Download Elevated Expression of Phosphatidylserine in the Outer Membrane

(CANCER RESEARCH 51. 3062-3066. June I, 1991]
Advances in Brief
Elevated Expression of Phosphatidylserine in the Outer Membrane Leaflet of
Human 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) in
the outer membrane leaflet of human tumor cells correlated with their
recognition by activated human monocytes. Three tumorigenic cell lines,
A375 melanoma and A431 and Colo-16 carcinomas, and a normal human
epidermal keratinocyte line (NHEK) were incubated with monocytes
activated 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. Semiquantitative analysis of PS in the outer leaflet of the cells revealed that
the tumorigenic cells expressed 3-7-fold more PS than did the nontu
morigenic NHEK. To determine whether enhanced PS expression on the
tumor cells was responsible for their recognition by activated monocytes,
NHEK were supplemented with exogenous!) supplied analogues of PS
and phosphatidylcholine. PS-labeled NHEK but not phosphatidylcholine-labeled nor control NHEK bound to activated human monocytes.
These results suggest a role for PS in monocyte recognition of tumor
cells.
Introduction
The macrophage plays many roles in diverse physiological
processes; it recognizes, phagocytoses, and ultimately disposes
of effete cells, cellular debris, and foreign invaders (1). Macro
phages also play an important role in host defense against
cancer (2) and infections (3). Normal, noncytotoxic blood
monocytes or tissue macrophages can be activated to become
tumoricidal subsequent to interaction with lymphokines, bac
terial products, or both (2). Although tumor cells are hetero
geneous with regard to many characteristics (4), they seem to
share susceptibility to destruction by activated macrophages. In
fact, activated macrophages can lyse tumor cells that are resist
ant to other immune effector cells such as T-cells or natural
killer cells or to chemotherapeutic drugs (1, 2). Moreover,
activated macrophages can discriminate between tumorigenic
cells, which they lyse, and nontumorigenic cells, which they do
not, even under cocultivation conditions (5).
Studies in many tumor systems have indicated that macrophage-mediated tumor cell lysis is independent of such tumor
cell characteristics as surface receptors, transplantation anti
gens, tumor antigens, species-specific antigens, cell cycle time,
expression of endogenous C type viruses, and metastatic poten
tial (for a review, see Ref. l). The exact mechanism that
regulates macrophage discrimination between normal and tu
morigenic cells is not known. The broad spectrum of tumor
cells susceptible to macrophage-mediated lysis suggests, howReceived3/18/91: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 in
accordance 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.
1 To »horn requests 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 target
cell recognition.
Membrane phospholipids are known to be asymmetrically
distributed between the two leaflets of the bilayer (6-9).
Whereas particular membrane phospholipids may show some
preference for either leaflet, PS1 is localized exclusively in the
inner leaflet of cells (6-10). The preservation of PS in the inner
leaflet of cells may play an important role in cell physiology
(11, 12) since its exposure in the outer leaflet of, e.g., sickled
RBC (13, 14) is associated with their recognition by mononuclear phagocytes (15). PS is also found in the outer leaflet of
activated platelets (16) and regulates hemostasis by serving as
a procoagulant surface (11, 12). In addition, RBC expressing
PS in the external leaflet (15, 17-19) or liposomes containing
PS (20) are rapidly bound to and phagocytosed by macrophages.
Collectively, these findings suggest that the maintenance of PS
asymmetry in cell membranes represents a homeostatic mech
anism that may differentiate normal from abnormal cells.
We have recently reported that tumorigenic, undifferentiated
murine erythroleukemia cells express 7-8-fold more PS in their
outer leaflet than do their differentiated counterparts (21).
These observations are extended here to the human system by
using three tumorigenic (melanoma, two squamous cell carci
nomas) and one normal (human epidermal keratinocytes) cell
line. Activated human blood monocytes bound to all three
tumorigenic lines to produce target cell lysis. In contrast, human
blood monocytes did not bind to or lyse normal keratinocytes.
Semiquantitative analyses of PS in the outer leaflet of the cells
by prothrombinase activity (21) showed that tumorigenic cells
expressed significantly higher levels of PS than did normal
keratinocytes.
