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Refractory Period Phenomenon in the Induction of Tissue Factor Expression on
Endothelial Cells
By Nathalie Busso, Stbphane Huet, Edwige Nicodeme, Jacques Hiernaux, and FranGois Hyafil
Tissue factor (TF) is the first factor of the extrinsic pathway of
coagulation. Normally, TF is not expressed on the surface of
endothelial cells. However, expression of TF can be induced
in these cells in response to stimulation by diverse inflammatory mediators such as interleukin-1p (IL-lp), tumor necrosis
factor-a (TNF-a), lipopolysaccharide (LPS), and phorbol 12myristate 13-acetate (PMA). We have studied the effect of
these mediators on the kinetics of the induction of TF-related
procoagulantactivity (PCA) on human umbilicalvein endothelial cells (HUVECs). PCA is transiently induced on HUVECs,
attaining a peak some 4 t o 8 hours after addition of inflammatory agents, with maximal accumulation of TF messenger
RNA (mRNA) occurring 3 to 5 hours earlier. Because the
expression of PCA by treated HUVECs returns t o basal levels
by 20 t o 30 hours, we examinedthe response of these cells t o
a second inflammatory stimulus. Continuous incubation of
cells with a single inflammatory agent for 24 to 48 hours
induces a hyporesponsive state with respect to the reinduction of TF expression by the same agent (14% of the initial
stimulation for IL-1p, 39% for TNF-a, 30% for LPS, and 7% for
PMA). Such a diminution in PCA was also observed in the
levels of TF mRNA. By contrast, pretreatment of HUVECs
with one agent did not dramatically affect the reinduction of
TF by any of the three other factors. We subsequently
focused our attention on the induction of the autologous
refractory period by IL-1p. De novo protein synthesiswas not
required during the preincubationof ECs for hyporesponsiveness to be observed. The establishment of the refractory
state did not depend on the downmodulation of IL-lp
receptor affinity or expression. Moreover, pretreatment of
HUVECs with IL-1p increased prostacyclin (PGI,) production
in response t o a second stimulation by IL-lp, although such
cells were unable t o reexpress TF under the same conditions.
This result suggests that distinct secondary messenger pathways are involved in TF induction and PGI, synthesis by IL-1p
in HUVECs.
Q 1991by The American Society of Hematology.
T
thrombogenesis, we evaluated the effects of inflammatory
mediators known to induce T F activity in ECs, such as
TNF-a, LPS, or PMA; in addition, such mediators present a
refractory period phenomenon with respect to the reinduction of TF activity. To identify common pathways in TF
induction, we studied the reinduction of TF activity by one
inflammatory agent on cells pretreated by an alternative
agent. Finally, we examined the mechanisms involved in the
IL-1p-induced refractory period.
ISSUE FACTOR (TF), a membrane-bound glycoprotein, initiates the extrinsic pathway of blood coagulation by serving as the receptor and essential cofactor for
factor VI1 and VIIa. Once formed, the factor VIIa-TF
complex activates factor IX and X, leading ultimately to
thrombin generation and fibrin accumulation.’.’ TF is constitutively expressed at different cellular sites that are not in
contact with blood.’ By contrast, TF is not usually expressed
by cells within the vasculature. However, both monocytes
and endothelial cells (ECs), two cell types that are in direct
contact with the blood, may be stimulated to express TF by
various inflammatory agents. On the basis of these observations, it has been proposed that constitutively expressed TF,
whose contact with blood is limited to events following
trauma and tissue destruction, is involved in hemostasis; by
contrast, T F induced on monocytes and ECs may be critical
in the thrombogenic states associated with different patholo g i e ~ . ~TF
. ~ activity can be induced in ECs by immune
complexes,’otumor-promoting phorbol esters such as phorbo1 12-myristate 13-acetate (PMA),” lipopolysaccharide
(LPS),’* thrombin,’’ tumor necrosis factor-a (TNF-a),’4.15
and interleukin-lp (IL-lp).I6 The regulatory mechanisms
controlling the induction of TF expression in ECs treated
with LPS, TNF-a, and PMA operate at both transcriptional
and posttranscriptional
One characteristic feature of T F activity on monocytes
and ECs is its transient induction, a peak occurring at 4 to 8
hours, followed by a return to basal levels after 24 hours.
Interestingly, it has been reported that human umbilical
vein ECs (HUVECs) that had been pretreated for 24 hours
with IL-lp became refractory to the reinduction of TF by a
second IL-1p stimulation.I6 Such refractory behavior may
have important pathophysiologic implications, because it
may serve to limit the extent of endothelium-related intravascular coagulation in the presence of an inflammatory
agent. However, the mechanisms underlying this insensitivity to further stimulation are poorly understood. To shed
new light on the mechanisms underlying the role of TF in
Blood, Vol78, No 8 (October 15). 1991: pp 2027-2035
MATERIAL AND METHODS
Reagents. Fibronectin and collagenase type I were from Sigma
(La VerpilliBre, France). Human platelet-free plasma and serum
were purchased from the Centre National de Transfusion Sanguine
(Les Ulis, France). Cell culture media were obtained from GIBCO
(Cergy-Pontoise, France), LPS from Escherichia coli serotype
0128:B12, and rabbit brain thromboplastin from Sigma. Goat
antiserum against human TF was generously provided by Dr J.H.
