Auto-Oxidation and Oligomerization of S on the Apoptotic Cell Surface Is Required for Mer -Mediated Phagocytosis of Apoptotic Cells This information is current as of October 2, 2021. Hiroshi Uehara and Emily Shacter J Immunol 2008; 180:2522-2530; ; doi: 10.4049/jimmunol.180.4.2522 http://www.jimmunol.org/content/180/4/2522 Downloaded from

References This article cites 53 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/180/4/2522.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

by guest on October 2, 2021 *average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Auto-Oxidation and Oligomerization of on the Apoptotic Cell Surface Is Required for Mer Tyrosine Kinase-Mediated Phagocytosis of Apoptotic Cells

Hiroshi Uehara and Emily Shacter1

Prompt phagocytosis of apoptotic cells prevents inflammatory and autoimmune responses to dying cells. We have previously shown that the blood anticoagulant factor protein S stimulates phagocytosis of apoptotic human B lymphoma cells by human monocyte-derived macrophages. In this study, we show that protein S must first undergo oxidative activation to stimulate phagocytosis. Binding of human protein S to apoptotic cells or to phosphatidylserine multilamellar vesicles promotes auto- oxidation of Cys residues in protein S, resulting in covalent, disulfide-linked dimers and oligomers that preferentially bind to and activate the human Mer tyrosine kinase (MerTK) receptor on the macrophages. The prophagocytic activity of protein

S is eliminated when disulfide-mediated oligomerization is prevented, or when MerTK is blocked with neutralizing Abs. Downloaded from Protein S oligomerization is independent of phospholipid oxidation. The data suggest that membranes containing phospha- tidylserine serve as a scaffold for protein S-protein S interactions and that the resulting auto-oxidation and oligomerization is required for the prophagocytic activity of protein S. In this way, apoptotic cells facilitate their own uptake by macro- phages. The requirement for oxidative modification of protein S can explain why this abundant blood protein does not constitutively activate MerTK in circulating monocytes and tissue macrophages. The Journal of Immunology, 2008, 180:

2522–2530. http://www.jimmunol.org/

poptosis is a physiological form of cell death that plays outer leaflet of the plasma membrane (10, 11). The PS exposure is an essential role in tissue and organ development and thought to be essential for the recognition and uptake of apoptotic A homeostasis in multicellular organisms. Rapid removal cells by phagocytes (12–14). of apoptotic cells by macrophages and certain types of neighboring Several different macrophage receptors have been identified as cells before the loss of plasma membrane integrity prevents the being involved in the phagocytosis of apoptotic cells (reviewed in leakage of potentially toxic and immunogenic cellular contents and Ref. 15). These include a PS receptor, integrins, CD14, CD36, and thereby prevents inflammation and autoimmune responses to cell receptors for oxidized low-density lipoprotein. One key macro- death (1, 2). This is thought to be one of the main physiological phage receptor is Mer tyrosine kinase (MerTK), which is a mem- by guest on October 2, 2021 advantages of death through apoptosis instead of necrosis, in ber of the TAM family of receptor tyrosine kinases (16). It is which cellular macromolecules may leak out and stimulate an in- expressed on epithelial cells and monocytic cells (17), both of flammatory response before removal of the cells from the tissue (3, which have phagocytic activity. The essential role of MerTK has 4). Engulfment of apoptotic cells by macrophages also triggers been demonstrated for the phagocytosis of photoreceptor outer production of anti-inflammatory and immunosuppressive cyto- segment cells by retinal pigment epithelium cells (18, 19). kines, further limiting an immune response (5, 6). Studies using In addition to these phagocyte surface molecules, a number of knockout mice have shown an association between autoim- soluble molecules have been identified that may control the inter- mune disease and abnormal clearance of apoptotic cells (7, 8). action between apoptotic cells and phagocytes (2). These include The removal of apoptotic cells involves apoptotic cell surface protein S, MFG-E8, , C1q, mannose-binding lectin, throm- ␤ molecules, phagocyte receptors, and soluble factors that modulate bospondin, pentraxin, 2-glycoprotein I, and surfactant A cell recognition and uptake (1, 9). Upon induction of apoptosis, and D. Opsonization of target cells with bridging molecules may cells lose the phospholipid asymmetry normally found in the expand the repertoire of potential phagocytic targets by allowing plasma membrane and expose phosphatidylserine (PS)2 on the classical phagocyte receptors that are customarily associated with infection and immunity to mediate recognition and uptake of ap- optotic cells and thereby limit the undesirable side effects from Laboratory of Biochemistry, Division of Therapeutic Proteins, Center for Drug Eval- uation and Research, Food and Drug Administration, Bethesda, MD 20892 exposure to dead cell debris (20, 21). We and others showed that the anticoagulant factor protein S is Received for publication June 28, 2007. Accepted for publication November 30, 2007. required for the efficient uptake of apoptotic lymphoma cells by The costs of publication of this article were defrayed in part by the payment of page macrophages in vitro (22, 23). Protein S, originally defined as an charges. This article must therefore be hereby marked advertisement in accordance anticoagulant protein that is a nonenzymatic for activated with 18 U.S.C. Section 1734 solely to indicate this fact. protein C, is a vitamin K-dependent, 68-kDa monomeric protein 1 Address correspondence and reprint requests to Dr. Emily Shacter, Center for Drug present at a concentration of ϳ25 ␮g/ml in the blood (24, 25). It Evaluation and Research, Food and Drug Administration, 29 Lincoln Drive, Building 29A, Room 2A-11, HFD-121, Bethesda, MD 20892-4555. E-mail address: [email protected] 2 Abbreviations used in this paper: PS, phosphatidylserine; MerTK, Mer tyrosine 2-methyl-propaimidaamide; (Ϯ)9-HODE, (Ϯ)-9-hydroxy-10E,12Z-octadecadienoic kinase; CFDA, carboxyfluorescein diacetate, succinimidyl ester mixed isomer; PI, acid; IAA, iodoacetamide; NEM, N-ethylmaleimide; TnCl, taurine chloramine; sMer, propidium iodide; PC, phosphatidylcholine; DOPS, dioleoyl PS; DOPC, dioleoyl PC; soluble Mer; LMV, large multilamellar vesicle; oxPLPS, oxidized PLPS; LDS, lith- PLPS, 1-palmitoyl-2-linoleoyl-sn-glycero-3-[phospho-L-serine]; AAPH, 2.2Ј-azobis- ium dodecyl sulfate. www.jimmunol.org The Journal of Immunology 2523