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. (South
San Francisco, CA). Activated Factor X (Factor Xa), L-serine, and LPS
were purchased from Sigma Chemical Co. (St. Louis, MO). Thrombin
and the thrombin-sensitive chromophore, S2238, were from Helena
Laboratories (St. Louis, MO), and prothrombin (Factor II) was ob
tained from Calbiochem (San Diego, CA). NBD-PC was purchased
from Avanti Polar Lipids (Birmingham, AL). NBD-PS was prepared
from NBD-PC by phospholipase D-catalyzed base exchange in the
presence of L-serine (22) and purified by thin-layer chromatography.
Factor V was isolated from bovine plasma (21) and was activated by
incubation with subcatalytic amounts of thrombin (0.02 unit) for 3 min
at 37°C.Small unilamellar vesicles were prepared from PC by sonication (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 human
epidermal keratinocytes; NBD. l-oleoyl-2-|[A'-(7-nitrobenz-2-oxa-l,3-diazol-4yl)amino]caproyl|; PS. phosphatidylserine; PC, phosphatidylcholine;
phate-buffered saline; HBSS, Hanks' balanced salt solution.
PBS, phos
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EXPRESSION OF MEMBRANE PS BY TUMOR CELLS
determined by the Limulus amebocyte lysate assay (sensitivity limit of
0.125 ng/ml; Associates of Cape Cod, Woods Hole, MA).
Cell Cultures. The A375 human melanoma cell line (23) and human
squamous cell carcinoma cell lines Colo-16 (24) and A431 (25) were
maintained as monolayer cultures in Eagle's minimal essential medium
supplemented with 5% fetal bovine serum, sodium pyruvate, nonessential amino acids, 2 mM L-glutamine, 2x vitamin solution, penicillin,
and streptomycin (complete Eagle's minimal essential medium). The
cells were incubated at 37°Cin a humidified atmosphere of 5% CO; in
air. NHEK and keratinocyte growth medium were purchased from
Clonetics Corporation (San Diego, CA). All cell lines were examined
for and were found to be free of Mycoplasma contamination.
Isolation and Culture of Human Peripheral Blood Monocytes. Mononuclear cells were separated by Ficoll-Hypaque centrifugation from
buffy coats (Gulf Coast Regional Blood Center, Houston, TX) obtained
from 400 ml of blood from healthy donors. The monocytes were isolated
from the mononuclear cells by counterflow centrifugal elutriation (26)
with a Beckman JE-6B elutriation rotor. Fractions containing mono
cytes were obtained at a speed of 3500 rpm and a flow rate of 42 to 48
ml/min. These fractions were washed with HBSS and were resuspended
in complete Eagle's minimal essential medium. More than 97% of the
cells were monocytes as determined by morphology, nonspecific ester
ase staining, and positive staining with monoclonal anti-human monocyte antibody Leu-M3 (Becton Dickinson, Mountain View, CA). Cell
viability was >98% as determined by trypan blue dye exclusion.
Binding Assay. Monocytes (5 x 105/well) were plated into 38-mirr
wells of round-bottomed plates. After 90 min, nonadherent cells were
removed 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 7interferon (10 units/ml) (activated monocytes). Target cells (A375,
A431, Colo-16, NHEK) in exponential growth were incubated for 24 h
with medium containing 0.3 ¿iCi/mlof [125I]iododeoxyuridine (specific
activity, 2200 Ci/mmol). The radiolabeled cells were washed twice with
HBSS to remove unincorporated radioisotope and then harvested by
brief trypsinization. After washing, 0.1 ml of cells (2 x 105/ml) in
medium without serum was added to control or to activated monocyte
monolayers. Target cells were also plated into plastic wells as an
additional control. The cultures were incubated at 37°Cfor 30 min and
then placed on a Mini-Orbital shaker (Bélico
Biotechnology, Vineland,
NJ). The cells were shaken for 1 min at an instrument setting of 5 and
washed twice with PBS. Adherent cells were lysed with 0.1 ml of 0.1 N
NaOH. The lysates were absorbed onto cotton swabs and radioactivity
was monitored in a gamma counter. Monocytes and target cells were
also processed for scanning electron microscopy (27).
