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EfferocytosisMediated−Plasminogen Activator Receptor

Phosphatidylserine and Opsonizes Urokinase High Molecular Weight Kininogen Binds

Qingyu Wu, Raymond B. Birge and Yi WuAizhen Yang, Jihong Dai, Zhanli Xie, Robert W. Colman,

http://www.jimmunol.org/content/192/9/4398doi: 10.4049/jimmunol.1302590March 2014;

2014; 192:4398-4408; Prepublished online 31J Immunol 

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0.DCSupplementalhttp://www.jimmunol.org/content/suppl/2014/03/30/jimmunol.130259

Referenceshttp://www.jimmunol.org/content/192/9/4398.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2014 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Journal of Immunology

High Molecular Weight Kininogen Binds Phosphatidylserineand Opsonizes Urokinase Plasminogen ActivatorReceptor–Mediated Efferocytosis

Aizhen Yang,* Jihong Dai,*,† Zhanli Xie,* Robert W. Colman,† Qingyu Wu,*

Raymond B. Birge,‡ and Yi Wu*,†

Phagocytosis of apoptotic cells (efferocytosis) is essential for regulation of immune responses and tissue homeostasis and is mediated

by phagocytic receptors. In this study, we found that urokinase plasminogen activator receptor (uPAR) plays an important role

in internalization of apoptotic cells and also characterized the underlying mechanisms. In a flow cytometry–based phagocytic

assay, uPAR-deficient macrophages displayed significant defect in internalization but not tethering of apoptotic cells. When

uPAR-deficient mice were challenged with apoptotic cells, they exhibited pronounced splenomegaly resulting from accumulation

of abundant apoptotic cells in spleen. Overexpression of uPAR in HEK-293 cells enhanced efferocytosis, which was inhibited by

Annexin V and phosphatidylserine (PS) liposome, suggesting that uPAR-mediated efferocytosis is dependent on PS. In serum

lacking high m.w. kininogen (HK), a uPAR ligand, uPAR-mediated efferocytosis was significantly attenuated, which was rescued

by replenishment of HK. As detected by flow cytometry, HK selectively bound to apoptotic cells, but not viable cells. In purified

systems, HK was specifically associated with PS liposome. HK binding to apoptotic cells induced its rapid cleavage to the two-

chain form of HK (HKa) and bradykinin. Both the H chain and L chain of HKa were associated with PS liposome and apoptotic

cells. HKa has higher binding affinity than HK to uPAR. Overexpression of Rac1/N17 cDNA inhibited uPAR-mediated efferocy-

tosis. HK plus PS liposome stimulated a complex formation of CrkII with p130Cas and Dock-180 and Rac1 activation in uPAR-

293 cells, but not in control HEK-293 cells. Thus, uPAR mediates efferocytosis through HK interaction with PS on apoptotic cells

and activation of the Rac1 pathway. The Journal of Immunology, 2014, 192: 4398–4408.

Efficient clearance of apoptotic cells by phagocytes (effer-ocytosis) is an essential mechanism for maintenance ofnormal tissue homeostasis and regulation of immune re-

sponses (1, 2). Phagocytes, such as macrophages, use a variety ofreceptors to recognize and internalize apoptotic cells. After engulfingan apoptotic cell, macrophages produce anti-inflammatory cytokines,

such as IL-10 and TGF-b, which prevent inflammation and tissuedamage. However, if apoptotic cells are not rapidly cleared, they willbecome secondary necrotic, causing the release of toxic intracellularAgs that induce tissue damage and production of proinflammatorycytokines. Dysfunctional efferocytosis is often associated withchronic inflammation, with a variety of pathological sequelae in-cluding autoimmune diseases and development of a necrotic corein atherosclerotic plaque (3, 4). Thus, elucidation of the efferocytosisprocess is critical for understanding tissue homeostasis and inflam-mation resolution.Phagocytic receptors that interact with apoptotic cells must

recognize specific eat-me signals on the apoptotic cells. Theomnipresent exposure of phosphatidylserine (PS) on a variety ofapoptotic cells suggests that PS is a general eat-me signal (1, 2).Phagocytes may recognize PS directly through the PS receptorssuch as T cell Ig and mucin domain-1/4 and stablin-2 (5, 6).Another pool of phagocytic receptors, such as av integrins andreceptor tyrosine kinase Mer, interact with PS via opsonins, in-cluding milk fat globule–enhanced GFP (EGFP) and growth ar-rest–specific 6, which bind to PS (1, 7). However, so far, it is stillsurprising that so little is known about how the single PS stimulusdirectly and indirectly transmits the eat-me signal to multiple PSreceptors and how a phagocyte reaps meaningful information fromsuch a general signal. Elucidation of these important issues relieson more comprehensive understanding of key players involved inthis process.Urokinase plasminogen activator receptor (uPAR; CD87) is a

multidomain GPI-anchored protein, implicated in many cellularprocesses, ranging from cell proliferation, motility, angiogenesis,wound repair, inflammation, and tumor invasion to metastasis (8,9). Moreover, the altered expression and function of uPAR areclosely related with inflammatory conditions and autoimmune

*Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, First AffiliatedHospital, Soochow University, Suzhou 215006, China; †Sol Sherry Thrombosis Re-search Center, Temple University School of Medicine, Philadelphia, PA 19140; and‡Department of Biochemistry and Molecular Biology, New Jersey Medical School,Rutgers University, Newark, NJ 07103

Received for publication September 25, 2013. Accepted for publication February 25,2014.

This work was supported in part by grants from the National Natural Science Foun-dation of China (30971491, 31201058, 81270592, and 81301534), the Priority Ac-ademic Program Development of Jiangsu Higher Education Institutions, and theNational Institutes of Health (AR057542 and AR063290).

A.Y., J.D., Z.X., and Y.W. performed research, collected data, analyzed and inter-preted data, and performed statistical analysis; J.D., Q.W., and Y.W. designed re-search; R.W.C. and Q.W. contributed vital reagents; and R.B.B. and Y.W. wrote thepaper.

Address correspondence and reprint requests to Dr. Yi Wu, The Sol Sherry Throm-bosis Research Center, Temple University School of Medicine, 3420 North BroadStreet, Philadelphia, PA 19140. E-mail address: yiwu@temple.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine;EGFP, enhanced GFP; HC, H chain; HK, high m.w. kininogen; HKa, two-chainform of HK; HK-B, biotinylated high-m.w. kininogen; IRES, internal ribosomeentry site; KKS, kallikrein–kinin system; LC, L chain; NBD-PC, 1-oleoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine;PC, phosphatidylcholine; PS, phosphatidylserine; SPR, surface plasmon resonance;suPAR, soluble human urokinase plasminogen activator receptor; TB, trypanblue; uPAR, urokinase plasminogen activator receptor; uPAR2/2, uPar-deficient;WT, wild-type.

