Elevated Expression of Phosphatidylserine in the Outer ......Elevated Expression of Phosphatidylserine in the Outer Membrane Leaflet of Human Tumor Cells and Recognition by Activated

(CANCER RESEARCH 51. 3062-3066. June I, 1991]

Advances in Brief

Elevated Expression of Phosphatidylserine in the Outer Membrane Leaflet ofHuman Tumor Cells and Recognition by Activated Human Blood Monocytes1

Teruhiro Utsugi, Alan J. Schroit, Jerome Connor, CorazÃ³nD. Bucana, and Isaiah J. Fidler2

Department of Cell Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

Abstract

We determined whether the presence of phosphatidylserine (PS) inthe outer membrane leaflet of human tumor cells correlated with theirrecognition by activated human monocytes. Three tumorigenic cell lines,A375 melanoma and A431 and Colo-16 carcinomas, and a normal humanepidermal keratinocyte line (NHEK) were incubated with monocytesactivated to the tumoricidal state by T-interferon and lipopolysaccharide.Activated human monocytes bound to and lysed all tumorigenic targets,while the nontumorigenic NHEK were neither bound nor killed. Semi-quantitative analysis of PS in the outer leaflet of the cells revealed thatthe tumorigenic cells expressed 3-7-fold more PS than did the nontumorigenic NHEK. To determine whether enhanced PS expression on thetumor cells was responsible for their recognition by activated monocytes,NHEK were supplemented with exogenous!) supplied analogues of PSand phosphatidylcholine. PS-labeled NHEK but not phosphatidylcho-line-labeled nor control NHEK bound to activated human monocytes.These results suggest a role for PS in monocyte recognition of tumorcells.

Introduction

The macrophage plays many roles in diverse physiologicalprocesses; it recognizes, phagocytoses, and ultimately disposesof effete cells, cellular debris, and foreign invaders (1). Macrophages also play an important role in host defense againstcancer (2) and infections (3). Normal, noncytotoxic bloodmonocytes or tissue macrophages can be activated to becometumoricidal subsequent to interaction with lymphokines, bacterial products, or both (2). Although tumor cells are heterogeneous with regard to many characteristics (4), they seem toshare susceptibility to destruction by activated macrophages. Infact, activated macrophages can lyse tumor cells that are resistant to other immune effector cells such as T-cells or naturalkiller cells or to chemotherapeutic drugs (1, 2). Moreover,activated macrophages can discriminate between tumorigeniccells, which they lyse, and nontumorigenic cells, which they donot, even under cocultivation conditions (5).

Studies in many tumor systems have indicated that macro-phage-mediated tumor cell lysis is independent of such tumorcell characteristics as surface receptors, transplantation antigens, tumor antigens, species-specific antigens, cell cycle time,expression of endogenous C type viruses, and metastatic potential (for a review, see Ref. l). The exact mechanism thatregulates macrophage discrimination between normal and tumorigenic cells is not known. The broad spectrum of tumorcells susceptible to macrophage-mediated lysis suggests, how-

Received3/18/91:accepted4/15/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part by Grants R35-CA 42107 and CA 47845 from the National

Cancer Institute.1To Â»hornrequests for reprints should be addressed, at the Department of

Cell Biology. Box 173, The University of Texas M. D. Anderson Cancer Center.1515 Holcombe Boulevard, Houston, TX 77030.

ever, that a uniform surface moiety could be involved in targetcell recognition.

Membrane phospholipids are known to be asymmetricallydistributed between the two leaflets of the bilayer (6-9).Whereas particular membrane phospholipids may show somepreference for either leaflet, PS1 is localized exclusively in theinner leaflet of cells (6-10). The preservation of PS in the innerleaflet of cells may play an important role in cell physiology(11, 12) since its exposure in the outer leaflet of, e.g., sickledRBC (13, 14) is associated with their recognition by mononu-clear phagocytes (15). PS is also found in the outer leaflet ofactivated platelets (16) and regulates hemostasis by serving asa procoagulant surface (11, 12). In addition, RBC expressingPS in the external leaflet (15, 17-19) or liposomes containingPS (20) are rapidly bound to and phagocytosed by macrophages.Collectively, these findings suggest that the maintenance of PSasymmetry in cell membranes represents a homeostatic mechanism that may differentiate normal from abnormal cells.