ϩ is known to bind to PS on cell surfaces in a Ca2 -dependent man- 30–50% apoptosis as assessed by two different techniques: Hoechst 33342 ner using a Gla domain at the N terminus of the protein. Aberrantly and PI staining followed by nuclear morphology assessment using fluo- low levels of protein S may lead to inefficient uptake of early rescence microscopy (4) or by two-color flow cytometry using a FACScan (BD Biosciences) following labeling of cells with FITC-annexin V in apoptotic cells and expose immune cells to potentially immuno- binding buffer (10 mM HEPES, 0.14 M NaCl, 5 mM KCl, 2.5 mM

genic cellular contents and thus trigger an autoimmune response CaCl2, 1 mM MgCl2 (pH 7.4)) followed by addition of PI as described (26). Deficiencies in protein S, either hereditary or acquired previously (13). through autoantibody formation, lead to excess thrombosis (27) Modification of protein S with iodoacetamide (IAA), and are associated with autoimmune diseases such as systemic N-ethylmaleimide (NEM), or taurine chloramine (TnCl) lupus erythematosus (28, 29). The newly discovered role of protein S in stimulating the phagocytosis of apoptotic cells re- Protein S was covalently modified by incubating with 100 mM IAA or 10 mM NEM in PBS for 30 min at room temperature. The reaction was veals a novel functional link between the coagulation system stopped by passing the solution through a Sephadex G-25 desalting col- and autoimmunity (26). umn. TnCl was prepared by mixing 10 mM taurine with 2.5 mM HOCl, In this report, we investigated the biochemical and molecular which results in immediate and quantitative consumption of all of the mechanisms for stimulation of phagocytosis by protein S. We HOCl into TnCl (30). This solution was diluted to 0.5 mM TnCl in PBS containing 1.5 mM CaCl and incubated with protein S alone (0.25 mg/ml) or demonstrate that in order for protein S to serve as a prophagocytic 2 protein S bound to PS vesicles at 25°C for 1 h. The sulfhydryl-specific cross- molecule, it must bind to membranous PS, either on the surface of linking of protein S was performed in PBS containing 0.5 mM BM(PEO)2 apoptotic cells or in artificial phospholipids vesicles. Protein S then under the same condition as described for oxidation with TnCl. The reactions undergoes oligomerization through oxidation of cysteine residues were stopped by desalting with Sephadex G-25. and formation of intermolecular disulfide bonds. This oxidative

Preparation of phospholipid vesicles Downloaded from activation of the protein is required for binding to and activation the macrophage MerTK. Thus, under physiological conditions, Large multilamellar vesicles (LMV) were prepared by mixing phosphati- dylcholine (PC), PS, and rhodamine-labeled phosphatidylethanolamine in protein S activates MerTK and stimulates macrophage phago- chloroform at a 70:30:0.5 molar ratio, or by mixing PC and rhodamine- cytic activity only in the presence of apoptotic cells, thereby labeled phosphatidylethanolamine at a 100:0.5 molar ratio, drying under a preventing circulating protein S from randomly activating mac- stream of N2 gas, and resuspending in PBS at a concentration of 5 mM by rophage activity. vigorous vortexing. DOPS and DOPC were used in all phospholipid vesicle

experiments unless otherwise indicated. http://www.jimmunol.org/ Oxidation of phospholipids was performed by incubation with 2 mM Materials and Methods AAPH. Phospholipid-LMV prepared as described above were suspended in Reagents and Abs PBS at 5 mM and incubated with 2 mM AAPH for 3–4 h at 37°C. Lipids

were extracted with chloroform:methanol (2:1, v/v), dried under N2 gas, The following reagents were purchased from the vendors indicated. and resuspended in PBS. Lipid oxidation was assessed by quantifying con- FBS, RPMI 1640, DMEM, HBSS, and PBS were from Mediatech. jugated dienes from the secondary derivative of the UV spectrum around FITC-conjugated annexin V and PE-conjugated annexin V were ob- 230 nm using (Ϯ)9-HODE as a standard (31). Approximately 15–18% of tained from BD Biosciences. Human protein S was obtained from En- PLPS fatty acid chains were estimated to be oxidized by the AAPH treat- zyme Research Laboratories. R&D Systems provided recombinant mu- ment, which is similar to the level achieved by others (32) while no con- rine gas6 protein (C-terminal 559-aa residues linked to an N-terminal jugated dienes were detected in DOPC or DOPS treated with AAPH.

hexameric His tag) and recombinant human Mer-Fc, which is a chi- by guest on October 2, 2021 meric recombinant protein containing the extracellular domain of Phagocytosis assay MerTK fused to the Fc region of human IgG. Protein assay reagent was obtained from Bio-Rad. Protein G Sepharose beads, ECL chemilumi- Phagocytosis was quantified as described previously (13). Briefly, human nescence reagent, and protease inhibitor mixture were obtained from mononuclear cells were obtained by elutriation of human peripheral blood GE Healthcare. Precast 4–12% Bis-Tris NuPAGE polyacrylamide gels, and cultured for 4–7 days in RPMI 1640 with 10% heat-inactivated FBS 6 carboxyfluorescein diacetate, succinimidyl ester mixed isomer (CFDA), and 100 ng/ml recombinant M-CSF at a cell density of 0.5–0.8 ϫ 10 propidium iodide (PI), Hoechst 33342, and Prolong anti-fade solution cells/well in 24-well culture plates. For phagocytosis of apoptotic cells, were obtained from Invitrogen Life Technologies. Phospholipids 1,2- BL-41 cells were labeled with 0.12 ␮g/ml CFDA in PBS for 20 min at dioleoyl-sn-glycero-3-[phospho-L-serine] (dioleoyl phosphatidylserine; 37°C before apoptosis induction. The CFDA-labeled cells were treated DOPS), 1,2-dioleoyl-sn-glycero-3-phosphocholine (dioleoyl phosphati- with 200 ␮g/ml etoposide to induce apoptosis. After washing with DMEM, ϳ ϫ 6 dylcholine; DOPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-[phospho-L- 1 10 cells in 0.5 ml of DMEM were incubated with adherent mac- serine] (PLPS), phosphatidylethanolamine-N-(lissamine rhodamine B rophages (ϳ2:1 target cells: macrophages) at 37°C for 1 h. The wells were sulfonyl) (rhodamine-labeled phosphatidylethanolamine) were obtained washed with HBSS to remove free target cells and then the macrophages from Avanti Polar Lipids. (Ϯ)-9-hydroxy-10E,12Z-octadecadienoic were labeled with PE -conjugated anti-CD11b and -CD14 in binding buffer acid [(Ϯ)9-HODE] and 2.2Ј-azobis-2-methyl-propaimidaamide (AAPH) were for 30 min at 4°C. Macrophages were recovered by incubating with 1% lidocaine in PBS containing 0.5% FBS, fixed with neutralized 2.5% form- obtained from Cayman Chemical. BM(PEO)2, a sulfhydryl specific homobi- functional cross-linker, was obtained from Pierce. All other chemicals were aldehyde in PBS, and quantified by two-color FACScan analysis. The cells obtained from Sigma-Aldrich. positive for both PE and CFDA are scored as macrophages that had in- Ab sources were: rabbit anti-human protein S and HRP-conjugated sec- gested target apoptotic cells. The phagocytic index (percent) was calculated ondary Abs were obtained from DakoCytomation; anti-phosphotyrosine as the percentage of PE-positive macrophages that are also CFDA positive. (clone 4G10) was obtained from Millipore; PE-conjugated anti-CD11b For most experiments, the data were expressed as fold increase in the and -CD14 were obtained from BD Biosciences; goat anti-human phagocytic index compared with the control. MerTK and goat anti-human Axl were obtained from R&D Systems. To measure uptake of rhodamine-labeled LMV, the macrophages were Rabbit anti-human phospho-MerTK (p-MerTK), specific to MerTK pro- labeled with 0.12 ng/ml CFDA in PBS for 20 min at 37°C before incu- tein phosphorylated on tyrosine residues 749 and 753, and rabbit anti- bating with 0.1–0.2 mM LMV at 37°C for 90 min. The macrophages were Tyro3 were obtained from FabGennix. The specificity and use of the collected as described above. CFDA-positive and rhodamine-positive cells anti-Axl and -Tyro3 Abs was addressed using positive controls for re- were scored as macrophages that had ingested LMV and calculated as a ceptor expression in cell lysates, using A431 lysates for Axl and PC12 percent of the total number of macrophages. lysates for Tyro3 expression. Preparation of whole cell lysates and membrane fractions and Cell lines, culture conditions, and apoptosis induction Western blot immunoassay Human BL-41 lymphoma cells were obtained as described previously (4) Whole cell lysates were prepared by lysing cells in a buffer containing 10 and maintained in RPMI 1640 supplemented with 10% heat-inactivated mM HEPES (pH 7.4), 0.5% Triton X-100, 0.5% Nonidet P-40, 0.14 M ␮ FBS, 2 mM L-glutamine, and 50 M 2-ME in a humidified chamber with NaCl, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, and protease inhibitor 5% CO2. To induce apoptosis, BL-41 cells were treated for 3.5–5 h with mixture (Roche Diagnostics) for 20 min on ice. The nuclei were removed 200 ␮g/ml etoposide in growth medium. The treatment typically induced by centrifugation at 14,000 ϫ g for 12 min at 4°C. To isolate membrane 2524 OXIDATION OF PROTEIN S FOR PROPHAGOCYTIC ACTIVITY fractions, the cells were first treated with digitonin buffer (10 mM HEPES

