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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3862-3874
Biologically Active Fas Antigen and Its Cognate Ligand Are
Expressed on Plasma Membrane-Derived Extracellular Vesicles
By
Joseph Albanese,
Sarkis Meterissian,
Maria Kontogiannea,
Catherine Dubreuil,
Arthur Hand,
Sandra Sorba, and
Nicholas Dainiak
From the Departments of Medicine and Surgery, Royal Victoria
Hospital, McGill University, Montreal, Quebec, Canada; the Department
of Pediatric Dentistry, University of Connecticut Health Center,
Farmington, CT; and the Department of Medicine, Bridgeport Hospital,
Yale University School of Medicine, Bridgeport, CT.
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ABSTRACT |
Exfoliation of plasma membrane components is a directed process that
consumes energy and requires active cell metabolism. Proteins involved
in regulating the survival and proliferation of eukaryotic cells are
released on exfoliated vesicles. We examine here whether the Fas
receptor and its cognate ligand (FasL) are present on vesicles shed
from high metastatic potential CX-1 cells and low metastatic potential
MIP-101 cells and from HuT 78 cells, respectively. Rates of exfoliation
at 2 hours and cumulative levels of extracellular vesicles in
serum-free medium conditioned by CX-1 cells are increased by 1.8-fold
and 1.6-fold, respectively, relative to that in medium conditioned by
MIP-101 cells. Although vesicles shed from both cancer cell lines
contain Fas antigen, the amount of Fas per vesicle and the percentage
of vesicles containing Fas are increased for vesicles isolated from
MIP-101 cells, relative to those from CX-1 cells, as determined by
immunogold particle labeling and electron microscopy and by
immunofluorescence microscopy and flow cytometry. Results of metabolic
labeling with 35S-methionine indicate that Fas biosynthesis
is reduced by up to 3.3-fold for CX-1 cells, relative to that of
MIP-101 cells, consistent with the finding of decreased Fas on vesicles
shed from the plasma membrane of CX-1 cells. Although mRNA for soluble
Fas receptor is detectable in both cell lines, depletion of shed
vesicles from serum-free medium by ultracentrifugation removes all
detectable biological activity. FasL is detected on vesicles exfoliated
from HuT 78 cells by immunoelectron microscopy and Western blot
analysis. FasL-bearing vesicles induce apoptosis of Fas-expressing
cancer cells at the same level as observed by treatment with monoclonal anti-Fas antibody. Furthermore, Fas-bearing extracellular vesicles from
MIP-101 but not from CX-1 cells protect the CX-1 cell line from
FasL-induced and anti-Fas-mediated apoptosis, indicating that Fas
present on shed vesicles is biologically active. We conclude that the
Fas antigen and its cognate ligand are exfoliated from the cell surface
in a bioactive configuration. Exfoliation may provide a mechanism for
long-range signal-directed apoptosis while maintaining Fas/FasL on a
membrane surface.
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INTRODUCTION |
FAS ANTIGEN (Fas; CD95 receptor), a
cysteine-rich type I transmembrane glycoprotein, is a member of the
tumor necrosis factor (TNF) family. Its molecular mass ranges from 45 to 52 kD.1-3 In common with other members of the TNF
receptor family, Fas spans the plasma membrane once; its intracellular
domain is distinct from that of any other member of the family. Many
cell types, including lymphoid, myeloid, hematopoietic progenitor,
epithelial, and endothelial cells, express Fas on the cell surface and
undergo apoptosis when treated with anti-Fas antibody4,5 or
the cognate ligand for Fas, Fas ligand (FasL; CD95L).6 In
addition to normal cells, many malignant cells of hematologic and
nonhematologic origin express Fas, which can be triggered to signal
apoptotic cell death after ligation of Fas at the plasma membrane by
FasL or by functional antibodies to Fas.7,8
FasL is a type II transmembrane protein with an apparent molecular mass
varying between 36 and 43 kD.9,10 Recently, a 30-kD variant
of FasL has been immunoprecipitated from extracts of plasma
membranes.11 It, too, belongs to the TNF family of cytokines.11,12 However, in contrast to the ubiquitous
expression Fas, FasL is predominantly expressed on the surface of
activated T lymphocytes, natural killer (NK) cells, and Sertoli cells.
Recent studies have shown that colon13 and
liver14 carcinomas, as well as melanoma15 cells
also express FasL that may trigger apoptosis of activated T cells, a
process that may enhance tumor cell survival.14-16 Recent
evidence suggests that the interaction of (membrane bound) FasL with
Fas involves oligomerization of three FasL molecules on the surface of
effector cells that subsequently bind Fas molecules (also present as
trimeric complexes) on the surface of target cells.17 In
this model, each ligand subunit interacts with two of the three Fas
molecules, resulting in formation of a receptor complex.18
An increased concentration of soluble Fas has been detected in serum of
patients with solid tumors,19 T-cell leukemias, or B-cell
leukemias.20 Moreover, soluble Fas released from COS cells
transfected with cDNA encoding for Fas protein lacking a membrane-spanning domain inhibits cell death induced by anti-Fas antibody.21 Together, these findings suggest that reduced
tumor cell apoptosis may occur as soluble Fas competes with tumor
cell-surface Fas for T-cell/NK cell-surface FasL.22
Similarly, increased serum levels of soluble FasL have been detected in
patients with large granular lymphocytic leukemia and NK cell
lymphoma.23 In these patients, conversion of cell-surface
bound FasL to its soluble form was attributed to the action of a matrix
metalloproteinase.23
Components of the plasma membrane of both normal and malignant cells
are continually exfoliated from the surface as plasma membrane-derived
vesicles.24,25 Exfoliation may play a role in malignant
cell survival by permitting tumor-specific cell surface antigens to be
shed from the plasma membrane, thus avoiding recognition by host immune
defence mechanisms.26 Several reports have suggested that,
in addition to cloaking themselves from the host's immune system,
tumor cells actively participate in downregulating the function of
antigen-presenting cells by releasing plasma membrane component-bearing
vesicles, thereby increasing their chance of survival.26-28
Recently, Dolo et al29 demonstrated that transforming growth factor- -bearing vesicles released from human breast
carcinoma cells inhibit the proliferation of specific target
lymphocytes that recognize cell surface tumor antigens.
