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* Centre d’Etude et de Recherche en Virologie et Immunologie, Institut National de la Santé et de la Recherche Médicale, Lyon, France; and
Unité Mixte de Recherche, Centre National de la Recherche Scientifique-bioMérieux SA, Ecole Normale Supérieure de Lyon, Lyon, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production by allogeneic T lymphocytes. LPC is most
effective in lipoprotein-deprived serum and can be inhibited by an
excess of native LDLs reflecting normal plasma conditions. Therefore,
by controlling the balance between native and oxidized lipoproteins and
the resulting production of LPC, the acute phase reactants may provide
a context of Ag presentation that is transiently favorable to immune
activation. Intralipid, a therapeutic lipid emulsion for parenteral
nutrition with unexplained immunomodulatory properties, also blocked
LPC activity. This opens perspectives for the understanding and
treatment of acute and chronic inflammatory
diseases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among other features, APR is accompanied by alterations in lipoprotein composition and is associated with oxidation of low density lipoprotein (LDL) (3, 4). Oxidative modification of LDL has been extensively studied for more than 20 years because it plays a central role in the pathogenesis of atherosclerosis (5). Epidemiologic studies have implicated infectious agents in the induction and development of atheromas, and many of the pathological features of this disease relate to dysregulated chronic inflammation. Interestingly, Chlamydia pneumoniae and chlamydial hsp60, an inflammatory Ag localized to atheromas, were found to induce cellular oxidation of LDL (6, 7). Increased levels of oxidized lipids in the serum and circulating LDL have been found in animal models of bacterial infection and inflammation (8). The host response to infection produces LDLs that not only contain more oxidized lipids but are also more susceptible to further oxidation ex vivo (8). Metabolic changes that contribute to LDL oxidation in vivo are far from being understood, but some clarification has been brought by comparative studies of high density lipoprotein (HDL) and LDL composition and function during homeostasis and APR. During homeostasis, LDL is protected from oxidative modification by HDL-associated enzymes, particularly paraoxonase, which destroys biologically active oxidized phospholipids (9). During APR, some acute phase reactants including serum amyloid A, ceruloplasmin, and apolipoprotein J transitory integrate HDL, which in turn loses its ability to control oxidation of LDL and production of oxidized phospholipids (10). Thus, homeostatic HDL maintains LDL in a noninflammatory state, whereas acute phase HDL favors the conversion of native LDL to proinflammatory oxidized LDL (oxLDL) and production of oxidized phospholipids. APR can be truly acute, as in the case of a viral or bacterial infection, but it may also become chronic and result in inflammatory diseases like atherosclerosis. By controlling the transient generation of proinflammatory oxLDL and bioactive oxidized phospholipids, APR may thus be part of a nonspecific innate immune response. We have recently shown that oxLDL promotes mature dendritic cell (DC) transition from differentiating monocytes (11), therefore supporting the hypothesis that some oxidized phospholipids generated during APR may signal the presence of a dangerous situation and favor the development of adaptive immunity. oxLDL have also been studied on various cell types on which it exerts several proinflammatory effects, which include production of monocyte chemoattractant protein-1 (12), M-CSF, and GM-CSF by endothelial cells (13), increased monocyte adhesion to and transmigration through the endothelial cell layer and lymphocyte chemotaxis (14, 15, 16).
