Cardiovascular Research (2009) 81, 216–225
doi:10.1093/cvr/cvn277
Leukotriene B4 enhances the activity of nuclear
factor-kB pathway through BLT1 and BLT2
receptors in atherosclerosis
Eva Sánchez-Galán1,2, Almudena Gómez-Hernández1,2, Cristina Vidal1,2,
José Luis Martı́n-Ventura1,2, Luis Miguel Blanco-Colio1,2, Begoña Muñoz-Garcı́a1,
Luis Ortega3, Jesús Egido1,2, and José Tuñón2,4*
1
Vascular Research Laboratory, Madrid, Spain; 2Autónoma University, Madrid, Spain; 3Department of Pathology, Hospital
Clı́nico, Madrid, Spain; and 4Department of Cardiology, Fundación Jiménez Dı́az, Avenida Reyes Católicos 2,
28040 Madrid, Spain
Received 11 March 2008; revised 27 September 2008; accepted 6 October 2008; online publish-ahead-of-print 13 October 2008
Time for primary review: 27 days
KEYWORDS
Leukotriene B4;
BLT1;
BLT2;
Inflammation;
NF-kB;
Atherosclerosis
Aims Leukotriene B4 (LTB4) is a powerful chemoattractant and pro-inflammatory mediator in several
inflammatory diseases, including atherosclerosis. It acts through its two membrane receptors, BLT1
and BLT2. The aim of this study was to determine the molecular mechanism involved in the proatherogenic effect of LTB4, BLT1 and BLT2 in atherosclerosis. Moreover, we characterized the expression of
5-lipoxygenase (5-LO) pathway and LTB4 receptors in blood and plaques from patients with carotid
atherosclerosis.
Methods and results In cultured monocytic cells, LTB4 induced a rapid phosphorylation of mitogenactivated protein kinases (MAPKs ERK1/2 and JNK1/2) and PI3K/Akt via BLT1 and BLT2 in a pertussis
toxin (PTX)-dependent mechanism (assessed via western blotting) and also increased nuclear factorkB (NF-kB) DNA binding activity (assessed via EMSA) in a MAPK- and reactive oxygen species-dependent
mechanism. Furthermore, LTB4 elicited interleukin-6, monocyte chemoattractant protein-1 and tumour
necrosis factor-a mRNA overexpression also via BLT1 and BLT2 by a PTX- and NF-kB-dependent mechanism (assessed by real-time PCR), promoting an inflammatory environment. When compared with healthy
subjects, patients with carotid atherosclerosis showed a significant increase in the expression of all
the components of the 5-LO pathway and BLT1 and BLT2 mRNA (real-time PCR) in peripheral blood
mononuclear cells and LTB4 plasma levels (ELISA). In these patients, an overexpression of 5-LO,
leukotriene A-4 hydroxylase (LTA4-H) and BLT1 was noted in the inflammatory region of carotid
plaques when compared with the fibrous cap (assessed by immunohistochemistry).
Conclusion The 5-LO pathway is enhanced in patients with carotid atherosclerosis. Furthermore, its
product LTB4 phosphorylates MAPKs and stimulates NF-kB-dependent inflammation via BLT1 and BLT2
receptors in cultured monocytic cells. The blockade of this pathway could be a novel and potential
therapeutic target in atherothrombosis.
1. Introduction
Leukotrienes are inflammatory lipid mediators derived from
the 5-lipoxygenase (5-LO) cascade of arachidonic acid.1 Leukotriene B4 (LTB4), one of the final products of this pathway,
is a potent chemoattractant and proinflammatory mediator
in several inflammatory diseases, including atherosclerosis.2
It is secreted by several cells such as monocytes, macrophages, mast cells, or neutrophils. LTB4 is present in
human atherosclerosis3 along with the components of the
5-LO pathway, 5-LO, 5-LO activating protein (FLAP) and
* Corresponding author. Tel: þ34 915504816; fax: þ34 915497033.
E-mail address: [email protected]
LTA4 hydrolase (LTA4-H), which are involved in the pathogenesis of this disorder.2,4 LTB4 binds to cell surface
G-protein-coupled receptors BLT1 and BLT2, generating a
variety of intracellular signals, such us calcium mobilization
or adenylate cyclase inhibition. BLT1 and BLT2 are, respectively, the high- and the low-affinity receptors. BLT1 is
preferentially expressed in leukocytes and monocytes,
while BLT2 is ubiquitously expressed.5,6
Apolipopotein E knockout (ApoE2/2) and low density
lipoproteins receptors knockout (LDLr2/2) mice treated
with BLT1 antagonists showed a reduced lipid accumulation,
monocyte infiltration, and size of atheroma.7 Moreover,
levels of adhesion molecules, such us CD11b, were
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2008.
For permissions please email: [email protected].
217
Intracellular mechanisms involved in the atherogenic effect of LTB4
reduced in mice treated with BLT1 antagonists.7 Macrophage
cell lines from BLT12/2 mice expressed BLT2 receptor and
exhibited chemotaxis to LTB4.8 In rat basophilic cells, LTB4
promotes conversion of monocytes to foam cells through
an enhanced expression of scavenger receptors (CD36) and
subsequent fatty acid accumulation.9 From these studies,
it was concluded that effects of LTB4 in chemotaxis and in
promoting foam cells are mediated by BLT1 and BLT2 receptors. Other studies in smooth muscle cells show that BLT1
expression was blunted by dominant-negative Ikkinase-B,
indicating a role for nuclear factor-kB (NF-kB). The involvement of BLT1 and BLT2 in atherosclerosis is not clear.
In this study, we have investigated the intracellular mechanisms involved in the atherogenic effects of LTB4 and their
possible regulation in a monocytic cell line (U-937). Moreover, we have examined LTB4 plasma levels and the 5-LO
pathway in peripheral blood mononuclear cells (PBMC) and
plaques from subjects with carotid atherosclerosis to
assess the in vivo relevance of our in vitro studies.
