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Departments of
*
Pharmacology and
Cell Biology, Chiron Technologies, Emeryville, CA 94608; and
Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, and Laboratory for Experimental and Clinical Immunology, University of Amsterdam, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Consistent with a theme of inflammation coincident with thrombosis are observations that anticoagulants may exhibit anti-inflammatory activities in vitro and in vivo (10, 11). For example, circulating levels of cytokines such as IL-6 and IL-8 in animal models of sepsis are significantly attenuated by therapeutic administration of the tissue factor pathway inhibitor (TFPI)2 (12, 13, 14). Subsequent investigations with the inhibitor of the extrinsic pathway of coagulation were aimed at understanding the basis for attenuation of an inflammatory cytokine response by TFPI (15). Ex vivo studies with human whole blood revealed that coagulation activation stimulated a rapid IL-8 response with delayed, lower IL-6 production. Moreover, the combined stimuli of coagulation activation and endotoxin induced an apparent synergistic IL-8 response (15).
The current in vitro studies were undertaken to further study the
potential vascular inflammatory response to coagulation activation and,
hence, to better understand anticoagulant regulatory mechanisms.
Analysis of the potential contribution of thrombin to cellular cytokine
responses was included, since thrombin has a wide spectrum of biologic
activity and anticoagulants typically act via inhibition of thrombin
generation (e.g. TFPI) or activity (e.g. hirudin). Proinflammatory
activities of thrombin may be linked to its ability to costimulate the
release of several inflammatory cytokines, including IL-1, IL-6,
monocyte chemoattractant protein (MCP), and TNF-
by leukocytes, and
melanoma growth-stimulating activity (GRO), MCP, and IL-8 by
endothelial cells (EC) (16, 17, 18, 19, 20, 21, 22, 23). Indeed, the previous whole blood
experiments studying TFPI inhibition of coagulation-stimulated cytokine
production implicated thrombin generation as a lead mediator (15).
Herein, we show that CD14+ monocytes are the cell type
responsible for the human blood cell IL-8 response to coagulation
activation in general and, specifically, to -thrombin. Moreover,
endothelial cells are shown, for the first time, to respond to
stimulation by either recalcified plasma or catalytically active
-thrombin with a dual IL-6 and IL-8 cytokine response in which
thrombin receptors participate.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Magnetic beads for depletion of CD14+ cells
were obtained from Dynal (Lake Success, NY). FITC-labeled Abs for flow
cytometry were obtained from Becton Dickinson (San Jose, CA).
Recombinant yeast hirudin and purified human -thrombin were obtained
from American Diagnostica (Greenwich, CT). Prothrombin was obtained
from Haematologic Technologies (Essex Junction, VT). Recombinant human
TFPI was expressed in Escherichia coli and purified and
refolded as previously
described.3 The TFPI was
formulated at 11 mg/ml in 2 M urea, 20 mM sodium phosphate (pH7.2), and
0.14 M NaCl. Since TFPI concentrations in the current experiments were
<20 µg/ml, the excipient was diluted sufficiently such that no
vehicle effect was ever observed under any experimental condition.
Thrombin receptor control (FSLLRN) or activating (SFLLRN) peptides were
obtained from Chiron Mimotopes (Victoria, Australia) with purity
>94%. Platelet activation by the agonist peptide at concentrations
5 µM, but not by the control peptide, was confirmed via classic
platelet aggregometry.
Cell cultures
The isolation of whole blood and selection of PBMC, and the
subsequent culturing of whole blood or PBMC, were performed as
previously described (15). Blood monocytes were enriched by layering
PBMC over NycoPrep medium and selecting the monocyte band as
described in the manufacturer’s supplied protocol (NycoPharm, Oslo,
Norway). Enriched monocytes were >90% CD45+, >80%
CD14+, and <5% CD67+ by flow cytometric
analysis and were cultured at 10,000 to 20,000 cells/well. Blood
polymorphonuclear leukocytes (PMN), cultured at 1–2 x
105 cells/well, were prepared by layering freshly isolated
EDTA blood over neutrophil isolation medium (NIM) according to the
manufacturer’s recommendations (Cardinal Associates, Santa Fe, NM) and
were >90% CD45+, 
75% CD14+, >85%
CD67+, <2% monocytes (side scatter) by flow cytometric
analysis. Cultured endothelial cells were either the immortalized human
EC-RF cell line (24) or early passage (p. 2–5) HUVEC obtained from
Clonetics (San Diego, CA). All results were reproducible with either
cell source. Endothelial cells were cultured in medium comprised of 1:1
medium 199 (Sigma, St. Louis, MO):RPMI 1640 (Irvine Scientific, Santa
Ana, CA), 10% FCS (Irvine Scientific), 10 µg/ml transferrin, and 10
ng/ml basic fibroblast growth factor (bFGF). Just before stimulation by
various factors, cultures were exchanged into serum-free medium
(OptiMEM, Life Technologies/BRL, Grand Island, NY). Coculturing of
isolated cells with coagulating plasma was accomplished by addition of
freshly isolated normal human citrated plasma, which was recalcified
and added at a 1:4 final concentration in microtiter wells containing
assay medium and cells. Fresh serum culturing was similarly performed
with the exception that the plasma was first recalcified for 20 to 30
min in sterile glass tubes, followed by ringing of the clot and
transfer of the serum to the microtiter wells. All cell culture
durations were 16 to 20 h, which was optimal when considering
either background stimulation or stimulated cytokine production
kinetics (Ref. 15 and data not shown). Notably, the concentration of
FCS in PBMC, monocyte, or EC cultures never exceeded 0.1%, precluding
the support of any significant endotoxin response. As an indication of
the magnitude of EC IL-6 and IL-8 production in response to a positive
control stimulus, LPS addition at a 1 ng/ml optimal concentration in
the presence of 5% FCS typically resulted in IL-8 production ranging
from 1000 to 3000 pg/ml.