These findings, coupled with accruing evidence for the participation of
Fas/FasL interaction in the maintenance of tissue homeostasis, prompted
us to investigate whether Fas and/or FasL are shed on plasma
membranes-derived vesicles from high (CX-1) and low (MIP-101)
metastatic potential human colorectal carcinoma cell
lines30 and from the cell-surface of HuT 78 cells, an
activated human T-cell line. Our results show that (1) Fas and FasL are released on shed vesicles, (2) Fas-bearing vesicles efficiently inhibit
FasL-and anti-Fas antibody-mediated apoptosis, and (3) FasL-bearing
vesicles induce cell death. These findings indicate that Fas and FasL
are present on vesicles in a bioactive conformation. Furthermore, our
finding that Fas-bearing vesicles inhibit FasL-bearing vesicle-mediated
cell death suggests that Fas/FasL recognition takes place when these
proteins are presented to each other as components of extracellular
vesicles.
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MATERIALS AND METHODS |
Cell cultures.
Human colorectal adenocarcinoma cell lines, CX-1 and MIP-101, were a
generous gift from Dr P. Thomas (Deaconess Hospital, Harvard Medical
School, Boston, MA). The HuT 78 cell line (American Type Culture
Collection, Rockville, MD) is an activated T-cell line obtained from a
patient with Sezary syndrome that is known to express FasL. Cell lines
were established in T225 cell culture flasks (Costar Corp, Cambridge,
MA) and regularly maintained, as described previously.30
Briefly, cells were cultured in RPMI-1640 (RPMI) medium (GIBCO BRL,
Grand Island, NY) supplemented with 10% fetal calf serum (FCS) and
containing 100 U/mL of penicillin and 100 mg/mL streptomycin (P/S)
(GIBCO BRL) in a humidified atmosphere containing 5% CO2
at 37°C. Before harvesting vesicles, cells were permitted to grow
under low serum conditions as follows: cells in confluent flasks were
washed three times with phosphate-buffered saline, pH 7.2 (PBS), and
gently detached with 15 mL PBS containing 15 mmol/L sodium citrate, pH
7.2. After centrifugation at 800g for 5 minutes, the citrate
solution was aspirated and the cells were resuspended in 20 mL RPMI
containing 8% FCS and P/S. Two milliliters of the cell suspension was
introduced into T225 culture flasks containing 50 mL of RPMI
supplemented with 8% FCS and P/S. Cells were allowed to reach
confluency and passaged once again in a serum concentration that was
further reduced by 2%. This process was repeated until the cells were
maintained in media containing 2% FCS and P/S. When cells were 60% to
70% confluent, media was aspirated, cells were washed three times with
PBS, and 200 mL of fresh, serum-free RPMI was added to each flask.
Flasks were returned into the incubator and cells allowed to grow for 16 to 18 hours. Typically, greater than 95% of cells were capable of
excluding trypan blue.
Cell surface labeling.
Eighteen hours before cell surface labeling, media in tissue culture
flasks was aspirated and the cells were washed three times with 30 mL
PBS. CX-1 and MIP-101 cells were incubated overnight in 100 mL
serum-free RPMI medium. On the day of the experiment, medium was
aspirated and the cells were washed three times with 30 mL PBS and
detached with 25 mL citrate solution. Cells were harvested by
centrifugation, resuspended in 2 mL PBS, and filtered through a 30-µm
nylon mesh (Falcon; Becton Dickinson, Franklin Lakes, NJ) to remove
clumps. The volume was adjusted to 3.5 mL with PBS and 1 × 107 cells were surface labeled by incubation with two
Iodo-Beads (Pierce, Rockford, IL) and 2 mCi 125I (Amersham
Life Science, Oakville, Ontario, Canada). The cells were gently mixed
by slowly inverting the 5-mL tube for 1 minute, and the reaction was
quenched by removing the beads. The cells were pelleted by
centrifugation for 5 minutes at 700g and washed three times
with 5 mL PBS. Radiolabeled cells were then cultured in serum-free RPMI
under the conditions described above.
Vesicle preparation.
Serum-free conditioned medium was decanted from flasks and centrifuged
at 2,000g for 10 minutes to remove cells and debris, as
described previously31. The supernatant was subjected to
ultracentrifugation at 100,000g for 12 hours at 8°C.
Supernatant was discarded and the vesicles were washed twice by
resuspending the pellet in PBS, followed by ultracentrifugation at
100,000g for 12 hours at 8°C. Vesicles were used
immediately or stored at  80°C until needed. Vesicles collected from 125I-labeled cells were resuspended in 5 mL
scintillation cocktail (Dupont, Kingston, Ontario, Canada) and the
amount of radioactivity (cpm) was determined by scintillation counting.
Viability assay.
Cell viability was correlated to the capacity of cells to reduce 3-(4,
5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide to formazan,
as described by the manufacturer of the MTT assay (Sigma Chemical Co,
St Louis, MO). Briefly, 2 × 105 cells were cultured
in wells of a 96-well microtiter plate (Becton Dickinson) for 24 hours
under the conditions described above. After the incubation period,
media was removed and replaced with 200 µL fresh RPMI medium
containing 10% FCS, cycloheximide (10 µg/mL), and 100 ng
isotype-matched antibody (IgM) as a control (Calbiochem Corp, LaJolla,
CA) or 100 ng murine monoclonal antihuman Fas antibody, CH. 11 (Kamiya
Biomedical Co, Thousand Oaks, CA). Twenty microliters of vesicles (40 µg total protein) prepared from CX-1 or MIP-101 cells and resuspended
in PBS was added to appropriate wells. Cells were
incubated for 24 hours, after which MTT reagent was added. The cells
were incubated for an additional 4 hours, and levels of formazan were
quantified with an enzyme-linked immunosorbent assay (ELISA) reader at
570 nm.
Relative resistance of CX-1 and MIP-101 cell lines to anti-Fas-induced
and FasL-induced cell death was determined in a similar fashion, with
the exception that RPMI medium was not replaced after the initial
24-hour incubation period; cycloheximide and antibodies were added to
the conditioned medium, as described above.
Fas biosynthesis.
Biosynthesis of Fas was measured using 35S-methionine, as
described previously.32 Briefly, CX-1 and MIP-101 cells
maintained in RPMI medium supplemented with 10% FCS were gently
detached by treatment with 15 mmol/L citrate-PBS solution and harvested by centrifugation at 800g for 5 minutes. Cells (1.5 × 107) were washed twice with PBS and resuspended in
methionine-free RPMI-1640 medium supplemented with 10% FCS at a
density of 5 × 105 cells/mL, and 300 µCi
35S-methionine (Amersham Life Science) was added. Cells
were incubated for 2 or 16 hours at 37°C in a humidified atmosphere
with 5% CO2. Trypan blue staining showed that, at the time
of harvest, greater than 95% of the cells excluded dye.
Preparation of RNA.