(pH 7.4), 0.01% digitonin, 0.14 M NaCl, 5 mM KCl, 2.5 mM CaCl2,1mM MgCl2, and protease inhibitor mixture for 5 min on ice followed by cen- trifugation at 14,000 ϫ g for 2 min. The supernatant (cytosolic fraction) was removed and the pellet was solubilized with lysis buffer followed by centrifugation to remove nuclei (14,000 ϫ g, 12 min). The supernatant of this fraction was used as a crude membrane fraction. Typically, ϳ20% of total protein was recovered in the membrane fraction. Protein concen- trations were determined using either the Bradford assay or the DC protein assay from Bio-Rad using BSA as a standard. SDS-PAGE was performed using precast 4–12% Bis-Tris gradient polyacrylamide gels and MOPS buffer (Invitrogen Life Technologies). Western blotting to nitrocellulose or polyvinylidene difluoride membranes was performed according to standard protocols. Protein S and Mer-Fc coprecipitation Protein S (50 nM), recombinant Mer-Fc (10 nM) and PS-LMV (0.5 mM) were incubated in 0.2 ml of binding buffer (see above) containing 0.1% BSA. After1honice, 1% ␤-D-octylglucopyranoside was added and the mixture was incubated an additional 20 min on ice. The mixture was cen- trifuged at 13,000 ϫ g for 12 min and then 10 ␮l of protein G-Sepharose beads were added to the supernatants. The suspension was kept at 4°C

overnight with gentle rotation. The beads were rinsed four times with bind- Downloaded from ing buffer with 0.1% Triton X-100 and 0.1% Nonidet P-40. Proteins bound to the beads were eluted by lithium dodecyl sulfate (LDS) loading buffer and separated by SDS-PAGE. Data analysis The FACScan results were analyzed using CellQuest Pro software (BD Biosciences). Most experiments and Western blots were performed at least http://www.jimmunol.org/ three times. Data are shown as the mean Ϯ SD. Statistical significance was assessed by the Student’s t test. Results Protein S stimulates uptake of PS-containing LMV As described previously, phagocytosis of apoptotic BL-41 cells by human monocyte-derived macrophages is enhanced significantly by the presence of FBS in the phagocytosis medium (22). Bio-

chemical fractionation of the FBS and immunodepletion studies by guest on October 2, 2021 revealed that protein S is the only serum component required for the FBS-dependent stimulation of phagocytosis. As shown in Fig. 1, A and B, purified human protein S fully replaces the phagocy- tosis stimulatory activity of the serum. Here, we further investi- gated the role of PS in protein S-stimulated phagocytosis by mea- FIGURE 1. Protein S (proS) stimulates the phagocytosis of apoptotic suring the ability of protein S to stimulate macrophage uptake of BL-41 cells and PS-LMV. FACScan analysis was used to assess phago- rhodamine-labeled LMV composed of PS and PC at a 30:70 ratio cytosis of CFDA-labeled apoptotic cells (detected in FL1) by PE-la- (PS-LMV), or PC alone (PC-LMV) (Fig. 1C). The uptake of LMV beled monocyte-derived macrophages (detected in FL2) that are stained with PE-labeled anti-CD11b and -CD14. Macrophages that have in- occurs primarily through phagocytosis and not through lipo- gested BL-41 cells are positive for both red and green fluorescence some fusion (33). Consistent with the results with apoptotic (upper right quadrant). A, Dot plot of phagocytosis of (a) control cells expressing PS, the addition of protein S increased the healthy BL-41 cells in complete medium (DMEM) containing 10% se- uptake of PS-LMV by 3-fold, resulting in ϳ22% rhodamine- rum, (b) apoptotic cells in plain medium (DMEM), (c) apoptotic cells in positive macrophages. Only 7% of macrophages became rhoda- DMEM containing 10% serum, and (d) apoptotic cells in DMEM plus mine positive in the absence of protein S. No uptake of PC-LMV 8 ␮g/ml human protein S. B, Quantitation of the data shown in A. The was observed, either in the presence or absence of protein S. These results are expressed as the fold increase in phagocytosis compared with results further confirm the essential role of PS in the protein S- the phagocytosis of control healthy cells incubated in the presence of dependent stimulation of phagocytosis of apoptotic cells and phos- 10% serum. Results of two independent experiments performed in du- Ϯ C pholipid vesicles. plicate are shown as mean SD. , Phagocytosis of rhodamine-labeled PS-LMV and PC-LMV in the presence or absence of protein S. The data Protein S stimulates phagocytosis through activation of MerTK show the mean Ϯ SD of three experiments conducted in duplicate. .p Ͻ 0.001 ,ء For protein S to stimulate phagocytosis, it must not only bind to apoptotic cells but must also bind to and activate the phagocytic machinery of macrophages. Previous reports have suggested that lysates. A ϳ160-kDa band corresponding to MerTK was detected protein S, like the homologous protein gas6, is a ligand for the while neither Axl nor Tyro3 proteins were detected under the con- TAM receptor tyrosine kinases such as MerTK and Tyro3 that are ditions used (data not shown). Based on these observations and expressed on monocytes and macrophages (34). gas6 shares 44% recent reports implicating the role of protein S in MerTK-mediated sequence identity with protein S (35). To investigate whether a clearance of outer segment by retinal pigment epithelium (18, 19), TAM tyrosine kinase plays a role in protein S-mediated phagocy- we focused our investigation on MerTK in macrophages. First, the tosis, we examined the level of expression of MerTK, Axl, and role of MerTK in protein S-mediated phagocytosis was assessed Tyro3 in the macrophages by immunoblot analysis of macrophage using a neutralizing Ab to MerTK. As shown in Fig. 2, addition of The Journal of Immunology 2525

