To the Surface of Apoptotic Cells Complement Regulator C4b

Vitamin K-Dependent Protein S Localizing Complement Regulator C4b-Binding Protein to the Surface of Apoptotic Cells

This information is current as Joanna H. Webb, Anna M. Blom and Björn Dahlbäck of September 27, 2021. J Immunol 2002; 169:2580-2586; ; doi: 10.4049/jimmunol.169.5.2580 http://www.jimmunol.org/content/169/5/2580 Downloaded from

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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 © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Vitamin K-Dependent Protein S Localizing Complement Regulator C4b-Binding Protein to the Surface of Apoptotic Cells1

Joanna H. Webb, Anna M. Blom, and Bjo¨rn Dahlba¨ck2

Apoptosis is characterized by a lack of inflammatory reaction in surrounding tissues, suggesting local control of complement activation. During the initial stage of apoptosis, cells expose negatively charged phospholipid phosphatidylserine on their surfaces. The vitamin K-dependent protein S has a high affinity for this type of phospholipid. In human plasma, 60–70% of protein S circulates in complex with C4b-binding protein (C4BP). The reason why protein S and C4BP form a high-affinity complex in plasma is not known. However, C4BP is an important regulator of the classical pathway of the complement system where it acts

as a cofactor in degradation of complement protein C4b. Using Jurkat cells as a model system for apoptosis, we now show protein Downloaded from S to bind to apoptotic cells. We further demonstrate protein S-mediated binding of C4BP to apoptotic cells. Binding of the C4BP-protein S complex to apoptotic cells was calcium-dependent and could be blocked with Abs directed against the phospholipid-binding domain in protein S. Annexin V, which binds to exposed phosphatidylserine on the apoptotic cell surface, could inhibit the binding of protein S. The C4BP that was bound via protein S to the apoptotic cells was able to interact with the complement protein C4b, supporting a physiological role of the C4BP/protein S complex in regulation of complement on the

surface of apoptotic cells. The Journal of Immunology, 2002, 169: 2580–2586. http://www.jimmunol.org/

poptosis is under physiological conditions a well-regu- charged phospholipids, e.g., activated protein C (apc)3 and protein lated process, characterized by a lack of induction of S (16). Protein S functions as cofactor to apc in the degradation of A inflammatory responses in the surrounding tissue (1). In factors Va and VIIIa (17, 18). The importance of protein C and viable cells, an asymmetric distribution of phospholipids between protein S is demonstrated by the fact that deficiency of either is the outer and inner leaflet of the membrane is maintained, the associated with increased risk of thrombosis (19, 20). Because an- negatively charged phosphatidylserine being predominantly local- ionic membrane surfaces (such as that provided by activated plate- ized in the inner leaflet (2). Erythrocytes and platelets were the first lets) have been shown to be equally competent in promoting pro- cells where this asymmetry was described (3, 4), but it was later and anticoagulant reactions (21, 22), it is likely that the surface of by guest on September 27, 2021 also found to be true for nucleated cells (5). During one of the first apoptotic cells is also able to promote anticoagulant reactions. stages of apoptosis, phosphatidylserine is transferred from the in- In plasma, protein S exists in two forms; ϳ30Ð40% circulates ner leaflet of the cell membrane to the outer leaflet (6, 7), resulting as free protein S, the remainder forming a 1:1 high-affinity (Kd in in the exposure of negatively charged phospholipid on the cell a range of 0.1Ð0.6 nM) complex with C4b-binding protein (C4BP; surface. This has been suggested to be a phagocytosis-stimulating Refs. 23Ð26). Only free protein S works as a cofactor to apc (27). signal to neighboring cells, resulting in the elimination of the ap- Protein S contains multiple domains, including an N-terminal optotic cell remnants. ␥-carboxy glutamic acid (Gla)-rich domain, which binds Ca2ϩ and Negatively charged phospholipid membranes, e.g., exposed on is responsible for the interaction with negatively charged phospho- activated platelets, bind vitamin K-dependent coagulation factors, lipids, a thrombin sensitive region, four epidermal growth factor thus supporting the assembly of procoagulant enzyme-cofactor (EGF)-like domains, and two laminin G-type domains. The G do- complexes (8Ð12). Several studies have also shown apoptotic cells mains are involved in the interaction with C4BP, especially resi- to have procoagulant properties (13Ð15). However, there are also dues 605Ð614 (28, 29), 420Ð434 (30, 31), and 447Ð460 (32). anticoagulant vitamin K-dependent proteins that bind negatively C4BP is an important regulator of the classical pathway of complement. It functions as a cofactor to factor I in the degradation of C4b (33). Furthermore, it inhibits the formation of the C3 conver- Division of Clinical Chemistry, Department of Laboratory Medicine, University Hos- tase (formed by C4b and C2a) and accelerates its decay (34). The pital Malmo¬, Lund University, Malmo¬, Sweden structure of C4BP resembles that of an octopus. It consists of seven Received for publication April 4, 2002. Accepted for publication June 19, 2002. identical ␣-chains, and one unique ␤-chain, the chains being held The costs of publication of this article were defrayed in part by the payment of page together in a central core. Several complement control protein charges. This article must therefore be hereby marked advertisement in accordance ␣ with 18 U.S.C. Section 1734 solely to indicate this fact. (CCP) modules compose each chain, the -chains having eight CCPs and the ␤-chain having three. Each ␣-chain binds a C4b, and 1 This work was supported by grants from the Swedish Research Council (07143), a Senior Investigator Award from the Swedish Foundation for Strategic Research, the a positively charged area on the interface between CCP1 and 2 is Cancer Foundation, the Network for Inflammation Research funded by the Swedish important for the binding (35). The ␤-chain binds protein S, the Foundation for Strategic Research, the Tore Nilson’s Trust, the Crafoord trust, the O¬ sterlunds Trust, Greta and Johan Kock’s Trust, and research funds from the Uni- main binding site being localized to CCP1 and involving a large versity Hospital Malmo¬. 2 Address correspondence and reprint requests to Dr. Bjo¬rn Dahlba¬ck, Division of Clinical Chemistry, Department of Laboratory Medicine, University Hospital Malmo¬, 3 Abbreviations used in this paper: apc, activated protein C; C4BP, C4b-binding pro- Lund University, S-205 02 Malmo¬, Sweden. E-mail address: Bjorn.Dahlback@ tein; CCP, complement control protein; 7-AAD, 7-amino actinomycin D; SAP, serum klkemi.mas.lu.se amyloid P component; EGF, epidermal growth factor; Gla, ␥-carboxy glutamic acid.