Ten million MIP-101 or CX-1 cells grown under serum-free conditions
were detached from T225 flasks and harvested by centrifugation at
800g for 5 minutes. The cell pellet was solubilized in 1 mL Trizol reagent (GIBCO-BRL) and 0.2 mL chloroform (Fisher Scientific, Montreal, Quebec, Canada) and then centrifuged at 12,000g for 15 minutes at 4°C, as instructed by the manufacturer. The aqueous phase was transferred to a new tube and 0.5 mL isopropanol was added.
After 10 minutes of incubation, the tube was centrifuged at
12,000g for 10 minutes at 4°C. The RNA precipitate was
washed with 1 mL 75% ethanol and centrifuged one more time, and the
pellet was allowed to dry for 15 minutes at room temperature. Isolated total RNA was dissolved in 30 µL diethyl pyrocarbonate
(DEPC)-treated water and quantified by UV spectroscopy
using an Ultrospec 3000 (Pharmacia Biotech, Baie D'Urfe, Quebec,
Canada).
Reverse transcriptase-polymerase chain reaction (RT-PCR).
First-strand cDNA synthesis was accomplished by using 1 µg total RNA
and random hexamer primer as substrates for Moloney-murine leukemia
virus (M-MuLV) reverse transcriptase (MBI-Fermentas). A 3 µL aliquot of cDNA was used as template for PCR amplification with
the following primer pair, 5 -GGA GCT GCC TCT TCT TCC-3 and 5-ACA CTA ATT GCA TAT ACT CAG AACTG-3, corresponding
to the complete CD95 (Fas antigen) coding region.33
Immunoprecipitation.
35S-methionine-labeled CX-1 and MIP-101 cells (1 × 107) were collected by centrifugation at 800g for 5 minutes. Pellets were dissolved in 1 mL 50 mmol/L Tris-HCl buffer, pH
8.0, containing 0.5% Triton X-100 (Sigma Chemical Co), 10 mmol/L EDTA,
and 150 mmol/L NaCl. Subsequently, suspensions were mixed by inversion while incubating at 4°C for 30 minutes. After solubilization, lysates were dialyzed against 0.05% Triton X-100 in Tris-HCl buffer at
4°C for 16 hours. The radiolabeled samples were cleared with goat
IgG (Becton Dickinson) immobilized on protein G beads (Pierce), and Fas
was immunoprecipitated by incubating the mixture with goat anti-Fas
(provided by Dr L. Owen-Schaub, University of Texas, MD Anderson Cancer
Center, Houston, TX) bound to protein G beads at 4°C for 2 hours.
Beads were washed with 0.05% Triton X-100 in Tris-HCl buffer three
times and resuspended in 75 µL of 2× sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer
(0.25 mol/L Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, and 0.1%
bromophenol blue). Samples were placed for 5 minutes in a water bath at
100°C and centrifuged in a minifuge for 10 seconds, and the
pelleted beads were discarded. A 15-µL aliquot of each sample was
retained to determine the amount (cpm) of 35S-methionine
incorporated in immunoprecipitated Fas. The remainder of each sample
was electrophoresed in a 7.5% polyacrylamide gel, and reactive bands
were visualized by autoradiography.
Protein assay.
Protein levels were determined using micro BCA assay (Pierce),
according to the protocol suggested by the manufacturer.
Electron microscopy.
Exfoliated vesicle samples were placed on a formvar-carbon coated 200 mesh copper grid that was glow-discharged just before application.
Samples were allowed to adhere for 1 minute and then wicked off with
filter paper and allowed to air dry. The grids were blocked with 2%
BSA in PBS for 30 minutes and then incubated for 1 hour with mouse
anti-Fas IgM (10 µg/mL). After three rinses in PBS and another block
for 10 minutes, the grids were incubated for 2.5 hours at 4°C on
drops of goat antimouse IgM labeled with 10-nm gold particles. The
grids were rinsed three times in PBS and then rinsed three times with
distilled water, stained for 1 minute with 2% ammonium molybdate,
dried, and examined and photographed in a Philips CM10 transmission
electron microscope (JOEL Ltd, Tokyo, Japan) at 60 kV.
Western analysis.
Expression of Fas and FasL was determined by Western blot analysis.
Solubilized plasma membranes or plasma membrane-derived vesicles were
immunoprecipitated and electrophoresed in SDS-7.5% polyacrylamide.
Resolved protein bands were transferred to nitrocellulose, blocked
overnight with Tris-buffered saline (20 mmol/L Tris buffer, 137 mmol/L
NaCI, pH 8.0) supplemented with 0.1% Tween-20 (TBS-T), and incubated
for 1 hour at room temperature with either murine polyclonal anti-FasL
antibody (BMS 140; Cedarlane Laboratories, Hornby, Ontario, Canada) or
rabbit polyclonal anti-FasL antibody (Cedarlane Laboratories) that was
diluted 1,000-fold. The nitrocellulose membrane was washed three times
with 50 mL TBS-T. Lanes treated with murine anti-Fas antibody were
incubated for 1 hour with rabbit antimouse IgG (H + L) (Jackson
ImmunoResearch Laboratories, Mississauga, Ontario, Canada) diluted
1,000-fold in TBS-T and then washed three times with TBS-T.
Subsequently, all lanes were treated for 30 minutes with horseradish
peroxidase-conjugated goat antirabbit antibody diluted 5,000-fold in
TBS-T. Blots were developed with chemiluminescence substrate (ECL;
Amersham Life Science).
Fas antigen was deglycosylated as follows. To 200 µL of plasma
membranes suspended in PBS was added sufficient SDS to give a final
concentration of 0.15%. The sample was boiled for 5 minutes and the pH
was adjusted to 7.8. After the addition of 3 U N-glyconase (Boehringer
Mannheim, Laval, Quebec, Canada) to the aliquot, the sample was
incubated at 37°C for 18 hours. Subsequently, an additional 3 U of
enzyme was introduced into the aliquot, and the sample was incubated at
37°C for 18 hours. Deglycosylated proteins were solubilized,
immunoprecipitated, and subjected to Western blot analysis, as
described above.
Immunofluorescent microscopy and flow cytometry.