2. Methods
2.1 Cell culture
U-937 monocytic cells [ATCC (TIB 202)] were cultured in RPMI supplemented with 10% heat inactived foetal bovine serum, 2 mmol/L
glutamine, 100 U/mL penicillin (GIBCO, BRL), at 378C in 5% CO2.
Before the experiments, cells were growth-arrested by incubation
in serum-free medium for 48 h and then replaced with fresh serumfree medium with or without (control cells) treatment. For the
inhibition experiments, U-75302 and Ly-2552837 (BLT1 and BLT2
selective antagonists, respectively) (Cayman Chemical, Ann Arbor,
MI, USA), Pertussin Toxin (PTX, Gai/0 protein inhibitor) (Sigma,
Saint Louis, MO, USA), MG-132, Bay117032, and parthenolide
(NF-kB inhibitors) (Calbiochem, La Jolla, CA, USA), PD98059, a
ERK1/2 inhibitor, SB203580, a p-38 MAPK inhibitor, SP-600125, a
JNK inhibitor, Wortmannin, a AKT inhibitor, and two different antioxidants, DPI and TIRON (Sigma, Saint Louis, MO, USA) were added
to the cultured medium 1 h before treatment with LTB4 (Cayman
Chemical, Ann Arbor). All the drugs used were prepared and
stored in dimethylsulfoxide (DMSO) at a final concentration of
1022 or 1023 mol/L at 2208C. To achieve the concentration used
for the different compounds, further serial 10-fold dilutions were
done in serum-free RPMI-1640 cell culture medium, which was
instantly added to cells yielding a final DMSO concentration
,0.1%. At the doses tested, neither vehicle nor the drugs produced
significant cell toxicity or apoptotic cell death (analysed by cell
morphology and flow cytometry; data not shown).
antibody at a final concentration of 1:50 (sc-372X, Santa Cruz Biotechnology) followed by FITC-conjugated antibody at a final concentration of 1:200 (Sigma, Saint Louis, MO, USA). The integrity of the
nuclei was confirmed with propidium iodide (IP) staining (1 mg/mL)
(Sigma, Saint Louis, MO, USA). Controls were stained with the secondary antibody alone (not shown). Coverslips were mounted in
FluorSave (Calbiochem, La Jolla, CA, USA) and examined by a
laser scanning confocal microscope (Leika).
2.4 Protein studies
To quantify protein levels, western blot analyses were also performed. Protein extracts from cultured cells were obtained by homogenization and centrifugation as previously described.11 Then,
samples were separated using a 8–12% SDS–polyacrylamide gel electrophoresis and were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). They were blocked in PBS
containing 0.1% Tween-20, 7.5% dry skimmed milk for 1 h at room
temperature and incubated in the same buffer with specific antibodies phospho-IKKab, IKKa, IKKb, phospho-IkBa, IkBa,
phospho-ERK1/2, ERK1/2, phospho-JNK1/2, JNK1/2, phospho-AKT,
AKT, phospho-p38, and p38 (Santa Cruz Biotechnology) overnight
at 48C. After washing, detection was made by incubation with
peroxidase-conjugated secondary antibody, and developed using
an ECL chemiluminiscence kit (Amersham Bioscience, Arlington
Heights, IL, USA). All the first antibodies were diluted 1:1000 and
secondary antibodies 1:5000. Proteins were quantified in all
samples by the BCA method in accordance with the manufacturer’s
directions (Pierce, Rockford, IL, USA) and a fixed amount of protein
(50 mg) was added in each lane to normalize for protein loading. The
quality of proteins and efficacy of protein transfer were evaluated
by Red Ponceau staining (not shown). To evaluate loading control,
total isoform of antibody in all cases was used.
2.5 Patients
Seventeen patients undergoing carotid endarterectomy in our Institution were studied. Patients with malignancies, inflammatory diseases, coagulation disorders, or those needing additional therapy
for chronic conditions different than atherosclerosis or its risk
factors were excluded. Informed consent was obtained before
enrollment. Table 1 shows the baseline characteristics of the
patients. Blood samples were collected the day of endarterectomy
before surgery, and from 19 healthy volunteers without significant
difference in age (55 + 9 years) and sex (12 men/7 women).
During surgery, carotid endarterectomy specimens were collected
for the study. The researchers who performed the studies at the laboratory were blind to the origin of the samples. The study was
Table 1 Baseline characteristics of patients with atherosclerosis
2.2 Analysis of nuclear factor-kB DNA
binding activity
NF-kB DNA-binding activity was determined as described.10 Briefly,
5-mg nuclear protein extracts binds to a [g-32P]- ATP-labelled oligonucleotide containing the NF-kB sequence (50 -AGTTGAGGGGAC
TTTCCCAGGC-30 ) (Promega, Madison, WI, USA). Complexes were
analysed by electrophoretic mobility shift assay (EMSA).10 Competition assays were performed by adding 100-fold excess of cold
probe before the labelled probe. For supershift, nuclear extracts
were incubated with 1.0 mg of p50 and p65 antibodies from Sta.
Cruz Biotechnology (Santa Cruz, CA, USA) 1 h before incubation
with labelled oligonucleotide.
2.3 Immunofluorescence
NF-kB localization was performed by indirect immunofluorescence.
Cells were fixed in paraformaldehyde 2% (Sigma, Saint Louis, MO,
USA), treated with 0.1% Triton X-100, and incubated with anti-p65
Age (mean + SD)
Sex (male/female)
Smoker
Never (%)
Past (%)
Present (%)
Diabetes (%)
Hypertension (%)
Hypercholesterolemia (%)
Statins (%)
Antithrombotic agents (%)
ACEIs (%)
Oral antidiabetics agents (%)
Insulin (%)
Calcium antagonists (%)
ACEIs, angiotensin converting enzyme inhibitors.