hydrophobic patch (36, 37). CCP2 also seems to contribute slightly measuring absorbance at 495 nm. Rates of FITC to protein were within to the interaction (38). recommended range (0.3Ð1.0; Ref. 51). The physiological role of the complex between C4BP and pro- Binding experiments tein S has remained an enigma. It has been suggested that protein ␮ S, due to its high affinity for negatively charged phospholipids, A total of 75 l of cells were incubated with protein S, C4BP, plasma purified C4BP/protein S complex, or protein S preincubated with C4BP (at localizes C4BP to areas where such phospholipids are exposed 37¡C for 30 min, in the presence of 2 mM CaCl2) in binding buffer in a final (39). The complex binds to synthetic phospholipid vesicles, but not volume of 100 ␮l. In time course studies, binding of protein S leveled of to platelet-derived microparticles, even though free protein S ef- at 25 min at room temperature, and remained stable at least until 60 min ficiently binds to these particles (40). (data not shown). Therefore, 25 min was chosen as incubation time. Cells ϫ It has been demonstrated that complement components attach to were then pelleted by centrifugation for 2 min at 350 g, and washed in binding buffer. Following a second centrifugation, 15 ␮g/ml FITC-labeled apoptotic cells, C1 arguably being the most important. C1q defi- Abs, HPS 54 for protein S, and MK 104 for all protein containing C4BP, ciency may predispose to autoimmunity in systemic lupus ery- annexin V labeled with PE (annexin V-PE; BD Biosciences, Mountain thematosus as a consequence of an impaired clearance of apoptotic View, CA), 5 ␮l10ϫ diluted, and 5 ␮l 7-amino actinomycin D (7-AAD; cells (41Ð44). Binding of C1 to apoptotic cells could result in ViaProbe; BD Biosciences) were added in binding buffer at a final volume of 100 ␮l. In control experiments, binding of FITC-labeled HPS 54 and activation of the early classical pathway unless efficient control 104 to corresponding proteins leveled off around 8 ␮g/ml and remained mechanisms exist. A possible regulation could be achieved by the stable up to 100 ␮g/ml (highest concentration tested). Following a 30-min localization of C4BP to the apoptotic cell surface. The aim of incubation in the dark at room temperature, samples were diluted with 100 the present investigation was to elucidate whether protein S has ␮l of ice-cold binding buffer and immediately analyzed in the flow cytom- the capacity to localize C4BP to the apoptotic cell surface. Using eter (FACSort; BD Biosciences). We also tested binding of the C4BP- Downloaded from protein S complex in the presence of human serum. The serum had been Jurkat cells, a human T lymphocyte line, as a model system for depleted of protein S and C4BP using HiTrap columns (5 ml; Amersham apoptotic cells, we demonstrate protein S-mediated binding of Pharmacia Biotech) coupled with HPS 54 and MK 104. Amounts of re- C4BP to apoptotic cells. The surface-bound complex could bind maining protein S and C4BP were tested using ELISAs for protein S and C4b, the main target for C4BP in the complement system. C4BP and were below detection levels. We then added back the C4BP- protein S complex (final concentration in serum 200 ␮g/ml) and incubated apoptotic cells for 30 min at 37¡C with 20% human serum where C4BP- Materials and Methods protein S had been added back. Cells were washed and binding detected in http://www.jimmunol.org/ Cells the flow cytometer using FITC-labeled MK 104 as described above. The apparent Kd for free protein S bound to the apoptotic cell surface Human Jurkat cells were cultured in RPMI, with 10% heat-inactivated was obtained from the plot of the increase in fluorescence as a function of