MIP-101 and CX-1 cells were cultured in T-75 flasks (Falcon; Becton
Dickinson), as described above. Cells were washed three times with PBS
and detached with 10 mL PBS-citrate solution. The cell suspension was
centrifuged at 300g for 5 minutes, and the pellet was then
resuspended in at 2 × 106 cells/mL PBS containing 1%
FCS (PBS-FCS). One hundred microliters (2 × 105
cells) of the cell suspension was transferred to plastic tubes (Falcon;
11.5 × 75 mm) and 200 ng of isotype-matched antibody (IgM) as a
control (Calbiochem Corp) or 200 ng murine monoclonal antihuman Fas
antibody (CH. 11) was added. The cells were mixed gently and placed on
ice for 20 minutes. After the incubation period, the cells were washed
twice with 2 mL PBS-FCS (centrifuged at 300g for 5 minutes),
and 100 µL (50 µg/mL) of goat antimouse IgG/IgM, (H + L)-fluorescein conjugated antibody (Pierce) was added. The cells were
mixed gently and the tubes were placed on ice for 20 minutes. The cells
were washed twice with PBS-FCS and harvested by centrifugation at
300g for 5 minutes. The cell pellet was resuspended in 50 µL
mounting fluid (1 part PBS and 9 parts glycerol), mounted on slides
under coverslips (Fisher, Montreal, Quebec, Canada), and sealed with
nail varnish. Cells were examined under fluorescence illumination using
an Optiphot-2 microscope (Nikkon, Mississauga, Ontario, Canada)
equipped with a 40× magnification objective. Photomicrographs
were obtained using a Nikon camera (model FX-35DX; Nikon,
Mississuaga, Ontario, Canada). Samples studied by flow cytometry were
prepared in identical fashion, except that, after the final wash, the
cell pellet was resuspended in 1 mL PBS before analysis.
Vesicles were harvested from serum-free medium conditioned by CX-1 or
MIP-101 cells and enumerated on a FACS VANTAGE sorter (Becton
Dickinson, Mountain View, CA) at the flow rate of 100 µL/min, side
scatter of 400 V, and FL1 of 700 V. Briefly, 5 × 106
vesicles were resuspended in 2 mL PBS-FCS, to which 100 ng mouse antihuman Fas IgM was added. Samples were incubated at 4°C for 30 minutes. Vesicles were washed as follows. The sample volume was
increased to 30 mL with PBS-FCS and vesicles were pelleted by
centrifugation at 100,000g at 4°C for 4 hours. Pellets were washed, resuspended in a final volume of 2 mL, and incubated at 4°C
for 30 minutes in the presence of rabbit dichloro
triazinyl amino fluorescein (DTAF)-conjugated antimouse IgM (20 µg/mL). Vesicles were washed, resuspended, and analyzed by flow
cytometry.
Statistical analysis.
Unless otherwise stated, means were compared using the Student's
t-test as calculated by Graphpad Prism version 2.0 statistical software (Graphpad Prism Inc, San Diego, CA).
| |
RESULTS |
Quantitative exfoliation from high (CX-1) and low (MIP-101) metastatic
potential cell lines.
The kinetics of exfoliation from well-differentiated and high
metastatic potential CX-1 cells was compared with that of poorly differentiated and low metastatic potential MIP-101 cells. As evident
from Fig 1A, CX-1 cells release more
125I-labeled protein in association with shed vesicles over
time (0 to 18 hours) than do MIP-101 cells. Cumulative radioactivity shed after 18 hours is 1.8-fold greater for CX-1 cells than for MIP-101
cells. When rates of exfoliation are compared (Fig 1B), both cell lines
exhibit the largest release of radioactivity (cpm) per hour after 2 hours of incubation, consistent with our results of kinetic studies for
shedding from normal human B cells and continuously maintained cell
lines.31 The rate of shedding from CX-1 cells
at 2 hours was 3.6-fold greater than that of shedding from MIP-101
cells.
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| Fig 1.
Kinetics of exfoliation from CX-1 and MIP-101 human
colorectal adeno-carcinoma cells. (A) Cumulative radioactivity (cpm)
released on shed vesicles collected from 1 × 107
surface-125I-labeled CX-1 ( ) and MIP-101 ( ) cells
after indicated incubation periods was quantified by scintillation
counting. (B) Rates of shedding were calculated by dividing total
radioactivity (cpm) released by the incubation time. Shown are the
means ± SD of three separate experiments. Note that shedding from
CX-1 cells is quantitatively increased relative to shedding from
MIP-101 cells.
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Visualization of Fas on shed vesicles.
Plasma membrane-derived vesicles isolated from medium conditioned by
CX-1 or MIP-101 cells were visualized by transmission electron
microscopy, and Fas antigen was detected on their surface by immunogold
particle labeling. Vesicle size and texture were heterogeneous,
findings typical for shed vesicles.24 As shown in
Figs 2 and
3, vesicles from both cell lines are
heterogeneous in shape and size (ranging from 0.05 to 0.5 µm) as well
as in distribution of Fas (arrowheads) on the shed vesicle surface. No
difference in vesicles released from MIP-101 versus CX-1 cells was
apparent by electron microscopy. However, vesicles derived from CX-1
cells (Fig 3) show relatively less immunogold particle labeling than do
vesicles shed from MIP-101 cells (Fig 2). The distribution of Fas on
vesicles derived from plasma membranes of MIP-101 cells was typical for
all fields that were examined. In contrast, vesicles shed from CX-1
cells failed to show the presence of Fas in the majority of fields that
were examined.
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| Fig 2.
Electron micrograph of immunogold-labeled Fas-bearing
vesicles shed from MIP-101 cells. Fas (arrowheads) was detected with gold particle-conjugated antimouse anti-Fas antibody. The micrograph is
at 57,000× magnification and the inset is at 68,000× magnification; scale bar = 0.2 µm.
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| Fig 3.
Electron micrographs of vesicles shed from CX-1 cells.
Large arrowheads point to gold particles in association with Fas
antigen on plasma membrane-derived vesicles. In (A), a few gold
particles (small arrowheads) not associated with vesicles can be seen
and apparently represent nonspecific background. Both micrographs are
at 92,000× magnification. Scale bar = 0.2 µm.
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Western blot analysis of detergent solubilized MIP-101-derived shed
vesicles and immunoprecipitated with anti-Fas antibody show two bands
with apparent molecular weights of 45 and 48 kD corresponding to
differentially glycosylated Fas protein
(Fig 4). Treatment of the vesicle extract
with N-glyconase before SDS-PAGE results in the disappearance of the
higher molecular weight band, indicating that, in fact, the proteins
immunoprecipitated with anti-Fas antibody are not unrelated proteins
but rather are glycoslyated isoforms of the same protein recognized by
the antibody.
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| Fig 4.
Western blot analysis of solubilized extracellular
vesicles harvested from MIP-101 conditioned medium. Vesicles extracted with n-octyl -D-glucopyranoside and treated with N-glyconase before
SDS-PAGE produce a single band with an apparent molecular weight of 45 kD when probed with anti-Fas antibody (N-gly). Vesicles extracted with
detergent and subjected to SDS-PAGE without N-glyconase treatment show
two bands with apparent molecular weights of 45 and 48 kD when probed
with anti-Fas antibody (C).
