Skip Navigation


Cardiovascular Research Advance Access first published online on April 14, 2008
This version [Corrected Proof] published online on May 12, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn087
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
79/3/537    most recent
cvn087v2
cvn087v1
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Dueñas, A. I.
Right arrow Articles by García-Rodríguez, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dueñas, A. I.
Right arrow Articles by García-Rodríguez, C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Selective attenuation of Toll-like receptor 2 signalling may explain the atheroprotective effect of sphingosine 1-phosphate

Ana I. Dueñas1, Mónica Aceves1, Isabel Fernández-Pisonero1, Cristina Gómez1, Antonio Orduña2, Mariano Sánchez Crespo1 and Carmen García-Rodríguez1,*

1 Instituto de Biología y Genética Molecular, CSIC-Universidad de Valladolid, C/Sanz y Forés s/n, 47003-Valladolid, Spain
2 Hospital Clinico Universitario, Valladolid, Spain

* Corresponding author. Tel: +34 983 184 841; fax: +34 983 184 800. E-mail address: cgarcia{at}ibgm.uva.es

Received 16 November 2007; revised 10 March 2008; accepted 31 March 2008

Time for primary review: 28 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: Vascular inflammation is a major atherogenic factor and Toll-like receptor (TLR) 2 ligands, including bacterial and serum lipoproteins, seem to be involved in atherogenesis. On this basis, we analysed the effect of lipoproteins and different lipid components on TLR2-dependent signalling.

Methods and results: In TLR2-transfected human embryonic kidney 293 cells and human monocytes, oxidized low-density lipoproteins inhibited nuclear factor (NF)-{kappa}B-driven transcriptional activity and chemokine gene expression in response to TLR2 ligands. Sphingosine 1-phosphate (S1P) and oxidized palmitoyl-arachidonoyl-phosphatidylcholine, but not lipoprotein-carried lysophospholipids, inhibited TLR2 activation. Silencing experiments in TLR2-transfected 293 cells showed that the S1P-mediated attenuation effect is mediated by S1P receptors type 1 and type 2. To address the physiological significance of these findings, additional experiments were performed in human peripheral blood monocytes and monocyte-derived macrophages. In both cell types, S1P selectively attenuated TLR2 signalling, as NF-{kappa}B and extracellular signal-regulated kinase activation, but not c-Jun amino terminal kinase phosphorylation, were inhibited by physiologically relevant concentrations of S1P. Moreover, the attenuation of TLR2 signalling was partially reverted by pharmacological inhibition of phosphoinositide 3-kinase (PI3K) and Ras pathways. In addition, S1P inhibited the chemokine gene expression elicited by TLR2, but not by TLR4 ligands.

Conclusion: These findings disclose a cross-talk mechanism between lipoprotein components and TLR in which engagement of S1P receptors exert selective attenuation of TLR2-dependent activation via PI3K and Ras signalling. A corollary to these data is that the negative cross-talk of S1P receptors and TLR2 signalling might be involved in the atheroprotective effects of S1P.

KEYWORDS Atherogenesis; Monocyte/macrophage; Lipid mediators; Toll-like receptor; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Atherosclerosis, once considered a disorder uniquely linked to cholesterol storage, is currently recognized as an inflammatory disease.1,2 Among the receptors involved in the inflammatory response, Toll-like receptors (TLR) have been associated with the development of atherosclerotic disease (for review see Vink et al.3). TLRs are pattern recognition receptors that activate the early host response to microbial components by triggering signalling cascades that promote the activation of transcription factors such as nuclear factor (NF)-{kappa}B and ultimately induce the expression of proinflammatory cytokines.4,5 Under certain circumstances, TLRs are also able to recognize some endogenous molecules, and there is in vivo evidence for the involvement of TLR signalling in hyperlipidaemia-induced atherosclerosis.6 It has been described that minimally oxidized low-density lipoproteins (LDL) can interact with TLRs and induce cytokine secretion,7,8 but also downregulate lipopolysaccharide (LPS)-induced expression of proinflammatory proteins,9 through a mechanism involving the mitogen-activated protein kinase (MAPK) cascade and the NF-{kappa}B route.10 Likewise, products of phospholipid oxidation, such as oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC) inhibit ligand activation of TLR2 and TLR4 receptors.11 Sphingosine 1-phosphate (S1P) is a lipid mediator associated with lipoproteins, especially with high-density lipoprotein (HDL) and also with LDL.12 In addition to acting as an intracellular second messenger, S1P triggers many biological responses via a family of G-protein coupled receptors expressed in cells from blood vessels and the immune system13–15 initially termed endothelial differentiation gene (EDG) and now renamed as S1P1–5 receptors.15 S1P is present in biological fluids at low-micromolar concentrations, but as lysophospholipids are not uniformly distributed in plasma, yet associated with lipoproteins, their concentration may be higher at cell surfaces.16 S1P receptors are coupled with both stimulatory and inhibitory pathways for adhesion molecule expression in endothelial cells,17 which might explain why S1P is involved in a variety of antiatherogenic actions, including cell survival, migration, and proliferation, but may also promote the expression of proatherogenic adhesion molecules.12,16 Several reports have demonstrated the role of TLR2 in atherogenesis.18 Both the TLR2 ligand peptidoglycan (PGN) and increased expression of TLR2 have been found in human atherosclerotic plaques.19,20 In addition, it has been described that TLR2 stimulation induces neointima and atherosclerotic plaque formation in mouse femoral arteries,21 and that endogenous TLR2 ligands have a central role on systemic inflammation in atherogenesis.22 As it is known that minimally oxidized LDL can interact with TLR47 and bacterial lipoproteins bind to TLR2,23 we studied the effect of lipoproteins and different lipid components on TLR2 activation. Our results show that S1P behaves as a negative regulator of TLR2 via negative cross-talk with S1P receptors, and suggest that the antiatherogenic actions of S1P12,16 could be explained, at least in part, by selective attenuation of TLR2 activation.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
2.1 Expression plasmids and reagents
cDNA encoding hemagglutinin-tagged human TLR2 was provided by Dr Michael Rehli (University of Regensburg, Germany). Pam3CSK4 was purchased from InvivoGen (San Diego, CA, USA). S1P, lysophosphatidylcholine (LPC), lysophosphatidic acid (LPA), platelet-activating factor (PAF), LPS from E. coli, thiobarbituric acid, 1,1,3,3-tetramethoxypropane, 2,6-di-tert-butyl-4-methyl-phenol, and PGN were from Sigma (St Louis, MO, USA). PAPC was purchased from Avanti Polar (Alabaster, AL, USA). LDL, HDL, GGTI-298, and farnesyltransferase (FPT) inhibitor II were from Calbiochem (San Diego, CA, USA). I{kappa}B{alpha}, β-actin, and non-phosphorylated extracellular signal-regulated kinase (ERK) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-SAPK/c-Jun amino terminal kinase (JNK) antibody was from Cell Signaling (Danvers, MA, USA), and the anti-human SAPK/JNK rabbit antibody (AF1387) was from R&D Systems (Minneapolis, MN, USA). TRIzol was from Life Technologies (Grand Island, NY, USA). Anti-phospho ERK antibody and pRL-TK (Renilla-luciferase expressing plasmid) were from Promega Inc (Madison, WI, USA). Limulus amebocyte lysate test was from Cambrex (Baltimore, MD, USA). Pre-designed small interfering RNA duplexes were from Ambion Inc. (Austin, TX, USA): S1P1 (no. 4143, no. 145848), S1P2 (no. 45076, no. 44984), S1P3 (no. 1959, no. 1875), S1P4 (no. 103833, no. 41882), S1P5 (no. 32856, no. 32769).