LDL is the main source of blood cholesterol in humans. It is a spherical particle of 20 nm with a hydrophobic core containing triglycerides and cholesteryl ester molecules and a surface monolayer of polar lipids (primarily phospholipids) and apolipoprotein B. Lysophosphatidylcholine (LPC) represents 1–5% of the total phosphatidylcholine content of LDL and is generated by oxidation and fragmentation of the polyunsaturated sn-2 fatty acyl residues of phosphatidylcholine, followed by the hydrolysis of the shortened fatty acyl residues by LDL-associated enzymes (17). Oxidative modification of LDL is associated with dramatic increased formation of LPC, which can reach 40–50% of the total phosphatidylcholine content (18). LPC content is also increased in circulating LDL after LPS treatment (8). LPC activates a wide range of cell types and is implicated in many aspects of the inflammatory response. It stimulates growth factor expression by endothelial cells (19, 20) and induces inflammation and leukocyte accumulation after intracutaneous injection in humans (21). LPC up-regulates adhesion molecules on endothelial cells and their production of monocyte chemoattractant protein-1 and stimulates proinflammatory cytokines synthesis (22, 23). LPC is also one of the mediators required in the cytotoxic response of human NK cells to tumor cells (24). In addition to its proinflammatory activity, LPC displays some contrasting immunoregulatory activities by binding to its high affinity receptor G2A on T cells (25). Interaction of LPC with G2A is thought to limit T cell responses by increasing the threshold of lymphocyte activation and proliferation. The effect of LPC on APCs such as DCs has not been investigated, leaving the question of its global function in the immune system open. In this paper, we asked whether the ability of oxLDL to favor monocyte-to-mature DC transition was mediated by LPC and investigated the possibility of control of this activity by native LDL and lipid nanoemulsion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LDL (1.025 
d 1.055 g/ml) was isolated from human
plasma of normolipidemic healthy individuals by ultracentrifugation.
The density of the plasma was raised to 1.025 g/ml using NaBr. After
ultracentrifugation at 100,000 rpm, 4°C for 4 h using a TL 100.4
rotor, the light fraction containing chylomycron, very low density
lipoprotein, and intermediate density lipoprotein was removed. The
density was adjusted at 1.055 g/ml with NaBr and, after
ultracentrifugation in the same conditions, the light fraction
containing LDL was collected, dialyzed extensively against 150 mM
NaCl/2.4 mM EDTA pH 7.2 at 4°C, filtered at 0.45 µm and stored
under nitrogen.
LDL oxidation
LDL concentration was adjusted at 1 mg/ml protein by dilution in PBS and was dialyzed at 4°C against PBS to eliminate EDTA. Cu2+-mediated oxidation was conducted at 37°C for 24 h by dialysis against 5 µM CuSO4/PBS. The reaction was stopped by addition of 40 µM butylated-hydroxy-toluene and extensive dialysis at 4°C against PBS containing 100 µM diethylendiamine pentaacetic acid. The oxidation degree was assessed by malonyl-dialdehyde (MDA) production measured by the LPO-586 assay (Oxis, Portland, OR), and hydrogen peroxide content was measured by PeroxOQuant quantitative peroxide assay (Pierce, Rockford, IL). Typically, native LDL preparations contained <1.5 ± 0.3 nmol MDA/mg total protein and 230 ± 82 nmol peroxides/mg total protein. After 24 h oxidation, oxLDL contained 16.7 ± 6.6 nmol MDA/mg total protein and 896 ± 295 nmol peroxides/mg total protein. Endotoxins in LDL or oxLDL were less than 0.6 pg/ml in final concentration as assessed by E-toxate test (Sigma-Aldrich, St Quentin-Fallavier, France).
Extraction of lipid and aqueous phases from lipoproteins
A total of 400 µl of methanol was added to 100 µl of LDL or oxLDL (1 mg/ml). After mixing, 100 µl of chloroform was added and samples were vortexed. Then, 300 µl of water was added for phase separation, and samples were vortexed vigorously and centrifuged for 1 min at 9000 x g. The upper phase (defined as the aqueous phase) was collected and dried in speed vacuum. The lower phase was mixed with 300 µl of methanol and centrifuged for 2 min at 9000 x g to pellet the proteins. Supernatant (defined as the lipid phase) containing the lipophilic molecules extracted in chloroform was collected and dried in speed vacuum. Dried products of both phases were resuspended in 100 µl of 150 mM NaCl/1 mM EDTA/10 mM Tris, pH 7.4.
Lipid emulsions
Nanoemulsions were prepared by the solvent displacement method
as described (26).
