Download Toll-like receptor 4 ligand can differentially modulate the

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Toll-like receptor 4 ligand can differentially modulate the
release of cytokines by human platelets
Fabrice Cognasse,1,2 Hind HamzehCognasse,2 Sandrine Lafarge,1,2 Olivier
Delezay,2 Bruno Pozzetto,2 Archie
McNicol3 and Olivier Garraud1,2
1
EFS Auvergne-Loire, Saint-Etienne, France,
2
GIMAP-EA3064, Université de Saint-Etienne,
Faculté de Médecine, Saint-Etienne, France, and
3
Department of Oral Biology, University of
Manitoba, Winnipeg, MB, Canada
Received 15 October 2007; accepted for
publication 29 November 2007
Correspondence: Professor Olivier Garraud, EFS
Auvergne-Loire and GIMAP-EA 3064,
Université de Saint-Etienne, Faculté de
Summary
Blood platelets link the processes of haemostasis and inflammation. This
study examined the immunomodulatory factors released by platelets after
Toll-Like Receptor 4 (TLR4) engagement on their surfaces. Monoclonal
anti-human FccRII Ab (IV.3)-treated human platelets were cultured with
TLR4 ligands in the presence or absence of blocking monoclonal antibody
to human TLR4. The release of sCD62p, epidermal growth factor (EGF),
transforming growth factor b (TGFb), interleukin (IL)-8, platelet
activating factor 4 (PAF4), platelet-derived growth factor, a, b
polypeptide (PDGF-AB), Angiogenin, RANTES (regulated upon
activation, normal T-cell expressed, and presumably secreted) and
sCD40L were measured by specific enzyme-linked immunosorbent assay.
TLR4 ligand [Escherichia coli lipopolysaccharide (LPS)] bound platelet
TLR4, which differentially modulates the release of cytokines by platelets.
It was noted that (i) sCD62p, IL-8, EGF and TGFb release were each
independent of platelet activation after TLR4 engagement; (ii) RANTES,
Angiogenin and PDGF-AB concentration were weaker in platelet
supernatant after TLR4 engagement; (iii) sCD40L and PAF4 are present
in large concentration in the releaseate of platelets stimulated by TLR4
ligand. The effects of LPS from E. coli on the modulation of secretory
factors were attenuated by preincubation of platelets with an anti-TLR4
monoclonal antibody, consistent with the immunomodulation being
specifically mediated by the TLR4 receptor. We propose that platelets
adapt the subsequent responses, with polarized cytokine secretion, after
TLR4 involvement.
Médecine, 15 rue Ambroise Paré, 42023 SaintEtienne cedex 2, France. E-mail:
[email protected]
Keywords: platelets, Toll-like receptor 4, lipopolysaccharides, cytokines,
inflammation.
Lipopolysaccharide (LPS), a cell wall component of all Gramnegative bacteria, is composed of an amphipathic lipid A
moiety, core oligosaccharides and a variable O-antigen polysaccharide domain. It is the natural ligand for Toll-like
receptor 4 (TLR4), the presence of which is essential for the cell
response to LPS. A link between TLR4 and innate immunity
has been described (Cook et al, 2004). The recognition of LPS
by the innate immune system results in an inflammatory
response characterized by the production of cytokines, such as
tumour necrosis factor alpha, interleukin (IL)-1b, IL-6 and
IL-8 (Akira & Takeda, 2004). Thus, TLR4 appears to be an
important factor for the detection of pathogens and the
induction of an adaptive immune response (Medzhitov et al,
1997).
Blood platelets, which are central to haemostasis, also have
profound effects on the regulation of the immune system,
linking innate (including inflammation) and adaptative
immunity (Tang et al, 2002; Elzey et al, 2003; Yeaman &
Bayer, 2006; von Hundelshausen & Weber, 2007). Furthermore, several studies have implicated LPS as a modulator of
platelet function. Shibazaki et al (1996) showed that LPS from
Escherichia coli induces a biphasic, organ- and strain-specific
accumulation of murine platelets, and proposed that this effect
is involved in the development of septic shock. Endo and
First published online 13 February 2008
ª 2008 The Authors
doi:10.1111/j.1365-2141.2008.06999.x Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 141, 84–91
Platelets, TLR4 Activation and Cytokines
Nakamura (1992) reported the LPS-stimulated accumulation
of 5-hydroxytryptamine (5-HT) in the liver, which was
temporally associated with both a fall in the circulating platelet
count and a reduction in the concentration of 5-HT in the
blood.