FIGURE 2. Blocking of MerTK with a neutralizing anti-MerTK Ab in- hibits protein S-dependent phagocytosis. Phagocytosis of apoptotic cells (AC) was performed in plain DMEM medium plus or minus protein S (2 ␮g/ml) and with or without the addition of goat anti-MerTK Ab (50 ␮g/ml) or normal goat IgG (NGS; 50 ␮g/ml). The anti-MerTK and normal IgG were present throughout the phagocytosis assays. The results are shown as .A statistical significance of p Ͻ 0.05 ,ء .(mean Ϯ SD (n ϭ 3 Downloaded from an anti-MerTK Ab completely inhibited the protein S-induced phagocytosis of apoptotic cells, indicating that MerTK is required for the protein S-dependent recognition and uptake of apoptotic BL-41 cells by macrophages. Because MerTK is known to be ac- tivated by tyrosine autophosphorylation, we investigated the effect of protein S on the activation of MerTK in macrophages coin- http://www.jimmunol.org/ cubated with apoptotic cells by measuring the level of tyrosine phosphorylation in MerTK protein immunoprecipitated from whole cell lysates. As shown in Fig. 3A, MerTK was rapidly tyrosine-phosphorylated after coincubation with apoptotic cells and protein S; phosphorylation was induced in Ͻ7 min and gradually decreased thereafter. Importantly, maximal phosphor- FIGURE 3. Incubation of macrophages with apoptotic cells and protein ylation of MerTK required the presence of both apoptotic cells S results in tyrosine phosphorylation of MerTK. A, Adherent macrophages and protein S; coincubation of macrophages with apoptotic cells were incubated with apoptotic BL-41 cells and protein S (2 ␮g/ml) for the by guest on October 2, 2021 alone or protein S alone resulted in no MerTK phosphorylation times indicated. Whole cell lysates were prepared with Triton X-100 and above the basal level (see blots in Fig. 3B and densitometric Nonidet P-40. Upper panel, Cell lysates immunoprecipitated with goat quantification in Fig. 3C). The results with protein S contrast to anti-MerTK Ab and immunoblotted with a monoclonal anti- phosphotyrosine Ab (lanes 1–5). Lane 5 is a lysate of apoptotic BL-41 those seen with the homologous serum protein gas6, which ac- cells. Lanes 6–8 show macrophage lysates obtained before or after coin- tivated MerTK even in the absence of apoptotic cells. The time cubation with apoptotic cells and immunoprecipitated with normal goat course of MerTK phosphorylation in response to protein S is IgG. Lower panel, A loading control prepared by stripping and reprobing consistent with the view that MerTK phosphorylation/activation the blot in the upper panel with a rabbit anti-MerTK Ab to show the precedes ingestion of the apoptotic cells (which takes ϳ20–30 amounts of total MerTK protein in each lane. B, Macrophage lysates were min; Refs. 4 and 13). prepared after 7 min of incubation with protein S alone, apoptotic cells alone, protein S and apoptotic cells, or recombinant gas6. As in A, immu- Protein S bound to apoptotic cells and to PS vesicles undergoes noprecipitates with anti-MerTK Ab were separated by SDS-PAGE, blotted, oxidative oligomerization and probed with anti-phosphotyrosine or goat anti-MerTK Abs. C, Densi- tometric analysis of the MerTK phosphorylation data shown in B. The The weak ability of protein S to stimulate MerTK activity in the intensity of the MerTK phosphorylation in each sample was normalized to absence of apoptotic cells suggested that protein S might be mod- the intensity of the total MerTK band in that sample and was then calcu- ified and activated in some way by apoptotic cells. To address this lated as the fold increase over the level of MerTK phosphorylation in possibility, we analyzed the SDS-PAGE/Western blot profile of control macrophages. The data are the mean Ϯ SD from three independent membrane-bound protein S following a 30-min incubation with experiments. Statistical significance is for comparisons to control macro- .p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .apoptotic cells. Proteins in the membrane fractions were analyzed phages by Western blot immunoassay using a polyclonal Ab against pro- tein S (Fig. 4A). Under nonreducing conditions, protein S bands at PS-LMV, although with the vesicles the dimer band was pre- ϳ130 and 210–220 kDa were detected in addition to the 68-kDa dominant and the trimer band was only weakly formed (Fig. monomer band. When the disulfide bonds were reduced with DTT, 4B). Because protein S did not bind significantly to PC, very these high molecular mass bands disappeared and two bands at little protein was recovered following incubation and centrifu- 68 and 72 kDa were seen. These match the bands seen with the gation with PC-LMV; only a faint monomer band was detected protein S starting material. Based on the molecular masses and under these conditions. the elimination of the high molecular mass bands by a disulfide The role of free cysteine residues in the formation of protein S bond reducing agents, we conclude that the 130- and 210- to dimers/oligomers on PS surfaces was confirmed by blocking the 220-kDa bands are disulfide-linked dimers and trimers of pro- cysteine residues with IAA, a sulfhydryl-reactive alkylating re- tein S, respectively. Disulfide-linked protein S dimers and tri- agent. The data in Fig. 4C show that IAA treatment of protein S mers were also formed in protein S that had been incubated with prevented dimer formation during incubation with PS vesicles. 2526 OXIDATION OF PROTEIN S FOR PROPHAGOCYTIC ACTIVITY

FIGURE 5. Protein S (proS) oligomerization by oxidized or nonoxi- dized phospholipid vesicles causes preferential binding to Mer-Fc. Western Downloaded from blot immunoassay of protein S coprecipitated with Mer-Fc in the presence or absence of different phospholipid vesicles (PL-LMV). PLPS denotes oxidizable, but unoxidized, palmitoyl linoleoyl PS vesicles; oxPLPS are the same vesicles oxidized with AAPH (see Materials and Methods). DOPS and DOPC are nonoxidizable dioleoyl PS and PC, respectively. SDS-PAGE was run under nonreducing conditions. The 68-kDa band cor-

responds to protein S monomer, which binds nonspecifically to protein G http://www.jimmunol.org/ beads. IAA-modified protein S was used for the sample in lane 9. The data are representative of three independent experiments.

