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Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1657-1664
Discordant Expression of Tissue Factor and Its Activity in Polarized
Epithelial Cells. Asymmetry in Anionic Phospholipid Availability as a
Possible Explanation
By
Carsten B. Hansen,
Bo van Deurs,
Lars C. Petersen, and
L. Vijaya
Mohan Rao
From the Department of TF/VIIa Research, Health Care Discovery, Novo
Nordisk A/S, Denmark; Structural Cell Biology Unit, the Department of
Medical Anatomy, The Panum Institute, University of Copenhagen,
Denmark; and the Department of Biochemistry, the University of Texas
Health Center at Tyler, Tyler, TX.
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ABSTRACT |
Recent studies have shown a discrepancy between the level of tissue
factor (TF) expression and the level of TF procoagulant activity on the
apical and basolateral surface domains of polarized epithelial cells.
The present investigation was performed to elucidate possible reasons
for the discordant expression of TF and its activity on the surface of
polarized epithelial cells using a human intestinal epithelial cell
line, Caco-2 and Madin-Darby canine kidney epithelial cells, type II
(MDCK-II). Functional activity of coagulation factor VIIa (VIIa) in
complex with TF was 6- to 7-fold higher on the apical than the
basolateral surface in polarized Caco-2 cells. In contrast, no
significant difference was found in the formation of TF/VIIa complexes
between the apical and basolateral surface. Confocal microscopy of
Caco-2 cells showed TF expression on both the apical and the
basolateral surface domains. Studies with MDCK-II cells showed that the
specific functional activity of TF expressed on the apical cell surface
was 5-fold higher than on the basolateral surface. To test whether
differential expression of TF pathway inhibitor (TFPI) on the apical
and basolateral surface could account for differences in TF/VIIa
functional activity, we measured cell-surface-bound TFPI activity in
Caco-2 cells. Small but similar amounts of TFPI were found on both
surfaces. Further, addition of inhibitory anti-TFPI antibodies induced
a similar enhancement of TF/VIIa activity on both surface domains.
Because the availability of anionic phospholipids on the outer leaflet
of the cell membrane could regulate TF/VIIa functional activity, we
measured the distribution of anionic phospholipids on the apical and
basolateral surface by annexin V binding and thrombin generation. The
results showed that the anionic phospholipid content on the basolateral
surface, compared with the apical surface, was 3- to 4-fold lower. Mild
acid treatment of polarized Caco-2 cells, which markedly increased the
anionic phospholipid content on the basolateral surface membrane,
increased the TF/VIIa activity on the basolateral surface without
affecting the number of TF/VIIa complexes formed on the surface.
Overall, our data suggest that an uneven expression of TF/VIIa activity
between the apical and basolateral surface of polarized epithelial
cells is caused by differences in anionic phospholipid content between
the two surface domains and not from a polar distribution of TFPI.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
TISSUE FACTOR (TF) is a cellular receptor
for plasma coagulation factor VIIa and formation of TF/VIIa complexes
on the cell surface triggers the coagulation cascade in
vivo.1 The TF/VIIa complex efficiently activates
coagulation factors IX and X. The resultant protease factor Xa (Xa)
activates prothrombin to thrombin, which in turn converts fibrinogen
into a fibrin matrix. TF pathway inhibitor (TFPI) functions as the
primary regulator of TF/VIIa activity during hemostasis.1,2
Feedback inhibition of the TF/VIIa complex is accomplished by the
formation of a stable quaternary complex of TF/VIIa/
Xa/TFPI.2
Normally, TF is constitutively expressed on the surface of many
extravascular cell types that are not in contact with the blood, such
as fibroblasts, pericytes, smooth muscle cells, and epithelial cells,
but not on the surface of cells that come in contact with blood, such
as endothelial cells and monocytes.3,4 However, a number of
pathophysiologic stimuli induces TF expression in both endothelial
cells and monocytes.5 Studies on the cellular distribution
of TF have shown a polar distribution of TF in human umbilical vein
endothelial cells and Madin-Darby canine kidney epithelial cells
(MDCK).6-9 Ryan et al6 reported that TF
activity was not expressed on the luminal cell surface of tumor
necrosis factor- (TNF-)-stimulated endothelial cells but rather
associated with subendothelial extracellular matrix. In contrast,
Mulder et al8 found that nearly all TNF--induced TF
activity was expressed on the endothelial cell surface rather than on
the subendothelial matrix. Further, these investigators8
also suggested that TF activity in endothelial cells was predominantly
located on the basolateral surface. However, Narahara et
al7 found that TF activity of interleukin-1 -stimulated
endothelial cells was almost exclusively located on the apical surface,
whereas little activity was found on the basolateral surface. Camerer
et al9 were the first to actually measure TF distribution
on the apical and basolateral surface domains of polarized cells grown
in a transwell system. They reported that in endothelial cells not only
TF activity, but also TF antigen, was localized primarily on the apical
surface. This study also showed that a large fraction of TF/VIIa
complexes formed on the basolateral surface of MDCK cells was not
functionally active, whereas a much smaller number of complexes formed
on the apical surface was highly active. The reason for the discordant expression of TF and its activity on polarized cells is, at present, unknown.
In the present study, we investigate possible reasons for the
discordant expression of TF and its activity on the surface of
polarized epithelial cells. Our data suggest that anionic phospholipids are not symmetrically exposed on the outer leaflet of the apical and
basolateral cell membrane domains of polarized epithelial cells and
that this differential availability of anionic phospholipids may
explain the polar expression of TF/VIIa functional activity.
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MATERIALS AND METHODS |
Reagents.
