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Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3413-3420
Tissue Factor and Factor VIIa Receptor/Ligand Interactions Induce
Proinflammatory Effects in Macrophages
By
Malcolm A. Cunningham,
Pauline Romas,
Paul Hutchinson,
Stephen R. Holdsworth, and
Peter G. Tipping
From the Centre for Inflammatory Diseases, Monash University,
Department of Medicine, Monash Medical Centre, Clayton, Victoria,
Australia.
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ABSTRACT |
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.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
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- , - , and - receptors.13 The
transmembrane domain consists of 23 amino acids. The intracytoplasmic
tail of TF is short, comprising only 21 amino acids, with 3 serine
residues that are potential phosphorylation sites.14 Unlike
PAR-13 and EPR-1,4,15 TF is not constitutively
expressed on leukocytes or endothelial cells. Adherence and
proinflammatory stimuli such as lipopolysaccharide (LPS) and
interferon- can induce TF expression.16
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.
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MATERIALS AND METHODS |
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.
The cellular composition of mononuclear cell preparations, assessed by
flow cytometry, after ficol density separation showed 75% ± 4%
macrophages, 20% ± 3% CD3+ T cells, and 5% ± 1%
CD19+ B cells. After adherence for 60 minutes, macrophages
were further enriched. This adherent population comprised 85% ± 2% macrophages, 12% ± 1% CD3+ T cells, and 3% ± 1% CD19+ B cells. Cell ratios did not alter significantly
with further adherence up to 24 hours. Macrophage-specific parameters
(below) were analyzed by selectively gating on macrophages, defined by their characteristic forward and 90° light scatter on flow cytometry.
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
The receptor specificity of the factor VIIa-induced response was
determined by incubation in the presence of a monoclonal mouse
antihuman TF antibody (5 µg/mL) or an irrelevant isotype-matched monoclonal antibody. Incubations were also performed in the presence of
2 mg/mL hirudin (Ciba Pharmaceuticals, Basel, Switzerland) to exclude
thrombin/PAR-1-mediated effects. This dose of hirudin abolished
thrombin (1 U/mL; Armour Pharmaceutical Co, Kaneke, IL) -stimulated
augmentation of macrophage ROS production. Incubations were also
performed in the presence of 0.2 U/mL dalteparin (Fragmin; Pharmacia
and Upjohn Pty Ltd, Rydalmere, New South Wales, Australia) to exclude
potential effects of endogenous factor Xa production via EPR-1. This
dose of dalteparin completely inhibited the augmentation of macrophage
ROS production induced by 5 µg/mL of purified human factor Xa
(American Diagnostica). The requirement for protein synthesis for PBMC
augmentation of factor VIIa-induced ROS production was assessed by
culture of PBMCs in the presence of 500 µmol/L cycloheximide
(Molecular Probes).
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 2 cell adhesion molecules, using a monoclonal antibody to the common integrin chain (CD18; 60.3; Bristol Myers Squibb Pharmaceutical Research Institute, Seattle, WA).28
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.
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RESULTS |
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).
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Table 1.
TF Expression and ROS Production by Nonadhered Human
Monocyte-Derived Macrophages After Density Sedimentation (Control) and
After Culture and Adherence Under Serum-Free Conditions (and the
Absence of Factor VIIa)
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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).
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| Fig 1.
ROS production by adhered human monocyte-derived
macrophages after 1, 2, 4, and 24 hours in serum-free culture in the
presence and absence of factor VIIa. (A) PMA-triggered ROS production.
(B) Spontaneous ROS production, measured without PMA triggering. ( )
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.
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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).
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| Fig 2.
The effect of increasing doses of factor VIIa on
PMA-triggered ROS production by monocyte-derived macrophages cultured
for 24 hours in serum-free media in the presence of LPS () and the
absence of LPS ().
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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.
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| Fig 3.