Copyright� 2014 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/14/$16.00

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disorders such as lupus and atherosclerosis (10–14) We and othershave reported that uPAR facilitates clearance of not only bacteriaand parasites (15, 16), but also apoptotic cells (17, 18). Given thatefferocytosis is critical for resolution of inflammation, and uPARexpression and function are modulated in many pathological pro-cesses, understanding the mechanisms for uPAR-mediated effero-cytosis is very important. In contrast, the study by Park et al. (17)has proposed uPAR as a negative regulator of phagocytosis of viablecells. These studies suggest that uPAR plays a differential role inengulfment of apoptotic cells and viable cells, and its function iscomplex in these processes (19). Therefore, it is necessary to furtherinvestigate the role of uPAR in efferocytosis and the underlyingmechanisms.In this study, using a uPAR-deficient (uPAR2/2) mouse model, we

present evidence that uPAR is required for internalization of apo-ptotic cells, which is mediated by a PS-dependent pathway. Highm.w. kininogen (HK), a known uPAR ligand in plasma, physicallybinds to PS and serves as an opsonin bridging the interaction ofuPAR with PS. Because HK is a critical component of the plasmakallikrein–kinin system (KKS; also named the plasma contact ac-tivation system), its involvement in phagocytosis of apoptotic cellsreveals a novel role for this system in regulation of innate immunityand apoptotic cell-associated procoagulant activity.

Materials and MethodsMice and reagents

Breeding pairs of uPAR2/2 mice on a C57BL/6 background were pur-chased from The Jackson Laboratory and housed in a specific pathogen-freefacility. Eight- to 10-wk-old mice were used in experiments, and wild-type(WT) littermates were used as controls. All of the animal protocols havebeen reviewed and approved by the Institutional Animal Care and UseCommittees of Soochow University and Temple University. Reagents werepurchased from Sigma-Aldrich unless otherwise specified.

Preparation of recombinant proteins and synthetic peptides

The recombinant soluble human uPAR (suPAR) protein was generated andpurified as previously described (20). Recombinant protein of human HKH chain (HC; aa 18–380) and L chain (LC; aa 390–644) was generated aspreviously described (21). The synthetic peptides GKD25 (GKDFVQPPTK-ICVGCPRDIPTNSPE), GCP28 (GCPRDIPTNSPELEETLTHTITKLNAEN),NAT26 (NATFYFKIDNVKKARVQVVAGKKYFI), KKY30 (KKYFIDFV-ARETTCSKESNEELTESCETKK), RET27 (RETTCSKESNEELTESCETK-KLGQSLD), LDC27 (LDCNAEVYVVPWEKKIYPTVNCQPLGM), VSP21(VSPPHTSMAPAQDEERDSGKE), GKE19 (GKEQGHTRRHDWGHEKQ-RK), HNL21 (HNLGHGHKHERDQGHGHQRGH), GHG19 (GHGLGHG-HEQQHGLGHGHK), FKL20 (FKLDDDLEHQGGHVLDHGHK), and HKH20(HKHGHGHGKHKNKGKKNGKH) were based on sequences in HK asdescribed previously (22, 23).

Cell preparation and transfection

Human full-length uPAR cDNA was subcloned into p-internal ribosomeentry site (IRES) 2–EGFP at the XhoI and SalI sites (18). Peritonealmacrophages were elicited from mice as previously described (24). Cultureof HEK293 cells and transfection using Lipofectamine 2000 (Invitrogen)were performed as previously described (18).

Preparation of liposomes and phospholipid-coated beads

Phosphatidylcholine (PC), PS, 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC), and 1-oleoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine (NBD-PC) were from Avanti-Polar Lipids. To prepare liposome containing 100% PC (PC liposome)or PC/PS (80:20 molecular percentage; PS liposome), the appropriateamounts of phospholipids were dissolved in chloroform in a glass tube.After being dried under nitrogen, phospholipids were suspended in PBSand vortexed (25, 26). To prepare phospholipid-coated beads, 6.5 mg/mlNucleosil 120-3 C18 beads (Macherey-Nagel) in 1 ml chloroform wereincubated with 2.5 mg/ml DOPC (PC beads) or mixed DOPC/PS (80:20molecular percentage; PS beads). After being dried under nitrogen, thebeads were resuspended in PBS and sonicated, followed by labeling with2.5 mM NBD-PC at 37˚C for 20 min. After centrifugation at 13,000 3 g

for 10 min, the beads were washed with ice-cold PBS twice to removeremaining NBD-PC and resuspended in PBS.

In vitro and in vivo phagocytosis assay

Apoptotic neutrophils or PS-coated beads were used as phagocytic targets.Studies using human blood were performed after approval by SoochowUniversity or Temple University Institution Review Boards with informedconsent in accordance with the Declaration of Helsinki. Human neutrophilswere collected and purified from peripheral blood as previously described(27). Mouse neutrophils were purified from bone marrow cells using anegative selection kit (Miltenyi Biotec). Apoptosis was induced by heatingat 42˚C for 60 min and incubation at 37˚C for 3 h. Apoptosis was evaluatedby an Apoptosis Detection Kit (BD Biosciences) and flow cytometry andverified as positive for Annexin V and ,10% positive to propidium iodidestaining. Apoptotic cells were purified by magnetic Annexin V microbeads(Miltenyi Biotec). Phagocytosis assay by flow cytometry was performed aspreviously described (7).

The in vivo phagocytosis assay was performed according to the methoddescribed by Morelli et al. (28) with slight modification. A total of 43 107

NBD-labeled PS beads were injected i.v. into mice. After 12 h, the micewere euthanized, and spleen was collected and digested with 400 U/mltype IV collagenase at 37˚C for 30 min. Splenic mononuclear cells wereisolated by density-gradient centrifugation at 300 3 g for 30 min andlabeled with biotin-conjugated anti-F4/80 Ab (eBioscience). F4/80-posi-tive macrophages were collected by anti-biotin magnetic microbeads (MiltenyiBiotec). Macrophages engulfing NBD-PS beads were measured by flowcytometry.

Spleen and thymus index and in situ identification of nuclearDNA fragmentation

Apoptotic cells (2 3 107/mouse) were i.v. injected into mice every day for1 wk. After mice were sacrificed, spleen and thymus were removed, blotteddry, and freed of blood clots. Both body and spleen or thymus wereweighed for calculation of spleen/thymus index (%) = (spleen/thymusweight [mg]/body weight [g] 3 100%) (29).