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Biosynthesis and cell-surface expression of Fas.
The relative distribution of Fas on the cell-surface of CX-1 and
MIP-101 cells was reevaluated and quantified using fluorescence microscopy. A discontinuous ring of fluorescence was observed on the
surface of CX-1 cells (Fig 5A), compared
with an intense fluorescence that was evident on MIP-101 cells (Fig
5B). Consistent with our findings in Fig 5A and B, flow analysis of
CX-1 and MIP-101 cells stained with IgM anti-Fas antibody and anti-IgG
FITC-conjugated antibody shows a 90-fold decrease (mean channel, 5.7 for CX-1 cells v 512.8 for MIP-101) in Fas antigen expression
at the cell surface of CX-1 cells relative to MIP-101 cells
(Fig 6). These results confirm our initial
observation that Fas may be downregulated in CX-1 cells, relative to
MIP-101 cells.
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| Fig 5.
Immunofluorescence microscopy indicates that CX-1 cells
(A) express lower quantities of cell-surface Fas than MIP-101 cells (B). Cells were labeled with mouse anti-Fas antibody and then treated
with antimouse FITC-conjugated antibody. Note that CX-1 cells (A) show
a discontinuous ring of fluorescence on their cell surface; in
contrast, MIP-101 cells (B) stain very intensely with secondary
antibody.
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| Fig 6.
Flow cytometry of MIP-101 and CX-1 human colorectal
carcinoma cells. MIP-101 (2 × 103) or CX-1 (2 × 105 ) were treated with murine anti-Fas antibody and then
stained with fluorescein-conjugated antimouse antibody as described in the Materials and Methods. MIP-101 (MIP-101) cells display greater fluorescence (mean channel, 512.76) than CX-1 (CX-1) cells (mean channel, 5.73), indicating greater expression of Fas antigen on the
latter cell line.
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To assess whether Fas is released at a high density on vesicles shed
from the surface of CX-1 cells (relative to that from MIP-101 cells),
extracellular vesicles were harvested, labeled with anti-Fas antibody
plus FITC-conjugated second antibody, and assessed by flow cytometry.
In general, labeling of vesicles shed from CX-1 cells was more
homogeneous than that of vesicles shed from MIP-101 cells. In two
separate studies, the mean ± SD percentage of labeled extracellular
vesicles from CX-1 cells was 1.95% ± 0.78%, whereas that for
vesicles from MIP-101 cells was 20.0% ± 3.1% (P < .01).
Together with electron micrographic results showing the presence of Fas
on very few vesicles shed from CX-1 cells (Fig 3), these observations
suggest that exfoliation plays a limited role in reducing the level of
cell surface Fas. This possibility prompted us to investigate rates of
Fas biosynthesis in CX-1 and MIP-101 cells that were labeled with
35S-methionine.
Figure 7 shows the amount of
radiolabeled-Fas specifically (cpm bound by anti-Fas minus cpm bound by
control antibody) precipitated from lysates obtained from CX-1 or
MIP-101 cells. After 6 hours, CX-1 cells synthesize 1.6-fold less Fas
than do MIP-101 cells. After 16 hours of incubation, CX-1 cells
synthesize 3.3-fold less Fas, relative to MIP-101 cells. These results
are supported by autoradiographs of 35S-labeled cells
showing the presence of molecular weight 45,000 and 48,000 proteins
(corresponding to the molecular masses of differentially glycosylated
Fas antigen) immunoprecipitated with anti-Fas antibody and subjected to
SDS-PAGE (Fig 8). Together, the results
suggest that reduced levels of Fas on extracellular vesicles may be the
result of diminished Fas synthesis and consequently, decreased amounts
of Fas on plasma membranes from which vesicles are shed.
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| Fig 7.
Synthesis of Fas by CX-1 and MIP-101 cells. CX-1 ( )
and MIP-101 ( ) cells were cultured in methionine-free medium for 6 or 16 hours with 300 µCi 35S-methionine. Cells were lysed
with Triton X-100 and the lysates were immunoprecipitated sequentially
with goat IgG and goat anti-Fas IgG antibody. Radioactivity in aliquots
(one fifth of the total sample) of each immunoprecipitated sample was
determined by scintillation. Note that Fas synthesis is greater for
MIP-101 cells than for CX-1 cells (P < .05). Shown are mean
values of a single experiment. Similar results were obtained in two
additional studies.
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| Fig 8.
Autoradiogram of 35S-labeled,
immunoprecipitated protein. Fas antigen immunoprecipitated from lysates
of MIP-101 (left) and CX-1 (right) cells incubated for 6 or 16 hours
with 300 µCi 35S-methionine were resolved on a 7.5%
SDS-polyacrylamide gel by electrophoresis. The autoradiogram was
obtained by exposing an x-ray film to the dried gel for 5 days at
80°C.
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|
Soluble receptor for Fas is produced by CX-1 and MIP-101 cells.
A soluble form of the receptor for Fas that lacks a transmembrane
domain may be released from tumor cells via the process of
secretion.21,22,34 Release of soluble Fas may provide a mechanism by which malignant cells escape
immunodestruction.35 To assess whether MIP-101 or CX-1
cells produce soluble Fas, both cell lines were examined for mRNA
encoding a soluble form of the receptor for Fas. Amplification of the
entire coding region showed two transcripts, corresponding to the
membrane-bound and soluble forms of Fas antigen
(Fig 9). RT-PCR showed that, although the major transcript appears to be 311 bp in size (membrane-bound form), a
minor transcript of 251 bp (soluble form) is also present in both cell
lines.33 No other bands were detected. It is noteworthy that mRNA for both forms of Fas appears to be less abundant in CX-1
cells, relative to that detected in MIP-101 cells, a finding that is
similar to the observation that CX-1 cells produce a lower level of
membrane-associated Fas receptor than do MIP-101 cells (Figs 5-8).
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| Fig 9.
Detection of Fas antigen splice variants in MIP-101 (MIP)
and CX-1 (C) cells by RT-PCR. Two transcripts of 311 and 251 bp corresponding to the membrane-bound form and soluble form of Fas antigen, respectively, are amplified after PCR of first-strand cDNA.
PCR products were electrophoresed in 2% agarose and visualized under
UV light after staining with ethidium bromide. Note that the intensity
of both bands is lower for CX-1 relative to MIP-101 cells, suggesting
decreased presence of FAS mRNA in the latter cell line. Also note that
the 251-bp band is less intense then the 311-bp band, suggesting that
the membrane-bound form of Fas predominates in CX-1 and MIP-101
cells.