Morrissey (Oklahoma Medical Research Foundation, Oklahoma
City, OK). Limulus assay was from Kabivitrum (Stockholm, Sweden). Recombinant human TNF-a(specific activity, 2 x lo7Ulmg)
was supplied by Genzyme (Boston, MA) and recombinant human
IL-1p (specific activity, 2.2 x lo7Ulmg) by the Glaxo Institute for
Molecular Biology (Geneva, Switzerland). All radiolabeled compounds and Hybond N nylon membranes were from Amersham
(Les Ulis, France). 6-keto-prostaglandin F,, (6-keto PGF,,) was
from Sigma and 6-keto PGF,, antiserum from Biosys (Compiegne,
From Laboratoires GLAXO, Centre de Recherches, Les Ulis,
France.
Submitted February 12,1991; accepted June 5, 1991.
Address reprint requests to Nathalie Busso, PhD, Laboratoires
GLAXO, 25 avenue du Qu’bec, 91951 Les UIis Cede+ France.
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.
0 1991 by The American Society of Hematology.
0006-4971191 I 7808-0029$3.00/0
2027
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2028
France). All the other chemicals were of the best commercial grade
available.
EC (HUVEC) cultures. ECs were isolated from human umbilical cords within 48 hours of delivery, according to a modification of
the method of Jaffe et al.” The umbilical veins were rinsed with
phosphate-buffered saline (PBS) without calcium and magnesium,
infused with 0.1% type 1 collagenase, and subsequently clamped
and incubated at 37°C for 7 minutes. The vein was rinsed twice with
culture media (RPMI 50%/M199 50%) containing 20% human
serum (HS) and antibiotics. Cells were recovered by centrifugation
and resuspended in culture media supplemented with 20% HS and
antibiotics; such cells were plated in 25-cm2 tissue culture flasks
precoated with purified human fibronectin (1 to 5 pg/cm2). When
cellular monolayers were confluent, cells from different cords
(three to seven) were trypsinized and pooled. For experiments,
second passage cells (HUVECs-P2) were subcultured into fibronectin-coated 96-well plates for the study of TF activity or into 75-cm2
tissue culture flasks for RNA extractions. The cells were characterized by their cobblestone appearance in monolayers and by positive
indirect immunofluorescence for detection of von Willebrand
factor (vWF). All tested agents were added onto confluent cell
monolayers in serum-free medium or in culture medium supplemented with serum. Cell viability was assessed by visual inspection
and confirmed in some experiments by vital staining of the cells and
cell counting.
TF assay. Procoagulant activity (PCA) was measured in a
single-stage clotting assay performed by robot (Biomek; Beckman,
Gagny, France). Briefly, HUVEC-P2 were grown to confluency in
96-well microplates and subsequently incubated with the agents to
be tested. They were then rinsed twice with calcium-free PBS and
sequentially frozen and thawed twice in 10 mmol/L Tris-HC1, 1
mmol/L EDTA pH 7.4 (80 kL/well) to lyse the cells. To each well,
80 pL of normal human citrated platelet-free plasma and 80 pL of
30 mmol/L CaCI, were added. Clot formation in the well was
monitored by determination of the optical density at 540 nm at
intervals of 35 seconds. The clotting time was determined for each
well as the time required to reach a fixed optical density. Milliunits
( m u ) of TF were arbitrarily defined by conversion from standard
curves (log-log plot) developed with a preparation of rabbit brain
thromboplastin. PCA was totally TF-dependent as assessed by use
of factor VII-deficient plasma instead of normal plasma or by
preincubation of cell lysates with anti-TF antibodies.
Prostacyclin (PGZJ assay. Samples of culture media were collected after cell incubation and stored at -20°C until assay of PGI,
was performed. Release of PGI, into the medium was quantitated
by radioimmunoassay (RIA) of its stable breakdown product,
6-keto PGF,,. Briefly, samples were diluted in RIA buffer (0.1
mol/L phosphate buffer, 0.1% gelatin, pH 7.4) and incubated with
15 pg of [3H]-labeled 6-keto PGF,, (157 Ciimmol) and 6-keto
PGF,, antiserum. After 30 minutes at room temperature followed
by an overnight incubation at 4”C, bound and free antigen were
separated by absorption of the free antigen to activated charcoal
(1% charcoal, 0.1% dextran in RIA buffer) for 10 minutes at 4°C.
The absorbed antigen was then separated by centrifugation at 40%
for 15 minutes at 4°C. The radioactivity of the supernatants was
measured in a p counter after addition of scintillation fluid. The
amount of 6-keto PGF,, was calculated from a standard curve
analyzed in parallel.
Dissociation of I L - l p from its receptor. Receptor-bound IL-lP
was removed by brief treatment (1 minute) of HUVEC monolayers
with an acidic buffer (0.15 mol/L NaCI, 50 mmol/L glycine, pH
3.2).
Estimation of protein synthesis. Protein synthesis in HUVECs
was quantified by determination of the incorporation of 3sSmethionine ( > 1,000 Ci/mmol) into trichloroacetic (TCA)-
BUSS0 ET AL
precipitable radioactivity. Briefly, cells grown to confluency in
96-well plates were incubated with 5 pCi/well of %-methionine in
normal medium containing 5% fetal calf serum (FCS), in the
presence or absence of the agents to be studied. After 4 hours, the
cells were washed three times and proteins precipitated by addition
of 15% ice-cold TCA to each well. Wells were then washed three
times and allowed to dry completely. All TCA-precipitated material was solubilized in 1 mol/L NaOH, transferred to vials containing scintillation fluid, and radioactivity counted in a p counter.