For in situ calculation of apoptotic cells accumulated in spleen, spleenwas immersion-fixed in 10% buffered formalin and embedded in paraffin.After tissue was sectioned and deparaffinized, it was stained using theTUNEL method (ApopTag fluorescein in situ apoptosis detection kit;Millipore). Nuclei were counterstained with DAPI to aid in visualization.The total DAPI-positive cells and cells with FITC-labeled apoptotic nucleiwere numerated under fluorescent microscope and analyzed with the KS400Image Analysis System (KS400; Zeiss, Heidelberg, Germany).

Amnis ImageStream Data Analysis

As we previously described (18), ImageStream data for the uptake of NBD-PC–labeled PS beads or apoptotic cells by PE-F4/80 labeled macrophageswere acquired using the Amnis ImageStream Analyzer instrument equippedwith the Amnis INSPIRE software. This software can distinguish the bindingand internalization of targets. The beads or apoptotic cells that overlappedwith two peripheral pixels of phagocytes were considered as bound but notinternalized; the rest was considered as internalized. The percentage ofcells for binding and internalization were analyzed with the Amnis IDEASsoftware.

Measurement of the binding of plasma proteins to apoptoticcells and liposomes

Single-chain human HK (Enzyme Research Laboratories), uPA, andvitronectin (R&D Systems) were biotinylated using the EZ-Link Sulfo-NHS-LC-Biotinylation Kit (Thermo Scientific) (30). Binding of bio-tinylated protein to cell surface was measured by flow cytometry. Bindingof HK to liposomes was analyzed using Western blot.

Surface plasmon resonance assay

The surface plasmon resonance (SPR) analysis was carried out with theBiacore X system (GE Healthcare, Pittsburgh, PA). PBS-Zn buffer (16 mMNa2HPO4, 3 mM NaH2PO4, 0.15 M NaCl, and 50 mM ZnCl2 [pH 7.4]) wasused as running buffer, and 7.4 mg/ml HK protein was diluted in 10 mMacetate (pH 4) and immobilized onto flow cell 2 of the CM5 sensor chip(GE Healthcare). The remaining activated sites on the chip surfaces wereblocked with a 35-ml injection of an ethanolamine hydrochloride solution(1 M at pH 8.5), followed by a 60-s wash with 2 M NaCl to remove anynonspecifically adsorbed materials. Approximately 3000 RU immobilizedHK protein was obtained. For all subsequent measurements, a flow rate of30 ml/min was used. PS liposome (PS/PC 1:9) or PC diluted with running

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buffer was allowed to bind to HK protein for 3 min to reach equilibrium,followed by 20 min of dissociation. The response curves were obtained bysubtraction of the signals over the reference surface from the binding re-sponse to HK immobilized surface and analyzed using BIAevaluationsoftware, version 2.0 (GE Healthcare).

Measurement of bradykinin production

Bradykinin was measured using an ELISA kit (Enzo Life Sciences,Farmingdale, NY).

Immunoprecipitation and immunoblotting

Immunoprecipitation and immunoblotting were performed as previouslydescribed (7).

Assay for Rac1 activation

GTP-loaded Rac1 was precipitated with GST-fusion protein expressinghuman PAK1 (GST-PAK1; aa 56–272) and detected by immunoblottingwith anti-Rac1 mAb (Millipore) (7).

Statistical analysis

The results are expressed as means 6 SD. In one-way ANOVA (formultiple groups) or Student t test (for comparisons between two groups),a p value ,0.05 was considered statistically significant. Unless statedotherwise, the data are from a single experiment representative of at leastthree separate experiments.

ResultsuPAR is required for the internalization of apoptotic cells bymacrophages

Our previous study using uPAR-overexpression cell lines haveshown that uPAR plays an important role in phagocytosis of apo-ptotic cells (18). Because uPAR overexpression failed to stimulateengulfment of viable cells, uPAR recognizes determinant signal onthe surface of apoptotic cells (18). Our initial motivation was toverify the function of uPAR using a genetic model and determinethe underlying mechanism. Tethering (binding) and internalizationof apoptotic cells by phagocytes are two key steps of efferocytosis.To verify the function of uPAR in efferocytosis, the binding andinternalization of apoptotic cells was compared between WT anduPAR2/2macrophages. As shown in Fig. 1A, the uPAR protein wasabsent in uPAR2/2 peritoneal macrophages (top panel), and itsmembrane expression was deficient on uPAR2/2 macrophages(bottom panel). In the flow cytometry–based phagocytosis assay,there was no remarkable difference in the association, includingbinding and ingestion, of apoptotic cells between WTand uPAR2/2

macrophages (Fig. 1Bi). To distinguish the role of uPAR in bindingand internalization of apoptotic cells, macrophages were treatedwith trypan blue (TB), which completely quenches fluorescence ofthe dye that stains unengulfed cells (Fig. 1Bii). Although whentreated with TB WT macrophages had similar internalization withthose without TB treatment, uPAR2/2 macrophages displayed sig-nificantly lower internalization of apoptotic cells than WT macro-phages by 60% (TB: +, Fig. 1Bi). This result suggests that uPAR isselectively required for internalization of apoptotic cells, but not thetethering. About 80% of WT macrophages internalized more thanone cell, but the percentage of phagocytic cells was of no differencebefore and after TB treatment. It has been suggested that uPAR onboth phagocytes and target cells regulates phagocytosis (17). Thus,we used this TB treatment method to examine the specific contri-bution of uPAR expressed on both macrophages and apoptotic cellsto internalization of apoptotic cells. As shown in Fig. 1C, whenviable neutrophils were used as targets, both WT and uPAR2/2

macrophages had low internalization capacity, and uPAR2/2 mac-rophages had a significantly stronger ingestion than WT macro-phages. However, when incubated with apoptotic neutrophils,uPAR2/2 macrophages displayed a dramatic reduction in inter-

nalization of apoptotic cells, regardless of the presence or absenceof uPAR on apoptotic cells (Fig. 1C). This result indicates thatuPAR is a bona fide receptor for internalization of apoptotic cells.Similarly to uPAR2/2 macrophages, when uPAR expression of hu-man monocyte cell line THP-1 cells was downregulated by smallinterfering RNA, their internalization of apoptotic cells was signif-icantly reduced (Supplemental Fig. 1). As shown in Fig. 1C, uPAR2/2

macrophages had greater phagocytosis of viable cells than WTmacrophages, and WT macrophages have greater phagocytosis ofviable uPAR2/2 cells than that of viable WT cells, which is con-sistent with the observations of Park et al. (17). The results fromthese two studies suggest that uPAR plays a distinct role in phago-cytosis of viable cells and phagocytosis of apoptotic cells.

