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Autocrine Loop of Endothelial Activation as an Intermediate Between Acute and Chronic Inflammation: an Experimental Model Involving Thrombin
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Institut National de la Santé et de la Recherche Médicale Unité 387, Hôpital Sainte-Marguerite, and
Immunotech, a Beckman Coulter Company, Marseille, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-. Addition of physiological
concentrations of exogenous soluble IL-6R (sIL-6R) to
thrombin-activated HUVEC was sufficient to increase the amounts of
MCP-1 produced, but not those of IL-8. These effects could be blocked
by anti-IL-6 or anti-sIL-6R blocking mAb, demonstrating the
existence of an autocrine loop of MCP-1 secretion, involving the
IL-6/IL-6R/gp130 complex on HUVEC. In addition, we identified
IL-8-activated neutrophils as a potential source of sIL-6R because
IL-8 induced IL-6R shedding from the neutrophil membranes and
increased in parallel sIL-6R concentrations in neutrophil
supernatants. Furthermore, addition of neutrophils to
thrombin-activated HUVEC significantly increased MCP-1 secretion, which
could be decreased by blocking IL-6. Thus, thrombin-activated
endothelium may induce a cascade of events characterized by IL-8
secretion, neutrophil local infiltration, and the release of IL-6R
from neutrophil membranes. sIL-6R may then complex with IL-6 and
increase the amount of MCP-1 produced by thrombin-activated
endothelium, favoring monocyte infiltration, and the transformation of
acute into chronic inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This in vivo evidence correlated with in vitro experiments
demonstrating that thrombin exerts proinflammatory activation on
various cells, notably on leukocytes and endothelial cells. Thrombin
has been shown to be directly chemotactic for both neutrophils and
monocytes, and to favor their adhesion to endothelium
(8, 9, 10, 11). These effects are greatly due to the ability of
thrombin to induce type I and type II endothelial activation. Indeed,
thrombin has been shown to induce rapid and transient type I
endothelial activation, independent of protein synthesis, consisting of
P-selectin expression, platelet activating factor, and prostacyclin
secretion (11, 12). Thrombin is also able to induce gene
transcription and protein synthesis of the major proinflammatory
mediators by endothelial cells. Thus, thrombin has been shown to induce
in a PAR-1-dependent way, the expression of the leuko-endothelial
adhesion molecules, endothelial-leukocyte adhesion molecule-1
(ELAM-1), ICAM-1, and VCAM-1, and to favor neutrophil and
monocyte adhesion to endothelium (13, 14). In addition,
thrombin induces endothelial production of the chemokines IL-8 and
monocyte chemotactic protein-1 (MCP-1), which favor leukocyte
activation and migration into inflamed tissues (13, 15, 16 , and our unpublished observations). Interestingly, on
endothelial cells, thrombin acts independently of the classical
proinflammatory cytokines IL-1 or TNF- and appears to directly
activate the transcriptional factor NF-
B (13, 14, 16, 17). Therefore, thrombin appears to be a complete
proinflammatory mediator able to induce vasodilatation, vessel
permeability, and leukocyte extravasation of both neutrophils during
acute inflammation, and mononuclear cells during chronic
inflammation.
IL-6 is also an important cytokine in the first steps of inflammation
(18, 19). IL-6 is a multifunctional cytokine secreted by
various cells, including endothelial cells, and is involved in the
acute phase response and in the immune response through T and B cell
activation. Interestingly, this cytokine demonstrated both pro- and
anti-inflammatory properties in vivo and in vitro
(20, 21, 22, 23, 24). IL-6 functions in cells are mediated through
fixation to a complex receptor, consisting in a transducing protein
gp130 and a ligand protein, the IL-6R (CD126) (25).
Whereas gp130 is present on almost all cell types, IL-6R is
expressed by a limited number of cells, including hepatocytes,
neutrophils, and mononuclear cells (26). However, through
membrane shedding or alternative splicing, IL-6R can be released by
these cells into a soluble form, sIL-6R, that can bound IL-6 and
protect it against enzyme inactivation (27, 28, 29). This
complex can then bind to gp130 on cell membranes and activate cells in
a mechanism called trans-signaling (30). It was
recently demonstrated that through trans-signaling, the
IL-6/sIL-6R complex can induce a proinflammatory phenotype in
endothelial cells that expresses gp130, but not IL-6R
(30, 31). In this study, we asked whether thrombin can
induce IL-6 secretion by endothelial cells, and we studied the possible
mechanisms controlling sIL-6R release by neutrophils, which could
lead to endothelial trans-signaling in an in vitro model
linking thrombosis and inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following materials were purchased: M199 and RPMI
1640 culture medium, FCS, PBS without Ca2+
and Mg2+ (BioWhittaker, Fontenay/Bois, France);
human -thrombin (1000 U/mg, controlled for the absence of HIV,
hepatitis, plasmin, plasminogen, and fibrin degradation products),
FMLP, purified endothelial supplement growth factor from bovine
pituitary gland, polymyxin B sulfate (Sigma, Saint-Quentin-Fallavier,
France); neutralizing rabbit anti-human TNF- polyclonal Ab
(Genzyme, Le Perray en Yvelines, France); and TRAP-14 (consisting of
SFLLRNPNDKYEPF and a control-"scrambled" peptide consisting of
NEFSLPKPFRYLNP; Neosystem Laboratories, Strasbourg, France).