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Biological effects of vesicles shed from CX-1 and MIP-101 on
anti-Fas-mediated cytotoxicity.
In previous experiments, we observed that CX-1 cells treated with
anti-Fas antibody are more sensitive to anti-Fas-mediated cell death
than are MIP-101 cells, as assessed by an MTT assay.36 Therefore, experiments were performed to determine whether vesicles shed from MIP-101 and CX-1 cells alter sensitivity to cell death induced by anti-Fas antibody. The finding of reversal of anti-Fas antibody-induced apoptosis would suggest that Fas present on shed vesicles is biologically active, analogous to the bioactivity of other
molecules expressed on the surface of shed vesicles.37-40
As shown in Fig 10, the viability of CX-1
cells after treatment with anti-Fas IgM alone (69.9% ± 0.7%) was
enhanced (P < .01) when these cells were treated
with anti-Fas in the presence of vesicles shed from MIP-101 cells
(94.9% ± 2.3%). In contrast, addition of anti-Fas IgM to CX-1
cells in the presence of vesicles from medium conditioned by CX-1 cells
did not increase cell viability, compared with treatment of CX-1 cells
with anti-Fas alone (74.2% ± 2.5% v 69.9% ± 0.7%,
respectively; P > .05). These results, together with
immunoelectron and immunofluorescence microscopy results showing high
Fas antigen density on vesicles derived from MIP-101 cells, indicate
that Fas shed on the surface of exfoliated vesicles is biologically
active. Moreover, the amount of activity present on shed vesicles
correlates with the amount of antigen that can be determined by
immunoelectron and immunoflourscence microscopy and by flow cytometry.
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| Fig 10.
Shed vesicles block anti-Fas-mediated CX-1 cell death.
Negative and positive controls were established by treating 2 × 105 CX-1 cells with 100 ng noncytotoxic, isotype-matched
antibody (IsoAb), or antihuman Fas IgM (Anti-Fas Alone), respectively. Viability of CX-1 cells was assessed in anti-Fas IgM-containing cultures prepared with 40 µg total membrane protein from vesicles shed from MIP-101 or CX-1 cells, as indicated. Note that, although Fas-mediated apoptosis is abolished in the presence of vesicles shed
from MIP-101 cells (relative to anti-Fas alone, P < .01), apoptosis is unaffected (P > .10) by the addition
of vesicles shed from CX-1 cells. Shown are the mean ± SD values in
three separate experiments.
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|
Bioactivity is undetectable in vesicle-free medium conditioned by
CX-1 or MIP-101 cells.
Because CX-1 and MIP-101 cells produce the soluble form of Fas (Fig 9),
it is possible that protection from anti-Fas-mediated apoptosis is due
to the release of soluble Fas receptor rather than to
vesicle-associated Fas. Therefore, serum-free medium conditioned by
CX-1 or MIP-100 cells was prepared and centrifuged under conditions that deplete extracellular vesicles but that do not remove soluble proteins of less than 100 kD.38 Vesicle-free medium
conditioned by either CX-1 or MIP-101 did not increase the viability of
CX-1 cells that were treated with anti-Fas IgM (65.6% ± 1.0% and
69.6% ± 2.0%, respectively), relative to cells treated with
anti-Fas IgM plus fresh RPMI medium (65.8% ± 3.4%;
Fig 11A). Although these results suggest
that physical presentation of Fas in association with a membrane
surface is important for expression of biological activity, they do not
exclude the possibility that small amounts of bioactive soluble Fas are
released from CX-1 and MIP-101 cells that are undetectable in the MTT
assay.33
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| Fig 11.
Conditioned medium from MIP-101 and CX-1 cells or HuT 78 fails to protect against anti-Fas-mediated apoptosis or induce
apoptosis, respectively. Serum-free medium conditioned by MIP-101,
CX-1, or HuT 78 cells was depleted of extracellular vesicles by
sequential centrifugation at 800g for 30 minutes and at
100,000g at 8°C for 12 hours. Negative and positive
controls were established by treating 2 × 105 CX-1 cells
with 100 ng noncytotoxic, isotype-matched IgM (IsoAb), or antihuman Fas
IgM (anti-Fas), respectively. (A) CX-1 cells were treated with 100 ng
antihuman Fas Ab and 100 µL medium conditioned by CX-1 (CX-1) or
MIP-101 (MIP-101) cells, respectively. Note that CX-1 and MIP-101
cell-conditioned medium fails to protect CX-1 cells from
anti-Fas-induced cell death. (B) Positive and negative controls were
established, as described above. CX-1 cells were treated 100 µL HuT
78 conditioned medium (HuT 78). Note that HuT 78 cell-conditioned
medium fails to induce apoptosis of CX-1 cells. Shown are the mean ± SD values in three separate experiments.
|
|
Visualization of FasL on shed vesicles.
To determine whether FasL is also released on shed vesicles, we
examined extracellular vesicles from HuT 78 cells which are known to
express high levels of FasL at their cell surface. Vesicles harvested
from HuT 78 cell-conditioned medium were labeled with rabbit anti-FasL
antibody and antirabbit immunogold-conjugated antibody and examined by
electron microscopy. As with vesicles derived from MIP-101 and CX-1
cells, vesicles isolated from HuT 78 cells are heterogenous in shape
and texture. As shown in Fig 12A,
vesicles labeled with immunogold particles (arrowheads) range in size
from 0.2 to 0.4 µm. In control experiments, no labeling was observed
for vesicles treated with immunogold-conjugated antibody alone (Fig
12B).
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| Fig 12.
Electron micrographs of vesicles shed from HuT 78 cells.
Arrowheads point to gold particles in association with FasL on plasma membrane-derived vesicles (A). No labeling is observed if vesicles are
treated with immunogold particle-conjugated antibody alone (B).
Micrograph (A) is 102,000× magnification and micrograph (B) is at
63,000× magnification.
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Detergent solubilized plasma membranes or vesicles derived from HuT 78 cells, immunoprecipitated with anti-FasL antibody and subjected to
Western blot analysis, show a 30-kD band
(Figs 13 and
14). The apparent molecular mass of
this protein is similar to that of FasL,11 consistent with
the notion that FasL protein on shed vesicles and on the plasma
membrane are similar in size. In contrast, FasL is not
immunoprecipitated from plasma membranes or vesicles derived from the
CX-1 colorectal cell line.
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| Fig 13.