TF messenger RNA (“A)
analysis. Total RNA was prepared
from 6 to 12 x lo6 cells using the guanidine thiocyanate/cesium
chloride method, and then electrophoresed and transferred onto
membranes as previously described.” Complementary DNA for
human TF was isolated from a human placental cDNA library in
Xgtll screened with an oligonucleotide probe. A total of 84 positive
clones was obtained. A 830-bp EcoRI fragment (up to the first
EcoRI site of the TF CDNA)’’-~~
was subcloned into pGEMl and
used as a probe. This TF-containing plasmid was radiolabeled to a
specific activity in excess of lo9dpmlpg using dCTP ~’cY.-~’P (3,000
Ci/mmol) by the random priming method (Multiprime DNA
labeling system; Amersham). Hybridization was performed for 3
hours at 65°C with 8 ng/mL of probe in the rapid hybridization
buffer (Amersham). The membranes were sequentially washed in
2X standard saline citrate (SSC), 0.1% sodium dodecyl sulfate
(SDS) (twice for 10 minutes), 1X SSC, 0.1% SDS (once for 15
minutes), 0.7X SSC, 0.1% SDS (once for 15 minutes), and finally in
0.5X SSC, 0.1% SDS (once for 15 minutes). Finally, the membranes were exposed (4 to 16 hours) for autoradiography using
intensifying screens.
RESULTS
Induction of TF activity by injlammatory agents. Unstimulated HUVECs displayed very low levels of PCA in our
standard clotting assay ( < 2 mU/4 x lo4cells). Stimulation
of the cells with IL-1p transiently increased PCA. Such
procoagulant activity was essentially due to TF induction
because this activity was not detectable when factor VIIdeficient plasma was used in the PCA assay. Moreover,
treatment of HUVEC-P2 lysates with antihuman TF antibodies completely prevented the IL-1p-induced PCA and
also blocked the low basal PCA expressed in unstimulated
cells (results not shown).
Kinetic analysis showed that TF activity attained a peak
between 4 to 8 hours and then declined to near basal levels
after 24 hours (Fig 1A). These results are in agreement with
the observations of Bevilacqua et
Short exposure (from
2 to 30 minutes) of HUVECs to 5 U/mL of IL-lp, followed
by washing and incubation of the cells in IL-lp-free
medium, resulted in a significant increase in TF activity by 4
hours (Fig 1B). These findings indicate that, after a short
incubation period, IL-lP is able to transduce a cascade of
intracellular events leading ultimately to increased TF
activity. IL-lp-induced TF activity was dose-dependent,
stimulation was observed with as little as 0.05 U/mL and
was maximal at 5 U/mL (Fig 1C). In addition to the effect
of IL-lp, we examined the effects of other inflammatory
mediators on TF activity. Our results are summarized in
Table 1. We observed that, under our assay conditions,
TNF-a, LPS, and PMA induced TF-related PCA. These
data agree with earlier observations (see for TNF-a, Bevilacqua et all4 and Nawroth and Stern,” for LPS, Colucci et
al,” and for PMA, Lyberg et all’).
-
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2029
TF INDUCTION ON ENDOTHELIAL CELLS
Table 1. Effect of Inflammatory Agents on the TF Activity of HUVECs
Maximal Effect
-
n
cn
16
A
Q)
0
P
0
12
?
3
8
.-c*
.->
4
Y
c
0
Fold Stimulation
IL-lP
TNF-a
LPS
PMA
5-30
5-30
5-30
20-50
Concentration*
5 U/mL
10 nglmL
1 FglmL
10 nmol/L
Time (h)t
4-8
4-6
4-6
4-8
*To determine the maximal effective concentration of different
inflammatory agents on TF activity induction, confluent HUVEC monolayers were incubated for 4 hours in medium containing 20% HS alone
or supplemented with IL-lP, TNF-a, LPS, or PMA at various concentrations.
tThe time at which the effect is maximal is determined by incubating
HUVECs with the indicated agent at its maximal effective concentration
for various periods up to 72 hours.
F
E
Agents
Q
L
I-
"
I
I
I
I
0
12
24
36
48
72
60
period (hours)
IL-'
40
B
30
20
10
L
0
I-
0
2
5
15 30 60 120180240
time to IL-l p (minutes)
h
-cn
Q)
0
P
0
T:
,
.-c,
>
.-
c
0
m
L
I-
4/
0
10
Ail these experiments were performed in the presence of
20% HS. However, comparable results were obtained in the
presence of 5% FCS or with serum-free medium. In
addition, none of the measured TF activities were due to
endotoxin contamination of the incubation media because
less than 12 pg/mL of LPS could be detected in incubation
samples by the Limulus lysate assay.
Effect of inflammatory agents on TF mRNA content.
Unstimulated cells contained undetectable levels of T F
mRNA. Exposure of cells for different times to either IL-1P
or TNF-a or LPS or PMA resulted in the induction of a
major 2.2-kb T F mRNA transcript. Induction of another
minor TF transcript of 3.1 kb was occasionally detected (Fig
2), in agreement with previous ~bservations.~~."