Recombinant human IL-8 and growth-related oncogene- (GRO-) were
obtained from R&D Systems (Abingdon, U.K.); IL-1R antagonist (IL-1Ra)
was a gift from Dr. C. A. Dinarello (University of Colorado Health
Sciences Center, Denver, CO); recombinant human IL-6, Chinese hamster
ovary-derived human sIL-6R, and the neutralizing anti-sIL-6R
mAb (clone PM-1) were provided by Dr. K. Yasukawa (Tosoh, Tokyo, Japan)
(32, 33). Neutralizing anti-human IL-6 mAb or
F(ab')2 (clone AH65) (34),
PE-conjugated anti-IL-6R mAb (clone M91) were obtained from
Immunotech (Marseille, France) and have been previously described
(34, 35).
Cell culture
HUVEC were obtained as previously described (13),
and used on passage 2 or 3. Cells were grown until confluent in 24-well
plates coated with 1% gelatin. The cells were then cultured in M199
containing 20% heat-inactivated FCS, 100 U/ml penicillin G, and 100
µg/ml streptomycin. Forty-eight hours before each experiment,
endothelial growth factor was withheld and the cells cultured in the
same medium containing 10% FCS for the first 24 h, then 5% for
the following 24 h. In some experiments, HUVEC were activated in
the absence of FCS. Thrombin, TRAP-14, the control peptide, IL-6, or
sIL-6R were then added to HUVEC, and after various culture times,
the supernatant from each well was collected, centrifuged, and stored
at -75°C before assay. In some experiments, IL-1Ra (10
µg/ml), anti-TNF- polyclonal Ab (10 µg/ml final dilution),
neutralizing anti-IL-6 mAb (100 ng/ml), or neutralizing
anti-sIL-6R mAb (2 µg/ml) was added to the culture. After
centrifugation, the supernatants were collected and stored at -75°C
for sIL-6R assay. All the experiments were performed in the presence
of polymyxin B (7 µg/ml) to prevent any biological effect due to
endotoxin contamination of the various reagents.
Polymorphonuclear cell (PMN) preparation
PMN were prepared from freshly drawn heparinized blood obtained
from healthy donors, as previously described (13). For
FACS analysis, cells were adjusted to 4 x
106 cells/ml in RPMI 1640 containing 10%
FCS and polymyxin B, then stimulated for 30 min to 24 h with
thrombin, IL-8, GRO-, or control medium, before FACS analysis. At
each time, supernatants were also collected and stored at -75°C for
sIL-6R assay. In some culture experiments, 106
fresh PMN were added to either unstimulated or overnight
thrombin-activated HUVEC, then after 24 h culture, chemokines were
measured in the supernatants.
FACS analysis
For IL-6R surface expression, PMN were directly stained with
PE-conjugated anti-IL-6R mAb (anti-CD126) or with a control
isotype (IgG1; Immunotech). Fluorescence was measured on a FACS
analyzer (XL; Coultronics, Margency, France). Expression of IL-6R
was studied by comparing staining with anti-CD126 mAb to that
obtained with the control IgG1. Cell surface IL-6R expression was
expressed as the percentage of mean fluorescence, as follows: [(mean
experimental anti-CD126 fluorescence - mean experimental IgG1
fluorescence)/(mean control anti-CD126 fluorescence - mean
control IgG1 fluorescence)] x 100.
RNA extraction
Unstimulated and thrombin-activated HUVEC in 25-cm2 culture flasks were directly solubilized in RNA extraction solution (RNAzol-B; BioProbe Systems, Montreuil, France). Total RNA was isolated using chloroform, and precipitated with isopropanol. RNA was quantified by spectrophotometry at 260 nm.