FCS, complemented with L-glutamine and antibiotics (growth medium). the protein S concentration. The Kd was calculated by fitting the data to the For the induction of apoptosis, cells were collected and resuspended at a following equation for a single site-binding isotherm using nonliner least concentration of 2 ϫ 106 cells/ml. Apoptosis was induced by addition of square regression analysis: anti-Fas Ab CD95 (CH-11 clone; Immunotech, Marseille, France), 200 ϭ ϩ ng/ml, to the growth medium and culture was continued for 5 h. This gave B Bmax/(1 Kd/[PS]) an ϳ50% apoptotic population and 50% live population of the cells gated (for gating details, see Analysis). After apoptosis induction, cells were har- The free concentration protein S [PS] was assumed to be equal to the added by guest on September 27, 2021 vested by centrifugation and washed in binding buffer (10 mM HEPES, concentration of protein S. The binding maximum, Bmax, was set to be the value where binding of protein S to the apoptotic cell seemed to saturate. 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2). Cells were then ϫ 6 resuspended in binding buffer, to a final concentration of 5 10 cells/ml. Preincubation using mAbs Proteins C4BP/protein S complex was preincubated with HPS 21 or 54 for 30 min at 37¡C at a molar ratio of 1:2. Then, the same protocol was followed as for Purified recombinant annexin V was purchased from BD PharMingen (San binding experiments. For protein S a different approach was used. Protein Diego, CA). Human protein S (45), C4BP, and the C4BP/protein S com- S labeled with FITC was preincubated with HPS 21 or 54 for 30 min at plex (46) and C4 (47) were purified from plasma as described in their 37¡C at a molar ratio of 1:1. A total of 75 ␮l of Jurkat cells were then respective references. To obtain C4BP without protein S, the purified incubated for 25 min at room temperature, in the dark, with 100 nM of the C4BP/protein S complex was dialyzed against 80% ethylenglycole, 10 mM protein S/mAb mixture in binding buffer in a final volume of 100 ␮l. This Tris-Hcl (pH 7.5) before being run through a heparin Sepharose (Amer- was then diluted in ice-cold binding buffer and immediately analyzed in the sham Pharmacia Biotech, Uppsala, Sweden) column equilibrated in the flow cytometer. same buffer. The column was washed with 10 mM Tris-HCl (pH 8.0) and C4BP was eluted with a gradient of 0Ð0.7 M NaCl in the same buffer. Annexin V inhibition Fractions containing C4BP were chosen and pooled after analysis using a screen agarose gel electrophoresis under native conditions. The protein Apoptotic Jurkat cells, 75 ␮l, were incubated for 25 min at room temper- pool was then dialyzed against 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, ature with protein S, 100 nM, and recombinant annexin V, 0, 3, or 100 nM and finally applied on a gel with coupled MK 104 as described previously in binding buffer at a final volume of 100 ␮l. Cells were then washed as (48). In this fashion, ␤-chain-containing C4BP can be separated from pro- described above and incubated with FITC-labeled HPS 54, 15 ␮g/ml, for tein S without losing its ability to bind protein S. The concentrations were 30 min at room temperature in the dark. Samples were then diluted in 100 determined by measuring absorbance at 280 nm. Extinction coefficients ␮l of ice-cold binding buffer and immediately analyzed in the flow (1%, 1 cm) used were 9.5 (protein S), 14.1 (C4BP), and 8.3 (C4). To obtain cytometer. C4b-like molecules, C4 was incubated with methylamine as described before (35). The C4(met) derivative was used throughout the study, but will Binding of C4b be called C4b for clarity. mAbs used were HPS 21 and 54, directed against For the C4b-binding, the same protocol was used as for binding experi- human protein S (49), and MK 104, against ␣-chain CCP1 of human C4BP ments, except that the C4BP/protein S concentration was kept to a final (50). For the labeling with FITC (isomer I; Sigma-Aldrich, St. Louis, MO), concentration of 250 nM, FITC-labeled C4b at increasing concentrations the proteins were dialyzed against 5 mM MOPS, 0.15 M NaCl, and then was added instead of MK 104-FITC, and annexin V-PE was not diluted. To diluted to 0.5Ð2.0 mg/ml in 5 mM MOPS, 0.15 M NaCl, 0.1 M sodium see whether binding of C4b to C4BP/protein S on the apoptotic cell could carbonate (pH 9.0), and 1 mM CaCl . A total of 50 ␮g of an FITC slurry 2 be inhibited by a blocking Ab, unlabeled MK 104 (3.3-fold molar excess (FITC at 10 mg/ml in DMSO) was added for every milligram of protein over C4b added) was added at the same time as C4b-FITC (300 nM) in a and the mixture was incubated for 60Ð120 min at room temperature in the separate experiment. dark. The reaction was terminated by the addition of 0.1 M NH4Cl (final concentration). FITC-labeled protein was separated from free FITC on a Analysis PD-10 column (Bio-Rad, Hercules, CA), equilibrated with 10 mM HEPES (pH 7.4), 140 mM NaCl. Final protein concentrations were determined by Flow cytometric results were analyzed using CellQuest software (BD Bio- measuring absorbance at 280 nm, and FITC-labeling was confirmed by sciences). Fluorescence was measured on a log scale and is expressed as 2582 C4BP AND PROTEIN S BIND TO APOPTOTIC CELLS geometric mean fluorescence, or percentage of maximal binding seen. A ing seen in each different experiment. The actual fluorescence in- gate was set around cells where no dramatic size changes were discernible, tensity/cell (geometric mean Ϯ SD) seen at highest concentration i.e., forward and side scatter were unchanged as compared with cells where of protein S was 2285 Ϯ 291. Binding was saturable, which en- no anti-Fas Ab had been added. Early apoptotic cells were defined as cells staining single positive for annexin V-PE. Late apoptotic and necrotic cells abled us to calculate an apparent Kd of the protein S association to ϳ were defined as cells staining double positive for annexin V-PE and the apoptotic cell. Free protein S demonstrated a Kd of 220 nM. 7-AAD; these cells were excluded from analysis. Live cells were defined For reference, the concentration of free protein S in human plasma as cells staining double negative for annexin V-PE and 7-AAD. There was is ϳ100 nM, which constitutes ϳ30% of total protein S (45, 46). no difference observed in the various binding studies between this live cell population and control cells, cultured without anti-Fas Ab. In all experi- Because our hypothesis was that protein S binds to the nega- ments, at least 10,000 cells were collected. tively charged phospholipids exposed early in apoptosis, which is what annexin V also binds to, the annexin V-PE was diluted 10 Results times to minimize competition between the two proteins. In control Protein S binds to apoptotic cells experiments, the annexin V reagent could be diluted 25-fold even at the highest concentration of protein S without loss of the signal, Jurkat cells, induced to undergo apoptosis with anti-Fas Ab, were i.e., the apoptotic population could still be distinguished from the mixed with protein S. Following incubation, cells were washed live population. As control, experiments were done in the same with ice-cold binding buffer and mixed with FITC-labeled mAb manner but with the exclusion of annexin V-PE and 7-AAD. Sim- HPS 54, directed against the first EGF-like domain on protein S. ilar protein S binding was observed, which ensures that protein S Annexin V-PE, which binds to negatively charged phospholipid did not bind to the annexin V. Furthermore, there was no binding