1-Palmitoyl-2-arachidonoyl-phosphatidylcholine and
L-
-lysophosphatidylcholine (Sigma-Aldrich) were diluted
in ethanol (100 mg in 2 ml of solvent). A total of 350 mg in 375 µl
of medium chain triglycerides (Société
Française des Oléagineux, St. Laurent-Blagny, France)
constituting the oily core of the emulsion were added. This ethanolic
solution was subsequently diluted to 15 ml with acetone. Solution was
dropped to 30 ml of an aqueous solution containing 84 mg of Pluronic
F68, a nonionic surfactant triblock copolymer
(poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene); BASF
Bioresearch, Ludwigshafen, Germany). The resulting mixture turned milky
as a result of nanoemulsion formation. After stirring for 1 h,
organic solvents were evaporated under reduced pressure at 40°C and
the solution concentrated to 5 ml. Mean diameters of prepared emulsions
and Intralipid 20% (Fresenius Kabi, Sevres, France) were determined by
photon correlation spectroscopy using a Malvern Zetasizer 3000HS
(Malvern Instruments, Worcs, U.K.). Composition and size of these
particles are reported in Table I
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The protein content of LDL, Intralipid 20%, LPC, or phosphatidylcholine (PC) emulsions was estimated by Coomassie Protein MicroAssay procedure (Pierce), and lipid composition was determined using cholesterol RTU, triglycerides enzymatic PAP 150, and phospholipids enzymatic PAP 150 kits from bioMérieux SA (Marcy l’Etoile, France).
Differentiation and treatment of monocyte-derived DCs
PBMCs were isolated from human peripheral blood by standard density gradient centrifugation on Ficoll-Hypaque (Amersham Pharmacia Biotech, Uppsala, Sweden), and then mononuclear cells were separated from PBLs by centrifugation on a 50% Percoll solution. Monocytes were purified by immunomagnetic depletion (Dynal Biotech, Oslo, Norway) using a mixture of mAbs: anti-CD19 (4G7 hybridoma; provided by Dr. Ron Levy), anti-CD3 (OKT3; American Type Culture Collection, Manassas, VA), and anti-CD56 (NKH1; Beckman Coulter, Fullerton, CA). Recovered monocytes were >90% pure as assessed by CD14 labeling. Monocytes were differentiated to immature DCs during 6 days with 40 ng/ml human recombinant GM-CSF and 250 U/ml human recombinant IL-4 in RPMI 1640 (Life Technologies, Rockville, MD) supplemented with 2 mM glutamine (Life Technologies), 10 mM HEPES (Life Technologies), 40 ng/ml gentamicin (Life Technologies), and 10% lipoprotein-deficient serum (LPDS; Sigma-Aldrich).
Differentiating monocytes were treated at day 5 by addition of
lipoproteins, phospholipids, or lipid emulsions to the medium at the
last day of differentiation. A total of 2 x
106 cells were treated with 10 µg/ml oxLDL or
LDL (total lipoprotein), with 20 µl of lipid or aqueous phases
extracted from oxLDL or LDL, or with 40 µM LPC or PC, either
dissolved in ethanol or emulsified with triglycerides (LPC or PC
emulsion). To block Gi proteins, day 5
differentiating monocytes were pretreated for 3 h with 100 ng/ml
pertussis toxin (PTX; Biomol, Plymouth Meeting, PA), and then the
medium was changed and cells were further incubated with 40 µM LPC
for 24 h as previously indicated. When mentioned, a
platelet-activating factor receptor (PAFR) antagonist (BN52021; Biomol)
was added 15 min before LPC. Competition experiments were performed by
concomitant adjunction of oxLDL (10 µg/ml) or LPC (40 µM) with
native LDL (50 µg/ml of proteins corresponding to 50 µg/ml
phospholipids) or Intralipid 20% (50 µg/ml phospholipids). At the
end of the differentiation (day 6), cells were harvested and analyzed.
Cell viability was superior to 90%. Endotoxin contamination of LPC was
excluded by lack of polymixin B (Sigma-Aldrich) inhibition.