Iodo-Gen was from Pierce Biochemical Co (Rockford, IL); sodium
[125I]iodide was from Amersham Corp (Arlington Heights,
IL); Chromozym X and Chromozym TH were from Boehringer Mannheim
(Indianapolis, IN); Dulbecco's modified Eagle's medium (DMEM) was
from GIBCO (Grand Island, NY); fetal bovine serum (FBS),
trypsin-EDTA, and penicillin-streptomycin were from BioWhittaker
(Walkersville, MD). Tissue-culture flasks and plates were from Becton
Dickinson (Bedford, MA). Transwell cell culture inserts were from
Corning Costar (Acton, MA). Other chemicals, reagent grade or better, were from Sigma (St Louis, MO) or Fisher Scientific
(Pittsburgh, PA).
Proteins.
Recombinant human VIIa was purified as described.10 Human
coagulation factor X and factor Va were obtained from Enzyme Research Laboratories (South Bend, IN). Factor Xa,11
prothrombin,12 human brain TF apoprotein,13 and
polyclonal rabbit anti-human TF IgG13 were prepared as
described previously. TF apoprotein was reconstituted in 60%
phosphatidylcholine, 40% phosphatidylserine vesicles as
described.14 Annexin V, purified from human placenta, was a
gift from Dr J.F. Tait (University of Washington, Seattle). Recombinant
TFPI15 was used as an antigen to raise monospecific polyclonal anti-TFPI antiserum in rabbits. The antiserum was heat inactivated at 56°C for 30 minutes and the IgG fraction was
separated by precipitation at 40% ammonium sulfate saturation followed
by DEAE-Affi-Gel blue chromatography. The IgG had specific activity of
more than 200 inhibitor units/mg protein.
Cell culture.
Caco-2 cells16 were maintained in DMEM (4.5 g/L glucose)
with GlutaMAX 1 supplemented with 10% FBS, 1% (vol/vol) non-essential amino acids and antibiotics. MDCK-II cells16 were
maintained in DMEM (4.5 g/L glucose) with GlutaMAX 1 supplemented with
5% FBS and antibiotics. Cells were seeded in 12-mm polycarbonate membrane transwells (0.4-µm pore size) at a density of 3 × 105 cells/cm2 (in parallel, cells
were also seeded at a same density in a clear polyester membrane
transwell for visual inspection). Medium was changed every second day.
Cells were maintained in transwells for a time period of more than 3 days after establishment of tight junctions to obtain well
differentiated monolayers. Establishment of epithelial cell integrity
and tight junction formation was assessed by transepithelial electrical
resistance (TER) measurements across the membrane using a MilliCell-ERS
ohmmeter (Millipore, Bedford, MA). Tight junction
formation was also demonstrated by the absence of radioactive tracer
leakage from either side of the cell monolayer (125I-VIIa
or 125I-annexin V). Cells were washed once on both sides
with buffer A (10 mmol/L HEPES, pH 7.45, 150 mmol/L NaCl, 4 mmol/L KCl,
and 11 mmol/L glucose) supplemented with 5 mmol/L EDTA and then washed twice with buffer B (buffer A supplemented with 5 mg/mL bovine serum
albumin [BSA] and 5 mmol/L Ca2+) before they were used in
the experiment. At well-differentiated monolayers, the cell number was
6 to 8 × 105 per transwell.
Radiolabeling of proteins.
VIIa and Annexin V were labeled using IODO-GEN-coated (Pierce) tubes
and Na125I according to the manufacturer's technical
bulletin and as described previously.17 The labeling
reaction was performed in tubes coated with 10 µg of IODO-GEN for 4 minutes on ice. The reaction was quenched by the addition of 1% KI,
and free iodine was removed by extensive dialysis against 150 mmol/L
NaCl, 10 mmol/L HEPES, pH 7.5. The concentration of the labeled
proteins was determined by measurement of absorbance at 280 nm.
Extinction coefficients (Ecm1%) of 13.2 and
6.0 were used for VIIa and annexin V, respectively.
Radiolabeled ligand binding to polarized cells.
Binding of 125I-VIIa to TF on the cell surface of polarized
Caco-2 cells was performed essentially as described.17
Cells in transwells were incubated for 2 hours at 4°C with 10 nmol/L of radiolabeled VIIa in buffer B added to each side in a final
volume of 500 µL. At the end of the incubation, cells were quickly
washed 3 times with ice-cold buffer B. Total surface bound
radioactivity was eluted from the apical and basolateral cell surface
by treating the cells with buffer A supplemented with 5 mg/mL BSA and
15 mmol/L EDTA. Radioactivity in eluates was measured by a gamma
counter (Cobra; Packard Instrument Co, Meriden, CT).
Nonspecific binding was determined in parallel duplicate wells in which
the cells were preincubated for 30 minutes with rabbit anti-human TF
IgG (100 µg/mL) before the addition of radioligand. TF-specific
binding was determined by subtraction of nonspecific binding from total binding. At 10 nmol/L of 125I-VIIa, the nonspecific binding
of factor VIIa to Caco-2 cell surfaces (to both apical and basolateral
surface domains) was about 40% of the total binding. Binding of
125I-VIIa to the cell surface of polarized MDCK-II cells
was performed essentially as described above except that nonspecific
binding was determined by adding 50-fold molar excess of unlabeled
factor VIIa because anti-human TF antibodies failed to block factor
VIIa binding to canine TF. Nonspecific binding of 125I-VIIa
to cell membrane in MDCK-II cells was in the range of 35 to 45% of the
total binding.
Binding of 125I-annexin V to Caco-2 cells was measured
essentially as described.18 Cells were incubated for 2 hours at 4°C with 10 nmol/L of radiolabeled annexin V in buffer B. Total cell-surface-associated (Ca2+-dependent)
radioactivity was recovered from the apical and basolateral surfaces by
eluting with EDTA containing buffer and the eluates were counted in a
gamma counter. Nonspecific binding was determined in parallel duplicate
wells in which the cells were preincubated for 30 minutes with 50-fold
molar excess of cold annexin V (500 nmol/L) before the addition of
radioligand. Annexin-V nonspecific binding, on both apical and
basolateral membranes, was 3% to 8% of the total binding.
Factor X activation assay.