Monocyte-mediated Cytotoxicity Assay. Monocyte-mediated cytotoxicity was assessed by measuring the release of radiation from DNA of
target cells as described previously (28, 29). Briefly, monocytes were
plated at the density of 1 x 105/38-mm2 well of flat-bottomed Microtest
with cold buffer and incubated with increasing concentrations of NBDPS for 20 min at 4°C.After washing, the amount of cell-associated
NBD-PS was quantified by fluorescence. The fraction of PS in the
outer leaflet was determined by its ability to be removed by "backexchange" with small unilamellar acceptor vesicles as described previ
ously (21, 30, 31). Aliquots of these NBD-PS containing RBC were
used as a standard for analysis of endogenous PS in the outer leaflet of
cells by prothrombinase assay.
Prothrombin-converting Activity Assay. NBD-PS containing RBC (2
x IO7cells/0.1 ml), tumor cells (0.5-1 x IO4cells/0.1 ml), and normal
cells (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 (0.8
unit) in Tris-NaCl buffer (50 m\i Tris-120 mM NaCl, pH 7.8; final
volume, 600 u\). After 3 min, the reaction was stopped by the addition
of EDTA to 15 mM. The thrombin-dependent chromophore S2238 was
then added (to 0.4 mM), and the rate of chromogen formation was
monitored at 405 nm with a Gilford response spectrophotometer using
appropriate kinetic software. The initial rate of thrombin conversion
activity, which is directly proportional to the amount of PS present on
the catalytic cell surface, was determined from the slopes of the absorbance. The absolute amount of PS present was determined by comparing
the rates of thrombin production to the rates generated by known
amounts of NBD-PS in RBC. To rule out that the trypsin treatment
might produce errors, A375 melanoma cells were incubated with 0.25%
trypsin for periods ranging from 1 to 10 min. PS content determined
with the prothrombinase activity assay was not affected as compared
to attached cells by the trypsin treatment (data not shown).
Insertion of Exogenous Fluorescent PS or PC into NHEK and A375
Melanoma Cells. Target cells were labeled with 0.2 mCi s'Cr for l h at
37°Cand washed with PBS. To inhibit transmembrane movement of
the exogenously added phospholipids, the cells were then incubated
with 2 mM pyridyldithioethylamine for 20 min on ice (30, 31). Aliquots
of 1 x IO6 cells in 2 ml of PBS were mixed with 4 ug of NBD-PS or
NBD-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 viability
was 90% (trypan blue exclusion).
The fraction of cell-associated NBD-lipid remaining in the outer
leaflet 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 with
activated monocytes was examined by measuring the ability of
monocytes to bind and lyse the target cells. In the first set of
experiments, the ability of activated human monocytes to bind
to tumorigenic or nontumorigenic cells was determined. As
Table 1 PS content of the outer leaflet of target cell membrane and binding to
and lysis by human monocytes
III plates (Falcon Plastics, Oxnard, CA) and allowed to adhere for 1.5
h at 37°C.At that time, nonadherent cells were removed by washing
with medium. The purity of the adherent monocytes was >98%. The
monocytes were activated as described above. Radiolabeled target cells
were harvested as described above and resuspended in medium, and
then IO4 cells were plated into wells containing adherent monocytes.
After 72 h the cultures were washed twice with HBSS. Adherent viable
cells were then lysed with 0.1 ml of 0.1 N NaOH. The lysates were
absorbed onto cotton swabs and radiation was monitored in a gamma
counter. The percentage of specific cytotoxicity mediated by monocytes
was calculated as
% of specific cytotoxicity =
A - B
x ¡00
toactivatedmonocytes(%)"48.1
activatedmonocytes(%r51
content'(ng/104
cells)75
CellsA375A431Colo-
cells(Mm2)202142131213
1.823.9
±
222±
1039
±
915±
+
7.436.2
±
634±
16NHEKBinding
1.86.9
±
12
±411
±0.8Cytotoxicitymediatedby
±1PS
±2Surfacearea1*
°In vitro binding of tumor or normal cells to activated monocytes was measured
after a 30-min incubation period using ['"IJiododeoxyuridine-labeled
target cells
as 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
where A is cpm in cultures of untreated monocytes and target cells and
B is cpm in cultures of activated monocytes and target cells.