and thus was used for early detection of apoptosis, and 7-AAD Downloaded from in the absence of protein S between mAbs and cells, in the pres- were also added. Samples were diluted in ice-cold binding buffer ence or absence of annexin V-PE/7-AAD (data not shown). and FACS analysis was immediately performed. Early apoptotic cells were defined as the population staining positive for annexin C4BP binds to apoptotic Jurkat cells in the presence of protein S V-PE (binding negatively charged phospholipid) and negative for To test whether C4BP binds to apoptotic cells and if protein S has 7-AAD (nucleic acid dye), live cells were defined as the population any influence on the interaction, FITC-labeled MK 104, directed that was negative for both. As shown in Fig. 1A, apoptotic cells against the ␣-chains of C4BP, was used for detection. Three dif- http://www.jimmunol.org/ bound protein S in a dose-dependent manner, whereas there was ferent forms of C4BP were tested. The first variant was the C4BP/ virtually no binding of protein S to live cells. Due to variation protein S complex purified from plasma. The second variant was between experiments with regard to fluorescence intensity/cell, we C4BP isolated from the purified C4BP/protein S complex on a have chosen to plot binding as mean percentage of maximum bind- heparin Sepharose column run in ethylene glycol. This breaks the strong hydrophobic bond between C4BP and protein S, and allows C4BP to be separated from protein S. This C4BP retains the ability to bind protein S. The third variant tested was C4BP incubated in vitro with protein S, to reconstitute the complex. All three forms of C4BP; i.e., in complex with protein S, free, and recomplexed with by guest on September 27, 2021 protein S, were tested for their ability to bind apoptotic cells. The results presented in Fig. 1B clearly show that C4BP/protein S complexes binds to apoptotic, but not to live Jurkat cells. Binding was dose-dependent, and C4BP by itself demonstrated almost no binding. For reference, the concentration of C4BP in human plasma is ϳ300 nM, the majority being ␤-chain containing and thus circu- lating in complex with protein S (52). Due to variation between experiments with regard to fluorescence intensity/cell, we have chosen to plot binding as mean percentage of maximum binding seen in each different experiment. The actual fluorescence intensity/cell (geometric mean Ϯ SD) seen at the highest concentration of C4BP-protein S complex was 465 Ϯ 175. To investigate whether the very slight binding observed by C4BP alone was due to small amounts of contaminating protein S (although not detected by Western blot), we repeated the experiment using FITC- labeled HPS 54 instead of the anti-C4BP Ab. The results were very similar to those obtained with the FITC-labeled MK 104, showing FIGURE 1. Binding of protein S and C4BP to Jurkat cells. Jurkat cells that indeed the binding of C4BP was due to trace amounts of were induced to undergo apoptosis and then incubated with C4BP with or protein S in the sample (data not shown). We also tested binding without protein S or with protein S alone. Cells were then washed and of the C4BP-protein S complex to apoptotic cells in the presence binding of protein was detected in the flow cytometer using FITC-labeled of serum, using detection with FITC-labeled MK 104. Also in the mAbs. Cells were gated to exclude cells in late apoptosis (positive for presence of serum there was a clear binding of the C4BP-protein 7-AAD). Cells positive for annexin V-PE are denoted apoptotic cells, cells S complex as shown in Fig. 2. negative for annexin V-PE are denoted live cells. Results are plotted as percentage of maximum fluorescent intensity seen (geometric mean). Each Binding of protein S and C4BP-protein S is dependent on the point consists of the mean of at least three different experiments Ϯ SD. A, phospholipid binding Gla-domain of protein S Binding of protein S. f, Live cells; Ⅺ, apoptotic cells. B, Binding of C4BP. f, Live cells, C4BP/protein S complex; Ⅺ, apoptotic cells, C4BP/protein FITC-labeled protein S and unlabeled C4BP/protein S (plasma pu- S complex; F, live cells, C4BP preincubated with protein S; E, apoptotic rified complex) were preincubated with mAbs HPS 21 and HPS cells, C4BP preincubated with protein S; Œ, live cells, C4BP without pro- 54. HPS 21 is directed against the Gla domain of protein S and tein S; ‚, apoptotic cells, C4BP without protein S. blocks binding of protein S to phospholipid, whereas HPS 54 is The Journal of Immunology 2583