Synthesis of the cDNA
A 25-µl reverse transcription mixture in strand buffer (25 mM Tris-HCl (pH 8.3), 37.5 mM KCl, 1.5 mM MgCl2) contained 4 µg RNA, 0.1 µg oligo(dT)12–18 (Pharmacia LKB Biotechnology, Orsay, France), 0.2 µmol DTT, 13 U of RNase inhibitor (Eurogentec, Angers, France), 400 µM dNTP (BioProbe Systems), 100 U of Moloney murine leukemia virus reverse transcriptase (SuperScript RT; Life Technologies, Eragny, France) and was incubated at 37°C for 60 min.
PCR
PCR amplification of the cDNA using GAPDH primers confirmed that equal amounts of RNA were reverse transcribed. For each condition, three different cDNA concentrations ranging from 32 to 6.4 ng of RNA equivalent concentrations were amplified in 25 µl containing 250 µM dNTP, 2 µM MgCl2, 0.25 U of Taq polymerase (Life Technologies), and 1 µM 5' sense and 5' antisense specific primers for MCP-1 and IL-6 for 30 cycles as previously described (13). Amplification consisted of 5 min at 94°C followed by 30 sequential cycles consisting of 1 min at 94°C, 1 min at 55°C, and 45 s at 72°C, then a final elongation cycle of 10 min at 72°C in a Crocodile II thermal cycler (Appligen, Illkirch, France). Products of PCR (10 µl) were electrophoresed in a 2% agarose gel (Nusieve; Tebu, Le Perray en Yvelines, France), and then, after ethidium bromide coloration, were quantified using densitometry on a gel imager EASY Herolab (Osi, Elancourt, France). As predicted, the amplification product (amplicon) for IL-6 was 260 bp and for MCP-1 was 274 bp.
RT-PCR specific primers
Specific primers were 5'-TCAATGAGGAGACTTGCCTG-3' (sense) and 5'-GATGAGTTGTCATGTCCTGC-3' (antisense) for IL-6; 5'-TCCAGCATGAAAGTCTCTGC-3' (sense) and 5'-TGGAATCCTGAACCCACTTC-3' (antisense) for MCP-1; and 5'-CCACCCATGGCAAATTCCATGGCA-3' (sense) and 5'-TCTAGACCGCAGGTCAGGTCCACC-3' (antisense) for GAPDH (Genset, Paris, France).
Cytokine assays
MCP-1 and IL-8 were measured using a specific ELISA (Quantikine,
R&D Systems). IL-6, sIL-6R, IL-1, IL-1, and TNF- were
measured using specific ELISAs from Immunotech.
Statistical analysis
Cytokine levels were expressed as the mean ± SEM of results obtained from at least three individual experiments performed in triplicate. The data were compared using the paired Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-
HUVEC were cultured in medium alone or with increasing
concentrations of thrombin for 24 h. As shown in Fig. 1
, A and B,
increased concentrations of IL-6 and MCP-1 were obtained with
increasing concentrations of thrombin compared with medium alone. A
plateau was observed for MCP-1 secretion, in the presence of 8 U/ml
thrombin. Thrombin-induced endothelial secretion of IL-6 and MCP-1 was
time-dependent, increasing over 24 h (Fig. 1, C and
D). In addition, TRAP-14, a PAR-1/PAR-2 agonist peptide,
induced both IL-6 (Fig. 1E) and MCP-1 (Fig. 1F)
secretion in a dose-dependent fashion, whereas a control-scrambled
peptide had no significant effect. When HUVEC were cultured with
thrombin and saturating concentrations of either IL-1Ra or neutralizing
anti-TNF- Ab, thrombin-induced IL-6 secretion was not modified
(data not shown, n = 4). Similarly, neither IL-1Ra nor
anti-TNF- significantly decreased thrombin-induced MCP-1
production (thrombin: 24 ± 2 ng/ml; + IL-1Ra (10 µg/ml):
21 ± 2 ng/ml; + anti-TNF-: 28 ± 3 ng/ml;
n = 4), whereas in parallel experiments, IL-1Ra
significantly inhibited IL-1-induced IL-6 and MCP-1 production by HUVEC
(86% inhibition, p < 0.01). In agreement with these
data, no significant concentration of IL-1 and TNF- could be
detected in thrombin-activated HUVEC supernatants (levels below the
detection limits of the assays: 15 pg/ml for IL-1, 50 pg/ml for
IL-1, and 5 pg/ml for TNF-, n = 4, data not
shown).
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Using RT-PCR, IL-6 mRNA was not detected in unstimulated HUVEC,
but steady-state levels rapidly increased after 1–2 h of thrombin
stimulation, reached a maximum after 6 h, and decreased to almost
baseline levels after 24 h (Fig. 2).