Polarized cells in transwells, after removing media and washing the
cells, were incubated with 10 nmol/L VIIa in buffer B (final volume,
500 µL) at 37°C for 15 minutes to allow VIIa binding to the
cell-surface TF. Unbound ligand was then removed, and the monolayer was
washed 3 times with buffer B. Activation of factor X on the monolayers
was initiated by adding 300 µL of buffer B containing 175 nmol/L
factor X. At various time intervals or at a fixed time (usually at 30 minutes), 20-µL aliquots were removed from apical and basolateral
sides of the well and added to 80 µL of stopping buffer, TBS (50 mmol/L Tris, 150 mmol/L NaCl, pH 7.5) containing 5 mmol/L EDTA and 1 mg/mL BSA. The amount of Xa generated was determined in a chromogenic
assay by transferring 50 µL of the above mixture to a microtiter
plate well and adding 50 µL of Chromozym X (2.15 mmol/L) to the well.
The absorbance at 405 nm was measured continuously in a microplate
reader (Molecular Devices, Sunnyvale, CA), and the initial rates of
color development were converted to Xa concentrations using an Xa
standard curve (85 pmol/L to 11 nmol/L). The rate of TF/VIIa dependent
activation of factor X on both the apical and basolateral surface was
linear up to 1 hour.
Fluorescence confocal microscopy.
Polarized Caco-2 cells were maintained in transwells. Cells on
transwell filters were fixed in 2% formaldehyde in phosphate buffer
(pH 7.2) and washed thoroughly. Nonspecific binding was blocked by
incubating the cell monolayer in blocking buffer (5% normal goat serum
in phosphate-buffered saline (PBS) in the absence or presence of 0.2%
saponin for permeabilization and increased accessibility of
antibodies). Cells were incubated for 60 minutes with rabbit anti-human
TF IgG (10 µg/mL) in blocking buffer on both sides of the cell
monolayer. As controls, monolayers were incubated with either blocking
buffer alone or preimmune IgG (10 µg/mL). After washing the cells in
PBS, they were incubated with fluorescein isothiocyanate
(FITC)-conjugated goat anti-rabbit IgG antibody (10 µg/mL) (Southern
Biotechnology, Birmingham, AL) for 60 minutes. After thorough washing
in PBS, transwell filters were rinsed briefly in distilled water and
mounted in Fluoromount G (Southern Biotechnology) containing 2.5 mg/mL
N-propyl-galleate (Sigma). Slides were examined with a Zeiss LSM 510 confocal laser scanning microscope using a C-Apochromat 63×, 1.2 water immersion objective (Carl Zeiss, Thornwood, NY), and the 488-nm
line of the argon laser for excitation of FITC. The cells were scanned and images saved at 1024 × 1024-pixel/8-bit resolution before importing into Adobe Photoshop (Adobe Systems, Mountain View, CA) for
compilation and direct printing.
Prothrombin activation assay.
Polarized cells in transwells were incubated with factor Va (20 nmol/L)
and Xa (2 nmol/L) for 15 minutes at 37°C, followed by prothrombin
(1,400 nmol/L). At various time points after the addition of
prothrombin, 20 µL subsamples were removed in 80 µL ice-cold
stopping buffer. The amount of thrombin generated in each subsample was
determined by transferring 50 µL of the above mixture to a microtiter
plate well and adding 50 µL of Chromozym TH (1.9 mmol/L) to the well.
The initial rates of color development in milliOD/min at
405 nm were measured continuously with a microplate reader and the
reading was converted to thrombin concentrations from a standard curve
made with human thrombin (4 mU/mL to 2 U/mL).
Elution of cell-bound TFPI and TFPI functional assay.
Cells were washed as described previously. Both compartments of
polarized cells in transwells were treated with 500 µL of 0.1 mol/L
glycine, pH 3.0, for 3 minutes. Eluates were removed and pH was
adjusted to 7.8 with 1 mol/L Tris, and assayed for TFPI
activity in a 2-step factor X activation assay. A 20-µL sample was
added to a microtiter plate containing 40 µL of reagent A (TBS
containing 1 mg/mL BSA, 90 pmol/L Xa, 3 nmol/L VIIa, 22 pmol/L human
TF, 15 mmol/L CaCl2). After a 30-minute incubation at room temperature, 100 µL of reagent B (TBS containing 1 mg/mL
BSA, 42 nmol/L factor X, 4 mmol/L CaCl2, and 0.86 µmol/L
Chromozym X) was added to each sample. At the end of a 20- to 30-minute reaction period, the absorbance was read at 405 nm in a microplate reader. A TFPI standard curve was obtained using dilutions of 0.125%
to 2% pooled normal human plasma or full-length recombinant TFPI (2.6 to 105 pmol/L).
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RESULTS |
Expression of TF/VIIa functional activity on polarized epithelial
cells.
Caco-2 cells were grown to polarity in the transwell cell-culture
system with distinct apical and basolateral cell-surface domains. Cell
polarity was assessed by measuring TER and only well-differentiated
cells with TER in the range of 400 to 600  cm2 were used.
In initial experiments, we added various concentrations of VIIa (0.1 to
40 nmol/L) to the apical and basolateral compartments, and the
expression of TF/VIIa procoagulant activity was measured in a factor X
activation assay. TF/VIIa activity was saturated at 10 nmol/L VIIa. As
shown in Fig 1A TF/VIIa functional activity was expressed primarily on the apical side of the polarized Caco-2 cells, with a 6- to 7-fold higher activity than on the basolateral surface. Addition of inhibitory polyclonal anti-TF IgG completely inhibited the expression of TF/VIIa functional activity on both surface
domains (data not shown). Similar experiments were also performed with
polarized MDCK-II cells with TER in the range of 100 to 200 cm2. In these cells, the apical surface exhibited a 2- to 3-fold higher TF/VIIa activity than the basolateral surface (data
not shown).
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| Fig 1.