The ligand and receptor specificity of factor
VIIa/TF-stimulated monocyte-derived macrophage ROS production after 24 hours of coincubation with LPS in serum-free media. Factor VIIa induced
significant augmentation of ROS production compared with the absence of
factor VIIa (control-no VIIa), but augmentation was not observed with
native factor VII or active site inactivated factor VIIa. Augmentation
of ROS production by factor VIIa was abolished in the presence of
anti-TF antibody (VIIa + anti-TF Ab) but was unaffected by a control
antibody (VIIa + control Ab). *P < .0001 compared with
control.
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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- inhibitor, U73122, but were unaffected by its
inactive analog (U73433) or the tyrosine kinase inhibitor, herbimycin
(data not shown). These Ca2+ fluxes were studied 10 minutes
after the completion of fluo-3 loading to minimize leakage of
Ca2+ into cytosolic compartments. The magnitude of the
Ca2+ fluxes induced by factor VIIa was consistent with
fluxes measured by other techniques that measure specific intracellular
Ca2+ in macrophages.29
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| Fig 4.
Ca2+ fluxes induced by factor VIIa in
monocyte-derived macrophages. (A) Cells equilibrated for 10 minutes
after fluo-3 loading. (B) Cells equilibrated for 15 minutes after
Fluo-3 loading. Solid circles and continuous lines represent the
response to addition of factor VIIa (2.5 µg/mL) added at 2 seconds
(arrowed). Open circles and dotted lines represent the response to
native factor VII (2.5 µg/mL). Triangles and broken lines represent
responses to factor VIIa (2.5 µg/mL) when the cells were coincubated
with anti-TF for 24 hours before the assessment of Ca2+
fluxes.
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Increasing the delay in analysis of Ca2+ fluxes after
fluo-3 loading or increasing the permeability of the cells results in higher apparent baseline Ca2+ concentrations and larger
apparent Ca2+ fluxes.29,30 In the current
studies, equilibration of monocyte-derived macrophages to 37°C for
15 minutes after fluo-3 loading resulted in higher baseline
Ca2+ concentrations (152 ± 64 nmol/L) and greater
apparent Ca2+ fluxes in response to factor VIIa (2.5 µg/mL) than cells equilibrated for 10 minutes (Fig 4B).
Ca2+ fluxes in cells equilibrated for 15 minutes did not
occur in response to factor VII or to factor VIIa in the presence of
anti-TF antibody.
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).
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| Fig 5.
Factor VIIa induced Ca2+ fluxes in
unstimulated U937 cells (), LPS-stimulated U937 cells (), and
U937 cells transfected with full-length ( ) and truncated TF DNA
sequences ( ) under a cytomegalovirus promoter. Profiles show a
representative response for each cell clone.
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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).
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| Fig 6.
Effect of anti-TF antibody and control antibody treatment
on the activation of rabbit elicited peritoneal macrophages in vivo.
Panels show production of ROS, expression of MHC II antigen (MHC II)
and the expression of the integrin chain (CD18) of leukocyte
adhesion molecules.
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| |
DISCUSSION |
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.
In addition to its recently described role in intracellular
signaling,31 ROS products are important effector molecules
for macrophage inflammatory functions. Macrophage ROS production is involved in the killing of intracellular pathogens,32
ischemia reperfusion injury (where ROS products are associated with
increased TF expression in coronary lesions),33 and
macrophage-mediated tissue injury.34,35 Factor VIIa, in the
absence of other coagulation proteins, augmented the capacity of
macrophages to produce ROS in a time- and dose-dependent manner. Thus,
direct TF/factor VIIa-mediated effects on macrophages have the
potential to enhance killing of intracellular pathogens and exacerbate
tissue injury in a variety of situations in which ROS are important mediators.
Previous studies have provided limited evidence for the capacity of TF
to modulate cellular functions such as increased poly(A) polymerase
mRNA production in fibroblasts,36 translocation of TF to
the caveolae in transformed human endothelial cells,37 and
enhanced metastatic potential of human melanoma cell
lines.38 A potential role in embryonic angiogenesis was
suggested by studies of TF gene in knock-out mice.39
However, failure of hemostatic function has been suggested as
alternative explanation for this observation,40 and recent
studies have demonstrated angiogenesis may proceed normally in some
TF-deficient mice.41 The current studies provide the first
evidence for direct modification of inflammatory cell function by
TF/factor VIIa-mediated signaling.
