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(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, CorazónD.
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 »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
37°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 37°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 37°Cfor 18-24 h with 0.2 ml
of medium
(control monocytes) or medium containing LPS (0.1 ¿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 ¿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
37°Cfor 30 min and
then placed on a Mini-Orbital shaker (BélicoBiotechnology,
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 37°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 ¡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 4°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 4°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 37°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 at37°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 4°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
±1.823.9±7.436.2±1.86.9±0.8Cytotoxicitymediatedby
activatedmonocytes(%r51
±222+915±
12±1PS
content'(ng/104cells)75
±1039±634
±411±2Surfacearea1*
oftarget
cells(Mm2)202142131213
°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 ±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 ± 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 ±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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EXPRESSION OF MEMBRANE PS BY TUMOR CELLS
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. 1£).
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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EXPRESSION OF MEMBRANE PS BY TUMOR CELLS
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.
3Ä).
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
C•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.
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EXPRESSION OF MEMBRANE PS BY TUMOR CELLS
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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on April 5, 2021. © 1991 American Association for Cancer
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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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