1. Art and Diseño

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C-Reactive Protein Induces Human Peripheral Blood Monocytes
to Synthesize Tissue Factor
By Jaroslav Cermak, Nigel S . Key, Ronald R. Bach, Jozsef Balla, Harry S . Jacob, and Gregory M. Vercellotti
The acute inflammatory response is frequently accompanied by serious thrombotic events. We show that C-reactive protein (CRP), an acute-phase reactant that markedly
increases its serum concentration in response t o inflammatory stimuli, induced monocytes to express tissue factor
(TF), a potent procoagulant. Purified human CRP in concentrations commonly achieved in vivo during inflammation (10 to 100 pg/mL) induced a 75-fold increase in TF
procoagulant activity (PCA) of human peripheral blood
mononuclear cells (PBM), with a parallel increase in TF antigen levels. CRP-induced PCA was completely blocked by
a monoclonal antibody against human TF but not by irrelevant murine lgG. Dot blot analysis showed a significant
increase of TF mRNA after 4 hours of incubation with CRP.
followed by a peak of PCA within 6 and 8 hours. Actinomycin D and cycloheximide blocked CRP-stimulated PCA.
T
HE INFLAMMATORY response includes phagocyte
margination and migration, chemoattractant generation, cytokine formation, and production of a heterogenous
group of proteins collectively called acute-phase reactants.
The most characteristic human acute-phase protein is Creactive protein (CRP).’ CRP, consisting of five identical,
noncovalently linked subunits of 23,000 molecular weight,2
is synthesized in the liver. Its serum concentration of less
than 1 jtg/mL can increase during the first 24 to 48 hours of
inflammation or tissue necrosis by several hundred-fold, a
response shown primarily to be due to interleukin-6 (IL-6)
ti mu la ti on.^.^ CRP is thought to amplify the host defense
system by potentiating its recognition capacities, as exemplified by its activation of the complement cascade and its
stimulation of various effector phagocytic cell^.^-^
The linkage between inflammation and coagulation may
well involve tissue factor (TF). T F is a membrane-bound
glycoprotein that initiates the extrinsic pathway of coagulation.’ The TF-factor VIIa (TF-FVIIa) complex activates FX
directly or indirectly via FIX activation, leading ultimately
to thrombin generation.’ “Resting” endothelial cells and
monocytes/macrophages express very low levels of procoagulant activity (PCA). However, in both these cell types, an
induction of T F expression with associated increased PCA
has been shown with exposure to various agents, including
inflammatory mediators such as endotoxin (LPS) or cyto-
kine^."*^
That CRP might also play a role in upregulating monocyte PCA, as suggested by Whisler et al,23is an attractive
possibility, because CRP is known to modulate other monoBecause
cyte function^^^-^^ through a specific re~eptor.*’~~’
CRP accumulates at sites of inflammation or tissue damage,
we questioned whether CRP might promote localized coagulation by stimulating monocytes to produce the specific procoagulant, TF. In the following studies, we used highly punfied human CRP to assay its effects on peripheral blood
monocyte and endothelial cell PCA and to discern mechanisms by which CRP promotes TF expression in these cells.
Blood, VOI 82, NO 2 (July 15). 1993: pp 513-520
suggesting that de novo TF protein synthesis was required. Endotoxin (LPS) contamination of CRP was excluded as the mediator of TF synthesis because: (1) CRP
was Limulus assay negative; (2) induction of TF PCA by
CRP was not blocked by Polymyxin B, in contrast t o LPSinduced PCA; (3)antihuman CRP IgG inhibited CRP-induced PCA, but not LPS-inducedPCA; (4)CRP was able t o
stimulate TF production in LPS-pretreated PBM refractory
t o additional LPS stimulation; and, (5) unlike LPS. CRP was
incapable of inducing TF in human umbilical vein endothelial cells. We suggest that CRP-mediated TF production in
monocytes may contribute to the development of disseminated intravascular coagulation and thrombosis in inflammatory states.
0 1993 by The American Society of Hematology.
MATERIALS AND METHODS
Reagents
Highly purified (>90%) human CRP was obtained from Sigma
CO(St Louis, MO) (catalogueno. C6 177), as well as endotoxin/lipopolysaccharide (LPS) (catalogue no. L3 129), lyophilized IgG fraction of rabbit serum against human CRP (catalogue no. C3257),
and TRIS [tris(hydroxymethyl)aminomethane].