MCP-1 mRNA was not detected in unstimulated HUVEC, and steady-state
levels increased after 2 h of thrombin stimulation, with a maximum
after 6 h, and maintained sustained levels over 24 h
(Fig. 2).
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, induced IL-6R shedding from
neutrophils
Because IL-6 in combination with sIL-6R has been reported to
activate endothelial cells (30), we looked for a possible
source of sIL-6R production in this model. To define a complete
inflammatory cascade, we asked first whether thrombin could induce
sIL-6R release from PMN. Neither significant shedding of IL-6R
when PMN were activated with thrombin for 30 min (Fig. 3A and Table I) nor significant sIL-6R secretion
when PMN were activated for 6 h (control medium: 719 ± 209
pg/ml vs thrombin: 742 ± 159 pg/ml, p = 0.7,
n = 4) or for 24 h (control medium: 768 ±
223 pg/ml vs thrombin: 716 ± 193 ng/ml, p = 0.5,
n = 6) was observed. We have previously reported
that thrombin induced an early and sustained secretion of IL-8 by HUVEC
(13). We asked whether IL-8 was able to induce IL-6R
shedding from PMN surface. This experiment was conducted on PMN
obtained from 15 healthy volunteers. Using FACS analysis, a significant
decrease of IL-6R expression on neutrophil membrane was observed,
after PMN were stimulated for 30 min with 10 ng/ml IL-8 (Fig. 3B and Table I). This loss of IL-6R expression on PMN
membranes (30–40% loss, p < 0.01, Table I) was
paralleled by a significant sIL-6R increase in IL-8-stimulated PMN
supernatants (160% increase, p < 0.05, Table I),
consistent with IL-6R shedding. Moreover, IL-8 dose-dependently
induced IL-6R shedding from neutrophil surface, reaching
significance for a concentration of 10 ng/ml (p
< 0.05, Table II). However, IL-8 effects
on IL-6R shedding were much weaker than those of FMLP, which was
used as a positive control in the same experiments (80–90% loss,
p < 0.01 and 285% increase, p <
0.05, Table I). Contrary to IL-8, GRO- did not appear to induce
significant IL-6R shedding (Fig. 3C) unless very high
concentrations (500 ng/ml) were used (Table I).
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increased thrombin-induced MCP-1 but not IL-8
secretion
Addition of various concentrations of sIL-6R (10–400 ng/ml) to
HUVEC stimulated with thrombin induced a dose-dependent significant
increase of endothelial MCP-1 secretion (Fig. 4A). MCP-1 secretion was
significantly higher (25% increase) with the addition of 200 ng/ml
sIL-6R, which is a concentration in the range of sIL-6R
concentrations observed in human fluids (32). The effects
of sIL-6R on thrombin-induced MCP-1 secretion appeared additive: 8
U/ml thrombin (23 ± 2 ng/ml), 5 ng/ml IL-6 + 200 ng/ml sIL-6R
(8 ± 1 ng/ml), 8 U/ml thrombin + 200 ng/ml sIL-6R (35 ±
1 ng/ml, n = 4, data not shown). On the contrary, no
significantly increased IL-8 secretion was observed in the same
experiments, when HUVEC were stimulated with thrombin in the presence
of 400 ng/ml sIL-6R (Fig. 4B). This was not due to the
low concentration of IL-6 present in thrombin-activated HUVEC, because
even in the presence of 30 ng/ml IL-6 and 200 ng/ml sIL-6R, we
observed no increase of IL-8 secretion by HUVEC (data not shown,
n = 3).
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(200 ng/ml) to various concentrations of
thrombin-activated HUVEC, in the presence or absence of FCS. In the
presence of FCS, 8 U/ml thrombin was necessary to significantly
increase MCP-1 secretion, whereas in the absence of FCS, significantly
increased MCP-1 secretion was observed with concentrations of thrombin
as low as 1 U/ml (Fig. 5, A
and B). These data are consistent with the presence of a
thrombin inhibitor in FCS. No increased IL-8 secretion was observed
whatever the condition used (Fig. 5, C and D).
Moreover, in the absence of FCS, a significant decrease of IL-8
concentration was observed at 8 U/ml thrombin (Fig. 5D).