64 + 8
13/4
21
21
58
41
50
63
75
87
47
19
19
35
218
approved by the Ethical Committee of the Fundación Jiménez Dı́az
in accordance with the principles outlined in the Declaration of
Helsinki.
2.6 Isolation of peripheral blood mononuclear cells
Twenty millilitres of blood were obtained from patients and healthy
controls. PBMC isolation was performed as described previously.10
Briefly, blood samples were diluted in phosphate-buffered saline
1:1, and the cells were separated in 5 mL Ficoll gradient (lymphocyte isolation solution; Rafer SL, Zaragoza, Spain) by centrifugation
at 2000 g for 30 min. PBMCs were collected, washed twice with cold
phosphate-buffered saline, and resuspended in appropriate buffer.
Approximately 95% of the cells were mononuclear cells (by flow
cytometry; data not shown).
E. Sánchez-Galán et al.
counterstained with haematoxylin. Negative controls using the corresponding IgG were included to check for non-specific staining (not
shown). Results are expressed as a percentage of positive staining
area in the shoulder and cap regions and quantified as described
previously.10
2.9.3 Southwestern histochemistry
The distribution and DNA-binding activity of NF-kB in situ was
detected using a digoxigenin-labelled double-stranded DNA probe
(Roche Molecular Biochemicals, Indianapolis, IN, USA) with a specific
NF-kB consensus sequence.10 Competition assays with 100-fold
excess of unlabelled probe were used as negative controls.
2.10 Statistical analysis
Plasma was obtained from blood drawn on EDTA after centrifugation
(2500 g for 15 min) and aliquoted to avoid freeze/thaw cycles. LTB4
was measured by ELISA (R&D Systems, Minneapolis, MN, USA), and
its concentrations were determined according to the manufacturer’s directions. The sensitivity of the LTB4 assay is ,20 pg/mL.
Intra- and inter-assay coefficients of variation were ,12%.
Statistical analysis was performed with SPSS 11.0 (SPSS Inc., Chicago,
IL, USA). Data were expressed as mean + SEM and analysed by
the Mann–Whitney U-test for in vivo studies and the t-Student
(Bonferroni) for in vitro experiments. The Kolmogorov–Smirnov test
was used to establish the parametric or non-parametric distribution
of LTB4 plasma levels. To meet the distributional assumptions, LTB4
plasma levels were log-transformed and expressed as median
(interquartile range). Statistical significance was defined as P , 0.05.
2.8 Gene expression studies
3. Results
2.7 ELISA
Total RNA from PBMC was isolated with TRIzolTM (Invitrogen,
Carlsbad, CA, USA) according to the manufacturer’s specifications
and quantified by absorbance at 260 nm in duplicate. cDNA was
synthesized with a high capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) using 2 mg of total RNA primed with
ramdon hexamer primers, following the manufacturer’s instructions.
Real time-PCR was performed using a fluorogenic TaqMan MGB probes
and primers designed by Assay-on-DemandTM gene expression
products (Applied Byosystem, Foster City, CA, USA). Assays on
demand used were: human 5-LO (Hs00167536_m1), human
FLAP (Hs00233463_m1), human LTA4H (Hs00251637_m1), human
BLT1 (Hs00175124_m1), human BLT2 (Hs001885851_s1), human MCP-1
(Hs00234140_m1), human IL-6 (Hs00174131_m1), and human TNF-a
(Hs00174128_m1). Quantitative RT–PCR was performed by 7500 Real
Time PCR System, and the relative quantification was carried out
with the Prism 7000 System SDS Software (Applied Biosystems).
The mRNA copy numbers were calculated for each sample with
the instrument software using Ct value (‘arithmetic fit point analysis
for the lightcycler’). Results were expressed in copy numbers, calculated in relation to control cells, after normalization against
GAPDH (Hs99999905_m1), and 18S eukaryotic ribosomal
(4310893E), as previously described.11 All primers, probes, software, and reagents were obtained from Applied Biosystems. All
measurements were performed in duplicate.
2.9 Histological analysis
2.9.1 Tissue sampling
Specimens were stored in paraformaldehyde for 18 h and later in
ethanol until being paraffin-embedded. The region of atheroma covering the bifurcation of the common carotid artery was chosen. We
studied the atherosclerotic plaques in two different areas: the
shoulder and the fibrous cap.10
2.9.2 Immunohistochemistry
Paraffin-embedded atherosclerotic plaques were dewaxed and rehydrated. The following polyclonal antibodies were used at a final concentration of 1:100 in PBS: FLAP (sc-28815) (Santa Cruz, CA, USA), 5-LO
(160402), LTA4-H (160250), BLT1 (120114), and BLT2 (120124) (Cayman
Chemical, Ann Arbor, MI, USA). A biotin-labelled secondary antibody was
used at a final concentration of 1:200 in PBS (Amersham Bioscience,
Arlington Heights). Then, ABComplex/HRP (DAKO) was added and
sections were stained with 3,30 -diaminobenzidine (DAKO) and
3.1 5-LO pathway in the blood of patients
with carotid atherosclerosis
First, we assessed the expression of 5-LO, FLAP, LTA4H, BLT1,
and BLT2 proteins in PBMC isolated from the blood of atherosclerotic patients. The results showed a significant increase
of 5-LO, FLAP, LTA4-H, BLT1, and BLT2 mRNA expression in
PBMC of atherosclerotic patients in relation to healthy controls (Figure 1A). We also observed an increase of LTB4
levels in the plasma from patients compared to the control
group (290 + 48 vs. 131 + 16 pg/mL; P , 0.005) (Figure 1B).