using FITC-labeled HPS 54, saturating concentrations of HPS 54 were used (see Materials and Methods), thus, no cross-linking was to be expected in those experiments. The binding of protein S to the apoptotic cell surface was Ca2ϩ-dependent, and addition of EDTA at the end of the incubation of FITC-labeled protein S and cells completely abrogated the binding (data not shown).

High concentrations of annexin V diminish the binding of protein S to apoptotic cells

FIGURE 2. Binding of the C4BP-protein S complex in the presence of As annexin V binds with a high afﬁnity to the negatively charged serum. Apoptotic cells were incubated with serum with or without the phospholipid exposed early in apoptosis (53), we tested whether an C4BP-protein S complex and binding of the complex was detected with equimolar concentration of annexin V could affect the binding of FITC-labeled MK 104. Filled line, cells incubated with serum depleted of protein S to the apoptotic cell. Annexin V, 100 nM was added to C4BP and protein S. Dotted line, cells incubated with depleted serum cells together with protein S, 100 nM. In addition, we also tested where C4BP-protein S had been added back. the effect of 3 nM unlabeled annexin V, which corresponds to the concentration of PE-labeled annexin V used in our binding experiments. Cells were washed and then incubated with FITC-labeled directed against the ﬁrst EGF-like domain in protein S (49). The

HPS 54 and 7-AAD. Cells were transferred to tubes containing Downloaded from proteins were then mixed with Jurkat cells induced to undergo ice-cold binding buffer and immediately analyzed in the ﬂow cy- apoptosis with anti-Fas Ab as before. Following incubation, cells tometer. In Fig. 4 we demonstrate that in the presence of annexin incubated with FITC-labeled protein S were immediately analyzed V, protein S binding to the apoptotic cells is diminished, strongly in the ﬂow cytometer while the cells mixed with the C4BP/protein suggesting that the interaction is between phosphoplipid and the S complex were washed and incubated with FITC-labeled MK 104 protein S Gla domain. As annexin V-PE was not included in this before analysis. Preincubation with HPS 21 blocked binding of experiment, it was not possible to selectively gate for the apoptotic http://www.jimmunol.org/ both protein S and C4BP/protein S, strongly suggesting that the population. Fig. 4B shows the same experiment where cells had Gla domain of protein S is what attaches both free protein S and not been induced to undergo apoptosis. However, it was clearly the C4BP/protein S complex to the apoptotic cell (Fig. 3, A and B). shown that annexin V is able to impair the protein S binding to In contrast, HPS 54 did not block binding. Rather, it seemed to apoptotic cells, demonstrating that the binding sites for protein S enhance the binding of FITC-labeled protein S (when the molar and annexin V on the apoptotic cells are the same. ratio between protein and Ab was 1:1) as compared with binding of protein S without preincubation with the mAb HPS 54 (data not shown). We have previously reported a similar phenomenon when C4b binds to apoptotic cells in the presence of the C4BP/protein studying protein S binding to synthetic phospholipid vesicles. The S complex by guest on September 27, 2021 explanation was then found to be cross-linking of two protein S To investigate whether the C4BP that was localized to the apo- molecules on the phospholipid surface by the divalent Ab (49). In ptotic cells could function to bind C4b, we preincubated cells in- our binding experiments using detection of cell-bound protein S duced to undergo apoptosis with the C4BP/protein S complex. The

FIGURE 3. Inhibition of binding using mAbs. Protein S and the C4BP/protein S complex were preincubated with mAbs HPS 21 (directed against the Gla- domain of protein S) and HPS 54 (directed against EGF1 of protein S) before being tested for binding to Jurkat cells. The protein S used was labeled with FITC and could be directly detected in the ﬂow cytometer. For detection of binding of the C4BP/protein S complex, FITC-labeled MK 104 was used in a second step. Cell count is plotted against ﬂuorescence intensity on a logarithmic scale. A and B, Apoptotic cells. C and D, Live cells. Black line, HPS 54 was used for preincubation; gray line, HPS 21 was used for preincubation. 2584 C4BP AND PROTEIN S BIND TO APOPTOTIC CELLS