Expression of cell-surface TF activity and antigen on
polarized epithelial cells. (A) TF activity: The apical and basolateral
surface domains of Caco-2 cells were incubated with VIIa (10 nmol/L)
for 15 minutes at 37°C and TF/VIIa functional activity on each side
was measured as the rate of Xa generation in the presence of 175 nmol/L
factor X. Data are the mean ± SD of 4 independent experiments
performed in duplicates. (B) Binding of 125I-VIIa to
cell-surface TF on polarized Caco-2 cells. Both compartments of
polarized Caco-2 cells were incubated with 10 nmol/L of
125I-VIIa in the presence or absence of polyclonal
anti-human TF IgG (100 µg/mL). VIIa bound to the apical ( ) and
basolateral ( ) surface was eluted by EDTA wash. The specific binding
to TF was determined as described in Materials and Methods. Data are
the mean ± SD of 5 independent experiments in duplicates. (C)
Specific functional activity of TF/VIIa complexes. Specific functional
activity of TF/VIIa complexes was calculated by relating the surface
TF/VIIa activity with the number of VIIa molecules bound to
cell-surface TF sites. Data are the mean ± SD of 4 independent
experiments in duplicates.
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Determination of TF/VIIa complex formation on polarized epithelial
cells.
To determine the number of TF/VIIa complexes formed on the apical and
basolateral surface of Caco-2 cells, we measured 125I-VIIa
binding to both sides of the polarized cell monolayers. Polarized
epithelial cells in transwells were exposed to
125I-VIIa (10 nmol/L) in the presence and absence of
anti-human TF IgG, and TF-specific VIIa binding was determined as
described in Materials and Methods. As shown in Fig 1B, no significant
difference was found in Caco-2 cells between the number of TF/VIIa
complexes formed on the apical side and the number of TF/VIIa complexes formed on the basolateral surface. 125I-VIIa binding
studies performed with MDCK-II cells showed that 2-fold more
125I-VIIa was bound to the basolateral surface compared
with the apical surface (data not shown).
The combined data of TF/VIIa activity and TF/VIIa complexes formed on
the two cell-surface domains (see Fig 1C) provide strong evidence for
the discordant expression of TF and the TF/VIIa functional activity in
polarized epithelial cells. These data establish that TF/VIIa complexes
formed on the basolateral surface of epithelial cells were less
functionally active than TF/VIIa complexes formed on the apical surface.
Localization of TF by confocal immunofluorescence microscopy.
Confocal microscopy imaging of polarized Caco-2 cells showed TF
expression on the apical as well as on the basolateral plasma membrane
domain (Fig 2). Different levels of TF
expression of individual cells within the monolayer combined with
varying heights of the individual cells caused the TF staining on the
apical membrane domain to appear heterogenous (Fig 2A and B). The
basolateral TF had a reticular pattern of staining (Fig 2C)
characteristic for basolateral antigens. The relatively strong lateral
signal is most likely caused by interdigitated lateral plasma
membranes. No TF staining was seen on the basal plasma membrane.
Although the data from confocal microscopy do not allow quantitative
comparisons of TF expression, they clearly showed TF antigen
localization on both apical and basolateral membrane domains. Very
little intracellular labeling was seen in permeabilized cells. Two
different sources of polyclonal rabbit anti-human TF IgG antibodies
gave the same results. Controls, including omission of primary
antibodies or use of preimmune IgG, showed minimal staining.
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| Fig 2.
TF localization on polarized Caco-2 cells. Cells were
processed for indirect immunofluorescence with polyclonal rabbit
anti-human TF IgG antibody followed by FITC-conjugated goat anti-rabbit
antibody. Images were acquired by confocal microscopy as described in
Materials and Methods. Cells were sectioned in the X-Y plane from the
apical to basal membrane surfaces at 0.5-µm increments. Panels (A)
through (G) represent nonpermeabilized cells. Panels (A) and (B)
represent single focal X-Y planes at 2-µm increments around the
apical membrane domain. Panels (C) and (D) represent single focal X-Y
planes at 2-µm increments through the center of the cells. Panel (E)
represents the basal membrane. Panels (F) and (G) show representative
X-Z confocal views of nonpermeabilized cells. In panels (H) through
(J), cells were permeabilized with 0.2% saponin. Panel (H) represents
TF-specific staining of the apical membrane domain. Panel (I)
represents mainly basolateral TF staining, 6 µm from plane in panel
(H). Panel (J) represents TF staining of the basal membrane, 14 µm
from plane in panel (H). Bars, 10 µm.
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TFPI expression on polarized Caco-2 epithelial cells.
A polarized expression of TFPI, the primary inhibitor of the TF/VIIa
complex, might explain the apparent discrepancy between expression of
TF/VIIa functional activity on the apical and basolateral surface
domains. To test this possibility, we examined the expression of TFPI
on polarized cells. After removal of the media and washing, polarized
epithelial cell monolayers on transwell membranes were treated with low
pH buffer (0.1 mol/L glycine, pH 3.0) for 3 minutes to elute
cell-surface-bound TFPI. The low pH eluate was assayed for TFPI
activity in a TF/VIIa inhibition assay. We found small but similar
amounts of TFPI in eluates derived from the apical and basolateral
sides of Caco-2 cells (Fig 3A). These
results suggest that Caco-2 cells synthesize low levels of TFPI and
that the TFPI was evenly distributed between the apical and basolateral surface domains.
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| Fig 3.
TFPI expression on polarized Caco-2 epithelial cells. (A)
Cell-surface-associated TFPI: The apical and basolateral cell surfaces
were treated with 500 µL 0.1 mol/L glycine pH 3.0 for 3 minutes to
remove cell-surface-associated TFPI. Eluates were removed from the
dish and pH was adjusted to 7.8 with 1 mol/L Tris and assayed for TFPI
activity. Data are the mean ± SD of 4 independent experiments. (B)
Effect of anti-TFPI on cell-surface TF/VIIa functional activity: Caco-2
cells grown in transwells were treated with control IgG (circles) or
with polyclonal anti-TFPI antibodies (100 µg/mL) (squares) for 30 to
60 minutes on both the apical (open symbols) and basolateral (closed
symbols) surfaces, and a Xa generation assay was performed as described
in the legend to Fig 1. Data are the mean ± SD of 5 independent
experiments.