3.2 Intracellular signalling induced by LTB4 through
BLT1 and BLT2 in human monocytic cells
To investigate the intracellular signalling pathways that
involve LTB4 activation, we analysed the MAPK and PI3K
pathways as well as G-proteins. LTB4 induced phosphorylation of ERK1/2, JNK1/2, AKT, and p38 in a time-dependent
mechanism (data not shown), peaking at around 15 min for
all kinases in monocytes (western blot). Pre-treatment
with U-75302 and Ly-255283 (BLT1 and BLT2 antagonists,
respectively) diminished phosphorylation of ERK1/2, JNK1/2
(Figure 2A), and AKT (Figure 2B), but did not modify the
p38 phosphorylation levels (Figure 2A). Moreover, we also
examined by western blot the effect of PTX, a specific
inhibitor of Gi/0-type G-proteins on LTB4-induced MAPK
and AKT pathway activation. Pre-treatment with PTX prior
to LTB4-stimulation, inhibited ERK1/2, JNK1/2 and AKT activation (Figure 2C). These results indicate that LTB4 induces
ERK1/2, JNK1/2, and AKT phosphorylation via BLT1 and BLT2
in a PTX-sensitive mechanism.
3.3 LTB4 increases NF-kB activity through BLT1
and BLT2 in human monocytic cells
To further investigate the mechanism involved in the inflammatory effects of LTB4, we studied NF-kB activation, which is
highly involved in the regulation of several pro-inflammatory
genes involved in atherosclerosis.
Intracellular mechanisms involved in the atherogenic effect of LTB4
219
Figure 1 The 5-LO pathway in the blood of atherosclerotic patients. (A) 5-LO/LTB4 pathway gene expression in peripheral blood mononuclear cells. 5-LO,
LTA4-H, FLAP, BLT1, and BLT2 gene expression was greater in patients with carotid atherosclerosis (n ¼ 17) than in healthy subjects (n ¼ 19) (real time-PCR).
Results are expressed as mean + SEM. *P , 0.05 vs. healthy. (B) LTB4 plasma levels (ELISA) were also higher in patients than in healthy controls. Black and
white bars represent patients and healthy subjects, respectively. Results are expressed as median (interquartile range).
LTB4 increased NF-kB DNA-binding activity in a dose- and
time-dependent mechanism with maximal effect observed
at 1027 mol/L, peaking at 90 min (data not shown). Pretreatment with U-75302 and Ly-255283 reduced LTB4-induced
NF-kB activation (Figure 3A). No additive effect was observed
when both antagonists were combined in cell culture
medium. Supershift assays confirmed that LTB4 activated
NF-kB complex as a p50/p65 heterodimer (Figure 3B).
Additional assays with immunofluorescence techniques
showed that, in control cells, a diffuse cytoplasmic immunofluorescence was seen with p65 antibody (Figure 3C). When cells
were treated with LTB4 (1027 mol/L) for 90 min, an intense
nuclear fluorescence was observed, showing nuclear translocation of NF-kB. Pre-treatment with U-75302 and Ly-255283 diminished the nuclear translocation of the p-65 subunit of NF-kB.
NF-kB activation involves phosphorylation of IkB by the
IkB kinase (IKK) complex, which results in IkB degradation.
In control cells, IkBa was found in the cytosolic fraction.
After 90 min of LTB4 stimulation, an increase in IKKab and
IkBa phosphorylation was observed (Figure 3D and E,
respectively). BLT1- and BLT2-specific antagonists reduced
IKKab and IkBa phosphorylation in monocytes (Figure 3D
and E, respectively). Moreover, the specific NF-kB inhibitors
MG-132, Bay-117032, and parthenolide reduced IkBa phosphorylation (data not shown).
Furthermore, we observed crosstalk between ERK and JNK
pathways in this process (Figure 4A). ERK activation was
affected by inhibition of JNK with SP600125. The same happened with JNK, which was inhibited by the ERK1/2 inhibitor
PD98059. Moreover, MAPK and NF-kB pathway are regulated
by redox mechanisms.1,12 In this sense, we pre-treated the
monocytes with two different antioxidants, DPI (an inhibitor
of flavoprotein-containing enzymes such as NADPH/NADPH
oxidase) and Tiron (an O2
2 scavenger) to determine the
role of reactive oxygen species (ROS). As shown in
Figure 4A and B, DPI and Tiron abolished LTB4-induced
MAPK pathways activation and NF-kB activation, respectively. These results show that LTB4 regulates MAPK and
NF-kB pathways in a ROS-dependent mechanism.
3.4 LTB4 induces proinflammatory factors through
BLT1 and BLT2 by a PTX- and NF-kB-dependent
mechanism in monocytic cells
In cultured monocytes, LTB4 upregulated IL-6, MCP-1, and
TNF-a expression at 3 h (Real Time-PCR). U-75302,
220
E. Sánchez-Galán et al.
Figure 2 LTB4 activates ERK1/2, JNK1/2, and PI3K/AKT via BLT1 and BLT2 in a PTX-dependent mechanism in U-937 cells. Cells were serum deprived for 24 h,
pre-treated with the BLT1 antagonist U-75302 (U; 1027 mol/L), the BLT2 antagonist Ly-255283 (Ly; 1027 mol/L) and Pertussis Toxin (PTX; 100nmol/L, a Gi/0 type
G-proteins inhibitor) for 1 h. Later, they were stimulated with LTB4 1027 mol/L for 15 min. The presence of increased phosphorylated proteins is considered as
MAPK pathway activation. Total isoform of antibody in all cases was used to evaluate loading control. Control cells refer to unstimulated cells, without treatment. (A) shows data, expressed as mean + SEM of five experiments (left panel), and a representative western blot (right panel). (B) shows data as mean + SEM
of five experiments (top panel) and a representative western blot (bottom). (C ) shows mean + SEM of five western blot experiments. *P , 0.001 vs. control;
#
P , 0.005 vs. LTB4.