2.2 Cells
Human embryonic kidney 293 and THP-1 cells were cultured in Dulbecco's Modified eagles's Medium and RPMI medium, respectively, supplemented with antibiotics, L-glutamine, and 10% Fetal bovine serum. Human monocytes were isolated from buffy coats of healthy volunteer donors by centrifugation onto Ficoll cushions and adherence to plastic dishes for 2 h as described.24 Differentiation of monocytes into macrophages was carried out by culture of adhered monocytes in the presence of 5% human serum for two weeks in Primaria six-well dishes. The investigation conforms with the principles outlined in the Declaration of Helsinki.

2.3 Modification of lipoproteins and lipid components
Oxidation of LDL was performed by incubating 0.1 mg of LDL protein/mL with 5 µM CuSO4 for 24 h at 37°C, as described.25 The extent of oxidation was determined by the thiobarbituric acid reactive substance (TBARS) assay26 based on the Slater and Sawyer method27 and was expressed as malondialdehyde equivalents using a standard curve of 1,1,3,3-tetramethoxypropane. All the reagents were endotoxin-free and the absence of LPS was confirmed by the Limulus amebocyte lysate test. Oxidation of PAPC and LPC was performed by air oxidation for 72 h at room temperature.

2.4 Transient transfection and luciferase assays
As previously described, 293 cells were transiently transfected with TLR2 expression plasmid, and the reporter plasmids NF-{kappa}B5xLuc and pRL-TK using the calcium phosphate method.28 24 h after transfection, cells were incubated overnight with TLR2 ligands (10 µg/mL PGN or 100 ng/mL Pam3CSK4), in the presence or absence of lipoproteins or lipid components, and then lysed and assayed for firefly- and Renilla-luciferase activities as described.28 Luminescence quantification was performed in a microplate luminometer equipped with a dual-injector system (EG&G Berthold, Germany).

2.5 RNA extraction and RNase protection assays
RNA isolated by the TRIzol method was used to analyse chemokine expression using a RiboQuant RNase protection assay from Pharmingen (San Diego, CA, USA), as previously described.28 Briefly, the hCK5 multiprobe template (including the chemokines RANTES, IP-10, MIP-1{alpha}, MIP-1β, MCP-1, IL-8, and the housekeeping genes L32, GAPDH) was labelled with ({alpha}-32P)UTP in the presence of T7 RNA polymerase and used for overnight hybridization with 3 µg of RNA. The hybridized RNA was digested with RNase and proteinase K, and the RNase-protected probes were purified and resolved on denaturing polyacrylamide gel electrophoresis (PAGE). Radiolabelled bands on the gel were acquired using the Personal Molecular Imager FX and quantified using the Quantity One Image Analyzer software (BioRad, CA, USA).

2.6 Silencing of sphingosine 1-phosphate receptors by small interfering RNA
siRNAs used in the experiments were the validated siRNA duplexes specific for S1P human receptors, and a negative silencer RNA control (Ambion, Inc). As indicated above, 293 cells were transiently transfected with siRNA specific for human S1P1–5 or control non-silencing siRNA or vehicle, along with TLR2 expression and reporter plasmid constructs. siRNA duplexes specific for S1P and control siRNA were used at 200 nM. After 24 h, transfected cells were either collected for RNA isolation followed by quantitative real-time RT-PCR to confirm the downregulation of S1P/EDG receptors mRNA expression, or activated overnight with Pam3CSK4 in the presence of S1P and used for luciferase assays as described above.

2.7 Real-time RT-PCR analysis
First-strand cDNA was synthesized from total RNA by the reverse transcription reaction, and later amplified by PCR. Primer sequences for human LPA receptors, S1P1, S1P2, and S1P4 were as described:30,31 for S1P3: sense 5'-CTTGGTCATCTGCAGCTTCATC-3' and anti-sense 5'-TCATTGTCAAGTGCCGCTCGAT-3'.; and for S1P5: sense 5'-ACAACTACACCGGCAAGCTC-3', anti-sense 5'-GCCCCGACAGTAGGATGTT-3'. cDNA was amplified in a PTC-200 apparatus equipped with a Chromo4 detector (BioRad) using SYBR Green mix containing HotStart polymerase (ABgene). β-Actin was used as a housekeeping gene to assess the relative abundance of mRNA.