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To further substantiate this conclusion, we tested the effect of
inhibitory polyclonal anti-TFPI IgG on the expression of TF/VIIa
functional activity on the apical and basolateral surface domains. Both
surfaces of the polarized cells were preincubated with anti-TFPI IgG
(or control IgG) for 60 minutes before they were used in determining
cell-surface TF/VIIa functional activity. The results (Fig 3B) showed
that addition of anti-TFPI IgG moderately enhanced the expression of
TF/VIIa proteolytical activity on both the apical and basolateral
surface. It is important to note that the addition of anti-TFPI IgG
enhanced TF/VIIa activity to the same extent on both surfaces and the
TF/VIIa functional activity on the basolateral surface was still
substantially lower than the TF/VIIa activity on the apical surface.
These data indicate that differential expression of TF/VIIa activity
could not be abolished by neutralization of TFPI inhibitory activity.
Expression of anionic phospholipids in polarized epithelial cells.
Because the availability of anionic phospholipids on the outer leaflet
of the cell membrane could regulate TF/VIIa functional activity,1 we evaluated the expression of anionic
phospholipids on polarized epithelial cells. We used 2 different
methods for assessment of anionic phospholipid expression on the outer
leaflet of the apical and basolateral surface domains in Caco-2 cells: (1) ability of the cell surface to support prothrombin activation in
the absence of exogenously added phospholipids, and (2) binding of
radiolabeled annexin V to the cell surface.
The initial rate of prothrombin activation on the basal surface was
3-fold lower than the initial rate observed on the apical surface
(Fig 4A), suggesting a lower availability
of anionic phospholipids on the basolateral surface. This observation
was further supported by the annexin V binding data in Fig 4B, which
showed a 3-fold lower annexin V binding to the basolateral surface
compared with the apical surface (190 ± 10 fmol/well on the
basolateral surface v 640 ± 70 fmol/well on the apical
surface). A lower availability of anionic phospholipids on the
basolateral surface compared with the apical surface was also observed
in MDCK-II cells. These cells exhibited a 6- to 8-fold lower
prothrombinase activity on the basolateral surface over the apical
surface (data not shown).
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| Fig 4.
Evaluation of anionic phospholipid availability by
thrombin generation and annexin V binding on the surface of polarized
Caco-2 epithelial cells. (A) Thrombin generation: Polarized cells in
transwells were incubated with factor Va (20 nmol/L) and Xa (2 nmol/L)
on both the apical and basolateral side for 15 minutes at 37°C
before prothrombin (1,400 nmol/L) was added. At various time points,
20-µL aliquots were removed from the apical or the basolateral side
and assayed for thrombin generated. Data are presented as initial rates
of thrombin generation (n = 4). (B) Annexin V binding: Polarized
Caco-2 cell layers were incubated with 10 nmol/L of
125I-annexin V on the apical and basolateral side at
4°C. Cell-surface-associated radioactivity was removed from the
apical () and basolateral () surfaces by washing with an
EDTA-containing buffer. Nonspecific binding was determined in parallel
duplicate wells in which the cells were preincubated for 30 minutes at
4°C with 50-fold molar excess of cold annexin V (500 nmol/L) before
adding 125I-annexin V (n = 3).
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Effect of mild acid wash on expression of TF/VIIa functional activity
and anionic phospholipids in polarized Caco-2 cells.
Polarized epithelial cells were treated with low pH buffer to elute
cell bound TFPI (and possibly other extracellular components bound to
the cell surface), and the formation of functionally active TF/VIIa
complexes on the cell membrane was subsequently measured. Mild acid
wash of cells slightly increased (1.4-fold) the TF/VIIa activity on the
apical surface. However, the same acid treatment markedly enhanced
(more than 5-fold) the expression of TF/VIIa functional activity on the
basolateral surface, thus abolishing the difference in TF/VIIa activity
between the basolateral and apical surface domains
(Fig 5).
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| Fig 5.
Effect of mild acid wash on TF/VIIa functional activity
on polarized Caco-2 cells. Caco-2 cells in transwells were treated for
3 minutes with a mild acid wash (0.1 mol/L glycine, pH 3.0) or with
buffer B (control). Cells were washed 3 times with buffer B after the
acid treatment and then assayed for TF/VIIa activity as described in
the legend to Fig 1. (Note: Polarized cell layers were tested for their
integrity after the assay by measuring TER and flow of iodine labeled
tracer across the cell layer. There was no loss of integrity after the
mild acid wash.) Data are the mean ± SD of 3 independent experiments
each in duplicates. Rate of Xa generation measured on the apical
surface was taken as 100%.
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To test whether the increased expression of TF/VIIa functional activity
on the basolateral surface after the mild acid wash is caused by an
increase in number of TF/VIIa complexes formed on the cell surface, we
determined the effect of mild acid wash on the number of TF/VIIa
complexes formed on the membrane by binding studies with radiolabeled
VIIa. The mild acid wash of cells did not increase TF-specific VIIa
binding to the either apical or basolateral cell surface (data not shown).
Next we examined the effect of the mild acid wash on the expression of
anionic phospholipids measured by annexin V binding to the cell surface
and by the prothrombinase activity on the cell surface. The annexin V
binding to the basolateral surface was increased by 6-fold after the
mild acid wash (190 ± 10 fmol/well in untreated cells to 1,120 ± 80 fmol/well in mild acid-treated cells) whereas only a 2-fold
increase in annexin V binding was observed on the apical surface after
the acid wash (640 ± 70 fmol/well in untreated cells to 1,220 ± 80 fmol/well acid-washed cells) (Fig 6A).