Determination of NBD-PS/RBC Standard Curve. RBC collected from
healthy donors were washed twice, resuspended in PBS (2 x IO8RBC/
ml), and treated with 2 mM pyridyldithioethylamine for 30 min at 4°C
to inhibit translocation of NBD-PS (30. 31). The cells were then washed
oftarget
were plated
into
triplicate wells containing activated monocytes. The cultures were terminated 72
h after plating of target cells. Data are the mean ± SE of 3 independent
experiments.
' PS content of the outer leaflet was determined by the prothrombinase
activity
assay as described in "Materials and Methods."
Data are the mean ±SE of 3
independent experiments normalized
d Average surface area calculated
to cell surface area.
from measurement
of the diameter
cells/group/experiment.
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EXPRESSION OF MEMBRANE PS BY TUMOR CELLS
shown in Table 1, all tumorigenic cells exhibited higher binding
to activated monocytes than that found for the normal NHEK
cells. Control experiments, where target cells were incubated
under the same conditions with control-nonactivated
mono
cytes or in the absence of any monocytes, demonstrated low
level binding (data not shown). The binding of suspended
monocytes to adherent A375 melanoma and NHEK cells and
the binding of suspended A375 melanoma and NHEK cells to
adherent monocytes was examined by scanning electron mi
croscopy. By 30 min of incubation, human blood monocytes
plated onto adherent target cells bound to the A375 melanoma
(Fig. 1, A and B) but not to the NHEK cells (Fig. 1C). Similar
findings were obtained when suspended target cells were plated
onto adherent monocytes. By 30 min of incubation, A375
melanoma cells bound to the monocytes (Fig. ID), whereas
NHEK cells did not (Fig. 1£).
To determine whether binding of macrophages to tumor cells
correlates with cytotoxicity, the ability of activated human
monocytes to lyse tumorigenic and nontumorigenic target cells
was determined. The data in Table 1 show that nontumorigenic
NHEK cells were not lysed (2% cytotoxicity), whereas A375
melanoma, A431, and Colo-16 squamous cell carcinoma cells
were lysed by activated monocytes with specific cytotoxicities
of 51, 22, and 15%, respectively.
The amount of PS present in the outer leaflet of target cell
membranes was assessed by measuring the initial rates of
thrombin production initiated by standard NBD-PS containing
RBC, 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 of
binding 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 standard
curve shown in Fig. 2, was corrected for cell surface area. This
compensation for differences in cell size normalized the data to
PS density (see Ref. 21). The results shown in Table 1 indicate
that the tumorigenic cells express 3-7 times more PS than do
nontumorigenic NHEK.
To further investigate the involvement of PS in monocytetarget cell recognition, "Cr-labeled NHEK and A375 mela
noma cells were supplemented with NBD-PS or NBD-PC, and
their propensity to be bound by activated monocytes was then
assessed. Back-exchange of NBD-lipid-treated cells at the ini
tiation of the binding experiments revealed that approximately
50% of the cell-associated lipid was localized at the outer leaflet.
As can be seen in Fig. 3, the binding of NBD-PS-labeled NHEK
to macrophages (31%) was markedly higher than that found
for NHEK labeled with NBD-PC (12%) or control NHEK (8%)
(Fig. 3/4). In contrast, no discernible differences in binding to
macrophages 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 young
and old cells, healthy and damaged cells, and nontumorigenic
and tumorigenic cells is not known. Several observations sug
gest that the expression of PS on the outer membrane leaflet
of cells could serve as a recognition moiety for macrophages.
For example, the insertion of an exogenous fluorescent PS
analogue (NBD-PS) into the outer leaflet of RBC facilitates
their uptake by macrophages (17) and clearance from the cir
culation after i.v. injection (18). Furthermore, experiments
using sickled RBC demonstrated that upon removal of oxygen,
PS is localized in both the inner and outer leaflets of the
membrane (13, 14), and these cells exhibited increased binding
to monocytes as compared with that of oxygenated, sickled
RBC (PS only in the inner leaflet) (15). Similarly, the recogni
tion of undifferentiated leukemic mouse cells by macrophages
could also be due to PS. Terminal differentiation of mouse
erythroleukemia cells is associated with a marked reduction of
binding to activated mouse macrophages (21, 32) and correlates
with a decrease in the expression of PS in the cells' outer leaflet
(21). Collectively, these data suggest that the expression of PS
in the outer leaflet of the cell may play a role in recognition
and subsequent removal by phagocytes (33).