Augmentation of ROS production by factor VIIa was specific for the
proteolytically active ligand. Responses did not occur with the zymogen
factor VII or with factor VIIa in which the enzymatic site had been
inactivated. Furthermore, the ability of a specific monoclonal antibody
to human TF to inhibit the factor VIIa-induced response demonstrates
its receptor specificity. Plasminogen has recently been demonstrated as
an alternative ligand for TF, which binds independently of factor
VIIa.42 Plasminogen is unlikely to contribute to the ROS
augmentation in the serum-free conditions of the current studies.
Macrophages have the capacity to produce a prothrombinase
complex,43 and in some reports their potential to produce
prothrombin has been suggested.44 The ability of factor
VIIa to augment ROS production in our studies was unaffected by the
presence of hirudin, excluding any role for thrombin in these effects.
The absence of serum and the use of recombinant factor VIIa in these studies exclude exogenous sources of factors, such as Xa, as ligands for their receptors. It is possible that monocytes may produce factor
Xa in culture. However, factor VIIa-augmented ROS production was
unaffected in the presence of the direct factor Xa inhibitor dalteparin,45 excluding a contribution of any endogenous
factor Xa/EPR-1-mediated effects to this response.
Maximal ROS responses to factor VIIa were induced at a concentration of
2.5 µg/mL, which is the same as that required for other effects
reported in fibroblasts and monocytes.36,46 Smaller responses were induced by factor VIIa at a concentration of 0.5 µg/mL, which is the concentration of factor VII in plasma. The local
concentration of factor VIIa generated after activation of factor VII
at the endothelial cell surface is unknown. The in vivo sensitivity of
this receptor/ligand system may also vary according to the local
density of TF expression and other factors. Therefore, it is difficult
to directly extrapolate the in vitro dose response curve to in vivo
conditions. However, the ability of anti-TF antibody to block
macrophage activation in vivo points to the in vivo physiological
relevance of TF/factor VIIa interactions.
Factor VIIa augmentation of ROS production was associated with rapid
induction of intracellular Ca2+ fluxes after
ligand/receptor binding. The short loading time for fluo-3 allows more
specific assessment of intracellular Ca2+ fluxes by
minimizing extracellular leakage of the fluorophore. These
intracellular Ca2+ fluxes are similar in magnitude to those
measured using flow cytometry after thrombin stimulation of human
endothelial cells47 and are consistent with other reports
of specific intracellullar Ca2+ fluxes.29
Because of its ability to measure specific cell-associated fluorescence, flow cytometry using fluo-3 produces lower apparent Ca2+ fluxes than does spectrofluorimetry.48
Increasing the loading time for fluo-3, which increases its
extracellular leakage, results in higher baseline cytosolic
Ca2+ concentrations and larger apparent Ca2+
fluxes.30,48
Inhibitor studies demonstrated the requirement for
phosho-inositol-specific phospholipase C, but not tyrosine kinase,
suggesting that TF/factor VIIa-induced Ca2+ fluxes in
macrophages are mediated by a G-protein-coupled
phospho-inositol-specific phospholipase C pathway. Binding of TF with
factor VIIa has also been demonstrated to induce intracellular
Ca2+ fluxes (but not functional effects) in human umbilical
vein endothelial cells (HUVECs) and Madin-Darby canine kidney
cells.30 Ca2+ fluxes induced in
monocyte-derived macrophages were ligand and receptor specific, as
demonstrated by the inability of native factor VII and inactivated
factor VIIa to induce Ca2+ fluxes and the ability of
anti-TF antibody to prevent factor VIIa responses. Specificity for
proteolytically active factor VIIa in initiating Ca2+
fluxes has also demonstrated in Madin-Darby cells30 and
TF-dependent metastatic behavior of Chinese hamster ovary (CHO)
cells.49 Although factor VII and factor VIIa bind to the
same site on TF, catalytic activation of factor VIIa by TF leads to
increased binding affinity and enhanced factor X
hydrolysis.50 These allosteric changes may be critical for
TF signaling.