Discussion Recently, there has been increased attention on the role of the complement system in apoptosis. Lack of C1q has been linked to the pathogenesis of systemic lupus erythematosus, as C1q deficiency might predispose to autoimmunity due to impaired clearance of apoptotic cells (41, 43). Assembly of complement proteins has been observed on apoptotic HUVEC cells (44), and complement has been suggested to partake in the clearance of apoptotic cells by macrophages (54). The C5b-9 membrane attack complex has been implied to play a role both as protector from apoptosis (sublytic complex; Refs. 55Ð57) and as an inductor of apoptosis (58Ð60). Thus, the complement system seems involved in many aspects of apoptosis. Our results demonstrate binding of C4BP/protein S complexes to apoptotic cells and provide new insight into the regulation of complement on apoptotic cells. In addition, binding of C4BP was totally dependent on protein S, because C4BP by itself did not display any binding. Protein S is shown to use the same binding FIGURE 4. Annexin V diminishes binding of protein S to apoptotic Downloaded from cells. Apoptotic Jurkat cells were incubated with 100 nM protein S and 0, site as annexin V, and binds to the cell surface exposed phospha- 3, and 100 nM annexin V. Cells were washed and bound protein detected tidylserine via its Gla domain. Furthermore, C4BP localized via using FITC-labeled HPS 54. Cell count is plotted against HPS 54-FITC protein S to the cell surface could still bind C4b, implying that fluorescence intensity on a logarithmic scale. Protein S binding: A, Apop- C4BP retains its physiological role also when attached to an apo- tosis-induced cells. Black line, in the presence of 100 nM annexin V; ptotic cell surface, because C4b will be cleaved by factor I when dotted line, in the presence of 3 nM annexin V; gray line, in the absence of C4b is associated with C4BP (61, 62). This is similar to our pre- annexin V. B, Noninduced (live) cells. Black line, in the presence of 100 http://www.jimmunol.org/ nM annexin V; gray line, in the absence of annexin V. vious results, where we saw that phospholipid vesicle-associated C4BP/protein S complex still could bind C4b (63). In addition, we could still detect binding of the protein S-C4BP complex to apoptotic cells when they were incubated with physiological concen- cells were washed, and FITC-labeled C4b was added in the pres- tration of C4BP-protein S in the presence of human serum, dem- ence of annexin V-PE and 7-AAD. C4b-FITC was found to bind onstrating that the affinity is sufficiently high for the complex to to apoptotic cells preincubated with the C4BP/protein S complex associate specifically with the apoptotic cells also when serum pro- as compared with the apoptotic cells that were not preincubated teins are present. We were interested to study the physiological with the C4BP/protein S complex (Fig. 5). Clearly, C4BP is able consequences of the binding of the protein S-C4BP complex to by guest on September 27, 2021 to bind C4b even when it is bound via protein S to apoptotic cells, apoptotic cells. However, our efforts to reliably and reproducibly demonstrating the physiological relevance of the C4BP/protein S induce and follow complement activation on these cells have been complex binding. The mAb MK 104 directed against CCP1Ð2of unsuccessful. Nonetheless, the present study demonstrates protein the C4BP ␣-chain (50), when added at the same time as C4b-FITC, S-mediated binding of C4BP to apoptotic cells and suggests a po- could block the binding between C4BP and C4b. We have previ- tential physiological role for the complex in vivo, in localizing ously described the binding site of C4b on C4BP to be localized to C4BP, via protein S, to apoptotic cells where C4BP can down- the interface of ␣-chain CCP1Ð2 (35). regulate complement activation. From a number of studies (41Ð44, 54), it appears that early complement components are important for the rapid clearance of apoptotic cells, but that the cell must be protected from assembly of later components in order not to provoke an inflammatory response triggered by the complement system. Apart from forming the cell lytic membrane attack complex, activation of complement yields an inflammatory response as anaphylatoxins (mainly C5a and C3a) and opsonins (mainly C3b) are generated. Thus, the presence of C4BP, a major inhibitor of the classical pathway, may be of