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from neutrophils
Addition of 200 ng/ml recombinant sIL-6R to thrombin-activated
HUVEC significantly increased MCP-1 secretion, and the increased MCP-1
production could be completely reversed by the addition of either
neutralizing anti-sIL-6R mAb or anti-IL-6 mAb (Fig. 6A), demonstrating an
autocrine loop of MCP-1 secretion involving IL-6 produced by
thrombin-stimulated HUVEC and exogenous sIL-6R. To evaluate whether
such an inflammatory loop involving IL-6 may exist in vivo with PMN as
a source of sIL-6R, we added freshly prepared PMN to
thrombin-activated HUVEC and measured MCP-1 secretion after a 24-h
culture. Addition of PMN to thrombin-activated HUVEC significantly
increased MCP-1 secretion, which could be partially decreased (25%
reduction) by adding anti-IL-6 neutralizing
F(ab')2 (Fig. 6B). As control
experiments, stimulation of fresh PMN with thrombin (8 U/ml), IL-6 (30
ng/ml), or IL-8 (30 ng/ml) did not induce MCP-1 production (levels
below the detection limits of the assay, n = 3, data
not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shedding from neutrophil
membranes.
Thrombin is known to induce endothelial MCP-1 and IL-8 secretion by
interacting with its PAR-1 receptor, independently of TNF- and
IL-1 (13, 15, 16). Thrombin has also been shown to
induce IL-6 secretion by fibroblasts and epidermal cell lines through
interaction with the PAR-1 receptor (36). In this study,
we observed that thrombin can directly increase mRNA levels and protein
synthesis of IL-6 in HUVEC, through interaction with a PAR, likely
PAR-1, in a LPS-, IL-1-, and TNF--independent way. IL-6 has
been reported in one study to directly induce HUVEC activation and
leuko-endothelial adhesion, but these data are controversial because
HUVEC express the gp130 transducer but not the IL-6R
(37). Alternatively, IL-6 is able to complex with
sIL-6R, then associate with gp130 homodimers and activate HUVEC for
chemokine production and adhesion molecule expression (30, 31). The same kind of data were observed in our model; addition
of physiological concentrations of exogenous sIL-6R to
thrombin-stimulated HUVEC was sufficient to increase MCP-1 secretion,
compared with HUVEC stimulated with thrombin alone, and this can be
completely reversed by anti-sIL-6R and anti-IL-6 blocking
mAbs, demonstrating the existence of an autocrine loop of MCP-1
secretion involving exogenous sIL-6R and IL-6 secreted by
thrombin-stimulated HUVEC. A similar observation has been made in the
case of adhesion molecule expression regulation in a model of
TNF--activated astrocytes (38). Interestingly, in the
same culture condition, we observed no increase in IL-8 secretion,
although other authors have found that the IL-6/sIL-6R complex
induces an autocrine loop of both MCP-1 and IL-8 secretion in
endothelial cells (30, 31). These differences are likely
due to the different concentrations of sIL-6R and FCS present in the
different experimental models. In the study by Modur et al.
(31), sIL-6R concentrations up to 1 µg/ml have been
used, and induced both HUVEC chemokine production as well as ELAM,
ICAM-1, and VCAM-1 expression, but these concentrations are by far in
excess compared with those of sIL-6R observed in human fluids, which
are rarely higher than 500 ng/ml (32, 39, 40). In the
study by Romano et al. (30), the sIL-6R
concentrations used are comparable to those used in our model and
induced both chemokine reduction and ICAM-1 expression, but not ELAM-1
or VCAM-1. However, in this study HUVEC were cultured in the presence
of 20% FCS and heparin.
However, our results may suggest that HUVEC are more sensitive to
stimulation by IL-6/sIL-6R for MCP-1 than for IL-8 secretion. A
similar observation has recently been made in mesangial cell lines
(41). This may be an important point in vivo, because the
pro- vs anti-inflammatory functions of IL-6 are still discussed.
Notably, studies performed in animals defective for the IL-6 gene have
shown that the absence of this cytokine favors on one hand,
neutrophilia, high circulating TNF- concentrations and lethality in
response to LPS challenge. In contrast, these animals have decreased
IgG and IgA concentrations and a weak ability to develop a Th-1
response against intracellular infectious agents (42).
Therefore, IL-6 properties might be rather anti-inflammatory during
endotoxemia and acute infection, and proinflammatory during chronic
intracellular infection (23, 42). Considering this
hypothesis, it is noteworthy that in our study, IL-6/sIL-6R is more
efficient in inducing MCP-1, a chemokine acting on mononuclear cells
rather than IL-8, which is more specific of neutrophils and favors
acute inflammation. Thus, the IL-6/sIL-6R complex may favor chronic
inflammation, activation of APCs, and finally the induction of an
immune response with inflammation solving. In agreement with
this point of view, the IL-6/sIL-6R complex has recently been shown
to protect T cells from apoptosis and to favor chronic inflammation
(43).
One important question in such a model in vivo is the potential source
of sIL-6R in situ. It has been suggested recently that the source of
serum sIL-6R could be the secretion of an alternatively spliced form
by hepatocytes, whereas in situ sIL-6R seems to be released by
shedding of the receptors expressed on the membranes of inflammatory
cells, notably neutrophils and monocytes (26, 27, 40).