vesicles that induce protein S cysteine oxidation (dimerization) are composed of dioleoyl PS and PC; oleic acid has only one unsat- FIGURE 4. Dimerization/oligomerization of protein S (proS) after urated bond and is extremely difficult to oxidize. This was con- binding to apoptotic cells or phospholipid LMV. A, Analysis of proS in- firmed in our experimental system by exposing DOPS and DOPC cubated with apoptotic BL-41 cells. As described in Materials and Meth- by guest on October 2, 2021 ods, BL-41 cells were induced to undergo apoptosis with etoposide (200 vesicles to the potent lipid oxidant AAPH and measuring forma- ␮g/ml) and then mixed with purified human protein S (8 ␮g/ml) in binding tion of conjugated dienes, which are a good measure of fatty acyl buffer for 30 min on ice. The membrane fraction was isolated and 10 ␮gof lipid peroxidation (31). Almost no conjugated diene formation was this fraction was separated by SDS-PAGE, transferred to nitrocellulose detected in dioleoyl vesicles (data not shown). A role for lipid membranes, and probed with rabbit anti-protein S. AC ϩ proS denotes the oxidation in promoting protein S dimerization was also ruled out membrane fraction from apoptotic cells incubated with protein S. Samples by creating oxidized lipid vesicles containing palmitoyl-linoleoyl in the left two lanes were nonreduced and in the right two lanes were PS (oxPLPS) and palmitoyl-linoleoyl PC; linoleic acid has two reduced with 50 mM DTT. B, Analysis of proS incubated with phospho- unsaturated bonds and is much more susceptible to oxidation than ␮ lipid LMV. proS (8 g/ml) was incubated with 0.5 mM vesicles made from oleic acid. Treatment of theses vesicles with AAPH resulted in PS-LMV or PC-LMV in binding buffer containing 0.1% BSA for1hon roughly 18% of the fatty acid chains being oxidized to conjugated ice. Following centrifugation, bound proteins were eluted with LDS-load- ing buffer and analyzed by Western blot immunoassay with anti-protein S dienes. However, incubation of protein S with oxidized PLPS ves- Ab as described for A. C, Effect of IAA modification on protein S dimer- icles resulted in the same level of protein S dimers as seen with ization induced by PS-LMV. Left panel, Native protein S or IAA-treated nonoxidizable DOPS vesicles (Fig. 4D). In contrast, a significant protein S (proS-IAA) was incubated with PS-LMV. Bound protein S was increase in protein S oligomer formation was observed when isolated and analyzed as described for B. D, Effect of PS oxidation on protein S bound to DOPS-LMV was treated either with the cys- protein S oligomerization. PLPS was oxidized (oxPLPS) with 2 mM teine oxidant TnCl or with the sulfhydryl-specific cross-linker AAPH as described in Materials and Methods. proS was incubated with BM(PEO)2 (Fig. 4E). Both the cross-linker and TnCl-induced either PLPS, oxPLPS, or DOPS and analyzed as described for B above. E, oxidative oligomerization were dependent on the presence of Protein S dimerization/oligomerization induced by chemical oxidation or PS-LMV; little oligomerization was observed when free protein cross-linking. Protein S was incubated with TnCl or the cysteine cross- S in solution was treated with TnCl. linker BM(PEO)2 in the presence or absence of DOPS-LMV as described in Materials and Methods. The data are representative of three independent Protein S dimer/oligomer formation is required for its experiments. MerTK-binding and activation and prophagocytic activity Subsequent studies tested whether the dimerization/oligomeriza- The source of the oxidation responsible for protein S oxidation tion of protein S is a prerequisite for its ability to bind to MerTK was investigated next. Previous studies with CD36 indicated that and stimulate macrophage phagocytic activity. First, the ability of its prophagocytic activity was stimulated by oxidized fatty acyl protein S monomer and oligomers to bind to MerTK was assessed chains in PS and, to a lesser extent, PC (36). The possibility that using recombinant MerTK that had been coupled to the Fc region lipid oxidation was also responsible for protein S oxidation in our of human Ig (Mer-Fc). Protein bound to Mer-Fc is precipitated experiments was considered unlikely given that the phospholipids using protein G-Sepharose beads. As shown in Fig. 5, protein S The Journal of Immunology 2527

oxPLPS vesicles or nonoxidizable DOPS vesicles were used, and only background levels of oligomerization and MerFc binding were seen with DOPC. Oxidation of the PS linoleoyl fatty acids with AAPH did not increase the level of protein S oligomers bound to Mer-Fc (Fig. 5, lane 6). Second, we prevented protein S oligomerization by pretreating the protein with IAA and then measured its ability to stimulate the phagocytosis of apoptotic cells. Fig. 6A shows that IAA modification of protein S blocked completely the ability of pro- tein S to stimulate the phagocytosis of apoptotic cells. IAA modification also blocked protein S-dependent phospholipid vesicle uptake by macrophages (Fig. 6B). A different sulfhydr- yl-blocking reagent, NEM, also completely inhibited the ability of protein S to stimulate phagocytosis (data not shown). Con- sistent with the role of MerTK in mediating protein S-stimu- lated phagocytosis, IAA modification inhibited the ability of protein S to stimulate MerTK phosphorylation during incuba- tion with apoptotic cells (Fig. 6C). Downloaded from

Discussion Protein S is present in the serum at concentrations (ϳ25 ␮g/ml; Ref. 25) that well exceed the concentrations required to activate macrophage phagocytosis in vitro (Ͻ0.5 ␮g/ml; H. Uehara and E.

Shacter, unpublished observations). Roughly 60% of protein S in http://www.jimmunol.org/ the plasma is bound to C4BP, which inactivates the procoagulant activity of protein S (24). However, this still leaves an excess of protein S to stimulate monocyte/macrophages via MerTK. Until now, a molecular mechanism for regulating the prophagocytic activity of protein S had not been well-understood. The findings FIGURE 6. Covalent blocking of the Cys residues in protein S abolishes presented here demonstrate that to stimulate macrophages, pro- its prophagocytic activity. A, Phagocytosis of apoptotic cells by macro- tein S must first undergo oxidative oligomerization on the ap- phages was conducted with 1 ␮g/ml of either native protein S or IAA- optotic cell surface. This requirement likely prevents circulat- modified protein S (proS-IAA). The data show the mean Ϯ SD from two ing protein S from constitutively stimulating monocyte/ by guest on October 2, 2021 Ͻ ء independent experiments conducted in duplicate. , p 0.01. B, Uptake of macrophage activities in tissues and in the circulation. Thus, PS-LMV by macrophages after addition of unmodified or IAA-treated pro- protein S does not serve simply as a passive bridge between tein S. The data show the mean Ϯ SD from three experiments conducted apoptotic cells and macrophages. Rather, a more complex p Ͻ 0.01. C, Tyrosine phosphorylation of MerTK following ,ء .in duplicate incubation of macrophages with apoptotic cells in the presence of protein mechanism exists in which protein S must first undergo Cys S or IAA-modified protein S was measured as described for Fig. 3. The oxidation and oligomerization on the surface of apoptotic cells Western blot immunoassay used Abs against MerTK protein or phospho- before it can stimulate phagocytosis. A potent cysteine oxidant MerTK, as indicated in the figure. D, Densitometric analysis of the blots, such as taurine chloramine does not induce oligomerization of such as that in C, was conducted as described for Fig. 3C. The data rep- protein S in solution (see Fig. 4E). It only occurs on the surface resent the mean Ϯ SD for three experiments. of apoptotic cells or PS vesicles. Hence, three essential com- ponents are required for serum to stimulate the phagocytosis of apoptotic cells: PS as the apoptotic cell receptor, MerTK as the dimer/oligomers that formed during incubation with PS vesicles macrophage receptor, and disulfide-linked protein S dimers or bound preferentially to Mer-Fc. These bands are only observed in oligomers as the catalysts. Interference with any of these com- coprecipitates from samples containing protein S, Mer-Fc, and PS ponents inhibits apoptotic cell clearance by macrophages. Ac- vesicles. There was some background binding of monomeric cording to this model, PS exposed on the outer surface of ap- protein S to the protein G beads in the absence of Mer-Fc (Fig. optotic cells is not only an “eat me” signal (37) but also 5, lanes 1 and 4) despite extensive washing and the use of provides a scaffold for intermolecular protein S interactions and different detergents (deoxycholate and SDS; data not shown). oxidative oligomerization of the protein. In this way, apoptotic There is some specific binding of monomeric protein S to cells enhance their own uptake by providing a platform for pro- Mer-Fc (Fig. 5, lane 3), but this does not stimulate MerTK tein S activation. A model for the proposed mechanism through activation (see Fig. 3). The most extensive binding to Mer-Fc which protein S stimulates phagocytosis of apoptotic cells is was with the protein S dimers and trimers, which are present in shown in Fig. 7. In this model, the binding of protein S dimers relatively low abundance but bind strongly to Mer-Fc protein. induces the dimerization and activation of the MerTK. MerTK Inhibition of disulfide bond formation by pretreating the protein dimerization and activation has been demonstrated in studies S with IAA significantly inhibited the level of dimers and tri- using rCD8-MerTK chimeric molecules (38). mers bound to Mer-Fc. The low level of dimers that bound even Formation of protein S dimers occurs through a spontaneous, with IAA treatment were present in the starting protein S prep- nonenzymatic auto-oxidation of cysteine residues and is indepen- aration (see Fig. 4A) and are actually enriched by binding to dent of lipid oxidation. Dimerization occurs when protein S is Mer-Fc. There was no difference in Mer-Fc binding when bound to DOPS vesicles that are resistant to oxidation, and the use 2528 OXIDATION OF PROTEIN S FOR PROPHAGOCYTIC ACTIVITY