Mediator analyses
Measurement of whole blood thrombin:antithrombin (TAT) complexes
as a sensitive index of coagulation activation and the measurement of
cell culture supernatant IL-6 or IL-8 levels was performed by sandwich
ELISA as previously described (15, 25, 26, 27). Elastase-1-antitrypsin
complexes, indicative of neutrophil activation and degranulation, were
measured via sandwich ELISA utilizing mAbs previously described (28).
Supernatants were also routinely analyzed for enzymatic activity
against chromogenic substrates, including Spectrozyme Xa and TH
(thrombin) (American Diagnostica) for correlation with TAT levels
and/or to evaluate residual activity of purified factors (prothrombin,
-thrombin, hirudin, TFPI) added to cultures singly or in
combination.
Data analysis
Values were expressed as mean ± SEM or SD as indicated.
When utilized, statistical analysis was perfomed with a statistical
software package (StatView; Abacus, Berkeley, CA). Differences between
a treatment group and control were assessed via ANOVA using Fisher’s
least significant difference, and significant difference was considered
at p 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Titrations of the
anticoagulant TFPI allowed graded levels of whole blood coagulation.
Significant IL-8 levels are detected when TAT levels, reflecting the
extent of coagulation activation, exceed 100 ng/ml, which is equivalent
to 
1 nM -thrombin formation.
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-thrombin was measured. As shown in
Table I, both recalcified plasma and
-thrombin stimulated an IL-8 response in the unfractionated PBMC
cultures, but not in cultures wherein CD14+ cells were
depleted. Moreover, control cultures consisting of the addition of
fresh serum, or comprised of TFPI addition to prevent clotting in the
recalcified plasma cultures, were unable to mount a significant IL-8
response (Table I). Therefore, peripheral blood CD14+ cells
were clearly responsible for producing IL-8 in response to the complex
coagulation stimulus or to pure -thrombin.
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A). Cytokine
contribution by polymorphonuclear leukocytes (PMN), the other type of
CD14+ blood leukocyte candidate, was excluded by culturing
neutrophils with recalcified plasma wherein coagulation stimulated
elastase release (33.1 ± 2.5 ng/ml fresh serum control vs
68.6 ± 0.1 ng/ml coagulation-stimulated) in the absence of any
detectable cytokine secretion (<10 pg/ml IL-8, IL-6, or TNF-).
Second, thrombin structural constraints for the monocyte IL-8 response
were studied. Addition of the direct thrombin inhibitor, hirudin,
attenuated IL-8 production stimulated by -thrombin stimulation,
implicating an essential contribution by the thrombin catalytic region
(Fig. 2B). Finally, characterization of the cytokine
response was extended to include evaluation of IL-6. As shown in Figure 2C, IL-6 production was reduced in magnitude relative to
IL-8 but was clearly stimulated by both coagulation activation
(recalcified plasma) and purified -thrombin. In addition, the
coagulation- or thrombin-stimulated cytokine response involved new
protein synthesis (Ref. 15 and data not shown). Interestingly,
prothrombin addition was capable of stimulating weak IL-8 and IL-6
responses, which were partially reduced by hirudin and may indicate low
level proteolytic release of thrombin during the culture period (Fig. 2, B and C).