We have recently reported that tumorigenic, undifferentiatedmurine erythroleukemia cells express 7-8-fold more PS in theirouter leaflet than do their differentiated counterparts (21).These observations are extended here to the human system byusing three tumorigenic (melanoma, two squamous cell carcinomas) and one normal (human epidermal keratinocytes) cellline. Activated human blood monocytes bound to all threetumorigenic lines to produce target cell lysis. In contrast, humanblood monocytes did not bind to or lyse normal keratinocytes.Semiquantitative analyses of PS in the outer leaflet of the cellsby prothrombinase activity (21) showed that tumorigenic cellsexpressed significantly higher levels of PS than did normalkeratinocytes.

Materials and Methods

Reagents. Eagle's minimal essential medium, HBSS, and fetal bovine

serum were purchased from M. A. Bioproducts (Walkersville, MD).Recombinant human -y-interferon was the gift of Genentech, Inc. (SouthSan Francisco, CA). Activated Factor X (Factor Xa), L-serine, and LPSwere purchased from Sigma Chemical Co. (St. Louis, MO). Thrombinand the thrombin-sensitive chromophore, S2238, were from HelenaLaboratories (St. Louis, MO), and prothrombin (Factor II) was obtained from Calbiochem (San Diego, CA). NBD-PC was purchasedfrom Avanti Polar Lipids (Birmingham, AL). NBD-PS was preparedfrom NBD-PC by phospholipase D-catalyzed base exchange in thepresence of L-serine (22) and purified by thin-layer chromatography.Factor V was isolated from bovine plasma (21) and was activated byincubation with subcatalytic amounts of thrombin (0.02 unit) for 3 minat 37Â°C.Small unilamellar vesicles were prepared from PC by sonica-

tion (20). Radioisotopes were obtained from New England Nuclear(Boston, MA). All the reagents except LPS were free of endotoxins as

1The abbreviations used are: LPS, lipopolysaccharide; NHEK. normal humanepidermal keratinocytes; NBD. l-oleoyl-2-|[A'-(7-nitrobenz-2-oxa-l,3-diazol-4-

yl)amino]caproyl|; PS. phosphatidylserine; PC, phosphatidylcholine; PBS, phosphate-buffered saline; HBSS, Hanks' balanced salt solution.

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EXPRESSION OF MEMBRANE PS BY TUMOR CELLS

determined by the Limulus amebocyte lysate assay (sensitivity limit of0.125 ng/ml; Associates of Cape Cod, Woods Hole, MA).

Cell Cultures. The A375 human melanoma cell line (23) and humansquamous cell carcinoma cell lines Colo-16 (24) and A431 (25) weremaintained as monolayer cultures in Eagle's minimal essential medium

supplemented with 5% fetal bovine serum, sodium pyruvate, nonessen-tial amino acids, 2 mM L-glutamine, 2x vitamin solution, penicillin,and streptomycin (complete Eagle's minimal essential medium). Thecells were incubated at 37Â°Cin a humidified atmosphere of 5% CO; in

air. NHEK and keratinocyte growth medium were purchased fromClonetics Corporation (San Diego, CA). All cell lines were examinedfor and were found to be free of Mycoplasma contamination.