some purified protein S preparations show a higher PS binding affinity and a 100-fold higher APC-independent anticoagulant activity (41). Annexin V, which is another PS-binding protein, also forms noncovalent oligomers upon binding to PS vesicles. Thus, oligomerization may be a common outcome for proteins bound to a PS surface (42). The data presented in this report show that disulfide-linked protein S dimers (and possibly oligomers) stimulate phagocy- tosis by activating macrophage MerTK and that protein S must undergo dimerization to stimulate MerTK. These conclusions are supported by the findings that a neutralizing Ab to MerTK inhibited protein S-stimulated phagocytosis, and that prevention of dimerization with SH-blocking agents (IAA or NEM) com- pletely inhibited the ability of protein S to stimulate both phagocytosis and MerTK tyrosine phosphorylation. In addition, activation of MerTK by protein S was only observed when the macrophages were coincubated with both protein S and apo- ptotic cells; very little MerTK activation was observed when the

FIGURE 7. Model for oxidative oligomerization of protein S leading to macrophages were incubated either with apoptotic cells alone or Downloaded from stimulation of the phagocytosis of apoptotic cells. The model depicts pro- with protein S alone. These findings are in contrast to the ho- tein S dimerization following binding to apoptotic cells that express exo- mologous serum protein and MerTK ligand gas6, which acti- facial PS. Protein S-coated apoptotic cells are recognized by macrophages vated MerTK even in the absence of apoptotic cells, thereby through the interaction between protein S dimers and MerTK on the mac- revealing a significant difference in biological activity between rophages, resulting in autophosphorylation of MerTK on Tyr residues. The protein S and gas6. line connecting two protein S molecules represent an S-S disulfide bond

Recently, Sather et al. (43) described the presence of a soluble http://www.jimmunol.org/ that is formed by auto-oxidation of Cys residues. form of Mer (sMer) in human plasma and culture medium, indi- cating that this might be a possible mechanism for regulating gas6- stimulated phagocytosis. sMer is a proteolytic cleavage product of of experimentally oxPLPS vesicles does not enhance dimer for- the MerTK receptor and consists of the extracellular domain of mation. Based upon these data, and the absence of any other ox- MerTK. It sequesters gas6 and blocks its ability to activate MerTK idizing reagents in the experiments with phospholipid vesicles, we and stimulate phagocytosis. This observation highlights an inter- conclude that protein S undergoes auto-oxidation when bound to esting and important difference between regulation of the biolog- membranal PS. The data do not rule out the possibility that oxi- ical activities of protein S and gas6. As demonstrated here, protein dizing species in apoptotic cells also contribute to protein S oxi- S oligomers are much more effective ligands for MerTK protein by guest on October 2, 2021 dation in the cell system, as has been proposed for other experi- than protein S monomers. It may therefore be predicted that cir- mental systems (32, 36). Addition of the protein disulfide culating protein S monomers can bypass the inhibitory effects of inhibitor bacitracin to the cell incubations did not inhibit either protein S dimer formation or phagocytosis of apoptotic cells, sMer and stimulate engulfment of apoptotic cells upon binding to suggesting that this key cell surface is not responsible for PS on the cell surface. catalyzing the observed disulfide bond formation (data not shown). The involvement of MerTK, gas6, and protein S in the phago- Taken cumulatively, the results suggest that binding of protein S cytosis of apoptotic cells in vivo has been studied in two dif- monomers to a PS scaffold aligns the cysteine residues in a way ferent rodent models. A role for MerTK was initially identified that promotes the dimerization/oligomerization by auto-oxidation. from studies of naturally occurring human and rat MerTK mu- The free cysteine residues that can potentially participate in the tations that caused a failure of phagocytosis of the outer seg- intermolecular disulfide bond are at positions 47, 72, 247, and 527 ment by retinal pigment epithelium, resulting in retinal degen- in the mature, secreted form of protein S. The Cys residues at 47 eration (44–46). Subsequent studies using MerTK knockout and 72 are localized between the PS-binding Gla domain and the mice revealed that macrophages lacking MerTK are unable to epidermal growth factor-like domain. Cys247 is localized between ingest apoptotic thymocytes, either in vivo or in vitro (8, 47). the fourth EGF-like domain and the SHBG domain, and Cys 527 Previous work showed that gas6 is a ligand for MerTK, Tyro3, is within the SHBG domain. Additional studies will be required to and Axl tyrosine kinases (34, 48, 49) and suggested that it stim- determine the specific disulfide bonding pattern that leads to acti- ulates photoreceptor outer segment phagocytosis by the retinal vation of protein S prophagocytic activity. pigment epithelium (50). However, more recent studies found The requirement for a PS scaffold for protein S activation mir- that gas6 knockout mice develop normal retina, suggesting the rors the role of protein S as a cofactor in the protein C anticoag- involvement of another molecule in MerTK-mediated phagocy- ulation system, which was the only known activity defined for tosis of outer segment (18, 19). Consistent with this finding and protein S before its prophagocytic activity was discovered (39). our previous findings, Hall et al. (18) showed that protein S When serving as an anticoagulant cofactor, the interaction between stimulates outer segment uptake and that protein S-dependent protein S and activated protein C also requires the presence of stimulation is abrogated in retinal pigment epithelial cells that a membrane surface containing PS (40). In both cases, the bind- lack MerTK activity. Furthermore, mouse protein S was found ing of protein S to PS involves the N-terminal Gla domain of to activate MerTK in isolated eyecups devoid of anterior eye the protein. It will be interesting to determine whether protein and neural retina (19). Our findings provide the molecular basis S oligomerization is essential for its role in supporting activa- for protein S binding to MerTK. Interestingly, the results may tion of protein C under physiological conditions. In this regard, explain why previous studies failed to detect human protein it is notable that noncovalent protein S multimers that exist in S-mediated activation of any of the human TAM kinases such The Journal of Immunology 2529 as MerTK and Tyro3 (51, 52); those experiments were con- 14. Williamson, P., and R. A. Schlegel. 2002. Transbilayer phospholipid movement ducted in the absence of a PS scaffold. and the clearance of apoptotic cells. Biochim. Biophys. Acta 1585: 53–63. 15. Wu, Y., N. Tibrewal, and R. B. Birge. 2006. Phosphatidylserine recognition by Dysregulation of protein S may have clinical impact that re- phagocytes: a view to a kill. Trends Cell Biol. 16: 189–197. lates to its role in stimulating the phagocytosis of apoptotic 16. Fantl, W. J., D. E. Johnson, and L. T. Williams. 1993. Signalling by receptor tyrosine kinases. Annu. Rev. Biochem. 62: 453–481. cells, most notably autoimmunity and sepsis (26). Protein S 17. Graham, D. K., T. L. Dawson, D. L. Mullaney, H. R. Snodgrass, and H. S. Earp. deficiency is a marker for the autoimmune disease systemic 1994. Cloning and mRNA expression analysis of a novel human protooncogene, lupus erythematosus (29). Whether this deficiency is the cause c-mer. Cell Growth Differ. 5: 647–657. 18. Hall, M. O., M. S. Obin, M. J. Heeb, B. L. Burgess, and T. A. Abrams. 2005. Both or the effect of lupus symptoms is not clear, but the relationship protein S and Gas6 stimulate outer segment phagocytosis by cultured rat retinal is consistent with the view that the efficient phagocytosis of pigment epithelial cells. Exp. Eye Res. 81: 581–591. apoptotic cells is required to prevent autoimmune reactions to 19. Prasad, D., C. V. Rothlin, P. Burrola, T. Burstyn-Cohen, Q. Lu, P. Garcia de Frutos, and G. Lemke. 2006. TAM receptor function in the retinal intracellular molecules released by dying cells. According to pigment epithelium. Mol. Cell Neurosci. 33: 96–108. our data, a possible cause and effect relationship between pro- 20. Hart, S. P., J. R. Smith, and I. Dransfield. 2004. Phagocytosis of opsonized ap- tein S deficiency and autoimmune disease should be examined, optotic cells: roles for “old-fashioned” receptors for antibody and complement. Clin. Exp. Immunol. 135: 181–185. possibly through targeted disruption of the protein S gene in an 21. Hoffmann, P. R., A. M. deCathelineau, C. A. Ogden, Y. Leverrier, D. L. Bratton, experimental animal model. In a different clinical setting, the D. L. Daleke, A. J. Ridley, V. A. Fadok, and P. M. Henson. 2001. Phosphati- dylserine (PS) induces PS receptor-mediated macropinocytosis and promotes binding of protein S to apoptotic cells in the circulation may clearance of apoptotic cells. J. Cell Biol. 155: 649–659. contribute to the progression of disseminated intravascular co- 22. Anderson, H. A., C. A. Maylock, J. A. Williams, C. P. Paweletz, H. Shu, and agulation during sepsis. In this situation, both proteins S and C E. Shacter. 2003. Serum-derived protein S binds to phosphatidylserine and stim- ulates the phagocytosis of apoptotic cells. Nat. Immunol. 4: 87–91. would be depleted through their binding to apoptotic cells and