FIGURE 1. uPAR is required for the internalization of apoptotic cells

in vitro. (A) Absence of uPAR expression in uPAR2/2 macrophages.

Macrophages were isolated from WT and uPAR2/2 mice (n = 3), pooled

together, and their expression of uPAR was measured by Western blot

analysis (Ai) and flow cytometry (Aii). The data are representative of three

experiments. (B) Internalization of apoptotic cells. Peritoneal macrophages

from WT and uPAR2/2 mice were incubated with apoptotic cells at 37˚C

for 15 min, followed by treatment with or without 0.04% TB to quench the

fluorescence of unengulfed apoptotic cells (Bi). Phagocytosis of apoptotic

cells was analyzed by flow cytometry. Association is indicated as per-

centage of binding and ingestion (TB:2) and ingestion (TB: +). Quench of

fluorescence signal of CFSE-stained apoptotic cells by TB was confirmed

by flow cytometry (Bii). (C) Viable or apoptotic neutrophils (53 105) from

WT or uPAR2/2 mice were incubated with adherent WT or uPAR2/2

macrophages (5 3 104) on 96-well plates at 37˚C for 15 min. After

macrophages were treated with TB, phagocytosis was analyzed by flow

cytometry. *p , 0.05, **p , 0.01, ***p , 0.001. SSC, side scatter.

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Amnis cellular imaging technology is able to distinguish thebinding and internalization of phagocytic targets by visualizinginteractions between phagocytes and target cells with confocalaccuracy and is capable of quantitating phagocytes with the boundand internalized targets. PS is major eat-me signal on apoptoticcells. We used this technique and PS beads as a second approach toverify the role of uPAR in PS-dependent internalization. Shown inFig. 2Ai are representative panels of peritoneal macrophagesbinding and engulfing NBD-labeled PS beads. Analysis by AmnisINSPIRE software indicates that uPAR2/2 macrophages exhibiteda higher capacity of binding PS beads than WT macrophages (58.265.7 versus 32.5 6 4.6%; p , 0.01; Fig. 2Aii). However, uPAR2/2

macrophages had significantly reduced internalization of PS beads(28.9 6 3.9 versus 60.8 6 8.4%; p , 0.01; Fig. 2Aii). Whenapoptotic cells were used as a target, uPAR2/2 macrophages alsoexhibited a significant decrease in internalization (78.9 6 3.4versus 30.2 6 7.3%; p , 0.01) and higher capacity of bindingthan WT macrophages (22.1 6 2.9 versus 68.8 6 7.2%; p , 0.01)(Fig. 2B). These results serve as additional evidence showing thatuPAR is indeed required for internalization of apoptotic cells.These data also suggest that apoptotic cells and PS-expressingbeads may bind to uPAR through HK and to other PS receptorssimultaneously, and the PS-mediated cross linking of uPAR withother receptors is required for the internalization. In the absence ofuPAR, macrophages have defects in internalization, but the targetsare still bound to other receptors and thus tethered to the surface ofmacrophages.

Deficiency of uPAR leads to accumulation of apoptotic cells inmouse spleen and decreases splenic macrophage efferocytosisin vivo

We next examined the in vivo function of uPAR in efferocytosis.After mice were challenged with i.v. injection of apoptotic cells,uPAR2/2 but not WT mice exhibited pronounced splenomegaly(Fig. 3Ai, ii), although WT and uPAR2/2 mice without injectionof apoptotic cells showed no significant difference in mean spleenindex at baseline (Fig. 3Aii). After mice received an injection ofapoptotic cells, the size of the thymus (mean thymus index) ofuPAR2/2 mice was not significantly different from that of WT(1.526 0.52 versus 1.676 0.33; p. 0.05), suggesting that spleenis probably the major site where uPAR is needed for the clearanceof injected apoptotic cells. As measured by a TUNEL assay, theaccumulation of apoptotic cells in uPAR2/2 spleens was increasedby ∼10-fold versus WT spleens (p, 0.01; Fig. 3Bi, ii). Moreover,as shown in Fig. 3C, in an in vivo phagocytosis assay, uPAR2/2

macrophages displayed a lower internalization of PS beads(Fig. 3Ci) or apoptotic cells (Fig. 3Cii) by ∼50% over WT mac-rophages, which is consistent with the in vitro observations(Fig. 1C). Because the exposure of a large number of apoptoticcells causes autoantibody production in mice, especially anti-PSAbs, autoantibodies in sera were measured with an ELISA assay.In sera from uPAR2/2 mice, the level of anti-PS Igs was signifi-cantly higher than that in WT mice (Supplemental Fig. 2), suggestingthat the impaired phagocytosis of apoptotic cells in uPAR2/2 micecauses autoantibody production in vivo.

uPAR mediates internalization of apoptotic cells throughrecognition of PS

The decrease in internalization of PS beads by uPAR2/2 macro-phages suggests that uPAR mediates efferocytosis through interac-tion with PS. To further dissect the mechanism underlying the roleof uPAR in efferocytosis, we examined uPAR-dependent eventsusing a cell line. We generated HEK293 cells overexpressing uPARand EGFP (uPAR-293 cells) using a bicistronic IRES vector, and

FIGURE 2. uPAR is required for internalization but not the binding of

PS beads. (A) Amnis ImageStream Data Analysis. Peritoneal macrophages

from WT and uPAR2/2 mice were incubated with NBD-labeled PS beads

at 37˚C for 20 min. After washing with PBS, macrophages were labeled

with PE-F4/80 Ab and evaluated using Amnis ImageStream Data Analysis,

as described in the Materials and Methods. Shown are the representative

output images after Amnis acquisition (original magnification 340) (Ai).

ImageStream data were acquired using the Amnis ImageStream Analyzer,

and the percentage of macrophages that bound (binding) and internalized

(internalization) PS beads was quantitated with the Amnis IDEAS soft-

ware (Aii). (B) As described above, apoptotic cells were used as a target

instead of PS beads, and peritoneal macrophages from WT and uPAR2/2

mice were labeled with CFSE prior to incubation with PKH26-labeled

apoptotic cells at 37˚C for 20 min. The percentage of macrophages binding

(Binding) and internalizing (Internalization) apoptotic cells were analyzed

with the Amnis IDEAS software. **p , 0.01.