We have shown the presence of certain Toll-Like Receptors
(TLR) on human platelets, and that the percentage of TLR
expression was significantly increased following activation, in
both permeabilized and intact platelets (Cognasse et al, 2005).
In addition, we have shown that stored platelets contain
molecules with known immunomodulatory competences and
differentially secrete them over time during storage (e.g. for
transfusion purposes) (Cognasse et al, 2006).
Finally, several groups (including ours) have recently
demonstrated that the TLR4 on platelets is functional
(Andonegui et al, 2005; Aslam et al, 2006; Patrignani et al,
2006; Cognasse et al, 2007a; Semple et al, 2007), prompting us
to study further the release of the secretory/immunomodulatory factors after the engagement of TLR4 on platelets.
Materials and Methods
Preparation of platelet concentrates
Platelet concentrates were prepared essentially as previously
described (Cognasse et al, 2005). Platelet concentrate bags of
290 ml (containing approx. 3Æ9 · 1011 platelets) were prepared
as pools of five buffy coats of whole blood samples from ABOidentical donors, with a single white blood cell (WBC)
filtration step according to routine blood bank manufacturing
procedures. The pooling of five buffy coats from ABOidentical donors to obtain platelet concentrate bags does not
allow conclusions to be drawn about the impact of gender or
genetic factors on the present findings.
The platelets used were tested for bacterial contamination in
routine bacterial growth tests and proved to be bacterial-free,
i.e., without bacteria growth capacity. The residual WBC count
was 0Æ025 · 106 ± 0Æ023 per tested platelet pool (Good
Manufacturing Practice procedure requires that WBC count
is below 106 per transfusion product, i.e. per platelet concentrate) (Council of Europe 2005).
Platelet concentrate mixes were obtained from the Auvergne–Loire Blood Bank and stored at 22 ± 2C under constant
agitation for 1 d prior to experimental investigation. It was
recently suggested that platelets stored at 4C retain superior
in vitro characteristics than those stored at 22C (Sandgren
et al, 2006, 2007), however, this remains controversial. Other
studies demonstrated that platelets undergo substantial
changes in both in vivo viability and in vitro properties when
they were exposed to temperatures below 20C for short
periods (Rao & Murphy, 1982; Bode & Knupp, 1994; Moroff
et al, 1994). Traditionally, platelets are stored at 22C and
consequently, in this study, platelet concentrates were
prepared according to routine blood bank manufacturing
procedures. This enabled a better understanding of TLR4
involvement in platelet physiology under normal storage
conditions.
Platelet stimulation
Platelets (3 · 1011 platelets/l) were incubated (20 min, room
temperature) with the anti-human FccRII monoclonal antibody
(MoAb) IV.3 (10 lg/ml; StemCell Technologies, Grenoble,
France) to saturate the free FccRII and block FccRII engagement
(Tomiyama et al, 1992). The platelets were then stimulated with
the TLR4 ligand LPS from E. coli (LPS E. coli; 0Æ5 lg/ml; 15 min,
room temperature) (Invivogen/Cayla, Toulouse, France), in the
presence or absence of a blocking anti-human TLR4 MoAb
(clone HTA125; 10 lg/ml; 30 min, room temperature) (Imgenex, San Diego, CA, USA), which blocked the activation of
monocytes by LPS (Paik et al, 2003). The optimal concentration
of LPS was determined by measuring their effects on platelets at
concentrations ranging from 0 to 2 lg/ml. The anti-human
TLR4 MoAb (clone HTA125, Imgenex) and the isotype control
MoAb (IgG2a, clone eBM2a, Nordic Immunological Laboratories, Tilburg, The Netherlands) were each used at a final
concentration of 10 lg/ml, and the TLR2 ligand Pam3CysSK4
(Invivogen/Cayla) was used at a final concentration of 1 lg/ml
(Damas et al, 2006).