Previous studies have demonstrated that mutation of the cytoplasmic
tail of TF inhibits the metastatic behavior of melanoma cell
lines38 and TF-dependent metastatic behavior of CHO
cells.49 In the current studies, a requirement for the
cytoplasmic tail of TF for factor VIIa-induced Ca2+ fluxes
in macrophages was demonstrated. Other investigators have suggested
that TF does not directly cause Ca2+ fluxes but realigns
factor VIIa and facilitates its interaction and signaling through other
(unidentified) cell surface receptors.30 However,
transfection of U937 cells demonstrated that factor VIIa cannot induce
Ca2+ fluxes when the cytoplasmic tail of TF is truncated to
leave only 5 amino acids in the intracellular domain. Cells expressing truncated TF fail to show Ca2+ fluxes despite levels of
cell surface TF expression similar to those of cells transfected with
full-length TF and LPS-stimulated U937 cells, which respond to factor
VIIa. This does not exclude the possibility that TF may signal by
coupling to a second intracellular receptor system, which has been
suggested by studies demonstrating association of TF with the Fc RI
chain homodimer in human monocytes.46
The in vivo capacity of TF to enhance macrophage activation was
demonstrated in rabbits. Blocking TF/factor VIIa interactions using an
antisheep TF antibody significantly reduced PMA-triggered ROS
production by the elicited peritoneal macrophages. In addition, blocking TF reduced expression of MHC class II and 2 integrin leukocyte adhesion molecules (indicated by reduced expression of the
common chain of these heterodimers), which are additional markers
of macrophage activation. Intraperitoneal administration of anti-TF
antibodies did not reduce macrophage recruitment into the peritoneum or
reduce macrophage viability. However, their reduced size and
granularity on flow cytometry is also an indication of a less activated
(more monocyte-like) phenotype. The lack of effect of TF inhibition on
the transmigration of monocytes into the peritoneum is consistent with
the observation that TF is not expressed on circulating
monocytes.2 These studies demonstrate that factor
VIIa/TF-mediated effects observed using human monocyte-derived macrophages in vitro have significant implications for macrophage activation in vivo.
In summary, this study demonstrates that the binding of cell surface
expressed TF to its active serine protease ligand, factor VIIa,
augments the proinflammatory functions of macrophages both in vivo and
in vitro. These findings demonstrate a role for the TF/factor VIIa
receptor/ligand interaction in augmentation of inflammatory processes.
Modification of the TF/factor VIIa interaction by the naturally
occurring inhibitor TFPI, blocking peptides, or antibodies may allow
novel therapeutic approaches to ameliorate human diseases such as
septic shock, crescentic glomerulonephritis, and atherosclerosis, in
which the induced TF expression on the macrophage cell surface may play
an important pathological role.
| |
ACKNOWLEDGMENT |
The authors thank Dr Chris Mitchell for critically reviewing the manuscript.
| |
FOOTNOTES |
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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T. Syrovets, M. Jendrach, A. Rohwedder, A. Schule, and T. Simmet
Plasmin-induced expression of cytokines and tissue factor in human monocytes involves AP-1 and IKK{beta}-mediated NF-{kappa}B activation
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[Abstract]
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H. Tapper and H. Herwald
Modulation of hemostatic mechanisms in bacterial infectious diseases
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C. T. Esmon
Introduction: are natural anticoagulants candidates for modulating the inflammatory response to endotoxin?
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M. A. Cunningham, E. Rondeau, X. Chen, S. R. Coughlin, S. R. Holdsworth, and P. G. Tipping
Protease-activated Receptor 1 Mediates Thrombin-dependent, Cell-mediated Renal Inflammation in Crescentic Glomerulonephritis
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TOP
ABSTRACT
INTRODUCTION