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the expression of uPAR on the cell surface was confirmed by flowcytometry (Fig. 4A). Compared with EGFP-293 cells that weretransfected with an empty IRES vector expressing EGFP alone(control mock), uPAR-293 cells exhibited a significantly highercapacity for internalization of apoptotic cells (Fig. 4B). However,

the expression of uPAR on uPAR-293 cells did not increase theinternalization of viable cells (Fig. 4B). Treatment of these cellswith suPAR significantly inhibited internalization of apoptotic cells(Fig. 4C), suggesting that the gain of functional phagocytosis isconferred by surface expression of uPAR. Using this cell model, wedetermined whether uPAR-mediated internalization of apoptotic cellsis dependent on PS. Annexin V is a 35-kDa phospholipid-bindingprotein and has a high binding affinity for PS in the presence ofcalcium (Ca2+). When apoptotic cells were preincubated with 50 mg/ml of unlabeled Annexin V and Ca2+, the binding of FITC-labeledAnnexin V was completely blocked (Fig. 4Di), suggesting that thebinding of Annexin V at the concentration of 50 mg/ml to PS onapoptotic cells became saturated. At the same concentration, AnnexinV significantly inhibited the engulfment by uPAR-293 cells in thepresence of Ca2+, but not the absence of Ca2+ (Fig. 4Dii). In addition,PS liposome, but not PC liposome, significantly inhibited uPAR-mediated engulfment of apoptotic cells (Fig. 4E). These resultssuggest that uPAR-mediated internalization of apoptotic cells isdependent on PS signals.

Plasma HK binds to PS and opsonizes uPAR-mediatedinternalization of apoptotic cells

To date, there are no data showing the direct interaction betweenuPAR and PS. In a cell-free system, we failed to detect recombinantsuPAR protein bound to PS liposome (data not shown). We pre-sumed that uPAR interacts with PS via opsonin(s) and thuscompared the internalization capacity of uPAR-293 cells in thepresence and absence of serum. As shown in Fig. 5A, the inter-nalization of apoptotic cells by uPAR-293 cells was completelyprevented in the absence of serum, suggesting that opsonin(s) inserum is necessary for uPAR-mediated efferocytosis. It has beenknown that uPAR has three ligands in plasma: uPA, vitronectin,and HK (31, 32). To test which protein(s) mediates uPAR effer-ocytosis, the assays were done using the serum depleted of eachprotein that was achieved by an immunoprecipitation method.Compared with normal serum, the internalization by uPAR-293cells was markedly compromised in the serum depleted of HK[HK(2); p , 0.01], which was reversed by replenishment of ei-ther HK or its cleaved form, two-chain HKa (Fig. 5B). In contrast,when uPAR-293 cells were cocultured with apoptotic cells in uPA-or vitronectin-depleted serum, there was no reduction in engulf-ment (Supplemental Fig. 3). Thus, HK is required for uPAR-mediated internalization of apoptotic cells.We next studied whether and how HK binds to apoptotic cells.

We have previously shown that HK binding to cell-surface ornegatively charged molecules is dependent on ZnCl2 (Zn2+) (31,33), and we began with investigation of the binding of HK toapoptotic cells in the presence and absence of Zn2+. As shown inFig. 5C, in the absence of Zn2+, few biotinylated HK (HK-B)bound to apoptotic cells. However, the addition of 50 mM Zn2+

increased the binding of HK-B by ∼18-fold (Fig. 5Ci, ii). When50 nM HK was incubated with mixed apoptotic cells and viablecells (Fig. 5Di), HK selectively bound to apoptotic cells that wereAnnexin V positive, but not to Annexin V–negative viable cells,whereas the binding of HK at this concentration was not saturable(Fig. 5Dii). The binding of HK to apoptotic cells was concentra-tion dependent (Fig. 5Dii), and the Michaelis constant was ,30nM. Thus, HK specifically binds to apoptotic cells. In contrast,vitronectin and uPA bound to both apoptotic cells and viable cells(Supplemental Fig. 4). SPR experiments were performed to in-vestigate the binding of PS liposome to immobilized HK. Eachconcentration used was diluted and injected in triplicate, andthe three curves were averaged to one curve per concentration(as shown) and analyzed using the BIAevaluation program (GE

FIGURE 3. uPAR2/2 mice exhibit impaired clearance of apoptotic

cells. (A) Apoptotic cells (2 3 107/mouse) were i.v. injected into WT and

uPAR2/2 mice (n = 7) every day for 7 d. Shown is the representative gross

appearance of spleens on day 8 (Ai). (Aii) The mean spleen index of WT

and uPAR2/2 mice that received injection of PBS (AC2) and apoptotic

cells (AC+) is shown. (B) Spleens of the above experiments were further

processed for paraffin-embedded sections. Apoptotic cells were detected

by TUNEL method. Nuclei were counterstained with DAPI. Representa-

tive images (original magnification 320) are shown (Bi). Scale bars, 100

mm. (Bii) TUNEL-positive cells were enumerated in 10 randomly chosen

high-power fields (hpf). (C) Internalization of PS-coated beads or apoptotic

neutrophils in vivo. As described in the Materials and Methods, 4 3 107

NBD-PC–labeled PS beads (Ci) or apoptotic neutrophils isolated from WT

or uPAR2/2 mice (Cii) were i.v. injected into WT and uPAR2/2 mice (n =

5). Six hours after injection, spleens were harvested and homogenized, and

F4/80-positive macrophages were purified. After treatment with or without

TB, internalization of PS-coated beads was analyzed by flow cytometry

(Ci). After treatment with TB, internalization of uPAR+/+ and uPAR2/2

apoptotic neutrophils was analyzed by flow cytometry (Cii). **p , 0.01,

***p , 0.001.

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Healthcare) as described in Materials and Methods. The increasein response units in all five traces after the injection of liposomesindicates the binding of the PS liposomes, but not PC liposomes,to the surface of the chip (Fig. 5E). Additional injections of PSliposomes (300 mM) did not increase the signal, indicating acomplete coverage of the surface with PS (data not shown). Theseresults demonstrate that HK is physically and specifically asso-ciated with PS, and HK may recognize apoptotic cells by directbinding to PS.HK has two functional domains, HC and LC, which are produced

by the cleavage. We further test which domain is involved in HKassociation with apoptotic cells. When 50 nM biotin-labeled HKwas incubated with apoptotic cells in the presence of recombinantHC or LC, both HC and LC inhibited the binding of biotin-labeledHK to apoptotic cells (Fig. 6Ai), suggesting that HK binds toapoptotic cells through both HC and LC. To examine whether HKdirectly binds to PS, HK was incubated with PC or PS liposome.As shown in Fig. 6Bi, HK bound to PS liposome was much moreabundant than that to PC liposome. Moreover, like HK, HC andLC bound to PS liposome directly (Fig. 6Bii). It has been shownthat D3 (273–376 aa) in HC and D5 (384–498 aa) in LC are majorsurface binding regions, and we thus used the peptide inhibitionmethod to map the binding domain of HK to apoptotic cells. Asshown in Fig. 6C, two D3 peptides, LDC21 and RET27, and oneD5 peptide, HKH20, dose-dependently inhibited the binding ofHK to apoptotic cells. However, a GKD25 and other D3 and D5peptides had no inhibitory effect (Fig. 6C and data not shown).