Flow cytometry and cytokine detection
Platelet surface expression was determined by flow cytometric
analysis of all events that were positive for CD41 (a platelet
surface characteristic) marker expression (gating). Platelets were
labelled with a PE-conjugated anti-human CD63 MoAb (BD
Biosciences, Le Pont de Claix, France) to determine platelet
activation status. The platelet TLR4 expression in the absence of
TLR4 ligand, as determined using the anti-human TLR4 MoAb,
was defined as 100% and the effects of TLR4 ligands expressed as
percentages of this control value (Fig 1). Figure 1 further shows
that E. coli LPS specifically and reproducibly bound to TLR4 and
not to e.g. TLR2.
The levels of soluble cytokines [sCD62p, epidermal growth
factor (EGF), transforming growth factor b (TGFb), IL-8,
platelet activating factor 4 (PAF4), platelet-derived growth
factor, a, b polypeptide (PDGF-AB), Angiogenin, RANTES
(regulated upon activation, normal T-cell expressed, and
presumably secreted) and sCD40L] were measured in triplicate
from aliquots of unstimulated (control) or LPS-stimulated
platelets by specific enzyme-linked immunosorbent assays
(ELISA) obtained commercially (R&D Systems Europe Ltd.,
Lille, France or Abcyss, Paris, France).
Statistical analysis
Inter-experiment comparisons in cytokine concentrations for
the different culture conditions were analysed by means of the
Wilcoxon paired test. All values were reported as means ± SD
and a P value <0Æ05 was considered to be significant.
ª 2008 The Authors
Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 141, 84–91
85
F. Cognasse et al
% of CD63 expression on CD41+
(A)
Relative % of TLR4 positive platelet
(B)
100
80
60
*
*
40
*
*
20
0
Unstimulated 0·5
1
1·5
Concentration of E.coli LPS (2 μg/mL)
2
100
80
*
60
40
No modulation of platelet cytokine release after TLR4
engagement
20
0
Medium
E.coli LPS
Isotype
control
IgG2a
Pam3CysSK4
Fig 1. Flow cytometric analysis of platelet marker expression (CD63
and TLR4) in the presence of E. coli lipopolysaccharide (LPS). Platelets
were pretreated (20 min, room temperature) with the anti-human
FccRII MoAb ‘‘IV.3’’. (A) Modulation of platelet CD63 expression in
the presence of various concentrations of LPS. The percentage of CD63
expression after gating for CD41 positive platelets was determined by
flow cytometry following incubation (15 min, room temperature) with
various concentration of E. coli LPS (0–2 lg/ml). (B) LPS binding to
platelet TLR4. The percentage of TLR4 expression after gating for
CD41 positive platelets was determined by flow cytometry following
incubation (15 min, room temperature) with: (i) Medium (Unstimulated control platelets); (ii) Lipopolysaccharide from E. coli (E.coli
LPS; 0Æ5 lg/ml); (iii) Isotype control IgG2a (IgG2a; 10 lg/ml); (iv)
Pam3CysSK4 (1 lg/ml). The percentage of TLR4 expression, in the
absence of ligand, was set to 100%, and platelet responses to TLR4
ligand expressed as percentages of the initial value. Values shown are
deduced from background levels. Data represent the mean ± SD of five
experiments. The asterisk represents statistical (Wilcoxon paired test)
differences (stimuli versus medium).
Results
Platelet marker expression CD63 and TLR4 in presence of
E. coli LPS
CD63, together with CD62p, is considered to be a reproducible
marker of platelet activation (Turner et al, 2005; Viisoreanu &
Gear, 2007). The level of CD63 on the surface of unactivated,
1-d storage platelets was 18Æ4 ± 2Æ7%, consistent with a mild
level of baseline activation that was probably a consequence of
86
the preparation procedure (Sandgren et al, 2007). Following
the addition of E. coli LPS (0Æ5 lg/ml for 15 min) there was
a 36Æ2 ± 5Æ1% (P < 0Æ05) increase in CD63 expression
(Fig 1A), indicating that the platelets were significantly activated by bacterial components. Increasing the concentration of
E. coli LPS (1–2 lg/ml) did not induce significantly more
CD63 expression on the platelet surface than that elicited by
0Æ5 lg/ml E. coli LPS (Fig 1A).