Hanks’ Balanced Salt Solution (HBSS), HEPES buffer solution,
and all cell culture media including RPMI 1640 were purchased
from GIBCO (Grand Island, NY). Heparin sodium was from LyphoMed (Melrose Park, IL); Polymyxin Band recombinant human
IL-lp (rIL- 1) were from Upjohn CO(Kalamazoo, MI); CS-depleted
serum was from Quidel CO (San Diego, CA); formaldehyde was
from Fisher Scientific (Pittsburgh, PA); and cycloheximide was
from NBCo (Cleveland, OH). Radiolabeled compounds and nylon
membranes were obtained from Amersham (Arlington Heights,
IL).
Murine monoclonal antibody (MoAb) against human TF (HTF1), polyclonal antibody against human TF (anti-HTF pAb), human
From the Department ofMedicine, University ofMinnesota, Minneapolis; and the Veterans Administration Medical Center, Minneapolis, MN.
Submitted November 30, 1992; accepted March 17, 1993.
Supported in part by grantsf o m the National Institutes of Health
(HL 33793), the Graduate School of the University of Minnesota,
and the Department of Veterans’Affairs. J.C. was supported by the
Charles Proshek Fellowship from the Minnesota Medical Foundation.
Address reprint requests to Gregory M. Vercellotti, MD, University of Minnesota, Department of Medicine/Division of Hematology, Box 480 UMHC, Harvard St at E River Rd, Minneapolis, MN
55455.
The publication costs of this article were defayed in part by page
charge payment. This article must therefore he hereby marked
“advertisement” in accordance with I 8 U.S.C. section I734 solely to
indicate this fact.
0 I993 by The American Society oJHematology.
0006-4971/93/8202-0004$’3.00/0
513
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514
CERMAK ET AL
FVIIa and FX, human TF standard, and bovine brain cephalin
were prepared as previously de~cribed?~-~’
All other reagents were
obtained from Sigma CO, unless otherwise specified.
All reagents, including CRP, were tested for endotoxin contamination by the Limulus assay (E-Toxate; Sigma CO)and were negative at levels from 0. I to 0.2 EU/mL (10 to 20 pg/mL).
Peripheral Blood Mononuclear Cells (PBM) and Peripheral
Blood Monocyte Isolation
Minimally heparinized (2 U/mL) blood from fasting healthy donors (after obtaining their informed consent according to the guidelines of the Committee on the Use of Human Subjects in Research,
University of Minnesota) was layered over Histopaque- 1077 and
centrifuged (500g)at 24°C for 30 minutes. The PBM collected from
the plasma/Histopaque interface were washed three times with
phosphate-buffered saline (PBS) and finally resuspended in RPMI
1640 1% fetal calf serum (FCS) at a concentration of 1 X IO6
cells/mL. The PBM contained an average of 80%lymphocytes and
20% monocytes as assessed by light microscopy and nonspecific
esterase staining. Contamination by polymorphonuclear leukocytes and platelets was less than 1%.
PBM at I X IO6 cells/mL in RPMI 1640 1% FCS were incubated with various reagents at 37°C in 5% CO, for the appropriate
times. After incubation, cells were washed once with fibrometry
buffer (0.16 mol/L NaCl 25 mmol/L HEPES buffer, pH 7.4). The
viability ofthe cells as assayed by Trypan blue exclusion was greater
than 95% after 6 hours of incubation and 92% after 18 hours of
incubation.
To prepare a relatively enriched population of peripheral blood
monocytes, PBM at 2 X IO6 cells/mL were plated onto plastic tissue
culture wells with RPMI 1640 + 1% FCS. After 2 hours of incubation, nonadherent cells were removed by washing five times with
medium. The adherent cells (approximately 75% to 80% monocytes and 20% to 25% lymphocytes) were incubated with various
reagents under the same conditions as PBM.
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Human Umbilical Vein Endothelial Cell (HUVEC)
Cultures
HUVEC were isolated and grown as previously described3’ and
used from passages 1 through 3 after reaching confluence. HUVEC
grown in 2-cm2 tissue culture wells (approximately 200,000 cells/
well) were incubated with various reagents in the same manner as
PBM; after the incubation, the cells were washed once with fibrometry buffer.
TF Assays
One-stage clotting assay. PBM cell pellet was resuspended in
0.5 mL fibrometry buffer, frozen at -7O”C, and thawed at 37°C
three times and sonicated. Peripheral blood monocytes and HUVEC were treated the same way. Cells were scraped from the bottom of tissue culture wells, frozen, and thawed three times before
sonication.
The clotting assay” consisted of 0.1 mL of cell sample and 0. I
mL of citrated normal human platelet-poor plasma. The reaction
was initiated in a fibrometer cup (Fisher Scientific CO)by the addition of 0.1 mL 25 mmol/L CaCI,. Results were expressed in arbitrary units per milliliter (U/mL) of T F by comparison of clotting
times with a standard curve obtained using a purified human TF
standard as previously de~cribed.’~
One unit of TF PCA equals 1 pg
of the TF standard. For HUVEC, the results were expressed in
U/mg of cell protein assessed by BCA protein assay (Pierce, Rockford, IL).
Two-stage clotting assay. PCA was measured on Coag-a-mate
XM (Organon Teknika, Durham, NC). In stage 1 of the assay, 20
pL of sample was incubated with 10 pL of 30 nmol/L human FVIIa
and 10 pL of I .5 pmol/L human FX at 37°C for at least 3 minutes.
The reaction was initiated by adding 20 pL of 25 mmol/L CaCI,,
and the sample was incubated for another 5 minutes. Stage 2 ofthe
assay was started by the simultaneous addition of 100 pL substrate
plasma (90 pL normal citrated bovine plasma plus 10 pL bovine
brain cephalin [6 mg/mL] in TRIS-buffered saline) and 100 pL 25
mmol/L CaCI,. Clotting time was recorded, and TFlevel was calculated from a standard curve obtained using a human brain TF standard.34
TF antigen enzyme-linked immunosorbent assay (ELISA). T F
antigen ELISA was performed as previously described.34 Briefly,
96-well microtiter plates were coated with 100 pL per well of IO
pg/mL HTF-I murine MoAb in a bicarbonate buffer (15 mmol/L
Na’CO,, 35 mmol/L NaHCO,, pH 9.6) and incubated overnight at
4°C. Wells were then filled with TBS (0.1 mol/L NaC1,0.05 mol/L
Tris, pH 7.5) containing 1% bovine serum albumin (BSA) to block
nonspecific sites and incubated again at 4°C for at least 4 hours.
One hundred microliters of sample for assay or TF standard diluted
in TBS containing 0.1% BSA, 0.1% Triton X-100, and 5 mmol/L
EDTA were added to triplicate wells. After an overnight incubation, the plates were extensively washed, 100 pL of anti-HTF polyclonal antibody at a final concentration of 0.2 pg/mL diluted in
TBS + I % BSA was added, and samples were incubated for 2 hours
at room temperature. After another wash, the samples were incubated with 100 pg goat antirabbit horseradish peroxidase-conjugated IgG (GAR-HRP; Bio-Rad, Richmond, CA) diluted 1:2,000 in
TBS + 1% BSA. After a further wash cycle, a chromogenic detection system was added for 30 minutes. The reaction was stopped
with the addition of 25% H2S04, and the sample absorbance was
read at 490 nm on a Dynatech MR 600 microplate reader (Dynatech Laboratories Inc, Chantilly, VA). TF antigen level was calculated from a standard curve obtained with diluted human T F standard in the range of 0 to 1,000 pg/mL.
TF mRNA analysis. T F mRNA content in PBM was analyzed
after treatment with CRP, LPS, serum, or RPMI 1640 1% FCS
alone. PBM cellular mRNA was isolated by the RNAzol method
(Tel-TEST, Inc, Friendswood, TX). Aliquots (4 pg) of total RNA
were dissolved in IO pL of H,O and mixed with 20 pL of 100%
formamide, 7 pL of 37% formaldehyde, and 2 pL of 20X SSC (3
mol/L NaCl + 0.3 mol/L sodium citrate), applied to slots on a
microsample filtration manifold (Minifold I; Schleicher and
Schuell, Inc, Keene, NH), and transferred onto nitrocellulose memb r a n e ~using
~ ~ UV cross-linking. The membranes were hybridized
at 42°C with a nick-translated ”P-labeled cDNA probe for human
TF.36Autoradiographs were obtained and quantitated by computer-assisted videodensitometry.”
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Statistical Methods
Statistical analysis was performed using the Student’s t-test. All
results are expressed as mean l?r I SD.
RESULTS
Induction of TF Expression on PBM and on Peripheral
Blood Monocytes
A significant increase in TF PCA of disrupted PBM was
found after 6 hours of incubation with either CRP (100
pg/mL) or LPS (10 pg/mL) ( P < .0001 for one-stage clotting
assay and P < .O 1 for two-stage clotting assay and TF antigen [Table 11). A difference between CRP- and LPS-induced TF expression was also significant ( P < .O 1 for all 3
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CRP INDUCES TISSUE FACTOR
515
Table 1. Induction of TF in PBM by Different Stimuli
Two-Stage
Clotting Assay
One-Stage
Clotting Assay
(U/W
Reagent
(UlW
Control
13.1k2.4
LPS (10pg/mL)
415.0t 46.4
CRP ( 1 0 0 ~ ~ n / m L ) 1,044.8rt 52.8
TF Antigen
ELSA
(pg/mL)
8.5 k 0.8
20.3 k 3.5
299.6f 13.0 373.3t 14.2
694.5f 28.9 726.1 t 17.5
_ _ _ ~
~
~
PBM at a concentration of 106/mL were incubated for 6 hours with
RPMl 1640 + 1 % FCS (control), LPS (10pg/mL), or CRP (1 00 pg/mL),
both in RPMl 1640 1 % FCS. The TF PCA was measured by one- or
two-stage clotting assay and TF antigen by ELISA. Results represent the
mean f 1 SD from five experiments performed in duplicates
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assays). In the two-stage clotting assay, PCA was undetectable when FVIIa was omitted from the assay (data not
shown). This result shows that the PCA is indeed TF-mediated. Most of the TF activity induced by CRP in mononuclear cells was encrypted, ie, CRP-treated (100 pg/mL)
mononuclear cells added intact to the one-stage clotting assay had 17%of the procoagulant activity (150 U/mL) compared with sonicated, disrupted CRP-treated mononuclear
cells (880 U/mL).'
Similarly, under the same conditions, a relatively
enriched population of peripheral blood monocytes exhibited a significant increase of TF PCA from a control value of
4.2 f 1.1 U/mL to 714.9 k 70.7 U/mL with LPS ( I O pg/
mL) and 1,495.8 f 267.9 U/mL with CRP (100 pg/mL)
(measured by the one-stage clotting assay).
To characterize CRP-induced PCA on PBM, the samples
previously treated with CRP for 6 hours were incubated
with a murine blocking MoAb (HTF-1) for I hour at room
temperature. This incubation decreased TF activity in the
one-stage clotting assay from 1,044.8 U/mL to 48.8 U/mL
(P < .OOOl)(Fig I). Incubation with an irrelevant murine
p
1200 1
-E
.
2.
1000
-
800
-
p c 0.0001
0.0001
1200
I
600
n
1I
7
-E
z
3
-
Ns
600-
n
U.
I-
U.
I-
I
C
IgG in the same concentration did not significantly affect
CRP-induced PCA. This further substantiates that the PCA
induced by CRP is TF.
To obtain evidence that CRP is the factor inducing TF
PCA, CRP (100 pg/mL) was pretreated with an IgG fraction
of rabbit serum against human CRP (200 pg/mL) for 30
minutes at 37°C. Pretreatment of CRP significantly decreased CRP-induced TF expression by PBM after 6 hours
of incubation (P < .OOOl). In contrast, antiserum against
human CRP had no effect on LPS induced TF expression
(Fig 2).
Under normal circumstances, the serum CRP concentration is less than 1 pg/mL, but can increase up to 1,000-fold
during acute infections or tissue n e c r o s i ~ . 'Therefore,
~,~~
we
examined a dose response of PBM exposed to varying CRP
concentrations for 6 hours; as shown in Fig 3, a concentration of CRP as low as 5 pg/mL significantly induces TF
PCA (P < .05), with a further increase in response up to 100
pg/mL. Levels of CRP 5 1 pg/mL did not induce TF PCA
(data not shown).
The time course of TF PCA induced in PBM by CRP
differed from that noted with LPS induction. As seen in Fig
4, a peak of CRP-stimulated PCA occurred between 6 and 8
hours and remained elevated even after 18 hours. In contrast, the LPS effect peaked between 4 and 8 hours and
tailed off at 18 hours.
Because CRP is able to induce complement activat i ~ n , ' *we
~ ,tested
~
whether CRP-induced TF PCA in serum
was dependent on complement. Using RPMI, C5-depleted
human serum, or heat-inactivated human serum (56°C for
30 minutes) as the diluent, we observed only a slight decrease in the induction of TF PCA by CRP. This result
suggests that complement activation does not substantially
400
-
200
RPMl
O+
CRP
RPMl
CRP/HTF-1
CRP/IgG
Fig 1. Effect of a blocking murine MoAb (HTF-1) on CRP-induced PCA in PBM. After 6 hours of incubation with CRP (100
pg/mL) in R P M l l 6 4 0
1% FCS or R P M l l 6 4 0
1% FCS alone,
PBM (106/mL) cells were incubated for 1 hour at room temperature
either with HTF-1 (200 pg/mL) (CRP/HTF-1) or with irrelevant murine IgG (200 pg/mL) (CRP/IgG). Results represent the mean 2 1
SD of three experiments in duplicates measured by the one-stage
clotting assay.
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+
UP
CRPl
CRP Ab
LPSl
LPS
CRP Ab
Fig 2. Effect of antihuman CRP antiserum on CRP- and LPS-in1% FCS)
duced TF PCA in PBM. PBM (106/mL in RPMl 1 6 4 0
were incubated for 6 hours either with CRP (100 pg/mL) or LPS (10
pg/mL), respectively, with CRP or LPS in the same concentration
preincubated with 200 pg/mLof purified rabbit antihuman CRP lgG
for 30 minutes at 37°C ([CRP/CRPAb], [LPS/CRPAb]). Results rep1 SD of three experiments in duplicates mearesent the mean
sured by the one-stage clotting assay.
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*
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516
CERMAK ET AL
1500
I
D < 0.0001
T-
100
25
50
10
5
CRPI
serum
RPMl
CRP concentration (pglml)
Fig 3. Effect of varying CRP concentrations (diluted in HBSS) on
PBM TF PCA after 6 hours of incubation. Results represent average
2 1 SD of three experiments in duplicates on PBM (lOg/mL) in
RPMl 1640
1% FCS as measured by the one-stage clotting assay.
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contribute to the effect of CRP on PBM T F synthesis (Fig
5).
Evidence That CRP and LPS Represent Two Different
Stimuli
As mentioned above, rabbit antiserum against CRP decreased CRP-induced T F expression, but did not affect
LPS-provoked PCA. To further assure that CRP-induced
PCA is distinct from that mediated by LPS, we coincubated
samples with Polymyxin B (10 pg/mL) for 6 hours. Polymyxin B, which binds and inactivates gram-negative endo-
1200
1
i
I
600
I&
I-
0
2
4
6
8
1.0
1.4
1.2
16
1.8
hours
Fig 4. Effect of different incubation times on CRP (100 pg/mL)
(0).
LPS (10 pg/mL) (01, and RPMl 1640
1% FCS (0)alone induced TF PCA in PBM (10*/mL in RPMl 1640
1%FCS) measured by the one-stage clotting assay. A representativeexperiment
is shown.
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CRPl
RPMl
CRPl
Hlserum
CRPI
CWepl.
serum
serum
Fig 5. Effect of serum complement on CRP-induced TC PCA in
PBM. CRP (100 pg/mL) was added to 10%human serum (CRP/
serum), RPMl 1640 (CRP/RPMI), 10% heat-inactivated serum
(CRP/Hlserum), or 10%C5-depleted serum (CRP/C5depl,serum)
and incubated with PBM (1OB/mL)for 6 hours. Results represent
the mean f 1 SD of three experiments in duplicates measured by
the one-stage clotting assay.
toxin (LPS),39did not significantly alter CRP-induced (10
pg/mL) T F activity (999.2 f 74.5 U/mL). In contrast, as
expected, LPS-induced (10 pg/mL) T F PCA (415.9 f 46.4
U/mL) decreased to less than 30%of initial values (P< .05)
in the presence of Polymyxin B ( 1 37.4 f 13 U/mL). Additional evidence was garnered by the fact that the CRP preparation used in these studies did not contain significant
amounts of endotoxin, as measured in the Limulus assay.
We also tested whether LPS-prestimulated PBM retained
the capacity to respond to CRP despite being desensitized to
further LPS s t i m u l a t i ~ n . ~Figure
~ , ~ ' 6 shows the effect of
repeated stimulation on PBM pretreated with LPS ( I O pg/
mL) with regard to T F synthesis. LPS-induced T F antigen
was elevated after 6 hours of incubation and became undetectable after 24 hours. At that time, LPS added in the same
concentration was not able to further increase TF antigen.
In contrast, CRP (100 pg/mL) stimulated T F antigen in
LPS-pretreated PBM, validating that LPS and CRP induced
T F via distinct pathways. Of interest, the amount of T F
induced by CRP in LPS-presensitized cells is blunted when
compared with that of naive mononuclear cells. This resembles Busso et al's4 observation that LPS or tumor necrosis
factor-a (TNF-a) restimulation of IL- 1(3-pretreated HUVEC results in less T F than control HUVEC.
Regulation of CRP-Induced TF PCA in PBM
Dot blot analysis of T F mRNA harvested from PBM after
4 hours of incubation with CRP (100 pg/mL) showed induction ofTF mRNA, whose increment, as judged by videodensitometry, was approximately 16-fold over that seen
within cells incubated with RPMI 1640 + 1% FCS and 1.5fold higher than after treatment with LPS (10 pg/mL)
(Fig 7).
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517
CRP INDUCES TISSUE FACTOR
DISCUSSION
.y
0
.
,
.
4
TREATMENT:
,
12
6
.
,
18
v,
,LPS
24
Tlmo (hra)
LPS
CRP
10pg/ml
30
4
100pg1ml
or
LPS lOpg/ml
Fig 6. Effect of repeated stimulation of PBM. PBM (106/mL in
RPMl 1640
1% FCS) were incubated for 2 4 hours with LPS (10
pg/mL). TF antigen expression was measured by ELISA at 0 , 6 , and
2 4 hours. After 2 4 hours, the cells were washed, resuspended in
RPMl 1640 2 1 % FCS, CRP (100 rg/mL) or LPS (10 rg/mL) was
added, and, after 6 hours of additional incubation, TF antigen was
recorded. Results of a representative experiment are shown. Similar results were obtained for these groups when TF PCA was measured by the one-stage clotting assay (data not shown).
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To examine the regulatory mechanisms, we also coincubated PBM with cycloheximide and actinomycin D. Both
cycloheximide and actinomycin D, at concentrations of IO
pg/mL, completely blocked CRP (100 pg/mL) induction of
T F PCA (one-stage clotting assay) in PBM ( 1 3.4 f 3.2 and
12.2 f 4.0 U/mL, respectively) compared with CRP alone
(1,044.8 f 52.8 U/mL), suggesting that de novo protein
synthesis was necessary for T F production.
Induction
TF Expression in H U VEC
Because both monocytes/macrophages and endothelial
cells are capable of synthesizing T F in response to inflammatory mediators such as LPS or cytokines, we tested the ability of CRP to induce TF PCA in endothelial cells. Both LPS
( 1 0 pg/mL) and rIL- 1 p (0.I pg/mL) induced TF expression
on HUVEC after 6 hours of incubation (Pe .01 when compared with RPMI 1640 + 1% FCS). In contrast, CRP (100
pg/mL) failed to increase HUVEC TF expression (Fig 8).
Fig 7. Dot blot analysis of TF
mRNA. PBM (in R P M l l 6 4 0
1% FCS) were incubated for 4
hours with CRP (100 rg/mL),
LPS (10 pg/mL), 10% human
serum, or RPMl 1 6 4 0
1%
FCS alone. mRNA was isolated
from 6 X 10' cells per each
group. Intensity of hybridization
signals was measured by videodensitometry and expressed as
the relative increase in comparison with RPMl 1640-treated
PBM.
In the genesis of thrombosis, Virchow first stressed the
importance of blood flow, the blood's propensity to clot.
and the integrity ofthe vessel wall, the last ofwhich could be
affected by inflammation.