The kinetics of induction were similar for IL-1P and LPS with a peak
at 1 hour, followed by a progressive decline in TF mRNA
levels. Incubation of cells with TNF-a and PMA gave
maximal TF mRNA induction at 2 and 4 hours, respectively. Thus, the transient increase in TF steady-state
mRNA levels was reflected some 3 to 5 hours later in a
transient increase in TF activity, observed after incubation
of HUVECs with inflammatoly mediators. Treatment of
cells with cycloheximide alone (10 p,g/mL) did not increase
TF mRNA level. Cycloheximide, in combination with LPS,
enhanced TF mRNA levels with respect to LPS alone (data
not shown). This may be due to stabilization of TF mRNA.
Observations consistent with this mechanism have been
reported."
Effect of repeated stimulation on TF expression: refractory
state phenomenon. The ability of activated ECs to respond
...J1 0-
10
1 0-
10
IL-1 p (Ulml)
10
10
Fig 1. (A) Time-course of the induction of TF activity by IL-lp in
HUVECs. Cells in triplicate culture wells were incubated in medium
containing 20% HS with 5 U/mL of IL-1s for up t o 72 hours. After the
various incubation periods, cells were washed twice and PCA, characterized as TF-dependent activity, was assayed in freeze-thawed
HUVEC lysates. (B) Effect of brief exposures of HUVEC cultures t o
IL-lp on the induction of TF activity at 4 hours. Cells were exposed for
various times t o 5 U/mL, extensively washed with PES, and reincubated in complete medium lacking IL-1p. After a total incubation
period of 4 hours, TF activity was measured. (C) Dose-dependencyof
the induction of TF activity by IL-lp in HUVECs. Cells were incubated
for 4 hours with various concentrations of IL-1s and TF activity
measured at the end of the incubation. One representative experiment of five independent experiments is shown throughout.
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BUSS0 ET AL
2030
A) IL1
B) TNF
-2.2
C 3 0 1H 2H 4H 8H 24H
C)LPS
kb
C 3 0 1H 2H 4H 8H
D) PMA
CI
-2.2 kb
C 3 0 1H 3H 6H
C 3 0 1 H 2H 4H 8H
to a second stimulation by an inflammatory agent was then
evaluated. Addition of fresh IL-1p for 4 hours to cells that
had been pretreated with IL-lp for 48 hours yielded a
significantly reduced restimulation, suggesting that ECs
had become “refractory” to the stimulatory signal (Fig 3A).
This phenomenon has also been observed by Bevilacqua et
al.”
To determine the concentration of the primary dose
necessary to render cells hyporesponsive, cells were incubated for 48 hours with different concentrations of IL-lp
(from 0.02 U/mL to 5 U/mL), washed, and then restimulated with a fixed dose of IL-lp (1 U/mL) (Fig 3B). The
induction of a refractory state was concentration-dependent, with clear-cut hyporesponsiveness at doses of IL-lp
2 1 U/mL during the first 48 hours of incubation. Therefore, a primary dose from 1 to 5 U/mL of IL-1 p was used in
all subsequent experiments.
The kinetics of recovery from the hyporesponsive state
after removal of the primary IL-lp dose were also determined. After an initial exposure of 48 hours to IL-lp, cells
were incubated for varying lengths of time with medium
alone and then rechallenged with IL-lp for 4 hours. Under
these conditions, cells maintained in medium alone for 24
and 48 hours were still not able to fully respond to a second
IL-lp stimulation (49% and 64%of a primary 4-hour IL-lp
stimulation, respectively; results not shown).
The kinetics of the development of TF hyporesponsiveness were subsequently examined. To observe a refractory
state on IL-lp restimulation at 24 hours, a primary incubation with IL-lp for at least 7 hours, followed by incubation
Fig 2. Effect of inflammatory
agents on TF mRNA content in
HUVECs. Confluent HUVEC
monolayers were incubated in
medium containing20% HS alone
(control cells, C) or supplemented
either with IL-l@ (A, 5 U/mL),
TNF-a (E, 10 ng/mL), LPS (C, 1
pg/mL), or PMA (D, 5 x lo-*
mollL) for the indicated times.
Monolayers were then washed
and total RNA extractedfrom the
cells. Equal amounts of RNA (10
Fg/track) were analyzed by
Northern blot and the filter hybridizedto a UP-labeledTF cDNA
probe.
in IL-1Qfree medium, was required. Maximal hyporesponsiveness was observed after an initial treatment of 24 hours
with IL-1p. Comparable hyporesponsiveness was observed
after 24 or 48 hours of initial treatment with I G l p (data not
shown).
We have examined whether other inflammatory agents
presented a similar refractory phenomenon with respect to
TF expression. Continuous incubation of HUVECs with
LPS for 48 hours led to a state of hyporesponsivenesswhen
the cells were rechallenged (4 hours) with fresh LPS (30%
of the primary stimulation). The same phenomenon was
observed with TNF-a (39% of the primary stimulation).
Among the inflammatory agents tested, PMA induced the
strongest refractory state that we have observed (only 7% of
a primary stimulation; Table 2).
The refractory state at the level of TF mRNA was then
studied. For all the inflammatory agents tested, decreased
TF mRNA steady-state levels were detected in restimulated cells when HUVECs pretreated with a specific factor
were compared with sham pretreated cells (Fig 4). Thus,
the observed refractory state, previously observed at the
level of TF activity, was accounted for by decreased
induction of TF mRNA.