Therefore, we asked whether thrombin could act as a complete
proinflammatory mediator in this model, and induce IL-6R release
from neutrophils, either through shedding or alternative mRNA splicing.
Thrombin did not significantly modify neutrophil membrane IL-6R
expression or sIL-6R concentrations after 30-min stimulation, nor
did it induce sIL-6R secretion in neutrophil supernatant after
12 h. Because FMLP, a potent neutrophil activator, induces the
shedding of IL-6R (26, 27, 31), we asked whether IL-8,
which is also a well-known neutrophil activator, was able to induce
IL-6R shedding. In all experiments performed in >15 healthy donors,
a significant, although moderate, decrease of IL-6R expression on
neutrophil membranes and a parallel increase of sIL-6R
concentrations in the supernatants were observed after 30-min
stimulation with IL-8, consistent with receptor shedding from PMN
membranes. On the contrary, GRO-, another chemokine acting on PMN,
was not able to induce IL-6R shedding unless very high
concentrations of this chemokine were used. Because IL-8 acts on PMN
through CXCR1 and CXCR2, whereas GRO- acts through CXCR2 unless high
concentrations such as 500 ng/ml are used (44), these data
are consistent with the fact that IL-8 induces IL-6R shedding
through activation of CXCR1. So far, bacterial toxins and C reactive
protein are the only physiological molecules known to induce IL-6R
shedding (45, 46).
This autocrine loop of MCP-1 secretion involving thrombin, IL-8, and
IL-6/IL-6R is likely to be present in vivo, because addition in
vitro of fresh PMN to thrombin-activated HUVEC significantly increased
MCP-1 secretion, which was 25% decreased by anti-IL-6 mAb.
Thrombin proinflammatory properties and the ability to induce
chemokine production may support leukocyte recruitment in tissues in
different autoimmune diseases, such as rheumatoid arthritis (RA) or GN,
in which both extravascular coagulation and inflammation exist.
Detection of fibrin, fibrinogen, and thrombin/antithrombin III
complexes in synovial fluids of RA has clearly established the
occurrence of extravascular coagulation in RA synovium
(47). Thrombin may play a role in RA pathogenesis through
induction of MCP-1 and chronic mononuclear cell infiltration as well as
induction of IL-8, which favors neutrophil recruitment during acute
flare-up of the disease. In addition to chemokines, RA synovial
fluids contain various proinflammatory cytokines, such as TNF-,
IL-1, and IL-6, and have also been shown to contain high concentrations
of sIL-6R (48). Therefore, all the conditions required
for the occurrence of the inflammatory cascade described in this study
are present in RA synovial fluids.
Similarly, PAR-1 is constitutively highly expressed on glomerular
endothelium, and epithelial and mesangial cells
(49). In animal models of immune-mediated GN, thrombin has
been shown to play an important pathogenic role consisting in crescent
formation, lymphocyte and macrophage infiltration, as well as fibrin
deposition (7). MCP-1 is an important cytokine in
immune-mediated GN, and it is likely that the ability of thrombin to
directly induce chemokine, especially MCP-1, secretion by endothelial
and mesangial cells plays an important role in kidney monocyte
infiltration (50). In addition, to infiltrate tissues,
monocytes interact with adhesion molecules, such as ICAM-1 and VCAM-1,
the former playing a critical role in crescent formation
(51). Endothelial ICAM-1 and VCAM-1 expression are induced
by thrombin through interaction with PAR-1, and allowed mononuclear
cell adhesion (14). Moreover, IL-6 has been shown to be
secreted by mesangial cells and to be involved in the severity of GN
(52), and the IL-6/sIL-6R complex increases mesangial
MCP-1 production (41). Thus, thrombin may induce IL-6
secretion and the autocrine loop of MCP-1 secretion involving IL-6 and
sIL-6R described in this study, which may be important in kidney
monocyte infiltration.
In conclusion, as shown in Fig. 7, this
study suggests that in acutely inflamed tissues containing neutrophils
IL-8 and IL-6, IL-8 may induce neutrophil IL-6R shedding, which
would complex with IL-6 and activate gp130-bearing cells, notably
endothelial cells, to induce a stronger MCP-1 secretion, thus
mononuclear cell recruitment and a shift toward chronic inflammation,
immune response, and possibly inflammation ending.