Similar results were also obtained for prothrombinase activity assay
(Fig 6B). The rate of thrombin generation was significantly increased
(3-fold) on the basolateral surface after mild acid wash whereas a
modest increase in rate of thrombin generation (1.5-fold) was observed
on the apical surface (Fig 6B). After mild acid treatment we did not,
however, obtain exactly the same rate of thrombin generation on the
apical as on the basolateral surface, as might be expected from the
annexin V binding data. The reason for this discrepancy is unknown, but
one could speculate that factors additional to anionic phospholipids
might affect the prothrombinase activity on cell surfaces.
View larger version (24K):
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| Fig 6.
Effect of mild acid wash on annexin V binding and
thrombin generation on polarized Caco-2 cells. (A) Annexin V binding:
The cells were treated with mild acid as described in the legend to Fig
5. Cells were then incubated with 10 nmol/L of 125I-annexin
V on the apical and basolateral side at 4°C. Total cell-associated
radioactivity was removed from the apical and basolateral surfaces by
washing with an EDTA-containing buffer. Nonspecific binding was
determined in parallel duplicate wells in which the cells were
preincubated for 30 minutes at 4°C with a 50-fold excess of cold
annexin V (500 nmol/L) before adding 125I-annexin V. Specific binding (total-nonspecific) on the apical () and
basolateral () surfaces are presented (n = 4). (B) Thrombin
generation: The polarized epithelial Caco-2 cells in transwells
were subjected to a mild acid wash (0.1 mol/L glycine, pH 3.0) or to
buffer B (control) on the apical or basolateral for 3 minutes. After
washing, the cells were incubated with factor Va (20 nmol/L) and factor
Xa (2 nmol/L) on both the apical and basolateral side for 15 minutes at
37°C before prothrombin (1,400 nmol/L) was added. At various time
points, 20-µL samples were removed from the apical (open symbols) or
the basolateral (closed symbols) side and assayed for the amount of
thrombin generated (n = 4).
|
|
| |
DISCUSSION |
Many cell types that do not normally come in contact with blood, such
as epithelial cells lining the body cavities, constitutively express
TF.4 Several types of epithelial cells form tight junctions separating the apical cell surface from the basolateral cell surface. Limited information is available on how TF is distributed and regulated
in such polarized cells. In the present study, using epithelial cells
grown on permeable filters where they polarize and form tight
junctions, we investigated the expression and regulation of TF on the
apical and basolateral cell-surface domains.
Binding studies with radiolabeled VIIa showed that TF antigen was
evenly distributed between the apical and basolateral membrane domains
of the polarized epithelial Caco-2 cells. Confocal microscopy also
showed both apical and basolateral localization of TF receptors. However, measurement of functional TF/VIIa activity revealed a highly
asymmetric distribution of the TF/VIIa activity, suggesting that
complexes formed on the basolateral membrane surface were much less
active than TF/VIIa complexes on the apical membrane surface. The
discordant expression of TF and its activity observed on the surface of
Caco-2 cells confirmed an earlier observation by Camerer et
al9 on polarized MDCK-I cells. Our data with Caco-2 cells
are, however, somewhat at variance with data reported by these
investigators9 showing that 88% to 94% of surface TF was
localized on the basolateral side of MDCK-I cells. We found an equal
distribution of TF antigen between the apical and basolateral surface
domains of Caco-2 cells. However, we should point out that we also
found a basolateral predominance (70%) of canine TF antigen in MDCK-II
cells. Camerer et al9 further reported that 73% to 83% of
surface TF antigen on endothelial cells was exposed on the apical
surface. The observed variance in TF antigen exposure between different
cell types may partly rely on differences in TF antigen sorting in
different cell types. In the absence of any known active sorting and
endocytosis signal sequence in the cytoplasmic domain, it is possible
that TF is subject to sorting by default pathways, which appear to
differ between different cell types.19
In our present studies, most TF activity was found to be associated
with the apical membrane surface of polarized epithelial cells. This is
in accordance with earlier observations on both endothelial and
epithelial cells,7,9 when the cells were grown on permeable
supports and shown to establish tight junctions and polarized cell
monolayers. Other reports6,8 on endothelial cell monolayers
which suggested a different localization of TF activity were performed
with cells grown on solid supports where the integrity of the cell
monolayer might be questionable, and such monolayers may therefore not
provide the best experimental model for studies of in vivo
endothelial cell features especially when it comes to proteins with a
polarized distribution.20
The discordant expression of TF and its activity observed on the
surface of Caco-2 cells suggests that most of the TF/VIIa complexes
formed on the basolateral surface were nonfunctional. These data extend
earlier observations made with other cell types which showed that the
majority of TF/VIIa complexes formed on the cell surface were
nonfunctional or cryptic.17,21-26 A number of mechanisms
have been proposed to account for the presence of nonfunctional TF/VIIa
complexes. It has been postulated that a limited availability of
anionic phospholipids on the outer leaflet of the cell membrane bilayer
could limit the number of physiologically functional TF/VIIa complexes
that could be formed.17,21,23 Camerer et
al9 raised the possibility that a polar distribution of
TF/VIIa inhibitors, such as TFPI, could account for the formation of
nonfunctional TF/VIIa complexes on the basolateral surface membrane
domain of epithelial cells. It has also been speculated that
localization of TF to specialized plasma membrane microdomains, such as
caveolae, might limit the function of TF.24 However, this
later explanation is unlikely to account for the present findings in
Caco-2 cells because these cells do not express caveolin-1 and are
devoid of invaginated caveolar structures.16 A putative model for TF encryption has recently been proposed by Bach and Moldow.25 They suggested that TF exists as a dimer which
binds VIIa but is unable to activate factor X, and that calcium influx into the cytosol (eg, induced by Ca2+ ionophores) triggers
a calmodulin-dependent process that causes the dimers to dissociate
into monomers capable of factor X activation.25,26 Because
there were no specific data in the present study that could exclude
this possibility, we cannot rule out the possibility that higher
TF/VIIa functional activity observed on the apical surface of polarized
cells could be due to the presence of a higher ratio of monomer TF on
the apical surface compared with the basolateral surface.