2.8 Immunodetection of I{kappa}B{alpha}, β-actin, ERK, and JNK proteins
Stimulation of 2 x 106 cells was done and later lysed with TNE buffer (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5 mM ethylene diamine tetraacetic acid, 0.01% NP-40, and a cocktail of protease and phosphatase inhibitors) for 10 min on ice. 50 µg of proteins per lane were analysed by SDS–PAGE followed by Western blotting. Proteins were detected using the enhanced chemiluminescence system from Amersham (Buckinghamshire, UK).

2.9 Statistical analysis
Results are expressed as mean ± SD. Data were analysed by ANOVA test using GraphPad Prism version 4 (GraphPad Prism Software, San Diego, CA, USA). Differences were considered statistically significant for P < 0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
3.1 Ox-LDL and HDL blunt TLR2-induced {kappa}B-driven transcriptional activity but exert different effects on chemokine gene expression in human monocytes
As it is known that minimally oxidized LDL can interact with TLR47 and bacterial lipoproteins bind to TLR2,23 we studied the effects of ox-LDL on TLR2 activation in 293 cells, which are devoid of both scavenger receptors and TLR.29,32 As shown in Figure 1, PGN stimulation of TLR2-transfected cells produced a significant enhancement of {kappa}B-driven transcriptional activity, which was reduced to 55% by treatment with 100 µg/mL of ox-LDL. A similar degree of inhibition was observed with native HDL at the same concentration. ox-LDL showed no effect on TLR4/MD2-transfected 293 cells activated with LPS (data not shown).


Figure 1
View larger version (30K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1 Effect of lipoproteins on Toll-like receptor 2 (TLR2) activation. (A) TLR2-transfected 293 cells were stimulated with peptidoglycan in the presence or absence of lipoproteins. Results are expressed as fold-increase of the {kappa}B-driven activity detected in cells treated with vehicle. Data represent mean ± SD of at least three experiments. Asterisk (*) indicates P = 0.0018 (LDLox), P = 0.0245 (HDL) when compared with cells treated with vehicle. Total RNA from THP-1 cells (B) and human monocytes (C) stimulated for 3 h with either 10 µg/mL PGN, 1 µg/mL LPS, or vehicle, in the presence or absence of 100 µg/mL lipoproteins were analysed using the chemokine mRNA assay. These are representative experiments of three with similar results.

 
To address the effect on chemokine mRNA expression, RNase protection assays were carried out in human monocytic cells. In THP-1 cells, PGN was a strong inducer of chemokine mRNA, whereas LPS was a less potent agonist (Figure 1B), which can be explained by the fact that THP-1 cells does not express CD14 in the absence of phorbol ester-induced differentiation.33 Densitometric analysis revealed that ox-LDL inhibited 35.1 ± 9.2% of MIP-1β, 37.6 ± 4.5% of MIP-1{alpha}, and 32.4 ± 2.6% of IL-8 mRNA levels induced upon TLR2 activation, and inhibited 60.9 ± 2.9% of MIP-1β, 59.8 ± 2.8% of MIP-1{alpha}, and 24.2 ± 4.4% of IL-8 mRNA levels induced upon LPS activation. Noteworthy, ox-LDL showed a clear inhibitory effect on the PGN- and LPS-mediated response (Figure 1B, rightmost lanes). Interestingly, ox-LDL reduced the PGN-induced expression of MIP-1β (25.3 ± 6.7% inhibition), MIP-1{alpha} (34.2 ± 9.8% inhibition), and IL-8 (28.6 ± 4.0% inhibition) (Figure 1C, lanes 2 and 11), whereas ox-LDL alone showed a slightly increasing effect (Figure 1C, lanes 1 and 10) when compared with the lack of effect of similar amounts of native LDL and HDL (Figure 1C, lanes 1, 4 and 7). In contrast, HDL showed a clear inhibitory effect on LPS-mediated mRNA induction (Figure 1C, lanes 3 and 6) of MIP-1β (37.4 ± 7.1%), MIP-1{alpha} (35.1 ± 1.4%), and IL-8 (19.2 ± 1.2%).

3.2 Effect of lipoprotein-carried lipid components on TLR2-dependent {kappa}B-driven transcriptional activity
To characterize the lipid moieties involved in the response to lipoproteins, we studied the effects of several phospholipids on TLR2-transfected 293 cells, which do express a series of lysophospholipid receptors as shown in quantitative RT-PCR experiments (Figure 2A). In transactivation experiments, marginal effects of LPC, ox-LPC, PAF, and LPA were observed on TLR2-dependent {kappa}B-driven transcriptional activity, whereas both ox-PAPC and S1P showed a significant inhibitory effect (Figure 2B). This finding is consistent with the hypothesis that PAPC and S1P could mediate a fraction of the inhibitory effect of lipoproteins on {kappa}B-activation, and agree with previous report on the ox-PAPC-mediated inhibitory effect of TLR2 and TLR4 activation.11 S1P showed a dose-dependent inhibitory effect that was observed in response to two different TLR2 ligands, namely PGN and the lipoprotein mimic Pam3CSK4 (Figure 2C). S1P did not significantly influence the {kappa}B-activity observed in the absence of ligands, suggesting that the observed effect is not because of an interference of S1P on the reporter assay. In contrast, 50 µM ceramide showed no effect on {kappa}B-driven transcriptional activity (data not shown).