23. Kask, L., L. A. Trouw, B. Dahlback, and A. M. Blom. 2004. The C4b-binding Downloaded from subsequent removal by monocytic cells (26). If this is correct, protein-protein S complex inhibits the phagocytosis of apoptotic cells. J. Biol. then supplementation of sepsis patients with both protein S and Chem. 279: 23869–23873. 24. Dahlback, B. 1991. Protein S and C4b-binding protein: components involved in the activated protein C would be a more effective therapy than ad- regulation of the protein C anticoagulant system. Thromb. Haemost. 66: 49–61. ministration of activated protein C alone (53). 25. Esmon, C. T. 2000. Regulation of blood coagulation. Biochim. Biophys. Acta 1477: 349–360. 26. Anderson, H. A., and E. Shacter. 2004. Natural anticoagulant proteins in the regu- Acknowledgments lation of autoimmunity: potential role of protein S. Curr. Pharm. Des. 10: 929–937. http://www.jimmunol.org/ We thank Howard Anderson, Baolin Zhang, Jee Chung, and other members 27. Kearon, C., M. Crowther, and J. Hirsh. 2000. Management of patients with he- reditary hypercoagulable disorders. Annu. Rev. Med. 51: 169–185. of the Laboratory of Biochemistry for many useful suggestions for this 28. Guermazi, S., V. Regnault, Y. Gorgi, K. Ayed, T. Lecompte, and K. Dellagi. research and manuscript. 2000. Further evidence for the presence of anti-protein S autoantibodies in pa- tients with systemic lupus erythematosus. Blood Coagul. Fibrinolysis 11: 491–498. Disclosures 29. Song, K. S., Y. S. Park, and H. K. Kim. 2000. Prevalence of anti-protein S The authors have no financial conflict of interest. antibodies in patients with systemic lupus erythematosus. Arthritis Rheum. 43: 557–560. 30. Weiss, S. J., R. Klein, A. Slivka, and M. Wei. 1982. Chlorination of taurine by References human neutrophils: evidence for hypochlorous acid generation. J. Clin. Invest. 1. Savill, J., I. Dransfield, C. Gregory, and C. Haslett. 2002. A blast from the past: 70: 598–607. by guest on October 2, 2021 clearance of apoptotic cells regulates immune responses. Nat. Rev. Immunol. 2: 31. Corongiu, F. P., and S. Banni. 1994. Detection of conjugated dienes by second 965–975. derivative ultraviolet spectrophotometry. Meth. Enzymol. 233: 303–310. 2. Krysko, D. V., K. D’Herde, and P. Vandenabeele. 2006. Clearance of apoptotic 32. Kagan, V. E., B. Gleiss, Y. Y. Tyurina, V. A. Tyurin, C. Elenstrom-Magnusson, and necrotic cells and its immunological consequences. Apoptosis 11: S. X. Liu, F. B. Serinkan, A. Arroyo, J. Chandra, S. Orrenius, and B. Fadeel. 1709–1726. 2002. A role for oxidative stress in apoptosis: oxidation and externalization of 3. Henson, P. M., and R. B. Johnston, Jr. 1987. Tissue injury in inflammation: phosphatidylserine is required for macrophage clearance of cells undergoing Fas- oxidants, proteinases, and cationic proteins. J. Clin. Invest. 79: 669–674. mediated apoptosis. J. Immunol. 169: 487–499. 4. Shacter, E., J. A. Williams, R. M. Hinson, S. Senturker, and Y. J. Lee. 2000. 33. Raz, A., C. Bucana, W. E. Fogler, G. Poste, and I. J. Fidler. 1981. Biochemical, Oxidative stress interferes with cancer chemotherapy: inhibition of lymphoma morphological, and ultrastructural studies on the uptake of liposomes by murine cell apoptosis and phagocytosis. Blood 96: 307–313. macrophages. Cancer Res. 41: 487–494. 5. Fadok, V. A., D. L. Bratton, A. Konowal, P. W. Freed, J. Y. Westcott, and 34. Stitt, T. N., G. Conn, M. Gore, C. Lai, J. Bruno, C. Radziejewski, K. Mattsson, P. M. Henson. 1998. Macrophages that have ingested apoptotic cells in vitro J. Fisher, D. R. Gies, P. F. Jones, et al. 1995. The anticoagulation factor protein inhibit proinflammatory cytokine production through autocrine/paracrine mech- S and its relative, Gas6, are ligands for the Tyro 3/Axl family of receptor tyrosine ␤ anisms involving TGF- , PGE2, and PAF. J. Clin. Invest. 101: 890–898. kinases. Cell 80: 661–670. 6. Voll, R. E., M. Herrmann, E. A. Roth, C. Stach, J. R. Kalden, and I. Girkontaite. 35. Manfioletti, G., C. Brancolini, G. Avanzi, and C. Schneider. 1993. The protein 1997. Immunosuppressive effects of apoptotic cells. Nature 390: 350–351. encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin 7. Botto, M., C. Dell’Agnola, A. E. Bygrave, E. M. Thompson, H. T. Cook, F. Petry, K-dependent proteins related to protein S, a negative coregulator in the blood M. Loos, P. P. Pandolfi, and M. J. Walport. 1998. Homozygous C1q deficiency coagulation cascade. Mol. Cell. Biol. 13: 4976–4985. causes glomerulonephritis associated with multiple apoptotic bodies. Nat. Genet. 36. Greenberg, M. E., M. Sun, R. Zhang, M. Febbraio, R. Silverstein, and 19: 56–59. S. L. Hazen. 2006. Oxidized phosphatidylserine-CD36 interactions play an es- 8. Scott, R. S., E. J. McMahon, S. M. Pop, E. A. Reap, R. Caricchio, P. L. Cohen, sential role in macrophage-dependent phagocytosis of apoptotic cells. J. Exp. H. S. Earp, and G. K. Matsushima. 2001. Phagocytosis and clearance of apoptotic Med. 203: 2613–2625. cells is mediated by MER. Nature 411: 207–211. 37. Grimsley, C., and K. S. Ravichandran. 2003. Cues for apoptotic cell engulfment: 9. Fadeel, B. 2004. Plasma membrane alterations during apoptosis: role in corpse eat-me, don’t eat-me and come-get-me signals. Trends Cell Biol. 13: 648–656. clearance. Antioxid. Redox. Signal 6: 269–275. 38. Zong, C., R. Yan, A. August, J. E. Darnell, Jr., and H. Hanafusa. 1996. Unique 10. Fadok, V. A., D. R. Voelker, P. A. Campbell, J. J. Cohen, D. L. Bratton, and signal transduction of Eyk: constitutive stimulation of the JAK-STAT pathway by P. M. Henson. 1992. Exposure of phosphatidylserine on the surface of apoptotic an oncogenic receptor-type tyrosine kinase. EMBO J. 15: 4515–4525. lymphocytes triggers specific recognition and removal by macrophages. J. Im- 39. Walker, F. J. 1980. Regulation of activated protein C by a new protein: a possible munol. 148: 2207–2216. function for bovine protein S. J. Biol. Chem. 255: 5521–5524. 11. Martin, S. J., C. P. Reutelingsperger, A. J. McGahon, J. A. Rader, 40. Walker, F. J. 1984. Protein S and the regulation of activated protein C. Semin. R. C. van Schie, D. M. LaFace, and D. R. Green. 1995. Early redistribution of Thromb. Hemost. 10: 131–138. plasma membrane phosphatidylserine is a general feature of apoptosis regardless 41. Sere, K. M., M. P. Janssen, G. M. Willems, G. Tans, J. Rosing, and of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J. Exp. T. M. Hackeng. 2001. Purified protein S contains multimeric forms with in- Med. 182: 1545–1556. creased APC-independent anticoagulant activity. Biochemistry 40: 8852–8860. 12. Fadok, V. A., A. de Cathelineau, D. L. Daleke, P. M. Henson, and D. L. Bratton. 42. Concha, N. O., J. F. Head, M. A. Kaetzel, J. R. Dedman, and B. A. Seaton. 1992. 2001. Loss of phospholipid asymmetry and surface exposure of phosphatidylser- Annexin V forms calcium-dependent trimeric units on phospholipid vesicles. ine is required for phagocytosis of apoptotic cells by macrophages and fibro- FEBS Lett. 314: 159–162. blasts. J. Biol. Chem. 276: 1071–1077. 43. Sather, S., K. D. Kenyon, J. B. Lefkowitz, X. Liang, B. C. Varnum, 13. Anderson, H. A., R. Englert, I. Gursel, and E. Shacter. 2002. Oxidative stress P. M. Henson, and D. K. Graham. 2007. A soluble form of the Mer receptor inhibits the phagocytosis of apoptotic cells that have externalized phosphatidyl- tyrosine kinase inhibits macrophage clearance of apoptotic cells and platelet ag- serine. Cell Death Differ. 9: 616–625. gregation. Blood 109: 1026–1033. 2530 OXIDATION OF PROTEIN S FOR PROPHAGOCYTIC ACTIVITY