Effect of sequentialstimdations with two different inflammatoty agents on TF expression. The preceding experiments
showed that cells pretreated with an inflammatory agent
“A” became refractory to the same agent with respect to
reinduction of TF activity. We also examined whether this
effect could be observed in response to stimulation with an
agent “B” on cells pretreated with agent “A.” Table 2
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2031
TF INDUCTION ON ENDOTHELIAL CELLS
A
"1
T
12
+
+
+
-
+
-
second incubation (4h)
B
-
-
first incubation (48h)
120,
- -
1
.
0.0
.
,
0.02
-
.
0.1
-
.
0.5
.
,
1
11-18 DOSES WRINQTHE FIRSTW INCUBAWN
.
.
.
5 Ulml
PERKID.
Fig 3. (A) Effect of stimulation with IL-lfl on TF activity: detection
of a refractory state phenomenon. Cells were incubated for 48 hours
with IL-lp (1 U/mL), washed, and then restimulated for 4 hours with
fresh IL-1s (1 U/mL). TF activity was measured in cell lysates at the
end of the second incubation. (B) Dose-dependency of the IL-l&
induced refractory state. Cells were incubated for 48 hours with
increasing concentrations of IL-le, and then restimulated for 4 hours
with 1 U/mL of IL-le. TF activity, measured at the end of the second
incubation, is expressed as a percentage of that detected for the initial
stimulation (mean 2 SD of four separate determinations).
was mediated through a decrease in TF mRNA level (Fig
5).
Study of the mechanisms of IL-I p-induced refractoty state.
One possible mechanism that might account for IL-1pinduced refractory state could arise from occupancy of
IL-1p receptors by the IL-1p added, or by the IL-1p added,
or secreted by ECs during the first incubation period, or
both." The concentration of IL-1p in our experiments was
2.6 to 13 pmol/L (1 to 5 U/mL), whereas the dissociation
constant of IL-1p on HUVECs is 81 pmol/L (N. Smithers,
personal communication, October 1990). Thus, it is unlikely
that the observed refractory state was due to a saturation of
all IL-1p receptor sites. This suggestion was confirmed by
treating HUVECs with an acidic buffer to remove receptorbound IL-1p before a second stimulation. Under these
conditions, HUVECs were unable to synthesize TF on
restimulation with IL-lp (Fig 6).
To assess the possible involvement of a negative feedback
regulation of IL-1p receptor in the induction of hyporesponsiveness, we characterised the expression and the binding
properties of IL-lp receptors in untreated HUVEC cultures and in cultures treated for 24 hours with l U/mL of
IL-1p. Unstimulated and stimulated cells showed no differences in IL-1p receptor mRNA levels (R. Lenstra, personal
communication, July 1990). Moreover, a preliminary experiment involving Scatchard analysis showed that a comparable number of IL-1p receptors with a similar affinity was
present at the surface of both untreated and treated cells,
thereby ruling out modulation of the receptor as a significant mechanism in the induction of the refractory state by
IL-1p (N. Smithers, personal communication, October
1990).
Alternatively, IL-lp-induced hyporesponsiveness could
be due to a nonfunctional IL-1p receptor, which would be
expressed at the same level and with the same affinity as the
functional one. If this was the case, we would then expect
that other biologic functions mediated by the binding of
IL-1p to its receptor would be affected. IL-1p induces PGI,
Table 2. Hyporesponsivenessof HUVECsAfter RestimulationWith
Inflammatory Agents
Restimulation (4 h)
summarizes these experiments. None of the combinations
of inflammatory compounds tested induced a refractory
state comparable with that observed after sequential stimulation with the same agent. Nevertheless, TF induction by
LPS or IL-lp on PMA-pretreated cells was significantly
decreased (up to 40%) with respect to sham-pretreated
cells. Partial unresponsiveness was also observed after
IL-1p stimulation on TNF-a-pretreated cells and conversely after TNF-a stimulation on IL-1p-pretreated cells.
In these cases, the decrease in TF activity was also significant. In addition, PMA stimulation of either TNF-a- or
IL-lp-pretreated cells resulted in a slight but not significant increase in TF induction with respect to untreated
cells.
In the case of the effect of IL-1p on PMA-pretreated
cells, we found that the decreased TF induction (as compared with TF induction by a primary IL-1p stimulation)
Pretreatment (48 h)
None
LPS
TNF-~i
IL-lP
PMA
LPS
TNF-a
IL-lP
PMA
100
100
ND
39 2 16*
6623*
9 9 k 14
100
ND
63 k 13t
14k8*
5 6 k 15t
100
ND
141 -t 40
132 k 7 5
7 2 10'
3 0 2 17'
114 2 40
802 10
5 5 k 17t
HUVECs were pretreated for 48 hours with an inflammatory agent,
washed twice, and then restimulated for 4 hours with the same or a
different agent. Doses used during pretreatment and restimulation
periodswere identical (LPS 0.5 pg/mL; TNF-a 10 ng/mL; IL-1p 1 U/mL;
PMA 5 x lo-' mol/L). Results are expressed as a percentage of a
primary stimulation. Values represent mean 2 SD of four to seven
independent experiments. Comparison between TF induction by an
inflammatory agent in mediator-pretreated cells and in sham-pretreated cells was performed with Student's t-test.
Abbreviation: ND, not determined.
*Significantly differentP I ,001.
tsignificantly different P I .01.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BUSS0 ET AL
2032
. _ . ..