Figure 2
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2 Effect of lipoprotein-associated lipid components on TLR2 activation. (A) mRNA isolation and quantitative RT-PCR of S1P/endothelial differentiation gene (EDG) receptors were performed as indicated in Methods. Data, expressed as arbitrary units (a.u.), represent mRNA levels of the indicated receptors normalized to β-actin expression. (B) TLR2-transfected 293 cells were stimulated with peptidoglycan in the presence of phospholipids [100 µM lysophosphatidyl choline (LPC), 100 µM ox-LPC, 100 µM lysophosphatidic acid, 20 µM ox-PAPC, 40 µM S1P, and 100 µM platelet-activating factor], and analysed as in Figure 1. Data are representative of three independent experiments. (C) Results, expressed as percent of inhibition of the NF-{kappa}B activity, represent mean ± SD of at least three experiments. Asterisk (*) indicates P < 0.0001 when compared with transfected cells treated with vehicle and corresponding dose of S1P.

 
3.3 S1P2 and S1P1 receptors are involved in the S1P-mediated attenuation of TLR2 activation
To address the type of receptor involved in the S1P effect, the expression of S1P1–5 receptors was selectively downregulated by siRNA in 293 cells co-transfected with pre-designed siRNA duplexes, together with TLR2 expression and NF-{kappa}B reporter plasmids. As demonstrated by quantitative RT-PCR, siRNA experiments significantly reduced endogenous mRNA levels of S1P receptors (Figure 3A, and data not shown). In transactivation experiments, S1P2 knock-down, but not a negative siRNA control, partially reverted the S1P-mediated attenuation of TLR2-induced NF-{kappa}B activation (Figure 3B). Results were confirmed with a second pair of siRNA duplex (data not shown). Downregulation of endogenous S1P1 also reduced, although at a lower extent, the S1P-mediated effect (Figure 3B). In contrast, downregulation of receptors S1P3–5 did not overcome the attenuation elicited by S1P (Figure 3B). Altogether these results suggest that S1P1 and S1P2 are the main receptors involved in the S1P-mediated attenuation of TLR2 activation. Since a complete blockade was not observed, most likely because of technical limitations, the possibility that a portion of S1P effect could not be mediated by receptors cannot be ruled out.


Figure 3
View larger version (34K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3 Receptors S1P1 and S1P2 are involved in the inhibition of TLR2 activation by S1P. 293 cells were transfected with either the corresponding target duplex or a negative siRNA control, along with TLR2 expression and reporter plasmids. (A) Downregulation of S1P receptors was confirmed by quantitative RT-PCR. Upper panel corresponds to the amplification of the indicated S1P gene; lower panel corresponds to the β-actin gene used for normalization purposes. (B) Transfected cells were stimulated and analysed as in Figure 2C. Asterisk (*) indicates P = 0.0006 (S1P2), P = 0.0026 (S1P1) when compared with cells transfected with siRNA negative control or mock.

 
3.4 Sphingosine 1-phosphate selectively attenuates Toll-like receptor 2 signalling in human mononuclear phagocytes
To test the physiological significance of the S1P-mediated inhibitory effect, additional experiments were conducted in human mononuclear phagocytes. The activation of TLR2 by Pam3CSK4 induced the degradation of the NF-{kappa}B inhibitor I{kappa}B{alpha} in human monocyte-derived macrophages. NF-{kappa}B activation was blocked by S1P at low micromolar concentrations. Statistically significant inhibition of TLR2-dependent NF-{kappa}B activation was observed with 1 µM S1P, which argues in favour of a receptor-dependent and physiologically relevant effect (Figure 4A). Similar effect was observed in monocytes and THP-1 cells, and when PGN was used as a stimulus (data not shown). This data agrees with the results observed in 293 transfected cells (Figure 2C), although much lower doses were required in macrophages, which could be explained by the fact that overexpression of TLR2 in transfected cells might increase the threshold for TLR2 attenuation.


Figure 4
View larger version (65K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4 S1P selectively inhibits TLR2-induced signalling. Human macrophages were incubated with Pam3CSK4 and the indicated S1P dose for the specified time. Cell lysates were analysed by Western blot. (A) Anti-I{kappa}B{alpha} and anti-phospho-ERK antibodies were used, and the uniformity of loading among the different lanes was confirmed with anti-β-actin and anti-ERK antibodies. (B) Anti phospho-Jun amino terminal kinase (JNK) antibody and JNK antibody were used. Data are representative of three independent experiments.

 
As activation of ERK is another consequence of the engagement of S1P/EDG and TLR, the involvement of this route was assessed with phospho-specific antibodies. While Pam3CSK4 and S1P alone induced the activation of ERK (data not shown), S1P attenuated TLR2-mediated ERK activation in a dose-dependent manner (Figure 4A). ERK phosphorylation was most prominent on the ERK2 isoform, was observed 20–30 min after TLR2 activation and later decayed. S1P only induced a slight phosphorylation of ERK2. Statistically significant attenuation of TLR2-dependent ERK phosphorylation was observed with 1 µM S1P (Figure 4A). Densitometry analysis of five experiments revealed that 1 µM S1P inhibited 43.5 ± 12.5% of TLR2-mediated ERK2 activation. Similar results were observed in monocytes and THP-1 cells (data not shown). As for JNK, another member of the MAPK family, phosphorylation of the JNK1 and JNK2 isoforms was observed 20–30 min after activation of human macrophages with Pam3CSK4 and S1P (Figure 4B). However, in sharp contrast to what was observed in the ERK route, S1P did not inhibit TLR2-induced JNK phosphorylation, even at concentrations as high as 40 µM (Figure 4B). A lack of effect was also observed in THP-1 cells and human monocytes (data not show).