In the present report, we determined whether a correlation
exists between the PS expression and interaction of human
tumor cells with activated human monocytes. Our results indi
cate that by 30 min, all three tumorigenic cell lines bound to
activated human blood monocytes, albeit to different degrees.
These results may be due to inter- and intratumoral heteroge
neity. A375 melanoma cells and A431 squamous carcinoma
cells were also lysed by the monocytes. Although Colo-16
squamous carcinoma cells bound to activated monocytes, they
were relatively resistant to lysis. These results agree with pre
vious reports (3, 33) showing that all tumorigenic cells bound
to activated monocytes but binding does not always result in
subsequent lysis (34). In contrast, nontumorigenic NHEK cells
did not bind to nor were they lysed by tumoricidal human blood
monocytes. Since PS expression in the outer leaflet of the 3
tumorigenic cell lines was 3-7-fold higher than the expression
of PS exposed on the outer membrane leaflet of the NHEK
cells, these data suggest a correlation between expression of PS
on the outer leaflet of target cell membranes and their recog
nition by monocyte-macrophages.
To investigate whether the inclusion of exogenous PS in the
outer leaflet bilayer of NHEK results in their recognition by
human monocytes, NBD-PS-labeled NHEK were incubated
C
•D
e
Z
0.4 -\
e
0.3-
25
50
NBD-PS
100
7
(ng/2x10
RBC)
Fig. 2. Standard curve generated from NBD-PS-labeled RBC. PDA-treated
RBC were labeled with increasing amounts of NBD-PS. The amounts of outer
leaflet NBD-PS in these cells were determined by direct fluorescence assaj versus
the initial rates of thrombin production (correlation of 0.98).
Incubation
time
(minutes)
Fig. 3. Binding of suspended NHEK (A) and A375 (B) cells to activated
adherent monocytes.
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EXPRESSION
OF MEMBRANE PS BY TUMOR CELLS
with activated monocytes. NHEK labeled with exogenous
NBD-PC served as controls. We used NBD-PC because PC has
a positive charge at its TV-substitution methylation site, and this
is the exact control for NBD-PS with a primary amine that is
also strongly positive (NH/). The results indicate that the
presence of PS, but not PC, on the outer leaflet of NHEK
directly correlated with binding to macrophages. Surprisingly,
the addition of PS to A375 cells did not increase the already
enhanced binding to monocytes. These results might suggest
that the recognition of tumor targets by monocytes ensues above
a critical threshold, similar to that observed for recognition of
PS-containing RBC ( 18).
In conclusion, the data presented here suggest that PS can
serve as a signal for triggering macrophage recognition as
manifested by binding to target cells (33). Although the binding
site on the macrophage surface responsible for recognition of
PS is not known, several possibilities exist. These include the
participation of autologous cytophilic antibodies (35, 36), com
ponents of the clotting cascade (16), and specific phospholipases (37, 38). These findings do not imply any lack of impor
tance for other and more developed mammalian recognition
systems, such as proteins and carbohydrate-regulated processes
(1), but do suggest that a relatively simple recognition mecha
nism involving phospholipids may also be important, a process
that has endured at least several steps of evolutionary
development.
Acknowledgments
We thank Dr. Dominic Fan for helpful suggestions, Kenneth Dunner,
Jr., for scanning electron microscopy, and Lola Lopez for expert help
in the preparation of this manuscript.
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Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1991 American Association for Cancer Research.
Elevated Expression of Phosphatidylserine in the Outer
Membrane Leaflet of Human Tumor Cells and Recognition by
Activated Human Blood Monocytes
Teruhiro Utsugi, Alan J. Schroit, Jerome Connor, et al.
Cancer Res 1991;51:3062-3066.
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