Plasma membranes extracted with detergent and subjected
to Western blot analysis using anti-FasL antibody (as described in the
Materials and Methods) show a single band with an apparent molecular
weight of 30 kD (H). Similar analysis of CX-1 plasma membranes shows no
reactivity (C).
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| Fig 14.
Detergent extracts of HuT 78 plasma membrane-derived
extracellular vesicles, immunoprecipitated with anti-FasL antibody and analyzed by Western blot, produce a 30-kD molecular weight band (H).
Detergent extracts of vesicles collected from CX-1 conditioned medium
fail to show the 30-kD molecular weight band (C).
|
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Effects of Fas ligand-expressing extracellular vesicles on cell
viability.
To test whether FasL-bearing vesicles derived from HuT 78 cells display
biological activity, CX-1 cells were incubated with FasL-associated
vesicles, and their viability was measured 24 hours later, using the
MTT assay. As shown in Fig 15, viability of CX-1 cells is decreased by incubation with FasL-bearing vesicles or
monoclonal anti-Fas antibody (positive control; P < .01).
Cells treated with either FasL-bearing vesicles or monoclonal anti-Fas antibody induce comparable decreases of the percentage of cell viability (69.4% ± 0.6% and 64.4% ± 0.6%, respectively;
P > .05). By contrast, viability is unchanged (98.83% ± 0.69%; P > .05) when CX-1 cells are incubated with
CX-1-derived extracellular vesicles (negative control). Vesicles shed
from the surface of MIP-101 cells (but not those from CX-1 cells)
restore nearly full viability (97.5% ± 0.8% v
93.9% ± 0.6%, for control CX-1 cells incubated with vesicles shed
from CX-1 cells v test CX-1 cells incubated with mixtures of
vesicles shed from Hut 78 cells and MIP-101 cells, respectively).
Furthermore, depletion of shed vesicles from serum-free medium
conditioned by HuT-78 cells results in removal of the biological
activity of FasL (Fig 11 B), suggesting that soluble FasL is either
inactive or present in an undetectable quantity. Together, these
results provide strong evidence that FasL on shed vesicles is
biologically active and that FasL interacts with its cognate receptor,
Fas, when the two proteins are presented together as components of
mixed populations of extracellular vesicles.
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| Fig 15.
Effect of FasL-bearing extracellular vesicles on CX-1
cell viability. Negative and positive controls were established by
incubating 2 × 105 CX-1 cells with 40 µg total membrane
protein prepared from vesicles shed from CX-1 cells (CX-1) or 100 ng
antihuman Fas IgM (Anti-Fas), respectively. Vesicles shed from Hut 78 cells suppress viability by nearly 40% (Hut 78 Alone). Whereas the
addition of 40 µg total membrane protein of vesicles shed from
MIP-101 cells restores nearly full viability (Hut 78 + MIP-101), the
addition of 40 µg total membrane protein of vesicles shed from CX-1
cells has a minimal effect (Hut 78 + CX-1). Note that the viability
of cells incubated with mixtures of vesicles prepared from Hut 78 cells and MIP-101 cells is increased, relative to that of cells incubated with mixtures of vesicles prepared from Hut 78 cells and CX-1 cells
(P < .01). Shown are the mean ± SD values in three separate experiments.
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| |
DISCUSSION |
Survival and differentiation signals are transmitted among eukaryotic
cells via interactions of growth factors released by effector cells in
the form of (1) soluble cytokines and/or (2) shed vesicle-bound
growth factors, with cognate receptors expressed on the target cell
surface.24,41 Alternatively, membrane-bound growth
regulators are presented to cognate receptors via direct physical
interactions at the cell surface, a process referred to as juxtacrine
communication.42 Comparably, apoptosis signals are
transmitted by soluble factors (eg, TNF- ), by perforin-containing vesicles, or by direct physical interaction of Fas ligand expressed on
cytotoxic T lymphocytes and NK cells with Fas receptor present on
target cells.43-45
Our finding that bioactive Fas and FasL are shed on vesicles derived
from the plasma-membrane of MIP-101 cells and HuT 78 cells,
respectively, is in accord with the notion that membrane-bound growth
factors, such as mBPA, macrophage-colony-stimulating factor (M-CSF), and flt3-flk2, are released on extracellular
vesicles where they express biological activity.46-49 The
release of cell-surface molecules on shed vesicles may provide a
mechanism for communication among cells that are not necessarily
adjacent to each other but in which long-lasting stimulation can be
achieved. Proximal interactions mediated by signals carried on
extracellular vesicles may be analogous to juxtacrine communication
between effector cells and target cells. Ligand-bearing extracellular
vesicles may permit long-range interactions while still maintaining
effector molecules concentrated on a membrane surface, thereby
restraining their dilution in the pericellular environment.
We determined here that vesicles shed from the surface of viable
MIP-101, CX-1, and HuT-78 cells display Fas of FasL. Because it is
possible that extracellular vesicles are themselves released from
potentially rare cells undergoing apoptosis during short-term culture,
it was important to distinguish shed vesicles from apoptotic bodies.
Unlike shed vesicles, apoptotic bodies are plasma membrane bound
structures that contain nuclear fragments and well-preserved organelles
such as mitochondria and endoplasmic reticulum.50,51 In
virtually all fields examined by electron microscopy, no evidence was
found for the presence of either nuclear material or organelles (intact
or partially degraded) within extracellular vesicles (Figs 2, 3, and
12). Accordingly, vesicles released from MIP-101, CX-1, and HuT-78
cells are morphologically identical to vesicles shed from normal human
lymphocytes, which, in previous studies, we have found to contain no
cytoplasmic markers.38,47
The release of proteins on exfoliated membrane vesicles is dependent on
intracellular processes, including mRNA synthesis and translation, as
well as posttranslational modification.31 Because shedding
from the cell surface requires energy and active cell metabolism and
occurs from distinct regions of the plasma membrane, exfoliation is a
directed (rather than random) process.46,47 Although the
mechanism of exfoliation is not completely understood, we have observed
that contractile proteins participate in the exfoliation of cytokines
in association with shed vesicles.52 Furthermore, the
observations that nonmetastatic cells shed less then their metastatic
counterparts,53 coupled with the findings that highly
metastatic cells exhibit fewer associations between actin and vinculin
and the plasma membrane,54 suggest that disruption of
cytoskeletal elements may be a prerequisite for extracellular vesicle
formation. Calpain, an intracellular calcium dependent protease,
demonstrates high specificity for anchor cytoskeletal elements,55 and its activation has been directly implicated with increased shedding of plasma membrane proteins.56
Phospholipases may also play a role in the release of vesicles from the
cell-surface. Before the release of plasma membrane-derived vesicles,
fusion must take place of closely approximated membranes at the base of
the vesicle. Phospolipases may mediate the fusion of adjacent regions
of the membrane by generating detergentlike molecules (lysophosopholipids) that enhance interactions among membrane structures.57
Exfoliation provides a mechanism of cell-cell signal trafficking for
not only cytokines but also other molecules, including the transferrin
receptor,58 C3 component of the complement system, fragment
crystallizable (Fc) domain of antibodies,59 as well as
class I and class II major histocompatibility (MHC)
antigens.40,60 It has been suggested that survival and
metastatic propensity of many tumor cell types are enhanced by
downregulation of cell-surface tumor antigens, thereby permitting
escape from immune recognition and destruction.57,61
Studies showing that, in some cases, high metastatic potential cell
lines shed greater amounts of plasma membrane-derived vesicles than do
their low metastatic potential counterparts have strengthened this
concept.62,63 The shedding patterns we observed for high
(CX-1) versus low (MIP-101) metastatic potential cells (Fig 1) are
consistent with this notion. Whether the increased level of shedding
from CX-1 cells protects against imunne recognition and/or
facilitates metastasis remains to be investigated.