We have previously shown a significant expression of TLR4
on the surface of CD41+ platelets (59 ± 2Æ1% of platelets)
(Cognasse et al, 2005). Therefore, the modulation of platelet
TLR4 expression in the presence of the ligand was examined
on CD41+ cells. Platelets were preincubated for 15 min with E.
coli LPS and analysed for TLR4 by flow cytometry. Incubation
of platelets with E. coli LPS led to a significant decrease of
49Æ2 ± 7Æ9% (P < 0Æ05) in the levels of TLR4 (Fig 1B). There
was no significant modification of TLR4 expression on the
platelet surface after stimulation with either the isotype control
Ab IgG2a or the specific TLR2 ligand, Pam3CysSK4 (Fig 1B).
CD62P (P-selectin) is a component of alpha granule membranes in resting platelets, which can be detected on the surface
of activated platelets (Metzelaar et al, 1993) and is subsequently cleaved in a soluble form (sCD62P) (Divers et al,
1995). The function of platelet TLR4 in ligand-induced
sCD62P release was examined, however incubation of platelets
with E. coli LPS had no significant effect on sCD62P release
(Fig 2A). Similarly there was no significant release of other
cytokines such as EGF (Fig 2B), TGFb (Fig 2C) and IL-8
(Fig 2D).
Diverse platelet cytokine release after TLR4 engagement
The engagement of TLR4 with E. coli LPS significantly
decreased the level of RANTES (86Æ1 ± 2Æ4 ng/ml vs.
103Æ6 ± 2Æ3 ng/ml, P < 0Æ05) (Fig 3A), possibly a result of its
sequestration in platelet alpha granules (Schmitt et al, 2000; El
Golli et al, 2005). Platelet incubation with E. coli LPS resulted
in a significant decrease of Angiogenin (51Æ8 ± 3Æ1 ng/ml vs.
57Æ8 ± 4Æ6 ng/ml, P < 0Æ05) (Fig 3B) and of PDGF-AB
(83Æ3 ± 5Æ5 ng/ml vs. 94Æ8 ± 6Æ1 ng/ml, P < 0Æ05) (Fig 3C).
The decrease in RANTES, Angiogenin and PDGF-AB
secretion may be the result of platelet cytokine or chemokine
receptor modulation. Several reports indicated that platelet
receptors bind and present cell-derived chemokines, and may
be involved in platelet activation, inflammatory or another
immune response.(Clemetson et al, 2000; Weber, 2005).
Alternatively Hu et al (1993) have shown that the angiogenin binding protein is a cell surface actin. A large number
of proteins can bind to actin and this may contribute to
several cellular functions, particularly cytokinesis (Hu et al,
1993).
ª 2008 The Authors
Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 141, 84–91
Platelets, TLR4 Activation and Cytokines
(A)
(B)
15
700
EGF ng/ml (x108 unit)
sCD62p ng/ml (x108 unit)
800
600
500
400
300
200
100
0
E.coli LPS
Unstimulated
(D)
100
90
80
70
60
50
40
30
20
10
0
IL-8 ng/ml (x108 unit)
TGFβ ng/ml (x108 unit)
5
0
Unstimulated
(C)
10
Unstimulated
E.coli LPS
E.coli LPS
1000
900
800
700
600
500
400
300
200
100
0
Unstimulated
E.coli LPS
Fig 2. No modulation of platelet cytokine release after TLR4 engagement. Platelets were pretreated (20 min, room temperature) with the anti-human
FccRII MoAb ‘‘IV.3’’ then incubated as previously reported and the levels of sCD62P (A), EGF (B), TGFb (C) and IL-8 (D) determined. Values shown
are deduced from background levels. Data (mean ± SD; n = 10 experiments) are expressed in ng/ml (·108 unit).
In contrast, platelet incubation with E. coli LPS resulted in
a significant increase in sCD40L levels (11Æ4 ± 1Æ4 ng/ml vs.
8Æ2 ± 1Æ7 ng/ml, P < 0Æ04) (Fig 3D) and of PAF4
(150Æ4 ± 6Æ9 ng/ml vs. 132Æ1 ± 11Æ8 ng/ml, P < 0Æ05) (Fig 3E).