Phenotype
Phenotype was analyzed by flow cytometry on a FACSCalibur (BD Biosciences, Franklin Lakes, NJ) using FITC-conjugated anti-CD14, -HLA-DR, and -CD80 and PE-conjugated anti-CD1a, -CD83, -CD86, and -CD40, all from Beckman Coulter.
Endocytosis
Differentiated cells, treated or not with 40 µM LPC as described above, were harvested at day 6, resuspended in 10% FCS medium, and incubated at 37°C for 30 min with 1 mg/ml Lucifer Yellow (Sigma-Aldrich) or 1 mg/ml FITC-T70-dextran (Sigma-Aldrich) or for 3 h with carboxylate-modified yellow-green FluoSpheres of 0.45 µm (Beads; Molecular Probes, Leiden, The Netherlands). Internalization was stopped on ice with cold PBS containing 0.1% BSA and 0.05% NaN3. Cells were washed three times at 4°C in this buffer, and their content in fluorescent probe was analyzed on a FACSCalibur (BD Biosciences).
Mixed lymphocyte reaction
Naive T lymphocytes were isolated from human peripheral blood.
PBMCs were isolated by density gradient centrifugation on
Ficoll-Hypaque. After depletion of monocytes on Percoll gradient, PBLs
were recovered in the dense fraction. T lymphocytes were purified by
immunomagnetic depletion using a mixture of mAbs: anti-CD19 (4G7),
anti-CD16 (3G8), anti-CD56 (NKH1), anti-glycophorin A
(11E4B7.6), and anti-CD14 (RMO52) (all from Beckman Coulter). T
lymphocytes were >95% pure as assessed by CD3 labeling. Primary MLRs
were conducted in 96-well flat-bottom culture plates. DCs were treated
or not at day 5 with 40 µM LPC or 10 µg/ml oxLDL in presence or not
of competitors as described above, collected at day 6, extensively
washed, and resuspended in RPMI 1640 supplemented with 2 mM glutamine,
10 mM HEPES, 40 ng/ml gentamicin, and 10% FCS (BioMedia, Boussens,
France). These cells were then cocultured with 2 x
105 allogeneic T cells in 200 µl of complete
culture medium at 1:5, 1:10, or 1:20 DC:T cell ratios. After 4 days,
150 µl of culture supernatant was analyzed for the presence of IL-2
and IFN- using cytokine-specific ELISA kits purchased from Endogen
(Woburn, MA).
Receptor analysis by RT-PCR
Total RNA was isolated using the RNeasy Mini kit (Qiagen, Courtaboeuf, France). One microgram of total RNA was reverse transcribed using the Thermoscript RT-PCR system (Life Technologies). cDNA was then used for PCR amplification of G2A receptor, PAFR, G-protein-coupled receptor 4 (GPR4), lectin-like oxLDL receptor (LOX-1), and ribosomal protein S12. The following primers were used to amplify G2A (5'-CGGTGGTTGTCATCTTCCTA-3' and 5'-TTAGCGGCCGCTCAGCAGGACTCCTCAATCAG-3') (27), GPR4 (5'-TAATGCTAGCGGCAACCACACGTGGGAG-3' and 5'-TCCAGTTGTCGTGGTGCA-3') (28), PAFR (5'-CTCGGGGTCATTGCTAATG-3' and 5'-TTCAGTGACCGTATCCGTG-3') (29), LOX-1 (5'-GGCTTGCTGCGACTCTAGG-3' and 5'-AGTGGGGCATCAAAGGAG-3') (30), and S12 (5'-GGAGGTGTAATGGACGTTA-3' and 5'-CTGAGACTCCTTGCCATAG-3') (29). The amplified products were analyzed by gel electrophoresis (414 bp for G2A, 258 bp for GPR4, 698 bp for PAFR, 569 bp for LOX-1, and 311 bp for S12).