The physiologic significance of circulation soluble Fas in sera from
patients with malignant tumors is unclear. One hypothesis is that its
presence impairs apoptotic death of cancer cells initiated by T and NK
cells. For example, Hughes and Crispe64 showed that a
soluble isoform of murine Fas (generated by alternative splicing of Fas
mRNA) physiologically limits apoptosis that is induced by Fas-Fas
ligand association. Nevertheless, the source of soluble Fas is not
always clear. Knipping et al20 reported that soluble Fas
harvested from medium conditioned by B-lymphoblastoid cells migrates to
the same position as the membrane-bound form of Fas when
electrophoresed in polyacrylamide. These investigators concluded that
soluble Fas is not generated by simple proteolytic cleavage from the
cell surface, but rather that soluble Fas is secreted into the medium,
possibly because it may lack a transmembrane domain.20
Whereas mRNA for soluble Fas is present in CX-1 and MIP-101 cells (Fig
9), results of vesicle-depletion experiments suggest that the soluble
form of the receptor may be biologically inactive, using the MTT assay
(Fig 11). On the other hand, it is well known that vesicles expressing
immune regulatory molecules are shed in vivo.65,66 Our
results showing that Fas is present on vesicles shed from the surface
of MIP-101 cells (Fig 2) raise the possibility that soluble Fas found
in human serum may be associated with circulating shed vesicles.
Indeed, methods used by Knipping et al20 to
isolate soluble Fas are nearly identical to those we have used for
isolation of vesicles from conditioned medium in this study and from
sera of leukemia patients.38,67 Moreover, vesicle-associated Fas is similar in molecular mass (45 and 48 kD; Fig
4) to soluble Fas found in human serum (48 to 52 kD20).
We observed that CX-1 cells express a lower quantity of cell-surface
Fas when compared with MIP-101 cells. This finding was confirmed by
immunofluorescence studies (Figs 5 and 6). We further show that CX-1
cells synthesize Fas at a lower rate than do MIP-101 cells (Fig 7).
However, low metastatic potential MIP-101 cells are more resistant to
anti-Fas antibody-mediated apoptosis than are CX-1 cells. This paradox
may be explained by our finding that MIP-101 cells release plasma
membrane-derived vesicles that contain a greater level of Fas than that
present on vesicles shed from the surface of CX-1 cells (Fig 3), even
though shedding rates are lower for MIP-101 cells (Fig 1). Thus,
MIP-101 cells may release vesicles bearing high levels of Fas that
effectively compete with cell surface Fas for binding of anti-Fas
antibody, thereby blocking Fas-mediated cell death. In such a scenario,
anti-Fas antibody added to a culture of MIP-101 cells is engaged by
vesicle-associated Fas and, despite elevated expression of cell surface
Fas (relative to CX-1 cells), MIP-101 cells escape anti-Fas
antibody-mediated apoptosis. This model predicts that extracellular
vesicles released from CX-1 cells having low levels of Fas will not
effectively protect against anti-Fas antibody-induced apoptosis, a
result that was observed in our studies (Fig 10). Moreover, vesicles
derived from MIP-101 cells (but not those from CX-1 cells) inhibit CX-1 cell death induced by FasL-bearing vesicles derived from HuT 78 cells
(Fig 15), activated T cells whose surface expresses FasL (Fig 13).
These results suggest that, like Fas expressed on MIP-101 cells, FasL
expressed on HuT 78 cells is released on extracellular vesicles and is
present in a bioactive conformation. Accordingly, vesicle-bound FasL
appears to be capable of interacting with vesicle-bound Fas or
cell-surface Fas.
Recently, Sato et al68 have shown that lymphoma cells
express and release soluble FasL into the circulation in vivo, an event
that may be associated with development of liver damage and
pancytopenia. Their results are consistent with an earlier publication
by Tanaka et al23 demonstrating that individuals with large
granular lymphocytic leukemia or NK cell lymphoma are susceptible to
systematic tissue damage resulting from the action of a metalloprotease
that cleaves cell-surface FasL and releases a soluble variant of the
ligand into the circulation of these patients. Our findings indicate
that FasL is also released from the cell-surface on exfoliated vesicles
(Figs 12, 13, and 14). Whether release of FasL on vesicles shed from
malignant cells occurs in vivo remains unknown.
In summary, our results provide evidence for an alternative mechanism
for the release of Fas and its natural ligand from the surface of cells
derived from human cancers. We show that a colorectal carcinoma cell
line (MIP-101) releases Fas on plasma membrane-derived vesicles that
are shed in vitro and that are capable of neutralizing apoptosis
induced by anti-Fas antibody or FasL. Furthermore, FasL is also
released on exfoliated vesicles in a bioactive conformation. These
findings add to the growing body of evidence suggesting that Fas
released from the cell-surface of tumor cells can potentially act to
evade detection by immune cells. The results support the notion that
exfoliation facilitates the presentation of signals for cell
survival/death on the membrane surface of effector cells.
| |
FOOTNOTES |
Submitted July 18, 1997;
accepted January 5, 1998.
Supported in part by the Bayer/Canadian Red Cross Society Research and
Development Fund and by Grant No. 00014-94-0049 issued to Georgetown
University from the Office of Naval Research in support of the
International Consortium for Research on the Health Effects of
Radiation.
Address reprint requests to Nicholas Dainiak, MD, Department of
Medicine, Bridgeport Hospital, 267 Grant St, Bridgeport, CT 06610.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract