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Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3413-3420
By
From the Centre for Inflammatory Diseases, Monash University,
Department of Medicine, Monash Medical Centre, Clayton, Victoria,
Australia.
The potential for tissue factor (TF) to enhance inflammation by
factor VIIa-dependent induction of proinflammatory changes in
macrophages was explored. Purified recombinant human factor VIIa
enhanced reactive oxygen species production by human monocyte-derived macrophages expressing TF in vitro. This effect was dose- and time-dependent, ligand- and receptor-specific, and independent of other
coagulation proteins. This receptor/ligand binding induced phospholipase C-dependent intracellular calcium fluxes. Transfection studies using a human monocyte-derived cell line (U937) demonstrated that an intact intracytoplasmic domain of TF is required for factor VIIa-induced intracellular calcium fluxes. The capacity of TF to
enhance proinflammatory functions of rabbit peritoneal-elicited macrophages (production of reactive oxygen species and expression of
major histocompatibility complex class II and cell adhesion molecules)
was demonstrated in vivo by treatment with an anti-TF antibody. These
data demonstrate that, in addition to its role in activation of
coagulation, TF can directly augment macrophage activation. These
effects are initiated by binding factor VIIa and are independent of
other coagulation proteins. These studies provide the first
demonstration of a direct proinflammatory role for TF acting as a
cell-signaling receptor.
TISSUE FACTOR (TF) IS A cell
surface-bound glycoprotein that binds both the zymogen, factor VII, and
the active serine protease factor VIIa.1 TF/factor VIIa
complexes activate the extrinsic coagulation pathway and are the major
in vivo initiator of coagulation. Under normal physiological
conditions, TF is expressed only at extravascular sites and
perivascularly in the adventitial layer of blood vessels.2
Coagulation may be initiated after the breach of vascular integrity by
contact of circulating factor VII and factor VIIa with constitutively
expressed TF or when systemic (eg, intravascular sepsis and
endotoxemia) or local inflammatory stimuli induce TF on
monocyte/macrophages or endothelial cells.
Potential interactions between coagulation and inflammation have
received renewed attention since the cloning of 3 inflammatory cell
surface-based receptors, proteinase activated receptor-1 (PAR-1),3 effector cell protease receptor-1
(EPR-1),4 and TF,5 that interact with
circulating serine proteases (thrombin, factor Xa, and factor VIIa,
respectively). A proinflammatory role for EPR-1 in vivo has been
suggested by the demonstration of factor Xa induced paw
edema6 and the prevention of graft-versus-host disease by
blocking the receptor7 in mice. Binding of thrombin to
PAR-1 triggers intracellular calcium fluxes8,9 and induces proinflammatory effects in vitro.10 Proliferative and
proinflammatory responses to thrombin have been demonstrated in
endothelial cells11 and macrophages,12
respectively. Direct proinflammatory effects of TF on cells have not
previously been demonstrated.
TF has an extracellular domain of 219 amino acids5 that
shares significant structural homology with the cytokine receptor superfamily and is most closely related to interleukin-10 (IL-10) and
interferon- Studies using anti-TF antibodies and tissue factor pathway inhibitor
(TFPI; which binds and inactivates factor Xa and TF/factor VIIa
complexes) provide evidence for proinflammatory effects associated with
extrinsic coagulation pathway activation. Treatment with an anti-TF
antibody inhibited coagulation and fibrin formation and reduced
inflammation in LPS-induced septic shock.17 In experimental glomerulonephritis (GN), treatment with anti-TF antibodies reduced indices of glomerular inflammation including proteinuria and major histocompatibility complex (MHC) class II expression in addition to
reducing glomerular fibrin deposition.18 Treatment with
TFPI reduced injury in spinal cord ischemia19 and
experimental GN.20 These studies suggest the potential for
TF to enhance inflammation by direct cellular activation in addition to
its procoagulant function.
Despite evidence suggesting close links between the activation of
inflammation and coagulation, no direct effects of TF on the function
of inflammatory cells have yet been demonstrated. Reactive oxygen
species (ROS) are inflammatory effector molecules produced by various
cells, including activated monocyte/macrophages. They have important
intracellular functions in cell signaling and killing pathogens and,
when released extracellularly, are potent effectors of inflammatory
tissue injury. In the current studies, the potential of TF/factor VIIa
to act as a cell signaling receptor/ligand system that induces
intracellular calcium fluxes and ROS production (as a marker of
inflammatory activation) was studied in monocyte/macrophages.
Human monocyte-derived macrophages.
Peripheral blood mononuclear cells (PBMCs) were prepared from healthy
human volunteers by density centrifugation (Ficoll-Hypaque; Pharmacia
Biotech, Uppsala, Sweden) of citrated venous blood. Cells were washed
twice in phosphate-buffered saline (PBS), resuspended at 1 × 106/mL in serum-free Dulbecco's Minimal Essential Medium
(DMEM), and incubated for 1 to 24 hours at 37°C in a 5%
CO2 atmosphere in the presence and absence of LPS. After
culture, supernatants were aspirated and adhered cells were harvested
by flushing with DMEM at 4°C. Cell viability, assessed by flow
cytometry using propidium iodide exclusion, was greater than 97% in
all experiments. LPS contamination of medium from LPS-free cultures was
undetectable (<15 pg/mL) using the limulus amebocyte lysis assay.
Measurement of TF expression.
TF expression was assessed by flow cytometry as previously
described.21 Cells were labeled with a monoclonal mouse
antihuman TF antibody (#4504; American Diagnostica, Greenwich, CT),
followed by a fluorescein isothiocyanate (FITC)-conjugated sheep
antimouse Ig antibody (Silenus, Hawthorn, Victoria, Australia). An
irrelevant isotype-matched mouse monoclonal antibody was used as a
control for the anti-TF antibody.
Measurement of ROS.
ROS production was assessed as described previously.22
After culture, PBMCs were resuspended at 5 × 105/mL
in PBS containing 4% fetal calf serum (FCS). Cells were incubated at
37°C for 10 minutes with phorbol myristate acetate (PMA; 500 ng/mL;
Sigma Chemicals, St Louis, MO) or normal saline (to assess spontaneous non-PMA-triggered ROS production), and then
dihydro-rhodamine 123 (100 ng/mL; Molecular Probes, Eugene, OR) was
added for an additional 10 minutes. Cells were placed on ice before
analysis by flow cytometry. PMA-triggered ROS production was expressed as the difference in mean fluorescence between PMA-stimulated and
saline-treated cells.
Factor VIIa-induced ROS augmentation.
PBMCs were incubated under the serum-free conditions described above
with purified human recombinant factor VIIa (American Diagnostica) at a
range of concentrations from 0 to 2.5 µg/mL in the presence or
absence of 0.5 µg/mL LPS (Sigma Chemicals). ROS production was
measured as described above. The ligand specificity of the response was
assessed by incubation with purified native factor VII (2.5 µg/mL;
American Diagnostica) and factor VIIa (2.5 µg/mL), in which the
active site was irreversibly inactivated with 1,5-dansyl-Glu-Gly-Arg
chloromethyl ketone (DEGRck; Calbiochem, San Diego, CA), as
previously described.23
Measurement of intracellular free calcium fluxes.
Intracellular Ca2+ fluxes were measured by flow cytometry
in PBMCs loaded with Pluronic F and Fluo-3 (Molecular Probes), as previously described.24 PBMCs (1 × 107
cells/mL) cultured with LPS (0.5 µg/mL for 24 hours, as described above) were incubated in RPMI medium (ICN Biomedicals, Aurora, OH)
containing 1% FCS, Pluronic F 127 (1 µg/mL), and Fluo-3 (3 µmol/L
in 0.25% dimethyl sulfoxide; Sigma Chemicals) for 45 minutes at
37°C to allow Fluo-3 loading. Cells were washed twice in RPMI at
room temperature to remove extracellular Fluo-3 and were then allowed
to equilibrate to 37°C for 10 minutes. At the end of this time,
stable baseline fluorescence over a 20-second period was confirmed by
flow cytometry. Baseline intracellular [Ca2+] was
calculated from the fluorescence at the end of this 20-second period.
Analysis was then interrupted to allow addition of potential agonists
(<2-second delay) and fluorescence signals were reacquired after a
20-second delay. Analysis was then continued for a further 70 seconds
(~90 seconds after the addition of agonists). The fluorescence units
were converted into Ca2+ concentrations by a nondisruptive
calibration procedure using a nonfluorescent calcium ionophore
Bromo-A23187 (10 µmol/L; Molecular Probes) followed by quenching with
manganese chloride (2 mmol/L; Sigma Chemicals), as previously
described.25 The following potential ligands were assessed:
factor VIIa (2.5 µg/mL), native factor VII (2.5 µg/mL), and
site-inactivated factor VIIa (2.5 µg/mL). Receptor specificity was
assessed by measurement of factor VIIa (2.5 µg/mL) -induced
Ca2+ fluxes in the presence of anti-TF antibody (5 µg/mL)
or control antibody. Ca2+ fluxes induced by factor VIIa,
factor VII, and factor VIIa in the presence of anti-TF antibody were
also assessed on cells that were allowed to equilibrate to 37°C for
15 minutes after fluo-3 loading. To investigate the role of second
messengers, factor VIIa-induced Ca2+ fluxes were studied in
the presence of a tyrosine kinase inhibitor (herbimycin A; 3 µmol/L;
Sigma Chemicals) or a phospholipase C inhibitor U73122 (5 µmol/L;
Biomol, Plymouth Meeting, PA) or its inactive analog (U73343; 5 µmol/L; Biomol) added 3 minutes before analysis of the basal
fluorescence level. Ca2+ fluxes were also measured in cells
incubated with these inhibitors for 24 hours.
Transfection of TF into U937 cells.
Oligonucleotide primers were used to obtain full-length and truncated
TF DNA constructs from human TF cDNA5 by the polymerase chain reaction (PCR) using Pfu polymerase. The truncated construct comprised the complete sequence for the extracellular and transmembrane regions and the sequence for only the first 5 amino acids of the intracytoplasmic domain to facilitate membrane anchoring. The sequence
for the terminal 16 amino acids of the 21 in the cytoplasmic tail was
deleted. These PCR products were cloned into a mammalian expression
vector (pcDNA3.1) containing a cytomegalovirus promoter, and the
sequences were confirmed by automated sequencing (ABI Prism; PE
Biosystems, Foster City, CA). Vector DNA (containing the TF cDNA
constructs) was linearized and transfected into a human monocytic cell
line (U937 cells) by electroporation. Transfected cells expressing
stable, high levels of TF on their cell membrane (assessed by flow
cytometry as described above) were selected by repeated passage of
cells in RPMI with G418 (400 µg/mL). The capacity of human factor
VIIa to induce intracellular Ca2+ fluxes was investigated
as described above. In contrast to PBMCs, U937 Ca2+ fluxes
were measured at 20°C.
Peritoneal elicited macrophages.
New Zealand White rabbits (2.0 to 2.3 kg; Monash University, Central
Animal Services, Clayton, Victoria, Australia) were injected intraperitoneally with 50 mL of 3.8% thioglycolate (Becton Dickinson, Cockeysville, MD) and treated intravenously with either functionally inhibitory sheep antirabbit TF globulin (n = 6) or normal sheep globulin (NSG; n = 6). Treatments were administered 6 hours (50 mg/kg),
30 hours (100 mg/kg), and 54 hours (50 mg/kg) after thioglycolate injection. Peritoneal exudate cells were harvested by lavage with Eagles medium (ICN Biomedicals) containing 1% FCS and 3.3% sodium citrate, 72 hours after administration of thioglycolate. Red blood cells were removed by water lysis for 30 seconds. Cells were then washed and resuspended in PBS with 4% FCS and counted using a hemocytometer. Peritoneal exudate cells were greater than 85% positive
by nonspecific esterase staining26 and had a viability of
greater than 97% by flow cytometry using propidium iodide exclusion. Flow cytometry was used to assess macrophage activation by their capacity to produce ROS after PMA stimulation (as described above), their expression of MHC class II using a monoclonal anti-rabbit MHC
class II antibody (2CAB12),27 and their expression of Study design and statistical methods.
Blood monocytes were collected from a group of 15 volunteers. All human
monocyte-derived macrophage experiments were performed in duplicate on
2 or more separate occasions on cells from a minimum of 5 individuals
for each parameter. Differences between groups were analyzed by the
ANOVA with post-hoc analysis of Fisher's protected least significant difference.
TF expression is upregulated during macrophage maturation.
Monocytes expressed low levels of TF (14 ± 3 mean fluorescence
units [mfu]) immediately after density separation (control). TF
expression increased significantly after adherence to plastic in the
absence of LPS (87 ± 16 mfu at 4 hours and 225 ± 9 mfu at 24 hours, both P < .001 compared with control). In the
presence of LPS (500 ng/mL), adherent monocyte-derived macrophages
showed greater enhancement of TF expression at 4 hours (116 ± 13 mfu) and 24 hours (512 ± 32 mfu, P = .047) as compared with
cells cultured in the absence of LPS. There was a significant
correlation between TF expression and PMA-triggered ROS production of
macrophages cultured under serum-free conditions (coefficient of
correlation, R2 = .849; P = .0239; Table 1).
Factor VIIa augments macrophage ROS production in a time- and
dose-dependent manner.
Adherence of monocyte-derived macrophages under serum-free conditions
resulted in a rapid initial augmentation of their PMA-stimulated ROS
production. At 1 hour in culture, ROS production was not significantly influenced by the presence of factor VIIa (2.5 µg/mL) or LPS
(Fig 1A). Subsequent ROS production in the
absence of factor VIIa and LPS remained stable for the following 23 hours. In the presence of LPS and absence of factor VIIa, ROS
production was also stable between 1 and 4 hours, but showed a small
significant increase at 24 hours (P < .008, compared with
that at 1, 2, and 4 hours). Factor VIIa in the absence of LPS induced a
marked increase in ROS production between 1 and 24 hours
(P = .0015). This effect was accentuated in the
presence of LPS, where ROS production was significantly augmented by
factor VIIa at 2 hours (P = .0004), 4 hours (P < .0001), and 24 hours (P < .0001) compared with 1 hour. In the
absence of PMA triggering, factor VIIa still produced a significant
increase in spontaneous ROS production at 24 hours (P < .001;
Fig 1B).
Factor VIIa-induced augmentation of ROS production is dose-dependent.
At 24 hours in the presence of LPS, there were significant increases in
ROS production with each increment of factor VIIa concentration from 0 to 0.5 µg/mL (P = .025), from 0.5 to 1.0 µg/mL (P = .001), and from 1.0 to 2.5 µg/mL (P < .0001). In the absence of LPS, significant increases in ROS production also occurred with each dose increment. The relative increase in response to the
maximal dose of factor VIIa (2.5 µg/mL) was similar in the presence
and absence of LPS (108% and 102%, respectively, above control;
Fig 2).
Factor VIIa-dependent ROS production in monocyte-derived macrophages
requires protein synthesis.
Culture in the presence of cycloheximide prevented factor VIIa
augmentation of ROS production at 2, 4, and 24 hours. Basal ROS
production by monocyte-derived macrophages (after 1 hour of adherence)
was 63 ± 5 mfu, and ROS production increased to 208 ± 5 mfu
after 24 hours of culture in the presence of LPS and factor VIIa (2.5 µg/mL). This increase was prevented by the addition of cycloheximide
(60 ± 8 mfu). These studies demonstrate that augmentation of ROS
production in the presence of factor VIIa requires a process involving
active protein synthesis by monocyte-derived macrophages. Because
cycloheximide is a nonselective inhibitor of protein synthesis, the
particular proteins involved in this process remain to be defined.
TF/factor VIIA augmentation of ROS is ligand and receptor specific.
Although factor VIIa (2.5 µg/mL) significantly augmented ROS
production, no change in ROS production was observed with native factor
VII or inactivated VIIa at the same concentration
(Fig 3). Anti-TF antibody abolished factor
VIIa-induced augmentation of ROS production, whereas no inhibition was
observed with a control antibody. The ability of anti-TF antibody to
abolish the response to factor VIIa and the absence of response to
inactivated factor VIIa demonstrates that this response is not
attributable to endotoxin.
Thrombin and factor Xa do not contribute to factor VIIa-induced ROS
production.
Experiments were performed in the presence of hirudin or dalteparin to
exclude a role for endogenous thrombin or factor Xa generation,
respectively, in the augmentation of monocyte-derived macrophage ROS
production by factor VIIa after 24 hours of serum-free culture with
LPS. The presence of hirudin did not effect factor VIIa augmentation of
ROS production (factor VIIa [2.5 µg/mL] + hirudin, 196 ± 14 mfu; factor VIIa + control, 205 ± 23 mfu). Hirudin alone in the
absence of factor VIIa did not effect ROS production (hirudin alone,
110 ± 13 mfu; control, 100 ± 10 mfu), but this dose of hirudin
abolished thrombin-induced augmentation of ROS production (thrombin [1
U/mL], 154 ± 11 mfu; thrombin + hirudin, 108 ± 8 mfu;
P < .001). Similarly, dalteparin, a direct factor Xa
inhibitor, did not inhibit augmentation of ROS production by high doses
of factor VIIa (2.5 µg/mL; factor VIIa + dalteparin, 219 ± 12 mfu; factor VIIa + control, 213 ± 5 mfu) or lower doses of factor
VIIa (0.5 µg/mL; factor VIIa + dalteparin, 146 ± 10 mfu; factor
VIIa + control, 126 ± 5 mfu). Dalteparin alone in the absence of
factor VIIa did not effect ROS production (dalteparin alone, 91 ± 7 mfu; control, 100 ± 10 mfu), but did abolish augmentation of ROS
production induced by 5 µg/mL of factor Xa (factor Xa alone, 127 ± 10 mfu; factor Xa + dalteparin, 100 ± 5 mfu; P = .03)
and 25 µg/mL of factor Xa (factor Xa alone, 185 ± 13 mfu; factor
Xa + dalteparin, 85 ± 7 mfu; P = .001).
TF/factor VIIa interactions induce intracellular calcium fluxes in
monocyte-derived macrophages.
Factor VIIa (2.5 µg/mL) induced rapid Ca2+ fluxes in
macrophage-derived monocytes from a baseline intracellular
Ca2+ concentration of 54 ± 8 nmol/L. The percentage of
responding cells was greater than 98%. Native factor VII (2.5 µg/mL)
and inactivated factor VIIa (2.5 µg/mL) did not induce
Ca2+ fluxes (Fig 4A). Factor
VIIa-induced Ca2+ fluxes were abolished in the presence of
anti-TF antibody but unaffected by control antibody (data not shown).
Ca2+ fluxes were also abolished in the presence of the
phospholipase-C-
The cytoplasmic tail of TF is required for factor VIIa-induced
Ca2+ fluxes.