In the course of inflammation, TF, an initiator of the
extrinsic coagulation pathway. may be expressed on cell surfaces that are not thrombogenic under normal circumstances, such as monocytes and endothelial cells. Various
inflammatory stimuli (eg, LPS. TNF, and IL-1) have previously been shown to increase TF expression on endothelial
cell^,'^-^^ whereas in human monocytes/macrophages. endotoxin?" activated complement." aggregated IgG,13and
TNFI4all have been reported to generate procoagulant activity, presumably by inducing T F neosynthesis.' Moreover,
an increase in circulating monocyte PCA in various inflammatory diseases (including Crohn's disease, meningococcal
sepsis, lupus erythrematosus, and rheumatic diseases) has
been previously
Rivers et a146 showed an increase in the expression of TF in monocytes from infants
with severe infection. We have recently obtained sera containing high levels of CRP ( 1 1.6 mg/dL) from a patient with
fevers, idiopathic retroperitoneal fibrosis, and deep venous
thrombosis that induced T F in normal monocytes. However, our anti-CRP antibody failed to inhibit TF induction
by this patient's sera. Defining a singular effect of CRP on
mononuclear cell tissue factor induction in sick patients'
sera may prove difficult because of concurrent serum elevations of IL-I, TNF, immune complexes, or other factors
that themselves can activate TF synthesis. We are planning
further TF studies with sera from ill patients who have high
serum CRP levels to further delineate CRP's role.
CRP was first reported by Tillet and Francis4' as a serum
factor possessing the capacity to precipitate a carbohydrate
derived from pneumococcal fraction C. CRP not only binds
this pneumococcal polysaccharide but also binds calcium.
phosphate monoesters. phosphoryl choline, galactose polymers. and p o l y c a t i o n ~CRP
~ * ~ is also able to bind polymorphonuclear leukocytes and to modulate neutrophil funct i o n ~and
~ ~has
. ~been
~ found to affect intracellular calcium
mobilization, superoxide production, and tumoricidal activity of peripheral monocytes/macrophages2e26via binding to
a specific surface receptor.28Ballou et aI5' recently showed
that CRP can induce TNF-(U,[email protected] IL-6 in monocytes
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RE AT1 U E
INTENS ITV-
16x
10 x
lx
CRP
LPS
RPMI
1.5~
Serum
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CERMAK ET AL
518
-.C
d
e
n
200-
p < 0.001
U
RPMl
LPS
rlL-1
Fig 8. Effect of various stimuli on TF PCA in HUVEC. Confluent
monolayers of HUVEC grown in 2-cmZ wells were incubated for 6
hours with RPMl 1640
1% FCS, CRP (100 pg/mL), LPS (IO pg/
mL), or rlL-I (0.1 pg/mL) (all reagents diluted in RPMl 1640 f 1%
FCS). The results represent the mean 2 1 SD from three experiments performed in duplicate and measured by the one-stage clotting assay.
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in a similar dose and time course as we have shown for TF.
Whether these cytokines play a role in the signal transduction for T F synthesis by CRP is now under investigation in
our laboratory.
Although induction of monocyte T F PCA in the course of
inflammation has previously been ascribed to agents such as
LPS, cytokines, or activated complement, the current study
shows that CRP may be equally important. The maximal
induced increment of TF PCA in PBM was greater than
75-fold over that of the control as measured by one- and
two-stage assay and corresponded with a parallel increase in
T F antigen. Likewise, Whisler et alz3previously described
increased PCA in PBM after coincubation of LPS or immune complexes with CRP and noted that CRP alone also
induced some PCA. Our experiments confirm these findings and show that the procoagulant activity reflects production of authentic T F because: ( I ) the activity is inhibited by
a blocking MoAb to T F and (2) the procoagulant activity
required FVIIa. However, we emphasize that because CRP
may increase up to 1,000-fold within 6 to 24 hours in response to infections or tissue destruction, CRP concentrations of 100 fig/mL, used in our experiments, are commonly achieved during the course of an infectious
episode. 1,3,38
Monocytes are considered to be the only circulating leukocyte capable of TF synthesis’’; however, CD4+ T-helper
lymphocytes may enhance the monocyte T F response to
stimuli such as LPS by both direct cell-cell contact as well as
via the production of stimulatory lymphokine^.'^,^^ Recently, a specific receptor for CRP has been described on
monocytes but not on peripheral blood lymphocyte^.'^ We
did not observe any significant differences in the results of
experiments performed on freshly isolated peripheral blood
mononuclear cells (containing approximately 20% to 25%
monocytes) or relatively purified (70% to 80%)peripheral
blood monocytes. However, our data do not exclude the
possibility that T lymphocytes are critical in mediating the
monocyte T F response to CRP.