Ly-2552837, and PTX significantly diminished LTB4-mediated
gene overexpression (Figure 5A). No additive effect was
observed when both antagonists were combined. Pretreatment with NF-kB inhibitors MG-132, Bay-117032, and
parthenolide significantly decreased IL-6, MCP-1, and
TNF-a mRNA induction caused by LTB4 in monocytes
(Figure 5B). These results suggest that LTB4 induced the
expression of pro-inflammatory cytokines IL-6, MCP-1, and
TNF-a, via BLT1 and BLT2 in a PTX- and NF-kB-dependent
mechanism.
human atherosclerotic plaques is characterized by an
increase in macrophage infiltration, COX-2 expression, and
NF-kB activation.10,13 A significant increase of 5-LO,
LTA4-H, and BLT1 expression in the shoulder when compared
with the cap of the lesions (Figure 6A) was observed. In contrast, FLAP and BLT2 expression was similar in both areas.
Proteins of the 5-LO pathway and BLT1 and BLT2 receptors
expression colocalized with active NF-kB (Figure 6B),
suggesting that they could all modulate the transcriptional
activity of NF-kB in human atheroma.
3.5 Expression of 5-LO/LTB4 pathway are
augmented in the shoulder region of carotid
atherosclerotic plaques
4. Discussion
To assess the in vivo relevance of our studies, we analysed
5-LO/LTB4 pathway in human atherosclerotic plaques. It
has been shown previously that the shoulder region of
Over the previous years, the leukotriene research field in
atherosclerosis has experienced a great advance. Genetic
studies have indicated a link between variants of FLAP,
LTA4-H, and 5-LO genes and the development of atherosclerosis and myocardial infarction.14,15 Moreover, in
Intracellular mechanisms involved in the atherogenic effect of LTB4
221
Figure 3 LTB4 increases NF-kB activity via BLT1 and BLT2 receptors in monocytic cells. (A) displays mean + SEM values from four different experiments and a
representative EMSA. Cells were pre-treated for 1 h with U-75302 (BLT1 antagonist), Ly-255283 (BLT2 antagonist), or both at doses of 1027 mol/L in all cases and
later stimulated with LTB4 at 1027 mol/L for 90 min. Control refers to cells without treatment. *P , 0.001 vs. control; #P , 0.005 vs. LTB4. (B) shows supershift
analysis of LTB4-induced p65/p50 NF-kB subunits. (C ) shows a representative confocal picture of NF-kB p65 nuclear translocation induced by LTB4 (1027 mol/L;
90 min) and the effect of U-75302 (BLT1 antagonist) and Ly-255283 (BLT2 antagonist) (1027 mol/L for both). Indirect immunofluorescence was performed using
p65 antibody and FITC-labelled secondary antibody (green staining). Nuclei were stained with propidium iodure (IP, in red). Merge refers to an overlapping of the
same images to stain for p65 antibody and for propidium iodure. Protein levels of p-IKKab, IKKa, IKKb (D) and p-IkBa and IkBa (E) were determined by western
blot. The upper panel shows data (mean + SEM) of five experiments. The low panel displays a representative western blot. *P , 0.001 vs. control; #P , 0.005
vs. LTB4.
patients with a specific risk to variants of FLAP and LTA4H,
DG-031, therapy with a FLAP inhibitor, leads to dosedependent suppression of biomarkers associated to an
increased risk of myocardial infarction.16 DG031 is now in
a phase III clinical trial for the prevention of myocardial
infarction (http://www.decode.com/).
Further in vivo studies performed on mice have shown
several connections among 5-LO, LTB4 receptors, hyperlipidaemia, and inflammatory chemokine production.7,17,18 The
plaque-forming cells, including macrophages and smooth
muscle cells, express the entire cascade of leukotriene biosynthetic proteins and receptors. In the present study, we
222
Figure 4 LTB4 induced ERK, JNK, and NF-kB activation in a MAPK- and ROSdependent mechanism. Quiescent U-937 cells were pre-incubated for 1 h with
PD98059 (ERK1/2 inhibitor) at 1026 mol/L, SB203580 (p-38 MAPK inhibitor) at
1026 mol/L, SP-600125 (JNK inhibitor) at 1026 mol/L, Wortmannin (AKT inhibitor) at 1026 mol/L, and two different antioxidants, DPI and TIRON at 1025 mol/L
for both. Later, monocytes were stimulated with LTB4 1027 mol/L for 15 min (A)
or 90 min (B). (A) Evaluation of interrelation among ERK, JNK, and ROS activation caused by LTB4. The figure shows data (mean + SEM) from four
western blot experiments. (B) LTB4 induced NF-kB activation in a MAPK- and
ROS-dependent mechanism. The figure shows data as mean + SEM of four
experiments and a representative EMSA. The specificity of the reaction was
established using competition assays with excess of unlabelled NF-kB oligonucleotide (Cold NF-kB; C). *P , 0.001 vs. control and #P , 0.005 vs. LTB4.
have identified the first evidence, to our knowledge, that
the proteins of the 5-LO pathway and BLT1 and BLT2 receptors are overexpressed in PBMCs of patients with atherosclerosis. We have demonstrated previously that NF-kB
activity, COX-2, and mPGES-1 are enhanced in PBMCs of
patients with carotid atherosclerosis when compared with
healthy controls.10,11,13,19 In our study, we have also noted
that LTB4 plasma levels are increased in these patients.
This could suggest the presence of an inflammatory state
in the circulating cells of these patients before they
migrate into the arterial wall. As a limitation, we must
acknowledge that plasma and PBMC data from patients
with atherosclerosis could have been influenced by the
stress of imminent surgery, a condition that was not
present in healthy controls. Nevertheless, to our knowledge,
there are no data in the literature supporting that stress by
itself may induce significant changes in the studied parameters. On the other hand, we must note that in our
study, the LTB4 concentrations measured by ELISA are 1–2
order of magnitude higher compared with those measured
by gas chromatography-negative ion chemical ionizationmass spectrometry (GC-MS)12 or immuno high-performance
liquid chromatography.20 We believe that this disparity in
E. Sánchez-Galán et al.
LTB4 concentration may be due to the different specificity
of the different techniques by LTB4. Furthermore, we performed the experimental protocol in accordance with specifications provided by the manufacturers, and the LTB4
concentrations present in our study are in accordance with
the results obtained by the manufacturers in their tests.