A
B
,
=--
..
C
-“F-r
- 2.2 kb
IL-1024 h
11-10 2 h
+ + - + +
LPS24h
LPS2h
+ + - + +
PMA20h+
PMA3h-
+ -
+ +
Fig 4. Effect of repeated stlmulatfon with an Inflammatory agent on lF mRNA content. Confluant HWEC monolayers were incubated in
medium containing 20% H S alone or supplemented with IL-lp (A, 5 UlmL), LPS (B, 1 pglmL), or PMA (C, 5 x lo-* mollL). After 20 to 24 hours,
cells were washed and reincubated for 2 to 3 hours as indicated, in medium containing the same agent at the same dose used during the first
incubation.Total RNA was extracted and analyzed by Northern blot using 10 pg/track of RNA. The filter was hybridized to a UP-labeled TF cDNA
probe.
production by ECs after a few hours of exposure.m It has
been hypothesized that such production results from the de
novo synthesis of enzymes involved in the pathway for PGI,
~ynthesis.”.~~
To evaluate PGI, production by HUVECs, we
measured 6-keto PGF,, in culture media after a primary
and a secondary stimulation by IL-lp. Figure 7 shows that
the content of PGI, in culture supernatants after IL-lp
treatment for 4 hours was increased about fivefold. Cells
cultured sequentially with IL-lp for 48 hours and then in
the absence of IL-lp for 4 hours produced more PGI, than
unstimulated cells, indicating that the effect of a primary
IL-lp stimulation persisted for at least 52 hours without
requiring the continuous presence of this cytokine. HUVECs pretreated for 48 hours with IL-lp produced more
PGI, than untreated cells after 4 hours of restimulation
-2.2
kb
with IGlp, thus showing the absence of cell hyporesponsiveness to restimulation. In addition, this latter observation
indicates that IL-1p receptors are still functional under
these culture conditions as repeated IL-lp stimulation is
able to further trigger PGI, synthesis.
To assess whether protein synthesis is required for
induction of the refractory state during initial stimulation,
cells were incubated for the first 24 hours with IL-lp in the
presence of 0.5 &mL cycloheximide,and then thoroughly
washed before a second stimulation with the same dose of
IL-lp (Table 3). Inhibition of protein synthesis during the
first 24 hours was approximately SO%, as measured by
%-methionine incorporation. HUVECs treated for 24
hours with IL-1s plus cycloheximide were still refractory to
the IL-1p-induced restimulation of TF activity. This finding
was not accounted for by a general toxic effect on biologic
functions of ECs nor by inhibition of IL-lp receptor
synthesis, as untreated HUVECs and HUVECs pretreated
for 24 hours with cycloheximide alone synthesized comparable levels of protein after 4 hours of incubation (see
radiolabeling results and data on expression of TF activity
after IL-lp stimulation).
These results suggested that de novo protein synthesis is
not required during the initial incubation for a refractory
period to be observed in conjonction with reinduction of
TF.
DISCUSSION
PMA20h
IL-101 h
+
+ +
+
-
Fig 5. IL-lp dmulatlon of PMA-pretreated HWECs: effect on TF
mRNA content. Confluent HUVEC monolayers were Incubated in
medium containing 20% HS alone or supplemented with 5 x lo-*
mol/L PMA. After 20 hours, the cells were washed and reincubated
for 1 hour in medium alone or medium supplemented with 5 U/mL of
IL-1p. Total RNA was extracted and analyzed by Northern blot using
10 pgltrack of RNA. The filter was hybridizedto a UP-labeledTFcDNA
probe.
The inflammatory agents IL-I@,TNF-a, LPS, and PMA
induced a transient increase in TF activity in HUVECs,
which attained a peak at 4 to 8 hours. Such activity was
preceded by a transient increase in the steady-state levels of
TF mRNA, with a maximum at 1 to 2 hours after stimulation for Lip,TNF-a,and LPS, and at 4 hours for PMA.
The timecourses of the induction of TF activity by these
agents are in agreement with previous report^.'^.'^^'^^'^^^^
However, some discrepancies exist concerning TF mRNA
induction. Thus, Scarpati and Sadler observed a maximal
response after 3 hours with both TNF-a and PMA on TF
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
TF INDUCTION ON ENDOTHELIAL CELLS
2033
T
- - + + - +
- - - - + +
- -k - -k + +
48
HOURS IL-18
ACIDIC TREATUEHT
4 HOURS IL-10
Fig6. Effect of an d d k treatment on the I L - l ~ n d u c e d
"ctoq
state. HWECs were incubatedwith or without 1 UlmL of IL-lg for 48
hours. The cells were washed and treated,where indkated, with an
acidic buffer to remove receptor-bound IL-1p. After washing, culls
were rechallengedwith 1 U/mL of IL-1p for 4 hours. At the end of this
recond incubation, the supernatants were removed, cells were
washed, and TF activity was measured in HWEC cell Ivsates.
mRNA lcvcls in HUVECs." Morcovcr, thcy found that
PMA was only cffcctivc at micromolar doscs, contrasting
with the cffcct of PMA on our cells at nanomolar doscs. In
thc samc ccllular modcl, Crossman et al found a timccoursc of induction of TF mRNA by LPS and PMA that was
almost identical, with a peak occurring during thc first 2
hours aftcr stimulation.'" In addition, thcy found that
cycloheximide alonc was able to inducc an incrcasc in TF
mRNA lcvcls aftcr 1 hour, thcrcby contrasting with its lack
of cffcct in our cxpcrimcnts and thosc of Scarpati and
Sadlcr."