The effect of S1P on TLR2-dependent expression of chemokine genes was studied in human monocytes. Low micromolar doses of S1P inhibited the expression of MIP-1{alpha}, MIP-1β, and IL-8 induced by the TLR2 ligand PGN (Figure 5, lanes 2 and 5). Densitometry analysis of three independent experiments revealed that S1P inhibited TLR2 activation: 56.8 ± 0.2% of MIP-1β, 49.9 ± 0.1% of MIP-1{alpha}, and 43.3 ± 2.7% of IL-8. On the other hand, when cells were activated with a TLR4 ligand either no inhibition or even increase of chemokine expression was observed (Figure 5, lanes 3 and 6). Taken together, these data suggest that S1P attenuates TLR2-induced chemokine expression, NF-{kappa}B activation, and ERK, but not JNK phosphorylation in mononuclear phagocytes.


Figure 5
View larger version (56K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5 S1P selectively inhibits TLR2-induced chemokine expression. RNA from human monocytes stimulated with peptidoglycan, lipopolysachharide, or vehicle in the presence or absence of S1P, was analysed as in Figure 1. Data are representative of three independent experiments.

 
3.5 PI3K and Ras pathways might be involved in the S1P-induced attenuation of TLR2 activation
Since the PI3K route has been implicated in the negative regulation of TLR activation,34,35 a pharmacological approach was used to address whether the PI3K route is involved in the S1P-mediated inhibitory effect. In human monocyte-derived macrophages, 100 nM wortmannin partially reverted the attenuation of TLR2-induced NF-{kappa}B activation elicited by S1P (Figure 6A, upper panel) and ERK phosphorylation (Figure 6A, middle panel), suggesting the involvement of the PI3K pathway. As several effects of S1P receptors type 1 and type 2 involve Ras and Rho signals,13 we tested their putative role on the S1P-mediated attenuation using specific inhibitors of both pathways. 50 µM FPT-inhibitor II was used to block Ras, and 10 µM geranylgeranyltransferase inhibitor (GGTI)-298 to inhibit Rho. Experiments performed in human macrophages showed that the Ras inhibitor partially reverted the S1P-mediated attenuation of NF-{kappa}B activation and ERK phosphorylation (Figure 6B), however the Rho inhibitor did not influence S1P effects (Figure 6B). The average of three experiments showed that 55.8 ± 6.9% of ERK phosphorylation and 43.0 ± 7.9% of I{kappa}B{alpha} degradation were reverted by the Ras inhibitor. Altogether, these results strongly suggest the involvement of PI3K and Ras pathways, but not Rho signalling, in the S1P-mediated attenuation of TLR2 activation.


Figure 6
View larger version (40K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 6 PI3K and Ras pathways are involved in the S1P-mediated attenuation of TLR2 signalling. (A) Monocyte-derived macrophages were treated with wortmannin or vehicle, and later stimulated with Pam3CSK4 in the presence or absence of S1P. Cell lysates were processed as described in Figure 4A. (B) Human macrophages were treated with GGTI-298, farnesyltransferase-inhibitor II, or vehicle, and later stimulated with Pam3CSK4 in the presence or absence of S1P. The blots shown are representative of three independent experiments.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
There is increasing evidence on the involvement of inflammatory reactions in atherogenesis. Epidemiological data have stressed the association of bacterial and viral infections with disease aggravation, whereas pathological studies have disclosed the recruitment and activation of macrophages into the vessel wall as a major factor in the progression of atheromatosis. Linkage between innate immunity and lipid metabolism has been provided by the recognition that the reverse cholesterol transport and the inflammatory phenotype of macrophages are regulated by liver X receptor (LXR) and retinoid X receptor, and infectious microorganisms can interfere with macrophage cholesterol metabolism through an inhibition of the LXR signalling pathway initiated by the activation of TLR3 and TLR4.36 On this basis, pathogen-associated molecular patterns can exert a double role on atherogenesis: blockade of cholesterol reverse transport and enhancement of the inflammatory response. The scenario of atherogenesis has been complicated by the finding that minimally oxidized LDL bind to TLR,7 and by the complex and varying composition of lipoproteins. Taking into account these data, we have addressed the effect of lipoproteins and their lipid moieties by studying signalling routes involved in the inflammatory response: the NF-{kappa}B and the MAPK pathways. LDL showed a weak inhibition of TLR2-induced {kappa}B activity, although it was not statistically significant. On the other hand, ox-LDL significantly inhibited the effect of TLR2 agonists in TLR2-transfected 293 cells. The effect on TLR2 activation was surprisingly similar in ox-LDL and HDL, but we consistently observed those results. Because of the complexity of lipoproteins, it has to be taken into account that their overall biological effect is the result of the sum of varying effects of several lipid components acting upon different receptors. In fact, although ox-LDL are generally associated with proatherogenic effects, they had also been associated with some anti-inflammatory effects, e.g. inhibition of IL-12 production in LPS-activated macrophages.25 The physiological relevance of these findings was further confirmed in human monocytes and macrophages by analysing chemokine mRNA expression and NF-{kappa}B activation. The implications of the reported negative cross-talk for cellular functions are currently under investigation. Preliminary studies showed that S1P is able to prevent TLR2-mediated apoptosis and to facilitate monocyte/macrophage adherence (unpublished results).

As oxidation of LDL can affect both the apoprotein and the lipid burden, we focused on the effect of different lipoprotein-carried lipid components on TLR2 activation. Whereas LPA, PAF, and LPC did not significantly affect TLR2 activation, we observed an inhibitory effect with ox-PAPC, which agreed with previous reports,11 and with S1P, a finding not previously reported. This result seems of interest, as S1P is one of the components of, HDL and LDL (for review see Okajima' study12). In human macrophages, significant attenuation of TLR2 signalling was observed with 1 µM S1P, which argues in favour of a physiologically relevant and surface-mediated receptor effect, as S1P concentrations in plasma and serum can vary from high nanomolar to low micromolar ranges, and the existence of S1P gradients in the vasculature is known.37 Moreover, as S1P is associated with lipoproteins, the concentration on the cell surface may be higher than the concentration in whole plasma.16