LTB4 is a potent pro-inflammatory mediator that activates
multiple leukocyte subsets leading to cell recruitment, production of ROS, and induction of gene expression.1,5,6,21 It
binds to specific heptahelical receptors of the rhodopsin
class, BLT1 and BLT2.5,6,22 These receptors interact with G
proteins in the cytoplasm, and induce different activities,
ranging from motility to transcriptional activation. BLT1 is
the high-affinity receptor that mediates most, if not all, of
its chemoattractant and proinflammatory actions. BLT2 is
the lower-affinity receptor that also binds other lipoxygenase products, but little is known about its functions. In
this paper, we investigated LTB4 signalling via BLT1 and
BLT2 and their contribution to inflammation in atherosclerosis. The NF-kB has a key role in inflammation and innate
immunity by regulating the expression of many proinflammatory and prothrombotic molecules.23,24 We have observed
that LTB4 significantly enhanced NF-kB DNA-binding activity.
We have demonstrated also that LTB4 induced nuclear translocation of p65 subunits. The blockade of BLT1 and BLT2
receptors with specific antagonists inhibited NF-kB DNA
binding activity in monocytes, suggesting that these receptors participate in NF-kB activation. Moreover, studies with
confocal microscopy showed that BLT1 and BLT2 inhibition
diminishes nuclear translocation of the p65 subunit. Furthermore, there was not an additive effect of selective BLT1 and
BLT2 inhibitors on NF-kB activation.
The activation of NF-kB involves phosphorylation of IkB by
the IKK complex, which results in IkB degradation. Our data
showed that the BLT1 and BLT2 antagonists reduced IKKab
and IkBa phosphorylation in monocytes. In addition, NF-kB
inhibitors MG-132, Bay-117032, and parthenolide also
reduced IkBa phosphorylation in monocytes, suggesting a
NF-kB-dependent mechanism of LTB4 activation in monocytes. Moreover, to study the mechanism by which NF-kB
is activated by LTB4, we investigated whether LTB4 activates MAPK pathways in monocytes cells. Some studies
show that LTB4 activates these pathways in several cell
types transducing different LTB4 responses such as proliferation and chemotaxis.25–28 In this work, we have found that
LTB4 induces a rapid activation of ERK1/2, JNK1/2, p38, and
PI3K/AKT, showing that these signalling pathways participate in the modulation of LTB4 actions. The inhibition of
BLT1 and BLT2 diminishes the activation of these pathways,
implicating both receptors in the modulation of LTB4
responses. Moreover, pre-treatment with PTX, also inhibited
ERK1/2, JNK1/2, and AKT activation, suggesting that LTB4
induces ERK1/2, JNK1/2, and AKT phosphorylation via BLT1
and BLT2, in a PTX-sensitive mechanism. In this sense, we
also have observed that LTB4 induced NF-kB activation in a
MAPK- and ROS-dependent mechanism. Therefore, we
must note that the inhibitory effect of antioxidants (DPI
and Tiron) gives indirect evidence on the role of ROS in
the effects evoked by LTB4; perhaps the measurement of
ROS-production is a more precise indicator of the ROS involvement in NF-kB activation induced by LTB4.
To further investigate the proinflammatory effect of LTB4
in monocytic cells, we noted that NF-kB dependent genes,
Intracellular mechanisms involved in the atherogenic effect of LTB4
223
Figure 5 LTB4 induces pro-inflammatory cytokines through BLT1 and BLT2 in a NF-kB-dependent mechanism in U-937. (A) Treatment with U-75302 (U, 1027 mol/L),
Ly-255283 (Ly, 1027 mol/L), and PTX (100 nmol/L) reduces the expression of IL-6, MCP-1, and TNF-a. (B) NF-kB inhibition with MG-132 (MG), Bay117032 (Bay), and
parthenolide (1027 mol/L, 1 h for all) significantly reduces LTB4-induced expression of IL-6, MCP-1, and TNF-a. Data are mean + SEM of four real-time PCR
experiments.*P , 0.001 vs. control; #P , 0.05 vs. LTB4.
such as IL-6, TNF-a, and MCP-1, are upregulated by LTB4,
and pre-treatment with NF-kB inhibitors diminished this
overexpression, suggesting a NF-kB-mediated transcriptional mechanism. Moreover, the blockade of BLT1, BLT2,
and Gai/0 protein, also inhibited IL-6, TNF-a, and MCP-1
mRNA expression induced by LTB4 in monocytes and no additive effect was observed when combining both antagonists.
This suggests that NF-kB activation and upregulation of
NF-kB-related genes by LTB4 is mediated to some extent
via BLT1 and BLT2 receptors in a PTX-sensitive mechanism.
Several studies have shown that BLT1 and BLT2 expression
depends on the cell type. Back et al.29 demonstrated that
BLT1 colocalizes with smooth muscle cells, endothelial
cells, and macrophages in human atheroma, whereas BLT2
protein was found only in macrophage areas, suggesting
that it may be involved in the vascular damage induced by
LTB4. Moreover, it has been reported that macrophages
from BLT12/2 mice show LTB4-induced chemotaxis
through the activation of a BLT2 receptor.8
It is interesting to note that Lundeen et al.25 and Kitaura
et al.30 reported that the addition of both combined antagonists to the culture medium had no additive effect on chemotaxis in mast cells and bone marrow-derived mast cells
(BMMC). The concentrations of U-75302 and Ly-255283
used in these works and in our study were selective for
BLT1 and BLT2 inhibition, respectively. In this sense,
Lundeen et al.25 proposed that BLT1 and BLT2 could function
together, as a heterodimer, completely inhibiting chemotactic responses. Our data suggest that both receptors may
work together also in the stimulation of cytokine expression,
as their combined blockade does not cause an additive
effect.