Isolation and Culture of Human Peripheral Blood Monocytes. Mono-nuclear cells were separated by Ficoll-Hypaque centrifugation frombuffy coats (Gulf Coast Regional Blood Center, Houston, TX) obtainedfrom 400 ml of blood from healthy donors. The monocytes were isolatedfrom the mononuclear cells by counterflow centrifugal elutriation (26)with a Beckman JE-6B elutriation rotor. Fractions containing monocytes were obtained at a speed of 3500 rpm and a flow rate of 42 to 48ml/min. These fractions were washed with HBSS and were resuspendedin complete Eagle's minimal essential medium. More than 97% of the

cells were monocytes as determined by morphology, nonspecific esterase staining, and positive staining with monoclonal anti-human mon-ocyte antibody Leu-M3 (Becton Dickinson, Mountain View, CA). Cellviability was >98% as determined by trypan blue dye exclusion.

Binding Assay. Monocytes (5 x 105/well) were plated into 38-mirrwells of round-bottomed plates. After 90 min, nonadherent cells wereremoved by washing. The monocyte monolayers (monocyte purity,>98%) were then incubated at 37Â°Cfor 18-24 h with 0.2 ml of medium

(control monocytes) or medium containing LPS (0.1 Â¿ig/ml)and 7-interferon (10 units/ml) (activated monocytes). Target cells (A375,A431, Colo-16, NHEK) in exponential growth were incubated for 24 hwith medium containing 0.3 Â¿iCi/mlof [125I]iododeoxyuridine (specific

activity, 2200 Ci/mmol). The radiolabeled cells were washed twice withHBSS to remove unincorporated radioisotope and then harvested bybrief trypsinization. After washing, 0.1 ml of cells (2 x 105/ml) in

medium without serum was added to control or to activated monocytemonolayers. Target cells were also plated into plastic wells as anadditional control. The cultures were incubated at 37Â°Cfor 30 min and

then placed on a Mini-Orbital shaker (BÃ©licoBiotechnology, Vineland,NJ). The cells were shaken for 1 min at an instrument setting of 5 andwashed twice with PBS. Adherent cells were lysed with 0.1 ml of 0.1 NNaOH. The lysates were absorbed onto cotton swabs and radioactivitywas monitored in a gamma counter. Monocytes and target cells werealso processed for scanning electron microscopy (27).

Monocyte-mediated Cytotoxicity Assay. Monocyte-mediated cytotox-icity was assessed by measuring the release of radiation from DNA oftarget cells as described previously (28, 29). Briefly, monocytes wereplated at the density of 1 x 105/38-mm2 well of flat-bottomed Microtest

III plates (Falcon Plastics, Oxnard, CA) and allowed to adhere for 1.5h at 37Â°C.At that time, nonadherent cells were removed by washing

with medium. The purity of the adherent monocytes was >98%. Themonocytes were activated as described above. Radiolabeled target cellswere harvested as described above and resuspended in medium, andthen IO4 cells were plated into wells containing adherent monocytes.

After 72 h the cultures were washed twice with HBSS. Adherent viablecells were then lysed with 0.1 ml of 0.1 N NaOH. The lysates wereabsorbed onto cotton swabs and radiation was monitored in a gammacounter. The percentage of specific cytotoxicity mediated by monocyteswas calculated as

% of specific cytotoxicity =A - B

x Â¡00

where A is cpm in cultures of untreated monocytes and target cells andB is cpm in cultures of activated monocytes and target cells.

Determination of NBD-PS/RBC Standard Curve. RBC collected fromhealthy donors were washed twice, resuspended in PBS (2 x IO8RBC/ml), and treated with 2 mM pyridyldithioethylamine for 30 min at 4Â°Cto inhibit translocation of NBD-PS (30. 31). The cells were then washed

with cold buffer and incubated with increasing concentrations of NBD-PS for 20 min at 4Â°C.After washing, the amount of cell-associated

NBD-PS was quantified by fluorescence. The fraction of PS in theouter leaflet was determined by its ability to be removed by "back-exchange" with small unilamellar acceptor vesicles as described previ

ously (21, 30, 31). Aliquots of these NBD-PS containing RBC wereused as a standard for analysis of endogenous PS in the outer leaflet ofcells by prothrombinase assay.