The attenuation of TLR2 activation elicited by S1P seems to be restricted to the NF-{kappa}B route and ERK phosphorylation, as no inhibitory effect was observed on the JNK pathway. This finding is consistent with a cross-talk mechanism involving routes shared by both stimuli and connected to PI3K and Ras signalling pathways. It has been reported that the small GTPase Ras and ERK are involved in the modulation of immune cell proliferation by S1P, whereas PI3K are involved in S1P-mediated survival of lymphocytes and other immune cells.15 Moreover, some inhibitory actions of S1P on adhesion molecule expression have been related to effects on the PI3K route in endothelial cells,17 where the balance of the effects on the NF-{kappa}B and PI3K pathways determines the overall activity of S1P. In keeping with this report and also with the concept that the PI3K route is one of the best characterized intracellular negative regulators of TLR activation,34,35 our data suggest that PI3K is involved in the inhibition of TLR2 activation by S1P. Notably, similar cross-talk effects of the parent EDG/LPA receptors with other receptors have been reported in astrocytoma cells, where LPA inhibited the Ca2+ transient, but not MAPK activation, elicited by epidermal growth factor.30 As to TLR4, another TLR known to play a role in atherosclerosis, S1P showed an effect different from that observed on TLR2 in human monocytes, as no impairment by S1P of LPS-induced chemokine expression (Figure 5) and NF-{kappa}B activation (data not shown) was observed, while S1P partially inhibited LPS-induced ERK phosphorylation (data not shown).

Results from siRNA studies could be extrapolated to macrophages since, with the exception of S1P4, the expression pattern of S1P receptors in macrophages and 293 cells is comparable. The most abundant receptor in macrophages, S1P2, is similarly expressed in 293 cells, S1P1 is even more expressed in 293 cells, and S1P3 is not detectable in macrophages (data not shown). In agreement with the S1P-mediated attenuation of TLR2-induced chemokine expression in human monocytes, S1P receptor signalling has been found to modulate inflammatory pathways in endothelial cells17,38 and in cooperation with scavenger receptor class B type I, S1P1 receptors have been involved in the HDL-induced inhibition of adhesion molecule expression.39 Moreover, activation of S1P1 receptor prevents monocyte–endothelial interaction in a type 1 diabetes mouse model.40 Regarding the S1P receptor subtype involved in the attenuation effect, RNA interference studies showed the involvement of S1P1 and S1P2. The S1P2 receptor has been shown as a negative regulator of PDGF-induced migration and proliferation of fibroblasts from S1P2-null mice,41 and of endothelial morphogenesis and angiogenesis through Rac downregulation.42 S1P1 is essential for vascular smooth-muscle cells to migrate and maturate in S1P1-null mice,43 and is a key receptor in endothelial barrier protection.44

In contrast to the well-known mechanism of TLR2 activation, its negative regulation is largely unknown. Our findings regarding S1P effect are reminiscent of a report showing cross-talk of TLR with adenosine receptors as a mechanism to fine-tune inflammatory responses.45 Another report has shown a mechanism to regulate inflammatory responses via negative cross-talk with TLR2 adaptor molecules by the novel tumour suppressor cylindromatosis.46 Our data underscore that the inflammatory response to lipoproteins follows the general paradigm of the response to microorganism components, since lipoproteins, as well as many pathogen-derived receptors, can include a heterogeneous mixture of components which can cross-link a diverse array of receptors, thereby allowing the scrutiny of the cargo at the phagocytic cell surface, and the ensuing integration of a response that can be inflammatory, non-inflammatory or anti-inflammatory.47

Taken collectively, the present data show an inhibitory effect of ox-LDL and HDL on TLR2 activation that is mimicked by S1P and ox-PAPC, and points to the lipoprotein-associated lipid burden as a major determinant of their overall effect on the TLR2 system. Moreover, our data suggest that the negative cross-talk of S1P receptors and TLR2 signalling could explain some of the S1P atheroprotective effects.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
Co-funded by FEDER-FSE 2000/2006, from Plan Nacional de Salud y Farmacia (SAF2004-01232, SAF2006/08031, Ministerio de Sanidad (FIS 03/1489), Red Recava and Red Brucelosis from Instituto de Salud Carlos III, and Fundación de Investigación Médica Mutua Madrileña (FMM). Instituto de Salud Carlos III supported post-doctoral contract fund to A.I.D.


   Acknowledgements
 
We are grateful to Centro de Hemoterapia y Hemodonación de Castilla y León (Valladolid, Spain) for providing buffy coats.

Conflict of interest: none declared.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 