Phosphorylation of extracellular signal-regulated kinase (ERK)
A total of 107 day 5 differentiating monocytes were starved for 1 h in RPMI 1640 medium without serum, containing 0.3% BSA (Sigma-Aldrich) before treatment with 40 µM LPC for 2, 5, 10, or 15 min. Cells were washed with cold PBS, and the pellet was lysed in 8 M urea/2% SDS/0.2 M Tris-HCl (pH 8.0)/100 mM DTT and boiled for 5 min. After centrifugation, the supernatant was analyzed on a 10% SDS-PAGE, and proteins were transferred on Immobilon-P (Millipore, Bedford, MA). Blots were saturated with 5% fat-free milk/TBS/0.1% Tween 20 for 1 h. Incubations with primary and secondary Abs were conducted in TBS/0.1% Tween 20/5% BSA. Anti-phospho-ERK and total-ERK were from Cell Signaling Technologies (Beverly, MA). Detection was performed using HRP-conjugated anti-mouse Ab (Amersham Pharmacia Biotech) with the ECL kit (Amersham Pharmacia Biotech).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that the presence of oxidized LDL during
monocyte differentiation to DCs yielded phenotypically and functionally
mature DCs (11). Optimal reactivity of monocytes was
obtained when oxLDL was added during late stages of differentiation in
GM-CSF and IL-4. Direct generation of mature DCs from differentiating
monocytes was best achieved when oxLDL was added at day 5 of
differentiation for 24 h. We also showed that FCS containing
native lipoproteins and native LDL added to LPDS were efficient
inhibitors of oxLDL activity, indicating that oxLDLs are active in
inflammatory conditions when the level of native LDL is reduced. Here
we used the same experimental procedure to screen for bioactive
molecules on differentiating monocytes cultured in LPDS to avoid
inhibition by native LDL. Because strong up-regulation of CD86 is a
constant feature induced by oxLDL, this marker was used as a first
screening readout to determine which component of oxLDL was responsible
for DC activation. Differentiating monocytes were treated for 24 h
with aqueous or lipid phases from native LDL or oxLDL. The oxidation
process generates various components, including oxysterols and modified
phospholipids, which are concentrated in the lipid phase, whereas the
aqueous phase may contain polar oxidized free fatty acids. Neither
total native LDL nor phases from native LDL could induce CD86
expression (Fig. 1, A–C). In contrast, lipid phase from oxLDL
strongly up-regulated CD86 expression, as did total oxLDL (Fig. 1, A and B). The aqueous phase from oxLDL had no
effect on CD86 expression (Fig. 1C). PC is the major
phospholipid component of native LDL, and its derivative LPC can
constitute as much as 50% of the total phospholipids of oxLDL.
Therefore, the effect of both PC and LPC was tested on day 5
differentiating monocytes. PC and LPC were also emulsified with
triglycerides to form particles of 300–400 nm constituted of
triglycerides covered with a monolayer of PC or LPC. Composition of
these emulsions is shown in Table I
. Both LPC and LPC emulsion induced
strong up-regulation of CD86 expression, whereas PC and PC emulsion did
not (Fig. 1, D and E). CD86 up-regulation by LPC
was dose-dependent with optimal induction for 40 µM LPC under these
experimental conditions (data not shown). Thus, LPC appeared to be one
of the bioactive molecules of oxLDL, and its activity could be vehicled
by LDL-like structures.
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As expected, monocytes differentiated to immature DCs in GM-CSF
and IL-4 in the absence of LPC (control) expressed a high level of CD1a
but no CD14 and an intermediate level of HLA-DR but no CD80, CD83, or
CD86 (Fig. 2A). Cells treated
with LPC up-regulated HLA-DR, CD83, CD86, and CD40, but CD80 was only
weakly induced. CD14 was not expressed, but CD1a remained high as it
did after oxLDL treatment (11). Thus, LPC treatment of
differentiating monocytes induced phenotypical characteristics of
mature DCs. Cell mortality measured by incorporation of propidium
iodide was not increased by LPC treatment (data not shown).
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B, LPC-treated cells
show a significant decrease in their endocytosis activities compared
with untreated cells corresponding to immature DCs (from 30% reduction
for macropinocytosis to 50% for pinocytosis and receptor-mediated
endocytosis).