Finally, to assess the in vivo relevance of our findings, we
analysed carotid human atheroma, and we observed a significant increase of 5-LO, LTA4-H, and BLT1 expression in
the shoulder when compared with the cap of the plaques.
In regard to this, we and others have shown previously
that the shoulder of human atheroma displays an increase
in macrophage infiltration, COX-2 expression, and NF-kB
activation.10,13,31 Additionally, on the one hand, symptomatic atherosclerotic plaques express elevated levels of
5-LO and LTB4, supporting the idea that LTB4 may be a
mediator of 5-LO-dependent plaque instability.32,33 On the
other hand, atherosclerotic lesions display more NF-kB
activity in coronary plaques responsible for an acute coronary syndrome that in stables ones.34 Here, we have demonstrated that active NF-kB colocalizes with LTA4-H and with
BLT2 in carotid atherosclerotic plaques, suggesting that
the 5-LO pathway favours NF-kB activation in situ, thereby
increasing their inflammatory state.
224
E. Sánchez-Galán et al.
Figure 6 Expression of 5-LO pathway in human carotid atheroma. (A) 5-LO, LTA4-H, and BLT1 expression are augmented in the shoulder of atherosclerotic
plaques (n ¼ 17). Representative examples of immunohistochemistry (Magnification 200). Results are expressed as mean + SEM. Black and white bars represent
shoulder and cap, respectively, *P , 0.005 and †P , 0.05 vs. shoulder. (B) Active NF-kB colocalizes with LTA4-H and BLT2. Blue colour shows active nuclear NF-kB.
Brown colour displays LTA4-H and BLT2 expression (Magnification 400).
In summary, our results demonstrate that patients with
carotid atherosclerosis display an enhancement of the leukotriene cascade in blood and in the vulnerable region of
the plaques that could contribute to increase the inflammatory burden in this disease. In cultured monocytes, LTB4
activate ERK1/2, JNK1/2, AKT, and NF-kB pathway via
BLT1 and BLT2 in a PTX-dependent mechanism, upregulating
the expression of pro-inflammatory cytokines involved in
atherosclerosis. Our data suggest that both receptors play
a role in the pro-inflammatory environment in atherosclerosis. Then, this pathway could be a novel therapeutic target
in atherosclerosis. Further studies are needed to confirm if
its inhibition is useful in the treatment of this disorder.
Acknowledgements
E.S.-G. is a fellow of Fundación Conchita Rábago. The authors would
like to thank Mar Gonzalez Garcia-Parreño for technical help with
the confocal microscopy.
Conflict of interest: none declared.
Funding
This work was supported by grants from SAF2004/06109, SAF2007/
63648, CAM (S2006/GEN-0247), Ministerio de Sanidad y Consumo,
Instituto de Salud Carlos III, Red RECAVA (RD06/0014/0035), Fondo
de Investigaciones Sanitarias (PI050451), Mutua Madrileña,
Fundación Ramón Areces, Sociedad Española de Arteriosclerosis y
Fundación Española del Corazón.
References
1. Peters-Golden M, Henderson WR Jr. Leukotrienes. N Engl J Med 2007;357:
1841–1854.
2. Mehrabian M, Allayee H. 5-lipoxygenase and atherosclerosis. Curr Opin
Lipidol 2003;14:447–457.
3. De CR, Mazzone A, Giannessi D, Sicari R, Pelosi W, Lazzerini G et al.
Leukotriene B4 production in human atherosclerotic plaques. Biomed
Biochim Acta 1988;47:S182–S185.
4. Spanbroek R, Grabner R, Lotzer K, Hildner M, Urbach A, Ruhling K et al.
Expanding expression of the 5-lipoxygenase pathway within the arterial
wall during human atherogenesis. Proc Natl Acad Sci USA 2003;100:
1238–1243.
5. Yokomizo T, Izumi T, Chang K, Takuwa Y, Shimizu T. A G-protein-coupled
receptor for leukotriene B4 that mediates chemotaxis. Nature 1997;
387:620–624.
225
Intracellular mechanisms involved in the atherogenic effect of LTB4
6. Yokomizo T, Kato K, Terawaki K, Izumi T, Shimizu T. A second leukotriene
B(4) receptor, BLT2. A new therapeutic target in inflammation and
immunological disorders. J Exp Med 2000;192:421–432.
7. Aiello RJ, Bourassa PA, Lindsey S, Weng W, Freeman A, Showell HJ. Leukotriene B4 receptor antagonism reduces monocytic foam cells in mice.
Arterioscler Thromb Vasc Biol 2002;22:443–449.
8. Subbarao K, Jala VR, Mathis S, Suttles J, Zacharias W, Ahamed J et al.
Role of leukotriene B4 receptors in the development of atherosclerosis:
potential mechanisms. Arterioscler Thromb Vasc Biol 2004;24:369–375.
9. Back M, Qiu H, Haeggstrom JZ, Sakata K. Leukotriene B4 is an indirectly
acting vasoconstrictor in guinea pig aorta via an inducible type of BLT
receptor. Am J Physiol Heart Circ Physiol 2004;287:H419–H424.
10. Martı́n-Ventura
JL,
Blanco-Colio
LM,
Muñoz-Garcı́a
B,
Gómez-Hernández A, Arribas A, Ortega L et al. NF-kappaB activation
and Fas ligand overexpression in blood and plaques of patients with
carotid atherosclerosis: potential implication in plaque instability.
Stroke 2004;35:458–463.
11. Gómez-Hernández A, Sánchez-Galán E, Martı́n-Ventura JL, Vidal C,
Blanco-Colio LM, Ortego M et al. Atorvastatin reduces the expression of
prostaglandin E2 receptors in human carotid atherosclerotic plaques
and monocytic cells: potential implications for plaque stabilization.
J Cardiovasc Pharmacol 2006;47:60–69.