  1. Libby P. Inflammation in atherosclerosis. Nature (2002) 420:868–874.[CrossRef][Medline]
  2. Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med (2005) 352:1685–1695.[Free Full Text]
  3. Vink A, de Kleijn DP, Pasterkamp G. Functional role for toll-like receptors in atherosclerosis and arterial remodeling. Curr Opin Lipidol (2004) 15:515–521.[CrossRef][Web of Science][Medline]
  4. Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol (2003) 21:335–376.[CrossRef][Web of Science][Medline]
  5. Beutler B, Jiang ZF, Georgel P, Crozat K, Croker B, Rutschmann S, et al. Genetic analysis of host resistance: toll-like receptor signaling and immunity at large. Ann Rev Immunol (2006) 24:353–389.[CrossRef][Web of Science][Medline]
  6. Bjorkbacka H, Kunjathoor VV, Moore KJ, Koehn S, Ordija CM, Lee MA, et al. Reduced atherosclerosis in MyD88-null mice links elevated serum cholesterol levels to activation of innate immunity signaling pathways. Nat Med (2004) 10:416–421.[CrossRef][Web of Science][Medline]
  7. Miller YI, Viriyakosol S, Binder CJ, Feramisco JR, Kirkland TN, Witztum JL. Minimally modified LDL binds to CD14, induces macrophage spreading via TLR4/MD-2, and inhibits phagocytosis of apoptotic cells. J Biol Chem (2003) 278:1561–1568.[Abstract/Free Full Text]
  8. Miller YI, Viriyakosol S, Worrall DS, Boullier A, Butler S, Witztum JL. Toll-like receptor 4-dependent and -independent cytokine secretion induced by minimally oxidized low-density lipoprotein in macrophages. Arterioscler Thromb Vasc Biol (2005) 25:1213–1219.[Abstract/Free Full Text]
  9. Mikita T, Porter G, Lawn RM, Shiffman D. Oxidized low density lipoprotein exposure alters the transcriptional response of macrophages to inflammatory stimulus. J Biol Chem (2001) 276:45729–45739.[Abstract/Free Full Text]
  10. Eligini S, Brambilla M, Banfi C, Camera M, Sironi L, Barbieri SS, et al. Oxidized phospholipids inhibit cyclooxygenase-2 in human macrophages via nuclear factor {kappa}B/I{kappa}B- and ERK2-dependent mechanisms. Cardiovasc Res (2002) 55:406–415.[Abstract/Free Full Text]
  11. Walton KA, Cole AL, Yeh M, Subbanagounder G, Krutzik SR, Modlin RL, et al. Specific phospholipid oxidation products inhibit ligand activation of Toll-like receptors 4 and 2. Arterioscler Thromb Vasc Biol (2003) 23:1197–1203.[Abstract/Free Full Text]
  12. Okajima F. Plasma lipoproteins behave as carriers of extracellular sphingosine 1-phosphate: is this an atherogenic mediator or an anti-atherogenic mediator? Biochim Biophys Acta (2002) 1582:132–137.[Medline]
  13. Spiegel S, Milstien S. Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat Rev Mol Cell Biol (2003) 4:397–407.[CrossRef][Web of Science][Medline]
  14. Saba JD, Hla T. Point-counterpoint of sphingosine 1-phosphate metabolism. Circ Res (2004) 94:724–734.[Abstract/Free Full Text]
  15. Rosen H, Goetzl EJ. Sphingosine 1-phosphate and its receptors: an autocrine and paracrine network. Nat Rev Immunol (2005) 5:560–570.[CrossRef][Web of Science][Medline]
  16. Nofer JR, Assmann G. Atheroprotective effects of high-density lipoprotein-associated lysosphingolipids. Trends Cardiovasc Med (2005) 15:265–271.[CrossRef][Web of Science][Medline]
  17. Kimura T, Tomura H, Mogi C, Kuwabara A, Ishiwara M, Shibasawa K, et al. Sphingosine 1-phosphate receptors mediate stimulatory and inhibitory signalings for expression of adhesion molecules in endothelial cells. Cell Signal (2006) 18:841–850.[CrossRef][Web of Science][Medline]
  18. Michelsen KS, Doherty TM, Shah PK, Arditi M. Role of toll-like receptors in atherosclerosis. Circ Res (2004) 95:E96–E97.[Web of Science][Medline]
  19. Laman JD, Schoneveld AH, Frans LM, van Meurs M, Pasterkamp G. Significance of peptidoglycan, a proinflammatory bacterial antigen in atherosclerotic arteries and its association with vulnerable plaques. Am J Cardiol (2002) 90:119–123.[CrossRef][Web of Science][Medline]
  20. Edfeldt K, Swedenborg J, Hansson GK, Yan ZQ. Expression of Toll-like receptors in human atherosclerotic lesions: a possible pathway for plaque activation. Circulation (2002) 105:1158–1161.[Abstract/Free Full Text]
  21. Schoneveld AH, Nijhuis MMO, van Middelaar B, Laman JD, de Kleijn D, Pasterkamp G. Toll-like receptor 2 stimulation induces intimal hyperplasia and atherosclerotic lesion development. Cardiovasc Res (2005) 66:162–169.[Abstract/Free Full Text]
  22. Mullick AE, Tobias PS, Curtiss LK. Modulation of atherosclerosis in mice by Toll-like receptor 2. J Clin Invest (2005) 115:3149–3156.[CrossRef][Web of Science][Medline]
  23. Lien E, Sellati TJ, Yoshimura A, Flo TH, Rawadi G, Finberg RW, et al. Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products. J Biol Chem (1999) 274:33419–33425.[Abstract/Free Full Text]
  24. Fernandez N, Alonso S, Valera I, Vigo AG, Renedo M, Barbolla L, et al. Mannose-containing molecular patterns are strong inducers of cyclooxygenase-2 expression and prostaglandin E2 production in human macrophages. J Immunol (2005) 174:8154–8162.[Abstract/Free Full Text]
  25. Chung SW, Kang BY, Kim SH, Pak YK, Cho D, Trinchieri G, et al. Oxidized low density lipoprotein inhibits interleukin-12 production in lipopolysaccharide-activated mouse macrophages via direct interactions between peroxisome proliferator-activated receptor-gamma and nuclear factor-{kappa}B. J Biol Chem (2000) 275:32681–32687.[Abstract/Free Full Text]
  26. Marchant CE, Law NS, Van D, Hardwick VSJ, Carpenter KL, Mitchinson MJ. Oxidized low-density lipoprotein is cytotoxic to human monocyte-macrophages: protection with lipophilic antioxidants. FEBS Lett (1995) 358:175–178.[CrossRef][Web of Science][Medline]