Although we showed a threefold increase of serum C5a
concentration when serum was incubated with CRP (100
pg/mL) in a recent s t ~ d y , CRP-induced
’~
monocyte T F expression does not only require complement activation (Fig
5); however, our results do not exclude the possibility that
activated complement components may add to the procoagulant effect of CRP.
Tebo and Mortensen” described a specific CRP receptor
distinct from the IgG receptor on human blood monocytes
and the human monocytic cell line U937. Excess of phosphorylcholine was unable to inhibit CRP binding to monocytes; similarly, in our experiments, even a 100-fold molar
excess of phosphorylcholine failed to inhibit CRP-induced
monocyte PCA (data not shown). The same investigators
also recently showed2’ that the CRP/CRP receptor complex
was internalized into an endosomal compartment in which
CRP was liberated and subsequently degraded. When U937
cells were exposed to CRP, a significant H 2 0 2production
and tumoricidal activity were detected after 8 hours.
We found that PBM TF mRNA significantly increases
after 4 hours of incubation with CRP, which was followed
by a peak of T F PCA at 6 to 8 hours. Such findings are
comparable to those published by Gregory et a1” who
showed induction of T F expression on isolated monocytes
exposed to LPS within 4 hours and peak PCA at 6 hours.
Similarly, in our studies, coincubation of CRP with both
cycloheximide and actinomycin D completely block CRPinduced T F PCA, suggesting that de novo synthesis of T F
protein is involved and that CRP induces monocyte T F
expression, presumably by acting at the transcriptional
level.
We rigorously excluded LPS contamination as a possible
artifact in these studies by showing (1) that all reagents (including CRP) were negative for significant levels of contaminating endotoxin as judged by the Limulus amoebocyte assay; (2) that Polymyxin B inhibited LPS-induced PCA and
had no effect on CRP-mediated PCA; (3) that a neutralizing
antibody against CRP had a selective blocking effect on
CRP-induced PCA; (4) that PBM pretreated with LPS were
able to increase T F in response to CRP despite desensitization to further LPS stimulation; and (5) that in cultured
HUVEC, we observed a failure to respond to CRP but not
LPS. Thus, we conclude from these data that CRP and LPS
represent two distinct stimuli.
It is evident that the vascular endothelium plays an important role in hemostasis and thrombosis, and that inflammatory agent-stimulated endothelium may affect the activation of the coagulation system. However, we were not
able to show a significant increase of HUVEC T F expression after exposure to CRP, in contrast to other inflammatory mediators, including LPS, IL-1, and TNF, which induce PCA in both monocyte/macrophages and endothelial
cells.9. IO.l4,20-22 we speculate that this intriguing difference
might be explained by the absence or considerably reduced
expression of the CRP receptor on cultured endothelial
cells.
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
CRP INDUCES TISSUE FACTOR
Our results suggest that increased monocyte TF expression during infection or tissue necrosis may be at least partially mediated by an increased CRP level. Thus, CRP-mediated monocyte PCA induction may play an important
role in altered microcirculation in inflammatory and necrotic tissue as well as contribute to the development of
disseminated intravascular coagulation in septic infections.
The ability of CRP to reinduce PCA in LPS-stimulated
monocytes in a phase in which they are refractory to further
LPS stimulation may be an important tool for the maintenance of a high monocyte PCA during inflammation. We
speculate that this enhanced TF production may better allow inflammatory cells to ward off bacteria by promoting
fibrin formation locally.
ACKNOWLEDGMENT
We thank Dr Henry Gewurz (Rush Presbyterian St Luke’s Medical Center, Chicago, IL) for kind advice; Dong Tuong and Theresa
Stella for technical assistance; and Linda Radtke for manuscript
preparation.
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1993 82: 513-520
C-reactive protein induces human peripheral blood monocytes to
synthesize tissue factor
J Cermak, NS Key, RR Bach, J Balla, HS Jacob and GM Vercellotti
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