Prothrombin-converting Activity Assay. NBD-PS containing RBC (2x IO7cells/0.1 ml), tumor cells (0.5-1 x IO4cells/0.1 ml), and normalcells (1-2 x 10" cells/0.1 ml) were incubated at 37Â°Cwith CaCl2 (6

mM), Factor Xa (0.2 unit), Factor Va (12 nM), and prothrombin (0.8unit) in Tris-NaCl buffer (50 m\i Tris-120 mM NaCl, pH 7.8; finalvolume, 600 u\). After 3 min, the reaction was stopped by the additionof EDTA to 15 mM. The thrombin-dependent chromophore S2238 wasthen added (to 0.4 mM), and the rate of chromogen formation wasmonitored at 405 nm with a Gilford response spectrophotometer usingappropriate kinetic software. The initial rate of thrombin conversionactivity, which is directly proportional to the amount of PS present onthe catalytic cell surface, was determined from the slopes of the absorb-ance. The absolute amount of PS present was determined by comparingthe rates of thrombin production to the rates generated by knownamounts of NBD-PS in RBC. To rule out that the trypsin treatmentmight produce errors, A375 melanoma cells were incubated with 0.25%trypsin for periods ranging from 1 to 10 min. PS content determinedwith the prothrombinase activity assay was not affected as comparedto attached cells by the trypsin treatment (data not shown).

Insertion of Exogenous Fluorescent PS or PC into NHEK and A375Melanoma Cells. Target cells were labeled with 0.2 mCi s'Cr for l h at37Â°Cand washed with PBS. To inhibit transmembrane movement of

the exogenously added phospholipids, the cells were then incubatedwith 2 mM pyridyldithioethylamine for 20 min on ice (30, 31). Aliquotsof 1 x IO6cells in 2 ml of PBS were mixed with 4 ug of NBD-PS orNBD-PC in 20 ii\ of ethanol (17). After 30 min incubation at 4Â°C,the

cells were washed and resuspended in PBS. At this time, cell viabilitywas 90% (trypan blue exclusion).

The fraction of cell-associated NBD-lipid remaining in the outerleaflet of the cells was determined as described for RBC (see above)except that 1% bovine serum albumin was substituted for vesicles.

Results

The interaction of tumorigenic or normal human cells withactivated monocytes was examined by measuring the ability ofmonocytes to bind and lyse the target cells. In the first set ofexperiments, the ability of activated human monocytes to bindto tumorigenic or nontumorigenic cells was determined. As

Table 1 PS content of the outer leaflet of target cell membrane and binding toand lysis by human monocytes

CellsA375A431Colo-

16NHEKBinding

toactivatedmonocytes(%)"48.1

Â±1.823.9Â±7.436.2Â±1.86.9Â±0.8Cytotoxicitymediatedby

activatedmonocytes(%r51

Â±222+915Â±

12Â±1PS

content'(ng/104cells)75

Â±1039Â±634

Â±411Â±2Surfacearea1*

oftarget

cells(Mm2)202142131213

Â°In vitro binding of tumor or normal cells to activated monocytes was measuredafter a 30-min incubation period using ['"IJiododeoxyuridine-labeled target cellsas described in "Materials and Methods." Data are the mean Â±SD of a repre

sentative experiment of five.'Target cells (I x 10") labeled with ['"Ijiododeoxyuridine were plated into

triplicate wells containing activated monocytes. The cultures were terminated 72h after plating of target cells. Data are the mean Â± SE of 3 independentexperiments.

' PS content of the outer leaflet was determined by the prothrombinase activityassay as described in "Materials and Methods." Data are the mean Â±SE of 3

independent experiments normalized to cell surface area.d Average surface area calculated from measurement of the diameter of 200

cells/group/experiment.

