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Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1657-1664
By
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.
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.
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- 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.
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.
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 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).
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
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).
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.
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.
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.
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).
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.
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.
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.
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TOP
ABSTRACT
INTRODUCTION
(TNF-
-stimulated
endothelial cells was almost exclusively located on the apical surface,
whereas little activity was found on the basolateral surface. Camerer
et al
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 
) 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.