Thc diffcrcnccs bctwccn our data and thosc rcportcd
lo
f
f
*z
1
lo
i 1200
T
'
T
l.0
+ - - +
- - - - +
- + - + +
Fig 7. Effect of repeated atimulltion with 11-1s on POI, s m h
by HWECs. HWECs were incubated in the prs~ms
or absence of 1
UlmL of IL-lg for 48 hours. The cells were warhed and tmated, where
indicated, wtth an acidic buffer. After washing, cells were mhalIenged with 1 U/mL of IL-lp for 4 hours. At the end of this second
incubation, the supematants were collected from the culture wellr
and tested for their content of &ketlo-PGF,, the stable PGI, metabolite, by a specific RIA.
prcviously may bc cxplaincd, at lcast in part. by diffcrcnccs
in EC culturc conditions or in thc numbcr of passagcs that
thc cells havc undcrgonc. Scarpati and Sadlcr, as wcll as
Crossman ct al, uscd mcdium supplcmcntcd with EC
growth factor (ECGF) and heparin.“," This aspcct may
constitutc an important diffcrcncc bccausc it has bccn
rcportcd that growth of HUVECs in thc prcscncc of ECGF
and hcparin dccrcascs thc inducibility of TF activity on
thcsc cclls." Thc rclativcly modcratc cffccts measured by
Scarpati and Sadlcrl- with PMA and T N F a on TF mRNA
lcvcls (cightfold and thrccfold of incrcasc, rcspcctivcly), as
wcll as thc rclativc inscnsitivity of thcir cells to PMA (only
cffcctivc at micromolar rangcs), could bc duc to cxtcnsivc
passaging of the cclls (cclls wcrc uscd bctwccn passagcs 15
and 20). Indccd. wc havc also obscrvcd that at thcsc
passagcs cclls wcrc far lcss rcsponsivc with rcspcct to TF
induction (data not shown).
Wc havc shown that thc transicnt TF induction sccn aftcr
an initial cxposurc of thc cclls to cithcr IL-IS. TNF-a, LPS.
or PMA is followcd by partial (for TNF-a and LPS) or total
(for IL-lp and PMA) hyporcsponsivcncss for TF rcinduction by a sccond homologous stimulation.
Wc havc also cxamincd thc qucstion as to whcthcr
prctrcatmcnt of thc cells with an agcnt "A" (IL-IS, TNF-a.
or LPS) leads to a rcfractory statc with rcspcct to the
rcinduction of thc cxprcssion of TF by a sccond agcnt "B"
(hctcrologous stimulation). In this lattcr casc, partial but
significant hyporcsponsivcncs.. has bccn obscrvcd aftcr
IL-l p stimulation on TNF-a-prctrcatcd cclls, and convcrscly aftcr TNF-a stimulation of IL-lp-prctrcatcd cclls.
Thc cxpcrimcnts that wc pcrformcd on PMA-prctrcatcd
cclls arc of intcrcst with rcspcct to thc mcchanism of action
of IL-IB, TNF-a. or LPS. I t is wcll known from studics of
difkrcnt biologic rcsponscs that phorhol cstcr trcatmcnt of
thc cells is followcd by a rcfractory pcriod during which a
sccond trcatmcnt has littlc. if any, cffcct. This loss of
rcsponsivcncss is accountcd for by downmodulation of
protcin kinasc C (PKC), thc major ccllular rcccptor for
phorhol cstcrs."'Thc fact that TF induction by IL-Ip and
LPS was significantly dccrcascd on PMA-prctrcatcd cclls
with rcspcct to untrcatcd cclls suggcsts a potcntial involvcmcnt of PKC in thc IL-Ip and LPS pathways. By contrast,
TNF-a was fully activc on cclls that had bccomc complctcly
unrcsponsivc to PMA, suggcsting that this cytokinc acts
through a PKC-indcpcndcnt mcchanism. Similar conclusions can bc drawn rcgarding thc sccondary mcsscngcr
pathways for IL-lp and TNF-a from studics of Pobcr ct al
on thc induction of cndothclial lcukocytc adhcsion molcculc-l (ELAM-I) in HUVECs."
Bccausc thc obscrvcd hyporcsponsivcncss is csscntially a
phcnomcnom of sclf-hyporcsponsivcncss (cclls prctrcatcd
with agcnt "A" arc rcfractory with rcspcct to TF induction
by thc samc agcnt "A," but rarcly and only to a limited
cxtcnt by a sccond agcnt "B')), wc havc focuscd our
attcntion on thc sclf-hyporcsponsivcncssinduced by IL-l p.