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shown in Table 1, all tumorigenic cells exhibited higher bindingto activated monocytes than that found for the normal NHEKcells. Control experiments, where target cells were incubatedunder the same conditions with control-nonactivated monocytes or in the absence of any monocytes, demonstrated lowlevel binding (data not shown). The binding of suspendedmonocytes to adherent A375 melanoma and NHEK cells andthe binding of suspended A375 melanoma and NHEK cells toadherent monocytes was examined by scanning electron microscopy. By 30 min of incubation, human blood monocytesplated onto adherent target cells bound to the A375 melanoma(Fig. 1, A and B) but not to the NHEK cells (Fig. 1C). Similarfindings were obtained when suspended target cells were platedonto adherent monocytes. By 30 min of incubation, A375

melanoma cells bound to the monocytes (Fig. ID), whereasNHEK cells did not (Fig. 1Â£).

To determine whether binding of macrophages to tumor cellscorrelates with cytotoxicity, the ability of activated humanmonocytes to lyse tumorigenic and nontumorigenic target cellswas determined. The data in Table 1 show that nontumorigenicNHEK cells were not lysed (2% cytotoxicity), whereas A375melanoma, A431, and Colo-16 squamous cell carcinoma cells

were lysed by activated monocytes with specific cytotoxicitiesof 51, 22, and 15%, respectively.

The amount of PS present in the outer leaflet of target cellmembranes was assessed by measuring the initial rates ofthrombin production initiated by standard NBD-PS containingRBC, A375, A431, Colo-16, and NHEK cells simultaneously.

Fig. 1. Scanning electron microscopy of monocyte binding to target cells. (A, B) Monocytes binding to adherent A375 melanoma cells after 30 min of incubation.Note a large number of round monocytes clustering on and around adherent melanoma cells. (Q Monocyte binding to adherent NHEK after 30 min of incubation.Note the lack of monocytes adhering to the NHEK. (D) Binding of suspended A375 cells (arrows) to adherent monocytes after 30 min of incubation. (E) Absence ofbinding of suspended NHEK to adherent monocytes after 30 min of incubation.

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The amount of PS, based on results obtained from the standardcurve shown in Fig. 2, was corrected for cell surface area. Thiscompensation for differences in cell size normalized the data toPS density (see Ref. 21). The results shown in Table 1 indicatethat the tumorigenic cells express 3-7 times more PS than donontumorigenic NHEK.

To further investigate the involvement of PS in monocyte-target cell recognition, "Cr-labeled NHEK and A375 melanoma cells were supplemented with NBD-PS or NBD-PC, andtheir propensity to be bound by activated monocytes was thenassessed. Back-exchange of NBD-lipid-treated cells at the initiation of the binding experiments revealed that approximately50% of the cell-associated lipid was localized at the outer leaflet.As can be seen in Fig. 3, the binding of NBD-PS-labeled NHEKto macrophages (31%) was markedly higher than that foundfor NHEK labeled with NBD-PC (12%) or control NHEK (8%)(Fig. 3/4). In contrast, no discernible differences in binding tomacrophages were found among control A375 melanoma cells(35%) and those labeled with NBD-PS (40%) or NBD-PC(33%) (Fig. 3Ã„).

Discussion

The main function of macrophages is to discriminate between"self" and "altered self by recognizing, phagocytosing, and

disposing of effete cells, cellular debris, and foreign invaders(1). How mononuclear phagocytes discriminate between youngand old cells, healthy and damaged cells, and nontumorigenicand tumorigenic cells is not known. Several observations suggest that the expression of PS on the outer membrane leafletof cells could serve as a recognition moiety for macrophages.For example, the insertion of an exogenous fluorescent PSanalogue (NBD-PS) into the outer leaflet of RBC facilitates

their uptake by macrophages (17) and clearance from the circulation after i.v. injection (18). Furthermore, experimentsusing sickled RBC demonstrated that upon removal of oxygen,PS is localized in both the inner and outer leaflets of themembrane (13, 14), and these cells exhibited increased bindingto monocytes as compared with that of oxygenated, sickledRBC (PS only in the inner leaflet) (15). Similarly, the recognition of undifferentiated leukemic mouse cells by macrophagescould also be due to PS. Terminal differentiation of mouse

0.4 -\

0.3-

25 50 7

NBD-PS (ng/2x10

100

erythroleukemia cells is associated with a marked reduction ofbinding to activated mouse macrophages (21, 32) and correlateswith a decrease in the expression of PS in the cells' outer leaflet

(21). Collectively, these data suggest that the expression of PSin the outer leaflet of the cell may play a role in recognitionand subsequent removal by phagocytes (33).