utmost importance to inhibit inflammation close to apoptotic cells. As we have shown in the present study that C4BP cannot bind in the absence of protein S, our results point toward the physiological relevance of the C4BP/protein S complex formation. Al- FIGURE 5. The C4BP/protein S complex increases binding of C4b to though it has been suggested before, this is the first time we show apoptotic cells. Increasing amounts of FITC-labeled C4b was mixed with that protein S is crucial to localize C4BP to an area where com- apoptotic Jurkat cells, either preincubated with the C4BP/protein S complement should be prevented. plex or just with buffer. Cells were then analyzed in the flow cytometer. Serum amyloid P component (SAP) has been shown to bind Increase in geometric mean fluorescent intensity/cell of cells preincubated apoptotic cells (64). It is conceivable that SAP may modulate the with the C4BP/protein S complex, as compared with the intensity seen function of apoptotic cell surface-bound C4BP, because SAP has where cells were preincubated with just buffer, is plotted against the amount of FITC-labeled C4b added (f). Each point represents the average been shown to have an effect on C4b-binding/factor I cofactor of three experiments Ϯ SD. When MK 104 was added to the cells at the function of C4BP (65). However, the interaction between C4BP same time as 300 nM C4b-FITC, the signal returned to that without C4BP/ and SAP is inhibited by the presence of phopshoethanolamine- protein S (F, indicated with an arrow). containing compounds (63, 65). Because SAP was shown to bind The Journal of Immunology 2585 to the apoptotic cells via phosphatidylethanolamine, it is possible 5. Devaux, P. F. 1991. Static and dynamic lipid assymetry in cell membranes. Bio- that SAP does not interact with C4BP bound to the apoptotic cell chemistry 30:1163. 6. Fadok, V. A., D. R. Voelker, P. A. Campbell, J. J. Cohen, D. L. Bratton, and surface, but rather binds directly to the cell surface. In the present P. M. Henson. 1992. Exposure of phosphatidylserine on the surface of apoptotic investigation, we observed that the C4BP-protein S complex could lymphocytes triggers specific recognition and removal by macrophages. J. Im- munol. 148:2207. bind to apoptotic cells in the presence of serum, indicating that the 7. Fadok, V. A., D. J. Laszlo, P. W. Noble, L. Weinstein, D. W. Riches, and complex can bind in the presence of SAP. Moreover, addition of P. M. Henson. 1993. Particle digestibility is required for induction of the phos- serum concentrations of SAP in the binding studies did not affect phatidylserine recognition mechanism used by murine macrophages to phagocy- tose apoptotic cells. J. Immunol. 151:4274. binding of the C4BP-protein S complex to apoptotic cells (data not 8. Kalafatis, M., N. A. Swords, M. D. Rand, and K. G. Mann. 1994. Membrane- shown). dependent reactions in blood coagulation: role of the vitamin K-dependent en- It has also been suggested that the death of T lymphocytes in zyme complexes. Biochim. Biophys. Acta 1227:113. 9. Gerads, I., J. W. Govers-Riemslag, G. Tans, R. F. Zwaal, and J. Rosing. 1990. atherosclerotic plaques may be considered beneficial if apoptosis is Prothrombin activation on membranes with anionic lipids containing phosphate, not accompanied by an inflammatory reaction (66). Our results and sulfate, and/or carboxyl groups. Biochemistry 29:7967. previous studies on Jurkat cells (42, 43) imply that strictly regu- 10. Thiagarajan, P., and J. F. Tait. 1990. Binding of annexin V/placental anticoagulant protein I to platelets: evidence for phosphatidylserine exposure in the pro- lated complement could be essential in the clearance of such cells. coagulant response of activated platelets. J. Biol. Chem. 265:17420. C4BP by itself did not bind apoptotic cells, while protein S did. 11. Bevers, E. M., P. Comfurius, J. L. van Rijn, H. C. Hemker, and R. F. Zwaal. 1982. Generation of prothrombin-converting activity and the exposure of phos- As apoptotic cells expose negatively charged phospholipids, as do phatidylserine at the outer surface of platelets. Eur. J. Biochem. 122:429. activated platelets, the apoptotic cell surface has been considered, 12. Bevers, E. M., P. Comfurius, and R. F. Zwaal. 1983. Changes in membrane phos- and also shown, to be procoagulant (13Ð15). Given our results and pholipid distribution during platelet activation. Biochim. Biophys. Acta 736:57.