These data suggest that HK bound to apoptotic cells through, atleast, its D3 (324–375 aa) and D5 (479–498 aa).

Association with apoptotic cells induces the cleavage of HKand production of bradykinin

As a major component of the plasma KKS, HK is responsible forbinding to activation surfaces for assembly of KKS components,leading to kallikrein activation and its cleavage of HK to two-chainHKa and bradykinin. We thus examined whether the associationof HK with apoptotic cells mediates its cleavage to HC and LC.As detected by Western blotting, incubation with apoptotic cellsresulted in the rapid cleavage of HK as early as 30 s and as afunction of time (Fig. 6D). In a reduced SDS gel, the cleavageproducts include three major fragments with molecular mass of62, 56, and 45 kDa, respectively, which represent HC (aa 18–380,62 kDa), LC (aa 390–644, 56 kDa), and a secondary 45-kDacleavage fragment of LC. Correspondingly, incubation of serumwith apoptotic cells, but not with viable cells, significantly in-creased the production of bradykinin (Fig. 6E). Thus, apoptoticcell membrane is a novel surface for activation of plasma KKS.

uPAR mediates efferocytosis through the p130Cas–CrkII–Dock-180–Rac1 pathway

Because uPAR is a GPI-anchored protein and localized in themembrane rafts, we thus tested whether the integrity of rafts isinvolved in uPAR efferocytosis. As shown in Fig. 7A, treatmentmethyl-b-cyclodextrin that depletes lipid in rafts significantly

FIGURE 4. PS is necessary for uPAR-mediated internalization of apoptotic cells. (A) HEK293 cells were transfected with pIRES2-EGFP plasmid

(EGFP-293 cells) or IRES2-EGFP-uPAR plasmid (uPAR-293 cells), and their expression of uPAR on membrane was measured by flow cytometry. (B)

EGFP-293 cells and uPAR-293 cells were incubated with PKH26-labeled viable or apoptotic cells for 2 h, respectively. Phagocytosis by green fluorescent

293 cells was evaluated after treatment with TB as described in the legend for Fig. 1. (C) suPAR inhibits phagocytosis of apoptotic cells in uPAR-293 cells.

uPAR-293 cells were incubated with PKH26-labeled apoptotic cells in the presence of 1 mg/ml suPAR or BSA for 2 h, which were subjected to the

phagocytosis assay. (Di) After preincubation with 50 mg/ml of unlabeled Annexin V or BSA plus 2.5 mM CaCl2 for 20 min, apoptotic cells were labeled

with FITC-conjugated Annexin V (FITC-AxV), followed by flow cytometric analysis. After preincubation with 50 mg/ml of unlabeled Annexin V in the

presence or absence of 2.5 mM CaCl2 for 20 min, apoptotic cells were cocultured with uPAR-293 cells, followed by treatment with TB and internalization

analysis using flow cytometry (Dii). (E) After preincubation with PBS (Ctrl), 50 nM PC liposome, or PS liposome for 20 min, apoptotic cells were

cocultured with uPAR-293 cells, followed by treatment with TB and internalization analysis using flow cytometry. *p , 0.05, ***p , 0.001.

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inhibited internalization of apoptotic cells by uPAR-293 cells,suggesting the dependence of uPAR-mediated efferocytosis onmembrane rafts. It has been known that membrane rafts are as-

sociated with intracellular signaling molecules complex for Rac1activation. Because Rac1 activation mediated by the p130Cas–CrkII–Dock-180 pathway is critical for internalization of apo-ptotic cells, and uPAR activates this pathway in other cells (34,35), we investigated whether Rac1 activation is required for uPARefferocytosis. In uPAR-293 cells expressing dominant-negativeRac1/N17 cDNA (Fig. 7Bi), their internalization capacity wassignificantly reduced (p , 0.01; Fig. 7Bii). Moreover, whenuPAR-293 cells were stimulated with PS liposome plus HK, CrkIIformed a complex with p130Cas and Dock-180, and an activeform of Rac1 was increased (Fig. 7C). However, HK or PS-liposome itself alone did not have such effect (Fig. 7D). Thesedata suggest that HK mediates PS–uPAR interaction and promotesclustering of uPAR in the plasma membrane, thereby enhancingthe intracellular p130Cas–CrkII–Dock-180 pathway leading toRac1 activation.

DiscussionOur present study shows that uPAR plays an important role ininternalization of apoptotic cells. The deficiency of uPAR causessplenomegaly resulting from impaired clearance of apoptotic cells.uPAR-mediated efferocytosis is dependent on PS pathway. More-over, we show that HK is a novel PS-binding protein and serves as anopsonin bridging uPAR on macrophages and apoptotic cells. uPARmediates internalization of apoptotic cells by activating p130Cas–CrkII–Dock-180–Rac1 pathway. However, its role in tethering ap-optotic cells is dispensable. Because uPAR is widely involved ina variety of vascular processes, its function in efferocytosis shouldprovide a novel insight into its function in vascular biology andpathological disorders.In our previous studies, we discovered that avb5 integrin is

important in engulfment of apoptotic cells (1, 7, 18). We positedthat integrins might dynamically form a cluster with its partnerreceptors in specialized membrane domains like membrane raftsthat activate small GTPases. We have shown that uPAR, which isa GPI-anchored protein localized in a lipid raft, is important for

FIGURE 5. HK binds to apoptotic cells and is re-

quired for uPAR-mediated internalization. (A) EGFP-

293 cells and uPAR-293 cells were cocultured with

apoptotic cells in the presence 10 or 0% FBS for 2 h.

Internalization of apoptotic cells was analyzed by flow

cytometry after treatment with TB as described in the

legend for Fig. 4. (B) The levels of HK in normal

serum (lane 1, inset) and HK-depleted serum (lane 2,

inset) were analyzed by immunoblotting. The uPAR-

293 cells were incubated with apoptotic cells in

presence of normal serum, HK-depleted serum [HK(2)],

HK-depleted serum replenished with 50 nM HK

[HK(2)+HK], or HKa [HK(2)+HKa], respectively.