TFPI is the primary inhibitor of the TF/VIIa functional activity. It is
possible that an asymmetric distribution of TFPI, ie, sorting
preferentially to the basolateral membrane, could limit the functional
activity of TF/VIIa complexes formed on this surface of Caco-2 cells.
Immunofluorescence studies of polar MDCK-I cell layers showed that TFPI
was essentially localized on the basolateral surface.9
However, our quantitative measurements of cell-bound TFPI and the
analysis of the effect of anti-TFPI on TF/VIIa functional activity
showed that a low level of TFPI is expressed in Caco-2 cells, where it
is evenly distributed between the apical and basolateral cell-surface
domains. Thus, it is unlikely that cell-bound TFPI plays a role in the
discordant expression of TF and its activity observed in Caco-2 cells.
The proteolytic activity of TF/VIIa complexes formed in suspension with
lipidated purified TF depends strongly on the presence of anionic
phospholipids, such as phosphatidylserine (PS).1,23,27 This
is also known to be the case for the procoagulant activity of TF on
cellular membranes. Bach and Rifkin23 reported that treatment of pericytes with calcium ionophore, which enhances the
expression of PS on the outer leaflet of cell membrane bilayer, enhanced the cell-surface TF/VIIa activation of factor X. Further, Le
et al21 showed that treatment of fibroblasts with
N-ethylmaleimide led to a 3- to 4-fold increase in the population of
functional TF/VIIa complexes on the intact cells, and that this was
accompanied by a substantial increase in the amount of anionic
phospholipids present on the outer layer of the cell membrane. The
increased TF/VIIa activity has also been observed on the surface of
apoptotic cells, which typically expose elevated levels of PS in the
exoplasmic leaflet.28,29 Our studies with polarized Caco-2
cells showed that the amount of anionic phospholipids on the
basolateral surface was 3-fold lower than the amount of anionic
phospholipids on the apical surface. Similar studies on polarized
MDCK-II cells showed an 8-fold higher prothrombinase activity on the
apical membrane domain compared to the basolateral domain. Thus, it is
likely that differences in content of anionic phospholipids between the apical and the basolateral surface are a general phenomenon of polarized cells that could account for the observed difference in the
functional state of TF/VIIa complexes formed on apical and basolateral
surface domains.
The above conclusion is further supported by the data obtained from
experiments in which polarized epithelial cells were mild acid-washed
and then analyzed for expression of TF/VIIa functional activity and
anionic phospholipid content. Our data showed that the mild acid
treatment markedly increased the expression of TF/VIIa functional
activity on the basolateral surface without affecting formation of
TF/VIIa complexes. The increase in TF/VIIa activity on the basolateral
cell surface was associated with an increase in the availability of
anionic phospholipids on the basolateral cell membrane. This change in
anionic phospholipid availability could be due to an increase in PS
exposure on the outer leaflet of the membrane. Alternatively, the mild
acid treatment could have led to dissociation of cell-membrane
components that influence accessibility to anionic phospholipids. It is
also possible that the mild acid wash might have removed or altered an
unknown cell-surface component that plays a role, by a different
mechanism, in regulating the expression of TF/VIIa functional activity
on the cell surface.
Overall, the cumulative data of the present study strongly support the
hypothesis that limited availability of anionic phospholipids on the
basolateral surface of polarized epithelial cells is responsible for
the nonfunctional property of TF/VIIa complexes formed on this surface domain.
| |
ACKNOWLEDGMENT |
We thank Dr Jan Stagsted (Institute of Medical Biochemistry, University
of Aarhus, Aarhus, Denmark) for his advice and support during the course of this work. Dr Usha Pendurthi (UT Health Center at
Tyler, TX) is greatly acknowledged for discussions and helpful suggestions. We also thank Todd Williams (UT Health Center at Tyler,
TX) for his technical assistance, and Dr Fritz von Bülow (University of Copenhagen, Copenhagen, Denmark) for help with the
confocal microscope.
| |
FOOTNOTES |
Submitted November 2, 1998; accepted April 26, 1999.
Supported in parts by Grant No. HL 58869 from the National Heart Lung
and Blood Institute. Supported in part by the The Danish Academy of
Technical Sciences (ATV) (to C.B.H.).
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.
Address reprint requests to Carsten B. Hansen, PhD,
Department of TF/VIIa Research, Novo Nordisk A/S, Novo Nordisk Park,
2760 Maalov, Denmark; e-mail: CBHa{at}novo.dk.
| |
REFERENCES |
1.
Rapaport SI, Rao LVM:
The tissue factor pathway: How it has become a "prima ballerina."
Thromb Haemost
74:7, 1995[Medline]
[Order article via Infotrieve]
2.
Broze GJ Jr, Girard TJ, Novotny WF:
Regulation of coagulation by a multivalent kunitz-type inhibitor.
Biochemistry
29:7540, 1990
3.
Drake T, Morrissey J, Edgington T:
Selective cellular expression of tissue factor in human tissues. Implications for disorders of hemostasis and thrombosis.
Am J Pathol
134:1087, 1989[Abstract]
4.
Fleck RA, Rao LVM, Rapaport SI, Varki N:
Localization of human tissue factor antigen by immunostaining with monospecific, polyclonal anti-human tissue factor antibody.
Thromb Res
59:421, 1990[Medline]
[Order article via Infotrieve]
5.
Camerer E, Kolsto AB, Prydz H:
Cell biology of tissue factor, the principal initiator of blood coagulation.
Thromb Res
81:1, 1996[Medline]
[Order article via Infotrieve]
6.