Each of these effects of E. coli LPS on immunomodulatory
factors was attenuated by preincubation with an anti-TLR4
MoAb (Fig 3), consistent with the effects being specifically
mediated by TLR4.
Discussion
CD40-CD40L interactions have been involved in inflammation
and thrombosis. Recent studies have showed clinical interest in
the use of sCD40L levels as a marker of thrombotic risk,
particularly for coronary syndrome (Heeschen et al, 2003;
Varo et al, 2003, 2006). Although sCD40L is emerging as
a promising marker of thrombotic risk, moderately little
attention has been given to the impact of sample processing
technique on the soluble CD40L assay (Varo et al, 2003, 2006;
Ahn et al, 2004). Thus we investigated the effect of blood
sampling techniques reported in the CD40L literature to help
clarify these issues. Elzey et al (2003) showed in transfusion
studies that platelet-derived CD154 alone is sufficient to
induce isotype switching and augments T lymphocyte function
during viral infection, thereby leading to enhanced protection
against viral rechallenge. Heddle (1999) demonstrated that
many chemokines which accumulate in platelet concentrates
after leucoreduction were associated with allergic transfusion
reactions. In addition, the intradermal injection of supernatants from activated platelets induces a deferred inflammatory
response, which is probably due to histamine release and is
likely to be due to platelet factor 4 (PF4) and macrophage
inflammatory protein alpha (MIP-1a) (Boehlen & Clemetson,
2001). It may be possible that, when transfused into patients,
these mediators cause allergic symptoms that are associated
with non-haemolytic transfusion reactions. Moreover, the
authors suggested that the mediators may cause other adverse
reactions; for example, the release of IL-8 during the preparation processes may synergise with other chemokines, such as
PF4 or RANTES (Boehlen & Clemetson, 2001).
Of note, CD14, the common LPS receptor on the surface of
mononuclear leucocytes (Jiang et al, 2005), was undetectable
on the surface of platelets by flow cytometry and the
incubation of platelets with an anti-CD14 MoAb did not
affect LPS binding (data not shown), as previously reported
(Stahl et al, 2006). However, this does not exclude the
possibility that soluble CD14 can participate in platelet
activation, as has been described for certain other cellular
systems (Pugin et al, 1993).
The delivery of proteins to the platelet plasma membrane,
including alpha granule proteins, seems to be controlled by the
nature of the ligand/receptor involved. The post-TLR4 regulation of platelets appears to be closely regulated; the current
study has shown that E. coli LPS favours the sequestration of
ª 2008 The Authors
Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 141, 84–91
87
F. Cognasse et al
(B)
120
*
*
100
80
60
40
20
0
Unstimulated
E.coli LPS
LPS block by LPS + IgG2a
α-TLR4
80
70
*
*
60
50
40
30
20
10
0
Unstimulated
LPS block by LPS + IgG2a
α-TLR4
E.coli LPS
(D)
120
*
*
sCD40L ng/ml (x108 unit)
PDGF-AB ng/ml (x108 unit)
(C)
ANGIOGENIN ng/ml (x108 unit)
RANTES ng/ml (x108 unit)
(A)
100
80
60
40
20
0
Unstimulated E.coli LPS
LPS block by LPS + IgG2a
α-TLR4
20
15
*
*
10
5
0
Unstimulated
E.coli LPS
LPS block by LPS + IgG2a
α-TLR4
(E)
PAF4 ng/ml (x108 unit)
200
*
*
150
100
50
0
Unstimulated
E.coli LPS
LPS block by LPS + IgG2a
α-TLR4
Fig 3. Various platelet cytokine release after TLR4 engagement. Platelets were pretreated (20 min, room temperature) with the anti-human FccRII
MoAb ‘‘IV.3’’, and then incubated as previously reported and the levels of RANTES (A), Angiogenin (B), PDGF-AB (C), sCD40L (D) and PAF4
(E) quantified by ELISA. Values shown are deduced from background levels. Data (mean ± SD; n = 5 experiments) are expressed in ng/ml (·108
unit). The anti-human TLR4 MoAb (a-TLR4) was used at a concentration of 10 lg/ml. Values shown are deduced from background levels. Data
(mean ± SD; n = 10 experiments) are expressed in ng/ml (·108 unit). The asterisk represents statistical (Wilcoxon paired test) differences (stimuli
versus medium).