The capacity to stimulate allogeneic T cells is another characteristic
of mature DCs. Therefore, the functional properties of LPC-treated
cells were further investigated in MLR. In contrast to control
untreated cells (immature DCs), LPC-treated cells could activate
allogeneic naive T cells inducing the release of IL-2 (Fig. 2C). Thus, addition of LPC during the late stage of monocyte
differentiation gave rise directly to mature DCs supporting allogeneic
T cell stimulation.
LPC action through G protein-coupled receptors
It has been recently shown that LPC is a high affinity ligand for
G2A receptor expressed on lymphocytes (25). LPC can also
bind with low affinity to GPR4 receptor, which has a wider expression
(28). Partial sensitivity to PAFR antagonists indicated
that some LPC effects could be mediated by the PAFR in various cell
types (33, 34). Finally, LOX-1, the lectin-like oxLDL
receptor that belongs to the scavenger receptor family, can mediate
LPC-induced oxLDL uptake in smooth muscle cells (35).
Expression of these LPC receptors was tested by RT-PCR. Total mRNA was
extracted from freshly isolated human monocytes, from differentiating
monocytes at day 5, and from immature DCs at day 6 of culture in GM-CSF
and IL-4. Total mRNA was also extracted when differentiating monocytes
were treated at day 5 with 40 µM LPC for 24 h. RT-PCR
amplification yielded a DNA fragment of the expected size for G2A,
PAFR, and GPR4 in undifferentiated monocytes as well as in
differentiating monocytes at day 5 and in immature DCs at day 6 (Fig. 3A). mRNA levels of G2A, PAFR,
and GPR4 remained constant throughout the differentiation and were
unchanged by LPC treatment. In contrast, LOX-1 mRNA was undetectable in
undifferentiated monocytes but was present at day 5 of differentiation
and in immature DCs. Although quantitative RT-PCR was not performed,
LOX-1 mRNA seemed to be further induced by LPC treatment (Fig. 3A).
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i proteins are sensitive to PTX, which blocks
Gi-coupled signaling. Fig. 3B shows
that up-regulation of CD86 by LPC was totally inhibited when cells were
preincubated with 100 ng/ml PTX for 3 h. Other phenotypical
modifications induced by LPC treatment and shown in Fig. 2A
were also inhibited by PTX (data not shown). These data indicated that
all LPC activity was mediated through G protein-coupled receptors,
therefore excluding scavenger receptors like LOX-1.
Fig. 3C shows that up-regulation of CD86 by LPC was reduced
by 40% when cells were preincubated for 15 min with the PAFR
antagonist BN52021 before stimulation with LPC. No specific antagonist
for G2A or GPR4 is currently available, but it has been shown that
phosphorylation of the ERK upon LPC treatment was dependent on G2A or
GPR4 expression (25). As shown in Fig. 3D,
Western blot analysis of differentiating monocytes exposed to LPC at
day 5 revealed a strong and transient increase in phospho-ERK occurring
within 2 min after LPC adjunction and decreasing rapidly to return to
its basal level after 15 min.
Th1-oriented response initiated by oxLDL and LPC and its regulation by lipids
The ability of maturing DCs to initiate Th1-type vs Th2-type
responses depends on the microenvironmental conditions of their
development. The question of to what extent the inflammatory mediators
oxLDL and LPC polarize Th cell development was addressed by analyzing
the cytokines secreted by allogeneic naive T cells in MLR. Fig. 4 shows that both oxLDL- and LPC-treated
cells stimulated the production of IFN- by T cells, whereas no IL-4
could be detected (data not shown). This indicated that oxLDL and LPC
may instruct DC to initiate Th1-biased responses.
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. In the presence of
native LDL, both oxLDL- and LPC-induced CD86 up-regulation were
strongly inhibited (73 and 87%, respectively) (Table II). Inhibition of DC maturation
correlated with the inhibition of DC ability to stimulate IFN-
secretion by allogeneic T cells. Therefore, as for oxLDL, native LDL is
an endogenous inhibitor of LPC.