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Footnotes |
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2 Abbreviations used in this paper: PAR, protease-activated receptor; s, soluble; TRAP, thrombin receptor-activating peptide; MCP-1, monocyte chemotactic protein-1; ELAM-1, endothelial-leukocyte adhesion molecule-1; GRO-, growth-related oncogene-; IL-1Ra, IL-1R antagonist; PMN, polymorphonuclear cell; RA, rheumatoid arthritis; GN, glomerulonephritis.
Received for publication October 6, 2000. Accepted for publication July 17, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  P. J. Wolters, C. Wray, R. E. Sutherland, S. S. Kim, J. Koff, Y. Mao, and J. A. Frank Neutrophil-Derived IL-6 Limits Alveolar Barrier Disruption in Experimental Ventilator-Induced Lung Injury J. Immunol., June 15, 2009; 182(12): 8056 - 8062. [Abstract] [Full Text] [PDF]
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 J. I. Borissoff, H. M.H. Spronk, S. Heeneman, and H. ten Cate Is thrombin a key player in the 'coagulation-atherogenesis' maze? Cardiovasc Res, June 1, 2009; 82(3): 392 - 403. [Abstract] [Full Text] [PDF]
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 Y. Banz, R. Rieben, C. Zobrist, P. Meier, S. Shaw, J. Lanz, T. Carrel, and P. Berdat Addition of dextran sulfate to blood cardioplegia attenuates reperfusion injury in a porcine model of cardiopulmonary bypass Eur. J. Cardiothorac. Surg., September 1, 2008; 34(3): 653 - 660. [Abstract] [Full Text] [PDF]
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 D. Chen, A. Carpenter, J. Abrahams, R. C. Chambers, R. I. Lechler, J. H. McVey, and A. Dorling Protease-activated receptor 1 activation is necessary for monocyte chemoattractant protein 1-dependent leukocyte recruitment in vivo J. Exp. Med., August 4, 2008; 205(8): 1739 - 1746. [Abstract] [Full Text] [PDF]
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 B. Rabe, A. Chalaris, U. May, G. H. Waetzig, D. Seegert, A. S. Williams, S. A. Jones, S. Rose-John, and J. Scheller Transgenic blockade of interleukin 6 transsignaling abrogates inflammation Blood, February 1, 2008; 111(3): 1021 - 1028. [Abstract] [Full Text] [PDF]
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A. Chalaris, B. Rabe, K. Paliga, H. Lange, T. Laskay, C. A. Fielding, S. A. Jones, S. Rose-John, and J. Scheller Apoptosis is a natural stimulus of IL6R shedding and contributes to the proinflammatory trans-signaling function of neutrophils Blood, September 15, 2007; 110(6): 1748 - 1755. [Abstract] [Full Text] [PDF]
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 C. J. Lockwood, F. Arcuri, P. Toti, C. D. Felice, G. Krikun, S. Guller, L. F. Buchwalder, and F. Schatz Tumor Necrosis Factor-{alpha} and Interleukin-1{beta} Regulate Interleukin-8 Expression in Third Trimester Decidual Cells: Implications for the Genesis of Chorioamnionitis Am. J. Pathol., October 1, 2006; 169(4): 1294 - 1302. [Abstract] [Full Text] [PDF]
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 W. A. Sands, H. D. Woolson, G. R. Milne, C. Rutherford, and T. M. Palmer Exchange Protein Activated by Cyclic AMP (Epac)-Mediated Induction of Suppressor of Cytokine Signaling 3 (SOCS-3) in Vascular Endothelial Cells. Mol. Cell. Biol., September 1, 2006; 26(17): 6333 - 6346. [Abstract] [Full Text] [PDF]
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 Z. Lin, A. Hamik, R. Jain, A. Kumar, and M. K. Jain Kruppel-Like Factor 2 Inhibits Protease Activated Receptor-1 Expression and Thrombin-Mediated Endothelial Activation Arterioscler. Thromb. Vasc. Biol., May 1, 2006; 26(5): 1185 - 1185. [Abstract] [Full Text] [PDF]
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 A. Tedgui and Z. Mallat Cytokines in Atherosclerosis: Pathogenic and Regulatory Pathways Physiol Rev, April 1, 2006; 86(2): 515 - 581. [Abstract] [Full Text] [PDF]
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 C.-Y. Yeh, J.-Y. Chen, and J.-S. Chia Glucosyltransferases of Viridans Group Streptococci Modulate Interleukin-6 and Adhesion Molecule Expression in Endothelial Cells and Augment Monocytic Cell Adherence Infect. Immun., February 1, 2006; 74(2): 1273 - 1283. [Abstract] [Full Text] [PDF]