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-thrombin was extended to human EC. Overnight
stimulation of EC by recalcified, clotting plasma, but not fresh human
serum, resulted in a dual IL-8 and IL-6 response (Fig. 3A). As observed with
monocyte cultures, stimulation of IL-8 or IL-6 responses by EC required
-thrombin concentrations >10 nM (Fig. 3, B and
C, and data not shown). The thrombin response was inhibited
by hirudin and was not significantly mimicked by prothrombin (Fig. 3, B and C). Therefore, monocytes and EC
share some features of coagulation- or -thrombin-stimulated cytokine
production (e.g., type/dose of stimuli and anticoagulant attenuation)
but exhibit some differences as well (e.g., that clotting plasma
stimulates EC IL-6 and IL-8 production, whereas the monocyte response
is predominantly IL-8).
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, the agonist (TRAP) peptide, but not
control peptide, at a 50-µM concentration mimicked stimulation of
both IL-8 and IL-6 production by a relatively low concentration of
-thrombin. The requirement for concentrations of agonist peptide in
the micromolar range is typical for many cellular responses for which
the thrombin receptor has been implicated (e.g., Refs. 8, 23, 29).
Accordingly, it appears that the "platelet-type" thrombin receptor
on human EC is capable of mediating a dual IL-8 and IL-6 cytokine
response to thrombin generated during coagulation activation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-thrombin represents a molecular contributor of
such a coagulation-stimulated cytokine program. A new link in understanding the nature of the cross-talk between the cytokine and coagulation cascades has evolved and is centered around IL-6. While activated coagulation components may support IL-6 production by various cell types as discussed herein, the generation of IL-6 may amplify coagulation. We demonstrated that neutralization of IL-6 during a chimpanzee endotoxin response resulted in attenuation of coagulation activation (31). More recently, it was shown that administration of IL-6 to cancer patients resulted in increased circulating levels of TAT complexes and prothrombin activation fragments (32). Whether IL-6 feedback on coagulation activation is direct or indirect via secondary mediators such as induced acute phase response proteins is unclear. Regardless, resolution of such a positive feedback loop underscores the importance of studying communication between the coagulation and cytokine pathways.
Results from the current studies provide additional insight into
mechanisms for amplification of cytokine production by coagulation
activation. Several lines of evidence suggest that the human monocyte
is the predominant cell type responsible for the rapid, strong
production of IL-8 and more delayed and reduced IL-6 release in
coagulating whole blood. First, fresh PBMC cultures incubated with
recalcified plasma recapitulate the cytokine response observed with
whole blood (Ref. 15; Fig. 1
, Table I). Second, selective depletion of
CD14+ cells from PBMC results in essentially complete
abrogation of the IL-8 response to coagulation activation (Table I),
and isolated CD14+ neutrophils do not release
proinflammatory cytokines in response to coagulating plasma. Last,
cultures of monocytes fractionated from fresh PBMC exhibit a cytokine
response to clotting plasma that is characteristic of either
coagulating whole blood or similarly stimulated whole PBMC. Provided
-thrombin represents a mediator for the coagulation-stimulated
response, identification of the monocyte as the key responsive cell
type is consistent with previous reports describing
-thrombin-stimulated IL-1, IL-6, MCP, and TNF release by cultured
PBMC or monocytes (16, 17, 18, 20, 23). Notably, the current direct
correlation of coagulation and thrombin activity along with
fractionation of the responsive cell type (Table II) has not been
previously reported.
Potential participation in a vascular proinflammatory response to
coagulation activation by cell types other than blood monocytes must be
considered. Indeed, human EC secrete both IL-6 and IL-8 in response to
incubation with clotting plasma but not fresh serum or anticoagulated
plasma (Fig. 3A). The potential participation of
smooth muscle cells in a vascular response to coagulation was not
studied in the current experiments. However, smooth muscle cells have
recently been shown to respond to low concentrations of -thrombin
with the production of IL-6 and MCP-1 (21, 33). An additional cellular
component related to a potential feedback relationship between the
cytokine and coagulation pathways can be envisioned via granulocyte
migration and activation in response to local IL-8 release.
The role of -thrombin as a key participant in coagulation-stimulated
proinflammatory cytokine responses by monocytes and EC is apparent from
a variety of data. First, the whole blood cytokine response is
attenuated by the direct thrombin inhibitor, hirudin (15). Second, the
dose-response for TAT formation in the whole blood IL-8 response and
the -thrombin-stimulated PBMC IL-8 response nearly overlap (i.e.,
minimal requirement of 10 nM -thrombin concentration
equivalent) (Figs. 1 and 2, Table I). Third, marked production of IL-6
and IL-8 by monocytes and EC occurs in response to recalcified,
clotting plasma but not fresh serum (Table I, Figs. 2 and 3),
implicating a role by biologically active coagulation proteases such as
thrombin and factor Xa (7). While this is the first report of an EC
proinflammatory cytokine response to coagulating plasma and consisting
of dual IL-8 and IL-6 production, -thrombin, which has been shown to
stimulate GRO and IL-8 production by cultured EC (19, 22), is
identified as a key contributor (Fig. 3, B and C;
Table II). In addition, limited evaluation of factor Xa potential as a
cytokine response-stimulating factor in PBMC and EC cultures revealed
some activity but was not of the magnitude of thrombin (Ref. 15; K.