After treatment with TB, the internalization of apo-

ptotic cells was evaluated by flow cytometry as de-

scribed in the legend for Fig. 4. (C) Apoptotic cells

were incubated with 100 nM HK-B in PBS with or

without 50 mM ZnCl2 at 4˚C for 15 min. After

washing with PBS containing 0.35% BSA and 50 mM

ZnCl2, the cells were stained with PE-avidin, and the

fluorescence intensity was quantitated by flow cy-

tometry. Representative data of HK bound to apo-

ptotic cells in the presence (Zn2+ plus) or absence (Zn2+

free) of zinc ions (Ci) and the mean fluorescence

intensity (MFI) from triplicate samples (Cii) are

shown. (D) Mixed apoptotic cells and viable cells

were incubated with 50 nM HK-B in Tyrode’s buffer

containing 0.35% BSA and 50 mM ZnCl2 at 4˚C for

15 min. After washing with PBS containing 50 mM

ZnCl2, the cells were labeled with PE-avidin and

nonsaturated amounts of FITC–Annexin V (AxV).

The intensity of PE fluorescence for HK-B bound to

apoptotic cells (FITC-AxV positive) and viable cells

(FITC-AxV negative) was analyzed by flow cytometry

(Di). Shown in (Dii) is the quantification of the per-

centage of PE-positive cells at the indicated concen-

trations of HK-B, representing HK-B–bound cells

(%). (E) SPR assay. PS liposome (Ei) or PC liposome

(Eii) at the indicated concentrations was allowed to

bind to HK-immobilized CM5 sensor chip to reach

equilibrium, followed by dissociation. The response

curves were analyzed using BIAevaluation software.

***p , 0.001.

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av integrin function in endothelial cells (30). Initially, we inves-tigated whether uPAR association with avb5 or avb3 integrins inlipid rafts could be a requisite for engulfment. Although we suc-ceeded in observing that uPAR is a novel phagocytic receptor,several lines of our data have indicated that uPAR’s function inefferocytosis is independent of integrins (18). Park et al. (17) re-ported that the loss of uPAR expression contributes to the en-hanced clearance of viable cells. Thus, the role of uPAR inefferocytosis and the underlying mechanisms are complex. In thisstudy, using TB treatment (Figs. 1–3) and the Amnis ImageStreamData Analysis (Fig. 2), we found that uPAR is required for theinternalization, but not for the tethering/binding, of apoptoticcells. It is likely that the discrepancy on the role of uPAR inefferocytosis results from a fact that uPAR plays distinct roles inphagocytosis of apoptotic cells and phagocytosis of viable cells. Itmight serve as an unconventional "do not eat me" to prevent the

phagocytes from engulfing healthy cells when the eat-me signalsdo not exist. When viable cells become apoptotic, uPAR mayrecognize eat-me signal PS and switch to a phagocytic receptor.We speculate that phagocytes recognize and bind to PS on apo-ptotic cells via uPAR and its partner PS receptors, but the PS-mediated cross linking of uPAR with other receptors is requiredfor the internalization. In the absence of uPAR, its partner PSreceptors may still bind to apoptotic cells but have defects in in-ternalization.The redistribution of PS to the external membrane surface is

a key element of apoptotic cell recognition and a molecular sig-nature that dying cells can be distinguished from viable cells (1). Inthis study, we provide evidence for the first time, to our knowl-edge, that uPAR-mediated efferocytosis is dependent on PS. BothAnnexin V and PS liposome significantly inhibit uPAR-mediatedphagocytosis of apoptotic cells (Fig. 4). Unlike other receptors

FIGURE 6. Both of HC and LC are involved in HK binding to apoptotic cells and PS. (A) HC and LC inhibit the binding of HK to apoptotic cells.

Apoptotic cells were incubated with HC and LC at the indicated concentrations at 4˚C for 20 min, followed by addition of biotin-HK (100 nM) and PE-

labeled avidin. The rest steps were as described in the legend for Fig. 5B. Shown are the measurements from three separate experiments. (B) HK binds to PS

liposome via its HC and LC. (Bi) Total of 5 mg of HK protein was incubated with PBS (2) or 50 nM PC or PS liposomes in Tyrode’s buffer containing

0.35% BSA and 50 mM ZnCl2 at 4˚C for 30 min. After ultracentrifugation at 55,000 rpm, the pellets were washed twice, solubilized by sample buffer, and

analyzed by immunoblotting with polyclonal anti-HK Ab. (Bii) Before (left panel) or after (right panel) 5 mg of HK, recombinant HC, or LC of HK were

incubated with 50 nM PS liposomes. After incubation with PS liposomes, the samples were ultracentrifuged, and the pellets were collected. Samples were

analyzed by immunoblotting with polyclonal anti-HK Ab. HKa serves as control for m.w. (C) In the presence of various peptides at the indicated con-

centrations, apoptotic cells were incubated with 100 nM HK-B, followed by staining with PE-avidin and quantitation by flow cytometry. (D) HK at 30 nM

was incubated with apoptotic cells in the presence of prekallikrein (30 nM) at 37˚C for the indicated time periods. Apoptotic cells were sonicated and

centrifuged at 800 3 g for 5 min. Pellets containing membrane fraction were analyzed by Western blotting using anti-HK Ab. (E) Bradykinin (BK)

production. Fresh human citrated cell-free plasma was incubated with 5 3 105 viable cells (Viable), apoptotic cells (Apoptotic), or equal volume of PBS

(control [Ctrl]) at 37˚C for 30 min, followed by measurement of BK production. *p , 0.05, **p , 0.01, ***p , 0.001.

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capable of directly recognizing PS, such as T cell Ig and mucindomain-1/4 (5), stabilin-2 (6), and brain angiogenesis inhibitor 1