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). This emulsion strongly antagonized
the phenotypic and functional maturation of DCs induced by LPC or
oxLDL, strongly inhibiting CD86 up-regulation and IFN- secretion by
allogeneic T cells (Table II). This suggests that lipid composition of
native LDL may determine its inhibitory potential. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production. Thus, LPC is one of the oxLDL components that
can promote mature DC generation. IFN- production during T-DC
interaction indicated that LPC is a Th1-promoting innate stimulus. Our
preliminary data have shown that IL-12 p70 secretion is induced by LPC,
a process that can be inhibited by an excess of native LDL (data not
shown). Compared with other type of stimulus like LPS, production of
IL-12 p70 is low but significant, suggesting a role for other
cytokines. In this context, IL-18 is of particular interest because it
has been shown to synergize with IL-12 to enhance IFN- production
(40, 41). Additional work is required to clarify the
relative contribution of various cytokines to the activation of T
lymphocytes by LPC-generated DCs.
Although LPC is an important lipid mediator of inflammation, its
initial mechanism of action is still poorly understood. GPR4 and G2A,
respectively low and high affinity receptors for LPC, have recently
been described (25, 28). In addition to these two
receptors, LPC action through the PAFR and LOX-1 has also been
suggested (33, 34, 35, 42). LOX-1 is a scavenger receptor with
a role in defense against Gram+ and
Gram- bacteria (43). However, this
receptor could be excluded because experiments using PTX as an
inhibitor of Gi showed that LPC action was
mediated through a G protein-coupled receptor. The PAFR is strongly
implicated in inflammatory reaction and is directly stimulated by
lipoteichoic acid, a Gram-positive cell wall component
(44). The PAFR antagonist BN52021 partially inhibited LPC
action, indicating that this receptor is involved in LPC action. Our
preliminary data suggest that this pathway may not be the initiator of
LPC effect but may rather amplify LPC action through other receptors
(F. Coutant, unpublished data). There is no specific inhibitor
or blocking Ab for GPR4 or G2A. However, it has been shown that ERK MAP
kinase activation by LPC was dependent on GPR4 or G2A expression
(25). Here we confirmed that LPC treatment of
differentiating monocytes resulted in ERK activation, therefore
strengthening the involvement of GPR4 or G2A in LPC-induced DC
maturation. Further studies are needed to address the possible
functional redundancy or complementarity among these three receptors.
It is tempting to speculate on the role of lysophospholipid receptors
and LPC in the control of innate and adaptive immunity. LPC
appears to favor the Ag-specific response by promoting mature DC
generation. LPC also appears to increase the threshold for
proliferation and activation of T cells (25). Therefore,
it is reasonable to assume that the highly controlled release of LPC
could be one mechanism to improve efficiency and safety of immunity by
increasing Ag presentation to naive T cells but limiting the extent of
the response and avoiding nonspecific activation of lymphocytes.
The high concentration of serum LPC (up to 100 µM) during homeostasis
suggests that the proinflammatory activities of LPC are blocked by
circulating activators. Albumin appears to be one of these inhibitors
as it binds to LPC and decreases some of its effects in vitro. Our
results shown in Table II indicate that native LDL is another inhibitor
of LPC explaining why LPC is more efficient in LPDS than in FCS.
Inhibition of LPC by native LDL can be mimicked by Intralipid, a stable
emulsion of triglycerides and phospholipids. Although the inhibition of
LPC by Intralipid remains to be further addressed, this supports the
notion that the ratio of phospholipids to lysophospholipids in the
direct environment of DCs may influence its maturation. Intralipid is
used as a source of energy to avoid wasting during septic shock. Our
data suggest that it may also help to limit inflammation.