Johnson unpublished observations). Finally, the qualitative nature of
the coagulation-induced whole blood or thrombin/PBMC cytokine response
is consistent (i.e., IL-8>IL-6>>TNF-; Ref. 15 and data not shown)
with a common mechanism.
While -thrombin may contribute significantly to a
coagulation-stimulated vascular proinflammatory cytokine response, some
elements of the biochemical mechanism for such a response are still
unclear. Hirudin significantly reduces the thrombin response by
monocytes, EC, and SMC (Figs. 2 and 3; Refs. 15, 16, 19, 22, 33), and
active site-modified thrombin is incapable of stimulating cellular
cytokine responses (16, 18, 21, 22), implicating a role of the thrombin
catalytic region. Morever, experiments with thrombin receptor agonist
peptides (TRAP) to invoke participation of the classic tethered ligand
receptor have yielded mixed results. TRAP has shown cytokine-inducing
activity by some cultured cells, including MCP-1 by mesangial cells and
monocytes (18, 23), GRO by HUVEC (19), IL-1 by LPS-costimulated
monocytes (20), and limited IL-6 production by SMC (21). However, TRAP
stimulation of cultured monocytes or EC does not always fully mimic
thrombin efficacy (e.g., Table II; Refs. 21, 23), suggesting that the
classic tethered ligand receptor may not be solely responsible for the
thrombin-stimulated PBMC or EC proinflammatory cytokine response.
Interestingly, a similar conclusion was proposed by Daniel et al. (34)
to explain platelet-derived growth factor (PDGF) production by EC
stimulated with -thrombin and diisopropylfluoro-phosphate
(DFP)-thrombin, but not hirudin-treated -thrombin. Potential
contribution by the loop B region of thrombin, reported to stimulate
monocyte biologic responses (35), was discounted, since we found the
reported peptide to be incapable of stimulating a PBMC IL-8 or IL-6
response (K. Johnson, unpublished observations). Accordingly, we
conclude that stimulation of a monocyte or EC proinflammatory (IL-6 and
IL-8) response by -thrombin requires specific domains including the
catalytic region, involves classic thrombin receptor participation, but
may include additional novel pathways. An additional participant may be
the recently discovered second thrombin receptor (PAR-3), which is
unresponsive to TRAP (36).
In summary, coagulation activation at local thrombotic sites is capable of stimulating a vascular proinflammatory cytokine response consisting of IL-8 and IL-6 release by pertinent cells, including blood monocytes and vascular EC. Thrombin is indicated as a key contributor to such an inflammatory response. Thrombin’s activity can be inhibited by hirudin and likely involves activation of thrombin receptors. Moreover, the coexistence of thrombin and IL-8 at a vascular site of inflammation can potentially amplify an IL-8 cellular response, since thrombin has been shown to cleave a major form of leukocyte- and EC-derived IL-8 (77-residue Ala-IL8) into a more potent neutrophil-activating 72-amino acid form (Ser-IL8–72) (37). Such potential cytokine production and modification at sites of clotting represents an interesting example of cross-talk between host inflammation and coagulation pathways.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: TFPI, tissue factor pathway inhibitor; GRO, melanoma growth-stimulating activity; MCP, monocyte chemoattractant protein; EC, endothelial cell; TAT, thrombin:antithrombin; TRAP, thrombin receptor agonist peptide.
3 T. J. Girard, C. Carr, R. Heeren, M. Gustafson, A. C. K. Chang, F. B. Taylor, Jr., L. B. Hinshaw, A. E. Mast, G. J. Broze, Jr., J. A. Stewart, B. D. Schwartz, W. F. Westlin, and G. R. Galluppi. 1997. Comparing full-length and truncated tissue factor pathway inhibitor in a primate sepsis model. Submitted for publication.
Received for publication June 6, 1997. Accepted for publication January 23, 1998.
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F. B. Taylor Jr, D. J. Stearns-Kurosawa, S. Kurosawa, G. Ferrell, A. C. K. Chang, Z. Laszik, S. Kosanke, G. Peer, and C. T. Esmon The endothelial cell protein C receptor aids in host defense against Escherichia coli sepsis Blood, March 1, 2000; 95(5): 1680 - 1686. [Abstract] [Full Text] [PDF]
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C. T. Esmon Introduction: are natural anticoagulants candidates for modulating the inflammatory response to endotoxin? Blood, February 15, 2000; 95(4): 1113 - 1116. [Full Text] [PDF]
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