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 J. R.S. Day, K. M. Taylor, E. A. Lidington, J. C. Mason, D. O. Haskard, A. M. Randi, and R. C. Landis Aprotinin inhibits proinflammatory activation of endothelial cells by thrombin through the protease-activated receptor 1 J. Thorac. Cardiovasc. Surg., January 1, 2006; 131(1): 21 - 27. [Abstract] [Full Text] [PDF]
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S. A. Jones Directing Transition from Innate to Acquired Immunity: Defining a Role for IL-6 J. Immunol., September 15, 2005; 175(6): 3463 - 3468. [Abstract] [Full Text] [PDF]
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M. I. Gomez, S. H. Sokol, A. B. Muir, G. Soong, J. Bastien, and A. S. Prince Bacterial Induction of TNF-{alpha} Converting Enzyme Expression and IL-6 Receptor {alpha} Shedding Regulates Airway Inflammatory Signaling J. Immunol., August 1, 2005; 175(3): 1930 - 1936. [Abstract] [Full Text] [PDF]
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 R. M. McLoughlin, B. J. Jenkins, D. Grail, A. S. Williams, C. A. Fielding, C. R. Parker, M. Ernst, N. Topley, and S. A. Jones IL-6 trans-signaling via STAT3 directs T cell infiltration in acute inflammation PNAS, July 5, 2005; 102(27): 9589 - 9594. [Abstract] [Full Text] [PDF]
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 P. C. W. Stone, F. Lally, M. Rahman, E. Smith, C. D. Buckley, G. B. Nash, and G. E. Rainger Transmigrated neutrophils down-regulate the expression of VCAM-1 on endothelial cells and inhibit the adhesion of flowing lymphocytes J. Leukoc. Biol., January 1, 2005; 77(1): 44 - 51. [Abstract] [Full Text] [PDF]
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S. W. Lindemann, C. C. Yost, M. M. Denis, T. M. McIntyre, A. S. Weyrich, and G. A. Zimmerman Neutrophils alter the inflammatory milieu by signal-dependent translation of constitutive messenger RNAs PNAS, May 4, 2004; 101(18): 7076 - 7081. [Abstract] [Full Text] [PDF]
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S. Srinivasan, M. E. Hatley, K. B. Reilly, E. C. Danziger, and C. C. Hedrick Modulation of PPAR{alpha} Expression and Inflammatory Interleukin-6 Production by Chronic Glucose Increases Monocyte/Endothelial Adhesion Arterioscler. Thromb. Vasc. Biol., May 1, 2004; 24(5): 851 - 857. [Abstract] [Full Text]
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 T. Shimizu, J. Nishihira, H. Watanabe, R. Abe, A. Honda, T. Ishibashi, and H. Shimizu Macrophage Migration Inhibitory Factor Is Induced by Thrombin and Factor Xa in Endothelial Cells J. Biol. Chem., April 2, 2004; 279(14): 13729 - 13737. [Abstract] [Full Text] [PDF]
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T. Minami, A. Sugiyama, S.-Q. Wu, R. Abid, T. Kodama, and W. C. Aird Thrombin and Phenotypic Modulation of the Endothelium Arterioscler. Thromb. Vasc. Biol., January 1, 2004; 24(1): 41 - 53. [Abstract] [Full Text] [PDF]
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R. B. Henderson, J. A. R. Hobbs, M. Mathies, and N. Hogg Rapid recruitment of inflammatory monocytes is independent of neutrophil migration Blood, July 1, 2003; 102(1): 328 - 335. [Abstract] [Full Text] [PDF]
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 T Cartmell, C Ball, A F Bristow, D Mitchell, and S Poole Endogenous interleukin-10 is required for the defervescence of fever evoked by local lipopolysaccharide-induced and Staphylococcus aureus-induced inflammation in rats J. Physiol., June 1, 2003; 549(2): 653 - 664. [Abstract] [Full Text] [PDF]
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S. M. Hurst, R. M. McLoughlin, J. Monslow, S. Owens, L. Morgan, G. M. Fuller, N. Topley, and S. A. Jones Secretion of Oncostatin M by Infiltrating Neutrophils: Regulation of IL-6 and Chemokine Expression in Human Mesothelial Cells J. Immunol., November 1, 2002; 169(9): 5244 - 5251. [Abstract] [Full Text] [PDF]
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 G. K. Tofaris, P. H. Patterson, K. R. Jessen, and R. Mirsky Denervated Schwann Cells Attract Macrophages by Secretion of Leukemia Inhibitory Factor (LIF) and Monocyte Chemoattractant Protein-1 in a Process Regulated by Interleukin-6 and LIF J. Neurosci., August 1, 2002; 22(15): 6696 - 6703. [Abstract] [Full Text] [PDF]
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