(36), recombinant uPAR protein did not bind to PS liposome andapoptotic cells (data not shown). Correspondingly, in the absenceof serum proteins, uPAR-mediated phagocytosis was completelyprevented (Fig. 5A). Thus, the role of uPAR in efferocytosis isdependent on opsonization of serum protein(s). Although the threeserum proteins uPA, vitronectin, and HK serve as ligands for uPAR(8), we found that only HK binds to apoptotic cells and PS lipo-some, and the binding was in a concentration-dependent manner(Fig. 5). Consistently, uPAR-mediated efferocytosis is inhibited inthe serum lacking HK (Fig. 5). Thus, HK serves as an opsonin foruPAR-mediated efferocytosis. This concept is supported by previ-ous observations. First, using two-dimensional gel electrophoresisand mass spectrometry, Anderson et al. (37) identified three proteinsin enriched factors from serum for stimulating macrophage effero-cytosis, which include albumin, protein S, and HK. Although HKis abundant in serum, they could not observe that kininogen is in-volved in efferocytosis in their system (37). It has been known thatHK that binds to the anionic surface is largely dependent on Zn2+,and kininogen is devoid of prophagocytic activity in their assay,probably due to no addition of Zn2+. In this study, we found that HKbinding to apoptotic cells is indeed Zn2+ dependent (Fig. 5). Sec-ond, Sugi and McIntyre (38) found that the Ab purified from plasmaof patients with antiphospholipid syndrome recognizes HK in thepresence of PS, suggesting that plasma HK forms a complex withPS in vivo, which becomes immunogenic. Third, we and others (30,31, 39–41) have previously shown that HK binds to a variety ofcells including leukocytes and endothelial cells via uPAR. Fourth,our recent study (42) using SPR has shown that compared with HK,the affinity of suPAR for HKa was 53-fold tighter. Because incu-bation with apoptotic cells induces HK cleavage to HKa (Fig. 6),the binding of HK to apoptotic cells causes its cleavage, which mayexpose its binding site for uPAR. This conformational change in HKbeing cleaved to HKa dynamically facilitates its more stable in-teraction with uPAR on phagocytes and increases the engulfment.Besides HK, plasma also contains low-m.w. kininogen, which hasthe exact same HC but no LC. However, LK does not bind to suPARin our BIAcore assay (42), indicating that uPAR binds to the LC ofHK. Consistently, in other cells such as endothelial cells, HKa bindto uPAR (43); moreover, HKa is associated with uPAR in lipid raftsvia its LC (domain 5). Because HK is abundant in circulation at 660nM, it readily serves as a sensor of apoptotic cells, especially whena large numbers of apoptotic cells persistently exist. Using a domainprotein and peptide inhibitory assay, we found that HK binds toapoptotic cells via its D3 and D5 regions.It has been known that Rac1 activation is the main signaling

pathway leading to phagocytosis of apoptotic cells (1, 2), its ac-tivation is dependent on localization in lipid rafts. Because thedominant-negative expression of Rac1/N17 inhibited uPAR-mediated engulfment (Fig. 7), uPAR internalizes apoptotic cellsby activating the Rac1 pathway. Supportively, the challenge ofuPAR by HK plus PS stimulated the complex formation of CrkIIwith p130Cas and Dock-180 and downstream Rac1 activation(Fig. 7). During the process of cell mobilization, Rac1 is mainlyactivated in the membrane rafts (44). Because uPAR is a GPI-anchored protein localized in lipid rafts, it might play a key rolein recruitment of other phagocytic receptors to membrane rafts,proximal to intracellular downstream signaling such as Rac1. Be-cause uPAR lacks a trans-membrane domain, the signal transduc-tion originating from this receptor, at least at the plasma membrane,requires lateral interactions with coreceptors (9). It awaits furtherinvestigation regarding which receptor(s) cooperates with uPAR andis responsible for mediating its signaling leading to Rac1 activation.Our results propose a novel concept that uPAR is important in

clearance of apoptotic cells and maintenance of vascular homeo-

FIGURE 7. Integrity of lipid rafts and Rac1 activation are required for

uPAR mediated-internalization of apoptotic cells. (A) After preincubation

with or without 2.5 mM methyl-b-cyclodextrin (MbCD) for 30 min, apo-

ptotic cells were cocultured with EGFP-293 or uPAR-293 cells. The inter-

nalization of apoptotic cells was analyzed by flow cytometry as described in

the legend for Fig. 4. (B) EGFP-293 or uPAR-293 cells were transfected with

empty vector (lane 1) or Rac1/N17 (lane 2) cDNA for 48 h. The over-

expression of Rac1/N17 was detected by Western blotting using anti-Rac1

Ab with b-actin serving as a loading control (Bi). Internalization of apoptotic

cells was analyzed (Bii). (C) EGFP-293 cells and uPAR-293 cells were

starved in DMEM plus 2% FBS for 8 h and incubated without (lanes 1 and

3) or with (lanes 2 and 4) 1.0 mM PS liposome plus 600 nM HK for 1 h in

basal DMEM containing 0.35% BSA and 50 mM ZnCl2. Cell lysates were

subjected to immunoprecipitation (IP) with anti-CrkII Ab (Ci) and Rac1

activity assay (Cii), respectively. The immunoprecipitates using anti-CrkII

Ab were analyzed by immunoblotting with mAb against p130Cas and

polyclonal Abs against CrkII and Dock-180. Active GTP-loaded form of

Rac1 and total Rac1 in cell lysates were detected by immunoblotting. (D)

After starvation in DMEM plus 2% FBS for 8 h, uPAR-293 cells were in-

cubated with PBS (control [Ctrl]), 600 nM HK, 1.0 mM PS liposome (PS), or

600 nM HK plus 1.0 mM PS liposome (HK+PS) for 1 h in DMEM con-

taining 0.35% BSA and 50 mM ZnCl2. Cell lysates were incubated with

GST-PAK1 conjugated with glutathione sepharose 4B beads, and active

GTP-loaded form of Rac1 (Pull down) and total Rac1 in cell lysates (Lysate)

were detected by immunoblotting. **p , 0.01, ***p , 0.001.

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stasis. Given that uPAR is frequently cleaved in atheroscleroticplaque, systemic bacterial infection, and cancer, the altered expressionof uPARmay contribute to accumulation of apoptotic cells, leading toinflammatory and autoimmune disorders and tissue damage (9, 45–48). This is helpful to explain the increase in the cleaved form ofuPAR in plasma associated with development of atheroscleroticlesions and production of inflammatory cytokines in plasma andplaques (10, 49).HK, as a major component of plasma KKS, possesses multiple

functions and plays an important role in thrombosis, inflammation,and fibrinolysis. Our current study reveals a novel function for theKKS system on its modulation of apoptotic cell clearance andinnate apoptotic immunity. So far, it has been known that bacteria(50), misfolded proteins (51, 52), collagen (52), and cellularreleasate such as heparin and polyphosphate (53) can activate theplasma KKS in vivo. Because incubation with apoptotic cellsinduces HK cleavage and bradykinin production (Fig. 6D, 6E),apoptotic cell membrane could be a new surface for plasma KKSactivation in vivo.In summary, we have demonstrated that uPAR plays an im-

portant role in internalization of apoptotic cells, which is mediatedby HK opsonization of uPAR interaction with PS. In the emergingpicture, this study raises the intriguing question as to whetheraltered activation of the plasma KKS and uPA–uPAR system andtheir cross talk contribute to the immune responses triggered byapoptotic cells. A further detailed molecular dissection of theseissues could lead to the better understanding of innate immunityand the development of novel therapeutic strategies that modulateinflammatory response.

AcknowledgmentsWe thank Xuemei Zhu for recombinant protein preparation.

DisclosuresThe authors have no financial conflicts of interest.

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