Ryan J, Brett J, Tijburg P, Bach RR, Kisiel W, Stern D:
Tumor necrosis factor-induced endothelial tissue factor is associated with subendothelial matrix vesicles but is not expressed on the apical surface.
Blood
80:966, 1992[Abstract/Free Full Text]
7.
Narahara N, Enden T, Wiiger M, Prydz H:
Polar expression of tissue factor in human umbilical vein endothelial cells.
Arterioscler Thromb
14:1815, 1994[Abstract/Free Full Text]
8.
Mulder AB, Hegge-Paping KS, Magielse CP, Blom NR, Smit JW, van der Meer J, Hallie MR, Bom VJ:
Tumor necrosis factor alpha-induced endothelial tissue factor is located on the cell surface rather than in the subendothelial matrix.
Blood
84:1559, 1994[Abstract/Free Full Text]
9.
Camerer E, Pringle S, Skartlien AH, Wiiger M, Prydz K, Kolsto AB, Prydz H:
Opposite sorting of tissue factor in human umbilical vein endothelial-cells and madin-darby canine kidney epithelial-cells.
Blood
88:1339, 1996[Abstract/Free Full Text]
10.
Thim L, Bjoern S, Christensen M, Nicolaisen EM, Lund-Hansen T, Pedersen AH, Hedner U:
Amino acid sequence and posttranslational modifications of human factor VII(a) from plasma and transfected baby hamster kidney cells.
Biochemistry
27:7785, 1988[Medline]
[Order article via Infotrieve]
11.
Rao LVM, Robinson T, Hoang AD:
Factor VIIa/tissue factor-catalyzed activation of factors IX and X on a cell surface and in suspension.
Thromb Haemost
67:654, 1992[Medline]
[Order article via Infotrieve]
12.
Bajaj SP, Rapaport SI, Prodanos C:
A simplified procedure for purification of human prothrombin factor-IX and factor-X.
Prep Biochem
11:397, 1981[Medline]
[Order article via Infotrieve]
13.
Rao LVM:
Characterization of anti-tissue factor antibody and its use in immunoaffinity purification of human tissue factor.
Thromb Res
51:373, 1988[Medline]
[Order article via Infotrieve]
14.
Rao LVM, Williams T, Rapaport SI:
Studies of the activation of factor VII bound to tissue factor.
Blood
87:3738, 1996[Abstract/Free Full Text]
15.
Pedersen AH, Nordfang O, Norris F, Wiberg FC, Christensen PM, Moeller KB, Meidahl-Pedersen J, Beck TC, Norris K, Hedner U:
Recombinant human extrinsic pathway inhibitor production isolation and characterization of its inhibitory activity on tissue factor-initiated coagulation reactions.
J Biol Chem
265:16786, 1990[Abstract/Free Full Text]
16.
Vogel U, Sandvig K, van Deurs B:
Expression of caveolin-1 and polarized formation of invaginated caveolae in Caco-2 and MDCK II cells.
J Cell Sci
111:825, 1998[Abstract]
17.
Le DT, Rapaport SI, Rao LVM:
Relations between factor VIIa binding and expression of factor VIIa/tissue factor catalytic activity on cell surfaces.
J Biol Chem
267:15447, 1992[Abstract/Free Full Text]
18.
Rao LVM, Tait JF, Hoang AD:
Binding of annexin V to a human ovarian carcinoma cell line (OC-2008). Contrasting effects of cell surface factor VIIa/tissue factor activity and prothrombinase activity.
Thromb Res
67:517, 1992[Medline]
[Order article via Infotrieve]
19.
Keller P, Simons K:
Post-golgi biosynthetic trafficking.
J Cell Sci
110:3001, 1997[Abstract]
20.
Langeler EG, van Hinsbergh VW:
Characterization of an in vitro model to study the permeability of human arterial endothelial cell monolayers.
Thromb Haemost
60:240, 1988[Medline]
[Order article via Infotrieve]
21.
Le DT, Rapaport SI, Rao LVM:
Studies of the mechanism for enhanced cell surface factor VIIa/tissue factor activation of factor X on fibroblast monolayers after their exposure to N-ethylmaleimide.
Thromb Haemost
72:848, 1994[Medline]
[Order article via Infotrieve]
22.
Drake TA, Ruf W, Morrissey JH, Edgington TS:
Functional tissue factor is entirely cell surface expressed on lipopolysaccharide-stimulated human blood monocytes and a constitutively tissue factor-producing neoplastic cell line.
J Cell Biol
109:389, 1989[Abstract/Free Full Text]
23.
Bach RR, Rifkin DB:
Expression of tissue factor procoagulant activity: Regulation by cytosolic calcium.
Proc Natl Acad Sci USA
87:6995, 1990[Abstract/Free Full Text]
24.
Mulder AB, Smit JW, Bom VJJ, Blom NR, Ruiters MHJ, Halie MR, van der Meer J:
Association of smooth-muscle cell tissue factor with caveolae.
Blood
88:1306, 1996[Abstract/Free Full Text]
25.
Bach RR, Moldow CF:
Mechanism of tissue factor activation on HL-60 cells.
Blood
89:3270, 1997[Abstract/Free Full Text]
26.
Bach RR:
Mechanism of tissue factor activation on cells.
Blood Coagul Fibrinolysis
9:S37, 1998
27.
Krishnaswamy S, Field KA, Edgington TS, Morrissey JH, Mann KG:
Role of the membrane surface in the activation of human coagulation factor X.
J Biol Chem
267:26110, 1992[Abstract/Free Full Text]
28.
Greeno EW, Bach RR, Moldow CF:
Apoptosis is associated with increased cell surface tissue factor procoagulant activity.
Lab Invest
75:281, 1996[Medline]
[Order article via Infotrieve]
29.
Bombeli T, Karsan A, Tait JF, Harlan JM:
Apoptotic vascular endothelial cells become procoagulant.
Blood
89:2429, 1997[Abstract/Free Full Text]
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ABSTRACT
INTRODUCTION