13. Bombeli, T., A. Karsan, J. F. Tait, and J. M. Harlan. 1997. Apoptotic vascular Downloaded from the fact that protein S has among the highest affinities of the vi- endothelial cells become procoagulant. Blood 89:2429. tamin K-dependent proteins for negatively charged phospholipids 14. Flynn, P. D., C. D. Byrne, T. P. Baglin, P. L. Weissberg, and M. R. Bennett. (67), the surface of the apoptotic cell may not be solely procoagu- 1997. Thrombin generation by apoptotic vascular smooth muscle cells. Blood 89:4378. lant, but also anticoagulant. In short, where there is coagulation, 15. Casciola-Rosen, L., A. Rosen, M. Petri, and M. Schlissel. 1996. Surface blebs on there is also anticoagulation. Casciola-Rosen et al. (15) showed apoptotic cells are sites of enhanced procoagulant activity: implications for co- that surface blebs of apoptotic cells are sites of enhanced proco- agulation events and antigenic spread in systemic lupus erythematosus. Proc. Natl. Acad. Sci. USA 93:1624. agulant activity. These surface blebs resemble microparticles 16. McDonald, J. F., A. M. Shah, R. A. Schwalbe, W. Kisiel, B. Dahlback, and http://www.jimmunol.org/ formed by activated platelets (68) to which we have shown protein G. L. Nelsestuen. 1997. Comparison of naturally occurring vitamin K-dependent proteins: correlation of amino acid sequences and membrane binding properties S to bind. We saw no binding of the C4BP/protein S complex to suggests a membrane contact site. Biochemistry 36:5120. these platelet-derived microparticles. The reason why these micro- 17. Walker, F. J. 1980. Regulation of activated protein C by a new protein: a possible particles differed from apoptotic cells in this regard is unclear. function for bovine protein S. J. Biol. Chem. 255:5521. 18. Walker, F. J., S. I. Chavin, and P. J. Fay. 1987. Inactivation of factor VIII by Considering that they both expose negatively charged phospholip- activated protein C and protein S. Arch. Biochem. Biophys. 252:322. ids, one could expect that they would display similar binding of not 19. Bertina, R. M. 1985. Hereditary protein S deficiency. Haemostasis 15:241. only free, but also C4BP-bound protein S. The explanation to this 20. Comp, P. C., R. R. Nixon, M. R. Cooper, and C. T. Esmon. 1984. Familial protein Sdeficiency is associated with recurrent thrombosis. J. Clin. Invest. 74:2082. may lie in the physiological role of the platelet microparticles, 21. Tans, G., J. Rosing, M. C. Thomassen, M. J. Heeb, R. F. Zwaal, and J. H. Griffin. foremost providing an area for coagulation or anticoagulation 1991. Comparison of anticoagulant and procoagulant activities of stimulated by guest on September 27, 2021 rather than complement activation. Because protein S loses its an- platelets and platelet-derived microparticles. Blood 77:2641. 22. Zwaal, R. F., P. Comfurius, and E. M. Bevers. 1998. Lipid-protein interactions in ticoagulant cofactor function when in complex with C4BP, binding blood coagulation. Biochim. Biophys. Acta 1376:433. of the complex to platelet microparticles would be without gain. In 23. Nelson, R. M., and G. L. Long. 1991. Solution-phase equilibrium binding interaction of human protein S with C4b-binding protein. Biochemistry 30:2384. contrast, on the apoptotic cell surface where complement compo- 24. Dahlback, B., B. Frohm, and G. Nelsestuen. 1990. High affinity interaction be- nents have associated, binding of the complex can be of great tween C4b-binding protein and vitamin K-dependent protein S in the presence of importance. It is conceivable that this surface may harbor both free calcium: suggestion of a third component in blood regulating the interaction. J. Biol. Chem. 265:16082. protein S, performing its anticoagulant function, and protein S with 25. Schwalbe, R., B. Dahlback, A. Hillarp, and G. Nelsestuen. 1990. Assembly of C4BP, regulating complement action. The potential role of protein protein S and C4b-binding protein on membranes. J. Biol. Chem. 265:16074. S in controlling coagulation on apoptotic cells is an important and 26. He, X., L. Shen, A. C. Malmborg, K. J. Smith, B. Dahlback, and S. Linse. 1997. Binding site for C4b-binding protein in vitamin K-dependent protein S fully interesting area of investigation, especially as patients suffering contained in carboxy-terminal laminin-G-type repeats: a study using recombinant from protein S deficiency are at an increased risk of thrombosis. factor IX-protein S chimeras and surface plasmon resonance. Biochemistry 36: In conclusion, we show that apoptotic cells bind protein S, and 3745. 27. Dahlba¬ck, B. 1991. Protein S and C4b-binding protein: components involved in that protein S can localize the complement regulatory protein the regulation of the protein C anticoagulant system. Thromb. Haemost. 66:49. C4BP to the surface of apoptotic cells, thus providing the potential 28. Walker, F. J. 1989. Characterization of a synthetic peptide that inhibits the interaction betwen protein S and C4b-binding protein. J. Biol. Chem. 264:17645. for local regulation of complement activation. In addition, the cell- 29. Weinstein, R. E., and F. J. Walker. 1990. Enhancement of rabbit protein S an- bound C4BP could still associate with C4b, strongly suggesting a ticoagulant cofactor activity in vivo by modulation of the protein S C4b-binding physiological role for the C4BP/protein S complex in vivo. protein interaction. J. Clin. Invest. 86:1928. 30. Ferna«ndez, J. A., M. J. Heeb, and J. H. Griffin. 1993. Identification of residues 413Ð433 of plasma protein S as essential for binding to C4b-binding protein. J. Biol. Chem. 268:16788. Acknowledgments 31. Ferna«ndez, J. A., J. H. Griffin, G. T. G. Chang, J. Stam, P. H. Reitsma, We thank Prof. Tommy Andersson (Division of Experimental Pathology, R. M. Bertina, and B. N. Bouma. 1998. Involvement of amino acid residues Lund University, Malmo¬, Sweden) for helpful discussions throughout this 423Ð429 of human protein S in binding to C4b-binding protein. Blood Cells Mol. study. Dis. 24:101. 32. Linse, S., Y. Ha¬rdig, D. A. Schultz, and B. Dahlba¬ck. 1997. A region of vitamin K-dependent protein S that binds to C4b-binding protein (C4BP) identified using bacteriophage peptide display libararies. J. Biol. Chem. 272:14658. References 33. Fujita, T., and V. Nussenzweig. 1979. The role of C4-binding protein and ␤1H 1. Israels, L. G., and E. D. Israels. 1999. Apoptosis. Oncologist 4:332. in proteolysis of C4b and C3b. J. Exp. Med. 150:267. 2. Bretcher, M. S. 1972. Assymmetrical lipid bilayer structure for biological mem- 34. Gigli, I., T. Fujita, and V. Nussenzweig. 1979. Modulation of the classical path- branes. Nat. New Biol. 236:11. way C3 convertase by plasma protein C4b binding and C3b inactivator. Proc. 3. Op den Kamp, J. A. 1979. 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