U937 cells expressed low constitutive levels of TF (49 ± 5 mfu),
but significantly enhanced their TF expression (250 ± 31 mfu;
P < .001) after 24 hours of culture in the presence of LPS. U937 cells transfected with cytoplasmic truncated tail or full-length TF DNA constructs under control of a cytomegalovirus promoter expressed
high constitutive levels of TF expression (212 ± 43 and 180 ± 22 mfu, respectively) that were not significantly different from the
levels in LPS-stimulated U937 cells. Factor VIIa (2.5 µg/mL) induced
intracellular Ca2+ fluxes in LPS-treated U937 cells and
cells expressing full-length TF but not in unstimulated U937 cells or
cells expressing TF in which the cytoplasmic tail had been truncated
(Fig 5).
Anti-TF antibody attenuates activation of peritoneal macrophages in
vivo.
In vivo inhibition of TF with a functionally inhibitory sheep
antirabbit TF antibody attenuated activation of rabbit macrophages recruited in response to intraperitoneal injection of thioglycolate. TF
antibody treatment did not affect the recruitment of cells (total cell
harvest at 72 hours: anti-TF-treated, 1.09 ± 0.11 × 107 cells; control, 1.06 ± 0.09 × 107
cells) or cell viability (>97% in both groups). However, the mean
cell size of peritoneal macrophages (measured by flow cytometry using
the mean forward angle light scatter signal) in rabbits treated with
anti-TF antibody was reduced by 40% (treated, 34 ± 4; control, 55 ± 6; P < .0001). Cell granularity (measured by the mean
right angle light scatter signal) was also significantly reduced
(treated, 23 ± 3; control, 27 ± 3; P = .04). These
characteristics indicate a less activated phenotype of recruited
peritoneal macrophages in rabbits after in vivo inhibition of TF.
Treatment with anti-TF antibody also significantly reduced macrophage
ROS production after PMA triggering (treated, 50 ± 15 mfu; control,
166 ± 15 mfu; P = .03). Expression of MHC II (treated, 23 ± 5 mfu; control, 80 ± 25 mfu; P = .04) and CD18
(treated, 43 ± 9 mfu; control, 189 ± 58 mfu; P = .02)
by elicited macrophages was significantly reduced in anti-TF
antibody-treated rabbits (Fig 6).
The current studies demonstrate that binding of TF to its natural
ligand, factor VIIa, results in proinflammatory phenotypic changes in
vitro in human monocyte-derived macrophages and in vivo in rabbit
peritoneal macrophages. Circulating monocytes do not express
TF.2 However, after maturation to a macrophage phenotype in
vitro by adherence both in the presence and absence of endotoxin, or in
vivo after recruitment across the peritoneal membrane, monocytes were
induced to express TF. In these TF-expressing cells, factor VIIa
augmented ROS production in vitro and anti-TF antibodies inhibited ROS
production in vivo.
The authors thank Dr Chris Mitchell for critically reviewing the manuscript.
Submitted December 11, 1998; accepted July 15, 1999.
Supported by grants from the National Health and Medical Research
Council of Australia (NH&MRC) and the Australian Kidney Foundation.
M.A.C. is the recipient of a NH&MRC Medical Postgraduate Research Scholarship.
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 correspondence to Peter G. Tipping, MD,
Monash University, Department of Medicine, Monash Medical Centre, 246 Clayton Rd, Clayton 3168, Victoria, Australia; e-mail:
peter.tipping{at}med.monash.edu.au.
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TOP
ABSTRACT
INTRODUCTION
, -
, and -
receptors.
)
Values for cells cultured in the presence of LPS; (
) values for
cells cultured in the absence of LPS. Continuous lines show values in
the presence of factor VIIa (2.5 µg/mL); dotted lines show values in
the absence of factor VIIa. ROS production is expressed as
mfu.
) and truncated TF DNA
sequences (
) under a cytomegalovirus promoter. Profiles show a
representative response for each cell clone.
RI