12. Wheelan P, Murphy RC, Simon FR. Gas chromatographic/mass spectrometric analysis of oxo chain-shortened leukotriene B4 metabolites. Leukotriene B4 metabolism in Ito cells. J Mass Spectrom 1996;31:236–246.
13. Gomez-Hernandez A, Martin-Ventura JL, Sanchez-Galan E, Vidal C,
Ortego M, Blanco-Colio LM et al. Overexpression of COX-2, Prostaglandin
E synthase-1 and prostaglandin E receptors in blood mononuclear cells
and plaque of patients with carotid atherosclerosis: regulation by
nuclear factor-kappaB. Atherosclerosis 2006;187:139–149.
14. Dwyer JH, Allayee H, Dwyer KM, Fan J, Wu H, Mar R et al. Arachidonate
5-lipoxygenase promoter genotype, dietary arachidonic acid, and atherosclerosis. N Engl J Med 2004;350:29–37.
15. Helgadottir A, Manolescu A, Thorleifsson G, Gretarsdottir S, Jonsdottir H,
Thorsteinsdottir U et al. The gene encoding 5-lipoxygenase activating
protein confers risk of myocardial infarction and stroke. Nat Genet
2004;36:233–239.
16. Hakonarson H, Thorvaldsson S, Helgadottir A, Gudbjartsson D, Zink F,
Andresdottir M et al. Effects of a 5-lipoxygenase-activating protein
inhibitor on biomarkers associated with risk of myocardial infarction: a
randomized trial. JAMA 2005;293:2245–2256.
17. Mehrabian M, Allayee H, Wong J, Shi W, Wang XP, Shaposhnik Z et al.
Identification of 5-lipoxygenase as a major gene contributing to atherosclerosis susceptibility in mice. Circ Res 2002;91:120–126.
18. Zhao L, Moos MP, Grabner R, Pedrono F, Fan J, Kaiser B et al. The 5lipoxygenase pathway promotes pathogenesis of hyperlipidemiadependent aortic aneurysm. Nat Med 2004;10:966–973.
19. Beloqui O, Paramo JA, Orbe J, Benito A, Colina I, Monasterio A et al.
Monocyte cyclooxygenase-2 overactivity: a new marker of subclinical
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
atherosclerosis in asymptomatic subjects with cardiovascular risk
factors? Eur Heart J 2005;26:153–158.
Shindo K, Miyakawa K, Fukumura M. Plasma levels of leukotriene B4 in
asthmatic patients. Int J Tissue React 1993;15:181–184.
Funk CD. Leukotriene modifiers as potential therapeutics for cardiovascular disease. Nat Rev Drug Discov 2005;4:664–672.
Tager AM, Luster AD. BLT1 and BLT2: the leukotriene B(4) receptors. Prostaglandins Leukot Essent Fatty Acids 2003;69:123–134.
Monaco C, Andreakos E, Kiriakidis S, Mauri C, Bicknell C, Foxwell B et al.
Canonical pathway of nuclear factor kappa B activation selectively regulates proinflammatory and prothrombotic responses in human atherosclerosis. Proc Natl Acad Sci USA 2004;101:5634–5639.
de Winther MP, Kanters E, Kraal G, Hofker MH. Nuclear factor kappaB signaling in atherogenesis. Arterioscler Thromb Vasc Biol 2005;25:904–914.
Lundeen KA, Sun B, Karlsson L, Fourie AM. Leukotriene B4 receptors BLT1
and BLT2: expression and function in human and murine mast cells.
J Immunol 2006;177:3439–3447.
Huang L, Zhao A, Wong F, Ayala JM, Struthers M, Ujjainwalla F et al. Leukotriene B4 strongly increases monocyte chemoattractant protein-1 in
human monocytes. Arterioscler Thromb Vasc Biol 2004;24:1783–1788.
Tong WG, Ding XZ, Talamonti MS, Bell RH, Adrian TE. LTB4 stimulates
growth of human pancreatic cancer cells via MAPK and PI-3 kinase pathways. Biochem Biophys Res Commun 2005;335:949–956.
Kanda N, Watanabe S. Leukotriene B(4) enhances tumour necrosis
factor-alpha-induced CCL27 production in human keratinocytes. Clin
Exp Allergy 2007;37:1074–1082.
Back M, Bu DX, Branstrom R, Sheikine Y, Yan ZQ, Hansson GK. Leukotriene
B4 signaling through NF-kappaB-dependent BLT1 receptors on vascular
smooth muscle cells in atherosclerosis and intimal hyperplasia. Proc
Natl Acad Sci USA 2005;102:17501–17506.
Kitaura J, Kinoshita T, Matsumoto M, Chung S, Kawakami Y, Leitges M
et al. IgE- and IgEþAg-mediated mast cell migration in an autocrine/
paracrine fashion. Blood 2005;105:3222–3229.
Cipollone F, Prontera C, Pini B, Marini M, Fazia M, De CD et al. Overexpression of functionally coupled cyclooxygenase-2 and prostaglandin E
synthase in symptomatic atherosclerotic plaques as a basis of prostaglandin E(2)-dependent plaque instability. Circulation 2001;104:921–927.
Cipollone F, Mezzetti A, Fazia ML, Cuccurullo C, Iezzi A, Ucchino S et al.
Association between 5-lipoxygenase expression and plaque instability in
humans. Arterioscler Thromb Vasc Biol 2005;25:1665–1670.
Qiu H, Gabrielsen A, Agardh HE, Wan M, Wetterholm A, Wong CH et al.
Expression of 5-lipoxygenase and leukotriene A4 hydrolase in human
atherosclerotic lesions correlates with symptoms of plaque instability.
Proc Natl Acad Sci USA 2006;103:8161–8166.
Ritchie ME. Nuclear factor-kappaB is selectively and markedly activated
in humans with unstable angina pectoris. Circulation 1998;98:
1707–1713.