This phcnomcnom is timc-dcpcndcnt (7 hours of incubation with IL-Ip is ncccssary to dctcct a rcfractory statc to a
sccond IL-Ip stimulation at 24 hours) and dosc-dcpcndcnt
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BUSS0 ET AL
2034
Table 3. Effects of Cycloheximide on TF Refractory Period Phenomenon
'6S-Methionine lncorporationt
(cpm x 10 -7
During
HUVEC Cultures
First Incubation
124 h)
Second Incubation'
(4 h)
First
Incubation
Medium
Medium
IL-I
IL-1
Medium
IL-1 (1 U/mL)
Medium
IL-I
28.4
28.4
27.9
27.9
2
Medium
Cycloheximide (0.5 bg/mL)
Cycloheximide
Cycloheximide IL-I
Cycloheximide IL-1
Cycloheximide
Medium
IL-I
Medium
IL-1
28.4
6.0
6.0
5.1
5.1
2
+
+
Second
Incubation
3
f3
f 4.1
2 4.1
26.5
29.1
26.5
27.8
2
f 0.5
2 1
2 2.3
3
3.7
26.0
27.1
23.5
22.9
2
f 0.7
0.7
0.2
2 0.2
2
2
2
0.4
f 0.9
2.1
2.5
2 2.2
2
2
TF Activity*
(mU/4 x
1.1
12.8
1.8
2.8
io4 cells)
2
0.2
f 0.7
2
0.3
f 0.3
1.1 f 0.1
0.9 f 0.1
7.1 2 0.8
3.2 2 0.3
2.8 f 0.3
'Cells were washed twice with prewarmed medium before the second incubation.
tProtein synthesis was determined by radiolabeling with "S-methionine as described in Materials and Methods.
STF activity was measured at the end of the second incubation period.
(maximal hyporesponsiveness after an initial IL-1p incubation at 5 U/mL). The refractory period observed in the case
of E - l p m l d be explained by several distinct regulatory
mechanisms that are as follows: (1) the saturation of IL-1
receptors by autocrine/paracrine mechanisms; (2) modulation of IL-1 receptor expression and/or affinity; (3) alteration of the IL-1 signal transducing pathway; (4) the
induction or activation of intracellular inhibitor(s) of TF
gene transcription; and (5) an increase in the rate of
degradation of TF mRNA. Some of these mechanisms may
be discounted on the basis of our studies. Several observations argue against a saturation of IL-1 receptors on
HUVECs. The concentration of IL-1p used in our experiments (2.6 to 13 pmol/L) does not saturate the IL-1
receptors on these cells that have a kd of 81 pmol/L (N.
Smithers, personal communication, October 1990), indicating that maximal receptor occupancy is not necessary for
the triggering of a maximal refractory state. Moreover, a
mild acidic treatment, which is known to remove bound
IL-lp, did not prevent the refractory period phenomenon.
A role for the IL-lp produced by stimulated HUVECs also
seems unlikely in view of the very low amounts that are
synthesized?' Finally, HUVECs that were refractory with
respect to IL-1p-induced TF expression continued to
express functional receptors, because they produced enhanced PGI, levels on restimulation. A downmodulation of
IL-1 receptor expression seems rather unlikely, because TF
mRNA levels were downmodulated whereas IL-1R mRNA
levels remained constant during hyporesponsiveness (R.
Lenstra, personal communication, July 1990). These data
were confirmed by a preliminary Scatchard analysis that
indicated that IL-lp-treated HUVECs presented a similar
number of receptors and a comparable binding affinity (N.
Smithers, personal communication, October 1990). Induciion of intracellular inhibitor(s) seems rather unlikely
because preincubation of HUVECs with IL-1p and cycloheximide did not prevent the refractory phenomenon. We have
no clues concerning the possible occurence of differential
TF mRNA stability, or of an alteration in the secondary
messenger pathway for IL-1p.
The refractory period phenomenon is not restricted to
the expression of TF on HUVECs. Other surface and
secreted mwlecules, which are chaTacteristic phenwtypic
markers of ECs, also present a refractory phenomenon with
respect to their induction. The regulation of the expression
of the cell adhesion molecule ELAM-1 by inflammatory
agents is comparable with our observations on TF induction. ELAM-1 is transiently expressed (presenting a peak at
4 to 6 hours) at the cell surface of IL-1p-, TNF-a-, or
PMA-stimulated HUVECS.~'As in the case of TF, cells
pretreated for 24 hours with one inflammatory agent were
hyporesponsive to the same agent with respect to ELAM-1
reinduction. When two different agents were used, crossinhibitions were observed with some combinations but not
with others, as in the case of TF. Comparable patterns of
cross-suppression are observed for TF and ELAM-1. These
findings suggest that the refractory period involved in
ELAM-1 and in TF expression may be comparable. Histamine establishes a desensitized state to further stimulation
of tissue-type plasminogen activator release33or to further
stimulation of PGI, production% by ECs. The refractory
period phenomenon observed for the modulation of various
functions by inflammatory agents is not restricted to ECs.
For example, it has recently been reported that LPS
induces a hyporesponsive state with respect to its own effect
on TNF-a production by a macrophage-like cell line.35This
observation can be related to the phenomenon of endotoxin
tolerance whereby animals treated with low doses of endotoxin become resistant to lethal
This observation is
at the basis of therapeutic strategies to prevent the occurrence of septic shocks, and illustrates the general interest of
understanding the regulatory mechanisms responsible for
the onset of the refractory period phenomenon in response
to inflammatory agents. In the case of TF, a clear understanding of the refractory period phenomenon might lead
to new ways of limiting extensive thrombogenesis.
ACKNOWLEDGMENT
We thank Dr J. Chapman for a critical reading of the manuscript.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2035
TF INDUCTION ON ENDOTHELIAL CELLS
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1991 78: 2027-2035
Refractory period phenomenon in the induction of tissue factor
expression on endothelial cells
N Busso, S Huet, E Nicodeme, J Hiernaux and F Hyafil
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