  27. Slater TF, Sawyer BC. The stimulatory effects of carbon tetrachloride and other halogenoalkanes on peroxidative reactions in rat liver fractions in vitro. General features of the systems used. Biochem J (1971) 123:805–814.[Web of Science][Medline]
  28. Duenas AI, Orduna A, Crespo MS, Garcia-Rodriguez C. Interaction of endotoxins with Toll-like receptor 4 correlates with their endotoxic potential and may explain the proinflammatory effect of Brucella spp. LPS. Int Immunol (2004) 16:1467–1475.[Abstract/Free Full Text]
  29. Rinninger F, Brundert M, Brosch I, Donarski N, Budzinski RM, Greten H. Lipoprotein lipase mediates an increase in selective uptake of HDL-associated cholesteryl esters by cells in culture independent of scavenger receptor BI. J Lipid Res (2001) 42:1740–1751.[Abstract/Free Full Text]
  30. Hernandez M, Barrero MJ, Crespo MS, Nieto ML. Lysophosphatidic acid inhibits Ca2+ signaling in response to epidermal growth factor receptor stimulation in human astrocytoma cells by a mechanism involving phospholipase C{gamma} and a G{alpha}i protein. J Neurochem (2000) 75:1575–1582.[CrossRef][Web of Science][Medline]
  31. Idzko M, Panther E, Corinti S, Morelli A, Ferrari D, Herouy Y, et al. Sphingosine 1-phosphate induces chemotaxis of immature dendritic cells and modulates cytokine-release in mature human dendritic cells for emergence of Th2 immune responses. FASEB J (2002) 16:625–627.[Free Full Text]
  32. Endemann G, Stanton LW, Madden KS, Bryant CM, White RT, Protter AA. CD36 is a receptor for oxidized low density lipoprotein. J Biol Chem (1993) 268:11811–11816.[Abstract/Free Full Text]
  33. Tamai R, Sugawara S, Takeuchi O, Akira S, Takada H. Synergistic effects of lipopolysaccharide and interferon-gamma in inducing interleukin-8 production in human monocytic THP-1 cells is accompanied by up-regulation of CD14, Toll-like receptor 4, MD-2 and MyD88 expression. J Endotoxin Res (2003) 9:145–153.[Medline]
  34. Fukao T, Koyasu S. PI3K and negative regulation of TLR signaling. Trends Immunol (2003) 24:358–363.[CrossRef][Web of Science][Medline]
  35. Liew FY, Xu D, Brint EK, O'Neill LA. Negative regulation of toll-like receptor-mediated immune responses. Nat Rev Immunol (2005) 5:446–458.[CrossRef][Web of Science][Medline]
  36. Castrillo A, Joseph SB, Vaidya SA, Haberland M, Fogelman AM, Cheng G, et al. Crosstalk between LXR and toll-like receptor signaling mediates bacterial and viral antagonism of cholesterol metabolism. Mol Cell (2003) 12:805–816.[CrossRef][Web of Science][Medline]
  37. Igarashi Y, Yatomi Y. Sphingosine 1-phosphate is a blood constituent released from activated platelets, possibly playing a variety of physiological and pathophysiological roles. Acta Biochim Pol (1998) 45:299–309.[Web of Science][Medline]
  38. Bolick DT, Srinivasan S, Kim KW, Hatley ME, Clemens JJ, Whetzel AA, et al. Sphingosine-1-phosphate prevents tumor necrosis factor-{alpha}- mediated monocyte adhesion to aortic endothelium in mice. Arterioscler Thromb Vasc Biol (2005) 25:976–981.[Abstract/Free Full Text]
  39. Kimura T, Tomura H, Mogi C, Kuwabara A, Damirin A, Ishizuka T, et al. Role of scavenger receptor class B type I and sphingosine 1-phosphate receptors in high density lipoprotein-induced inhibition of adhesion molecule expression in endothelial cells. J Biol Chem (2006) 281:37457–37467.[Abstract/Free Full Text]
  40. Whetzel AM, Bolick DT, Srinivasan S, Macdonald TL, Morris MA, Ley K, et al. Sphingosine-1 phosphate prevents monocyte/endothelial interactions in type 1 diabetic NOD mice through activation of the S1P1 receptor. Circ Res (2006) 99:731–739.[Abstract/Free Full Text]
  41. Goparaju SK, Jolly PS, Watterson KR, Bektas M, Alvarez S, Sarkar S, et al. The S1P2 receptor negatively regulates platelet-derived growth factor-induced motility and proliferation. Mol Cell Biol (2005) 25:4237–4249.[Abstract/Free Full Text]
  42. Inoki I, Takuwa N, Sugimoto N, Yoshioka K, Takata S, Kaneko S, et al. Negative regulation of endothelial morphogenesis and angiogenesis by S1P(2) receptor. Biochem Biophys Res Commun (2006) 346:293–300.[CrossRef][Web of Science][Medline]
  43. Liu YJ, Wada R, Yamashita T, Mi YD, Deng CX, Hobson JP, et al. Edg-1, the G protein-coupled receptor for sphingosine-1-phosphate, is essential for vascular maturation. J Clin Invest (2000) 106:951–961.[Web of Science][Medline]
  44. Garcia JGN, Liu F, Verin AD, Birukova A, Dechert MA, Gerthoffer WT, et al. Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement. J Clin Invest (2001) 108:689–701.[CrossRef][Web of Science][Medline]
  45. Lukashev D, Ohta A, Apasov S, Chen JF, Sitkovsky M. Cutting edge: physiologic attenuation of proinflammatory transcription by the G protein-coupled A2A adenosine receptor in vivo. J Immunol (2004) 173:21–24.[Abstract/Free Full Text]
  46. Yoshida H, Jono H, Kai H, Li JD. The tumor suppressor cylindromatosis (CYLD) acts as a negative regulator for toll-like receptor 2 signaling via negative cross-talk with TRAF6 and TRAF7. J Biol Chem (2005) 280:41111–41121.[Abstract/Free Full Text]
  47. Stuart LM, Ezekowitz RA. Phagocytosis: elegant complexity. Immunity (2005) 22:539–550.[CrossRef][Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
79/3/537    most recent
cvn087v2
cvn087v1
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Dueñas, A. I.
Right arrow Articles by García-Rodríguez, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dueñas, A. I.
Right arrow Articles by García-Rodríguez, C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?