Sensing the danger is a crucial function of APCs, especially for DCs that become activated and initiate primary immune responses (45, 46). DCs can be activated by exogenous pathogen-derived molecules or endogenous signals released by cells that are stressed, infected, or necrotic (47, 48). Our data suggest that oxidation of lipoproteins and production of LPC may be one inducible endogenous signal of danger that is released during the APR. The concentration of albumin drops during the APR, whereas oxidation of native LDL is actively promoted. As a consequence, the concentration of LPC is increased, whereas its two inhibitors are rendered less effective. Thus, the functionally available concentration of LPC is tightly controlled by the acute phase reactants in response to tissue injury. How DCs and T cells integrate LPC signal through a family of G protein-coupled receptors to promote the initiation of an immune response while limiting the extent of the response may thus be a major issue for the understanding and control of inflammatory and autoimmune diseases.
Besides the basic research observation that LPC promotes development of mature DCs, the data presented in this study may have practical implications in the field of cell therapy. Moreover, identification of blocking molecules in Intralipid and native LDL may prove valuable for the design of lipid nanoemulsions with anti-inflammatory properties.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Vincent Lotteau, Centre d’Etude et de Recherche en Virologie et Immunologie, Institut National de la Santé et de la Recherche Médicale Unité 503, 21 Avenue Tony Garnier, F-69007 Lyon, France. E-mail address: lotteau{at}cervi-lyon.inserm.fr
3 Abbreviations used in this paper: APR, acute phase response; LDL, low density lipoprotein; HDL, high density lipoprotein; oxLDL, oxidized LDL; DC, dendritic cell; LPC, lysophosphatidylcholine; MDA, malonyl-dialdehyde; PC, phosphatidylcholine; LPDS, lipoprotein-deficient serum; PTX, pertussis toxin; PAFR, platelet-activating factor receptor; GPR4, G-protein-coupled receptor 4; LOX-1, lectin-like oxLDL receptor; ERK, extracellular signal-regulated kinase.
Received for publication March 15, 2002. Accepted for publication June 3, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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for IFN- production from human T cells. Int. Immunol. 12:151.This article has been cited by other articles:
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  E. Ibeas, L. Fuentes, R. Martin, M. Hernandez, and M. L. Nieto Secreted phospholipase A2 type IIA as a mediator connecting innate and adaptive immunity: new role in atherosclerosis Cardiovasc Res, January 1, 2009; 81(1): 54 - 63. [Abstract] [Full Text] [PDF]
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 T. Lu, C. Newton, I. Perkins, H. Friedman, and T. W. Klein Cannabinoid Treatment Suppresses the T-Helper Cell-Polarizing Function of Mouse Dendritic Cells Stimulated with Legionella pneumophila Infection J. Pharmacol. Exp. Ther., October 1, 2006; 319(1): 269 - 276. [Abstract] [Full Text] [PDF]
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 S. Bluml, S. Kirchberger, V. N. Bochkov, G. Kronke, K. Stuhlmeier, O. Majdic, G. J. Zlabinger, W. Knapp, B. R. Binder, J. Stockl, et al. Oxidized Phospholipids Negatively Regulate Dendritic Cell Maturation Induced by TLRs and CD40 J. Immunol., July 1, 2005; 175(1): 501 - 508. [Abstract] [Full Text] [PDF]
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 P. A. VanderLaan and C. A. Reardon Thematic review series: The Immune System and Atherogenesis. The unusual suspects:an overview of the minor leukocyte populations in atherosclerosis J. Lipid Res., May 1, 2005; 46(5): 829 - 838. [Abstract] [Full Text] [PDF]
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 R. Ramoner, T. Putz, H. Gander, A. Rahm, G. Bartsch, C. Schaber, and M. Thurnher Dendritic-cell activation by secretory phospholipase A2 Blood, May 1, 2005; 105(9): 3583 - 3587. [Abstract] [Full Text] [PDF]
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F. Coutant, S. Agaugue, L. Perrin-Cocon, P. Andre, and V. Lotteau Sensing Environmental Lipids by Dendritic Cell Modulates Its Function J. Immunol., January 1, 2004; 172(1): 54 - 60. [Abstract] [Full Text] [PDF]
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