In the present report, we determined whether a correlationexists between the PS expression and interaction of humantumor cells with activated human monocytes. Our results indicate that by 30 min, all three tumorigenic cell lines bound toactivated human blood monocytes, albeit to different degrees.These results may be due to inter- and intratumoral heterogeneity. A375 melanoma cells and A431 squamous carcinomacells were also lysed by the monocytes. Although Colo-16squamous carcinoma cells bound to activated monocytes, theywere relatively resistant to lysis. These results agree with previous reports (3, 33) showing that all tumorigenic cells boundto activated monocytes but binding does not always result insubsequent lysis (34). In contrast, nontumorigenic NHEK cellsdid not bind to nor were they lysed by tumoricidal human bloodmonocytes. Since PS expression in the outer leaflet of the 3tumorigenic cell lines was 3-7-fold higher than the expressionof PS exposed on the outer membrane leaflet of the NHEKcells, these data suggest a correlation between expression of PSon the outer leaflet of target cell membranes and their recognition by monocyte-macrophages.

To investigate whether the inclusion of exogenous PS in theouter leaflet bilayer of NHEK results in their recognition byhuman monocytes, NBD-PS-labeled NHEK were incubated

Câ€¢D

eZ

e

RBC)

Incubation time (minutes)Fig. 2. Standard curve generated from NBD-PS-labeled RBC. PDA-treatedRBC were labeled with increasing amounts of NBD-PS. The amounts of outerleaflet NBD-PS in these cells were determined by direct fluorescence assaj versus Fig. 3. Binding of suspended NHEK (A) and A375 (B) cells to activatedthe initial rates of thrombin production (correlation of 0.98). adherent monocytes.

3065




with activated monocytes. NHEK labeled with exogenousNBD-PC served as controls. We used NBD-PC because PC hasa positive charge at its TV-substitution methylation site, and thisis the exact control for NBD-PS with a primary amine that isalso strongly positive (NH/). The results indicate that thepresence of PS, but not PC, on the outer leaflet of NHEKdirectly correlated with binding to macrophages. Surprisingly,the addition of PS to A375 cells did not increase the alreadyenhanced binding to monocytes. These results might suggestthat the recognition of tumor targets by monocytes ensues abovea critical threshold, similar to that observed for recognition ofPS-containing RBC ( 18).

In conclusion, the data presented here suggest that PS canserve as a signal for triggering macrophage recognition asmanifested by binding to target cells (33). Although the bindingsite on the macrophage surface responsible for recognition ofPS is not known, several possibilities exist. These include theparticipation of autologous cytophilic antibodies (35, 36), components of the clotting cascade (16), and specific phospholi-pases (37, 38). These findings do not imply any lack of importance for other and more developed mammalian recognitionsystems, such as proteins and carbohydrate-regulated processes

(1), but do suggest that a relatively simple recognition mechanism involving phospholipids may also be important, a processthat has endured at least several steps of evolutionarydevelopment.

Acknowledgments

We thank Dr. Dominic Fan for helpful suggestions, Kenneth Dunner,Jr., for scanning electron microscopy, and Lola Lopez for expert helpin the preparation of this manuscript.

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1991;51:3062-3066. Cancer Res Teruhiro Utsugi, Alan J. Schroit, Jerome Connor, et al. Activated Human Blood MonocytesMembrane Leaflet of Human Tumor Cells and Recognition by Elevated Expression of Phosphatidylserine in the Outer

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Elevated Expression of Phosphatidylserine in the Outer ......Elevated Expression of Phosphatidylserine in the Outer Membrane Leaflet of Human Tumor Cells and Recognition by Activated