RANTES, Angiogenin and PDGF-AB while enhancing sCD40L
and PAF4 release. It therefore seems plausible that different
sets of ‘‘polarized’’ cytokines are released after specific platelet
TLR4 engagement. Previous studies in dendritic cells have
shown that different LPS molecules may induce distinct
patterns of immunity while acting on the same receptor
(Pulendran et al, 2001). Thus, it can be hypothesized that the
nature and/or intensity of engagement of platelet membrane
TLR4 determines a preferential cytokine pattern production/
release, an issue that deserves further exploration. Meanwhile,
88
the data (Fig 3) suggests that sCD62p, IL-8, EGF and TGFb
release are each independent of platelet activation involving
TLR4, while RANTES, Angiogenin and PDGF-AB, each of
which are less abundant in the platelet releasate, are prone to
sequestration after the engagement of TLR4. An important
issue, with clinical implications in transfusion medicine (Khan
et al, 2006), concerns the conditions after which PAF4 and
sCD40L are present in large concentrations in platelet stimulated by TLR4 ligand. These cytokines exert potent effects on
the vascular epithelia and on inflammation in general (Prescott
ª 2008 The Authors
Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 141, 84–91
Platelets, TLR4 Activation and Cytokines
et al, 2000; Phipps et al, 2001; Danese & Fiocchi, 2005; Fillon
et al, 2006), and the amounts of sCD40L that are secreted
during platelet storage are compatible with biological effects on
immune circulating cells of the platelet concentrate transfusion
recipient (Cognasse et al, 2006, 2007a,b,c; Corash et al,
2006a,b). To date, the platelet stimulus that connects vascular
inflammation with vascular occlusion and thrombosis in sickle
cell disease has been elusive (Bennett, 2006). However the
current study suggests that the stimulus, LPS, is the same as for
other inflammatory conditions involving platelets and CD40/
CD40L, such as systemic lupus erythematous (Delmas et al,
2005; Nagasawa et al, 2005) and atherosclerosis (Chakrabarti
et al, 2005; Lee et al, 2006).
There has been a plethora of reports in recent years suggesting
that a number of endogenous molecules can activate the innate
immune system (Tsan & Gao, 2004), while TLRs and other
Pattern Recognition Receptors sense external danger (Pasare &
Medzhitov, 2004; Martinon & Tschopp, 2005). These endogenous molecules, particularly heat shock protein 70 (HSP70)
(Dybdahl et al, 2002), can induce the release of proinflammatory cytokines. Taken together, there is now a clear indication
that almost all sets of immune cells can exhibit, upon differential
stimulation, patterns of polarized cytokine secretion (Mosmann, 2000). Therefore, we propose that platelets can similarly
respond by differentially releasing polarized cytokines. However
in contrast to other immune cells, which differentially respond
by gene reorganization, platelets must thus use a process of
differential exocytosis that is as yet still unclear. It can be
speculated that, in a given local environment, platelets may
contribute with other cell types to the maintenance of a coherent
cytokine pattern. In conclusion, we propose that TLR expression
on platelets serves to sense TLR ligands, primarily bacterial,
according to the infectious non-self model of revised danger
theory (Gallucci & Matzinger, 2001).
Acknowledgements
The authors gratefully acknowledge the invaluable help of
Dr Patricia Chavarin and of Mrs Sophie Acquart and Françoise
Boussoulade in processing the platelet concentrate bags, and of
Sophie Danini, and Anne Zabinski for experimental procedures. They also thank Mr Philip Lawrence for kindly revising
the manuscript. They also thank Dr J.M. Cavaillon, Head of
Unit ‘‘Cytokines & Inflammation’’ Pasteur Institut (France)
for fruitful discussions and critical reading of this manuscript.
This study was supported by the EFS Auvergne-Loire and the
‘‘Association pour la Recherche en Transfusion’’ (contract
ART-No 43-2006).
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