1285) Google Scholar, 15McNeil H.P. Simpson R.J. Chesterman C.N. Krilis S.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4120-4124Crossref PubMed Scopus (1622) Google R.A.S. R. J. Immunol. 1995; Google Scholar). This the possibility that enhanced thrombosis in autoimmune disorders may be the of cell and by antibody-dependent binding of to seen in 6 and to the A has been suggested for autoimmune against proteins that bind PE T. 1996; 84: Google Scholar). In it that β2GPI not thrombosis but is involved in the recognition and clearance of cells that to membrane lipid This include not cells but also as of platelets and cell C. for critical analysis and and for