44. Feng, W., D. Yasumura, M. T. Matthes, M. M. LaVail, and D. Vollrath. 2002. 49. Varnum, B. C., C. Young, G. Elliott, A. Garcia, T. D. Bartley, Y. W. Fridell, MerTK triggers uptake of photoreceptor outer segments during phagocytosis by R. W. Hunt, G. Trail, C. Clogston, R. J. Toso, et al. 1995. Axl receptor tyrosine cultured retinal pigment epithelial cells. J. Biol. Chem. 277: 17016–17022. kinase stimulated by the vitamin K-dependent protein encoded by growth-arrest- 45. Gal, A., Y. Li, D. A. Thompson, J. Weir, U. Orth, S. G. Jacobson, E. Apfelstedt-Sylla, specific gene 6. Nature 373: 623–626. and D. Vollrath. 2000. in MERTK, the human orthologue of the RCS rat 50. Hall, M. O., A. L. Prieto, M. S. Obin, T. A. Abrams, B. L. Burgess, M. J. Heeb, retinal dystrophy gene, cause . Nat. Genet. 26: 270–271. and B. J. Agnew. 2001. Outer segment phagocytosis by cultured retinal pigment epithelial cells requires Gas6. Exp. Eye Res. 73: 509–520. 46. Nandrot, E., E. M. Dufour, A. C. Provost, M. O. Pequignot, S. Bonnel, K. Gogat, 51. Godowski, P. J., M. R. Mark, J. Chen, M. D. Sadick, H. Raab, and D. Marchant, C. Rouillac, B. Sepulchre de Conde, M. T. Bihoreau, et al. 2000. R. G. Hammonds. 1995. Reevaluation of the roles of protein S and Gas6 as Homozygous deletion in the coding sequence of the c-mer gene in RCS rats unravels ligands for the Rse/Tyro 3. Cell 82: 355–358. general mechanisms of physiological cell adhesion and apoptosis. Neurobiol. Dis. 7: 52. Nagata, K., K. Ohashi, T. Nakano, H. Arita, C. Zong, H. Hanafusa, and 586–599. K. Mizuno. 1996. Identification of the product of growth arrest-specific gene 6 as 47. Seitz, H. M., T. D. Camenisch, G. Lemke, H. S. Earp, and G. K. Matsushima. a common ligand for Axl, Sky, and Mer receptor tyrosine kinases. J. Biol. Chem. 2007. Macrophages and dendritic cells use different Axl/Mertk/Tyro3 receptors in 271: 30022–30027. clearance of apoptotic cells. J. Immunol. 178: 5635–5642. 53. Hoffmann, J. N., J. M. Fertmann, and K. W. Jauch. 2006. Microcirculatory 48. Ohashi, K., K. Nagata, J. Toshima, T. Nakano, H. Arita, H. Tsuda, K. Suzuki, and disorders in sepsis and transplantation: therapy with natural coagulatory in- K. Mizuno. 1995. Stimulation of sky receptor tyrosine kinase by the product of hibitors antithrombin and activated protein C. Curr. Opin. Crit. Care 12: growth arrest-specific gene 6. J. Biol. Chem. 270: 22681–22684. 426–430. Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021