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Am J Physiol Cell Physiol 295: C905-C914, 2008. First published July 16, 2008; doi:10.1152/ajpcell.00544.2007
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VASCULAR BIOLOGY

LPA modulates monocyte migration directly and via LPA-stimulated endothelial cells

Cindy Gustin, Martine Van Steenbrugge, and Martine Raes

Laboratory of Biochemistry and Cellular Biology, Unit of Research on Cellular Biology (URBC), Facultés Universitaires Notre-Dame de la Paix (FUNDP), University of Namur, Namur, Belgium

Submitted 15 November 2007 ; accepted in final form 11 July 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Lysophosphatidic acid (LPA) is a bioactive lysophospholipid ligand present in oxidized low-density lipoprotein. The effects of LPA were investigated, first separately on endothelial cells (EC) and monocytes. Using Ki16425 (an LPA1 and LPA3 receptor antagonist), GW9662 [a peroxisome proliferator-activator receptor (PPAR{gamma}) antagonist], and pertussis toxin (that inhibits Gi/o), we demonstrate that LPA enhances IL-8 and monocyte chemoattractant protein-1 expression through a LPA1-, LPA3-, Gi/o- and PPAR{gamma}-dependent manner in the EAhy926 cells. The effect of LPA on chemokine overexpression was confirmed in human umbilical vein endothelial cells. LPA was able to enhance monocyte migration at concentrations <1 µM and to inhibit their migration at LPA concentrations >1 µM, as demonstrated by using a chemotaxis assay. We then investigated the effects of LPA on the cross-talk between EC and monocytes by evaluating the chemotactic activity in the supernatants of LPA-treated EC. At 1 µM LPA, both cell types respond cooperatively, favoring monocyte migration. At higher LPA concentration (25 µM), the chemotactic response varies as a function of time. After 4 h, the chemotactic effect of the cytokines secreted by the EC is counteracted by the direct inhibitory effect of LPA on monocytes. For longer periods of time (24 h), we observe a monocyte migration, probably due to lowered concentrations of bioactive LPA, given the induction of lipid phosphate phosphatase-2 in monocytes that may inactivate LPA. These results suggest that LPA activates EC to secrete chemokines that in combination with LPA itself might favor or not favor interactions between endothelium and circulating monocytes.

lysophosphatidic acid; endothelial cells; monocytes; chemotaxis

LYSOPHOSPHATIDIC ACID (LPA) is a potent bioactive lipid mediator with multiple cellular effects (21) produced by activated platelets or by enzymatic cleavage of membrane phospholipids (3, 12). The concentration of LPA in the serum is estimated at 2 to 20 µM, whereas the concentration in plasma is considerably lower (80 nM to 0.7 µM) (10). LPA evokes many cellular responses by binding to five specific G protein-coupled receptors (GPCRs) known as LPA1-5 that have been cloned and shown to be expressed in most mammalian cells and tissues (for a recent review see Ref. 20). Another GPCR (P2Y5) involved in the maintenance of human hair growth has been recently described as a member of a subgroup of LPA receptors, including LPA4 and LPA5 (28). Moreover, the McIntyre group (19) has shown that LPA is also a high-affinity ligand for the peroxisome proliferator-activator receptor {gamma} (PPAR{gamma}), suggesting the participation of LPA in intracellular signaling and cell regulation.

LPA has recently attracted much interest due to its multiple roles in physiological and pathological conditions since LPA is enriched, for instance, in activated platelets and ovarian cancer cells, suggesting potential roles in cancer (33) but also in inflammatory diseases such as atherosclerosis. The evidence that LPA has athero- and thrombogenic activities has increased substantially in recent years (for a review see Ref. 35). Indeed, Siess and co-workers (36) have demonstrated that LPA is also formed in oxidized LDL (oxLDL) and accumulates in the intima of human atherosclerotic lesions. It also stimulates endothelial adhesion molecule expression and subsequent monocyte-endothelial interactions, a key process in the onset of the early atherosclerotic lesion (32). Moreover, Lin et al. (17) have recently demonstrated that LPA enhanced IL-8 and monocyte chemoattractant protein-1 (MCP-1) expression in human umbilical vein endothelial cells (HUVEC) through a Gi/o-, Rho- and nuclear factor (NF){kappa}B-dependent mechanism, favoring endothelial cell-leukocyte interactions through the induction of these two chemokines. But, in vivo, the evolution of the initial atherosclerotic lesion relies on a complex interplay between in particular the endothelial cells acting as a secretory tissue, and monocytes responding to them by transmigration. So, given its multiple sources of production in this context, it is important to unravel the interactions between endothelial cells and monocytes, all together in the presence of bioactive LPA.

It is well documented that several cell types migrate in response to LPA, including human fibroblasts (2), hepatoma cells (15), and breast carcinoma cells (24, 41). However, responses of endothelial cells were more variable, depending on the cell strain and the extracellular matrix used (26, 27). The role of specific LPA-receptors in cell migration has also been studied in Jurkat T cells where a limited role for LPA1 and a major role for LPA2 in cell migration were observed using a trans-Matrigel migration assay (45). Finally, Shida et al. (34) found that LPA was a strong chemoattractant for human colon carcinoma DLD1 cells that express exclusively the LPA1 receptor. On the other side, several authors also describe an inhibitory effect of LPA on cell migration. Peyruchaud et al. (29) showed that the chemotaxis of osteosarcoma cells toward platelet-derived growth factor is inhibited by the addition of LPA. Zhou et al. (46) also showed that a preincubation with LPA during 30 min decreases the migratory response of peripheral blood mononuclear cell to MCP-1 of about 50% (46). All these data suggest that cell migration in response to LPA is cell-type specific, but with differences in the fine tuning according to the LPA-receptors expressed at the cell surface, the extracellular matrix used in the assay, as well as the LPA concentration and stimulation time used. Given these contradictory data, we wanted to study the chemotactic effect of LPA alone and in conditioned media of LPA-treated endothelial cells on monocyte migration to better understand the interplay between these two cell types when exposed to LPA, a pro-atherogenic lipid. So, to study the capacity of LPA to induce chemotactic factors, we chose the endothelial EAhy926 cells, a well-characterized human vascular endothelial cell line displaying various characteristics typical of human endothelial cells, such as synthesis of von Willebrand factor, PGI2 and thrombomodulin (5, 9, 37). Moreover, we already demonstrated that LPA enhances the secretion of pentraxin-3, a pro-inflammatory protein by EAhy926 cells as well as by primary HUVEC cells, with similar fold inductions in a time- and concentration-dependent manner (13). In this work, we present evidence that LPA enhances IL-8 and MCP-1 expression in both EAhy926 and HUVEC cells, through Gi/o-, LPA1-, LPA3- and PPAR{gamma}-dependent pathways. We then evaluated the effect of LPA on monocyte migration since, except for one previous study reporting the haptotactic migration of human monocytes induced by very high micromolar concentrations of LPA (300 µM) (46), the activation of human monocytic cells by LPA has been poorly documented up to now. With the use of THP-1 monocytic cells, our data show that monocyte migration is not only induced by the chemokines secreted by the endothelial cells, but that it is also fine tuned by the monocytes themselves, according to the LPA concentration and incubation time used, suggesting a complex interplay between both cell types in pro-inflammatory conditions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemical reagents and antibodies. 1-Oleoyl-LPA, fatty acid-free BSA, GW9662, monoclonal anti-{alpha}-tubulin antibody, and pertussis toxin (PTX) were purchased from Sigma (St. Louis, MO), Ki16425 {3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5- isoxazolyl] benzylsulfonyl) propanoic acid} was synthesized and kindly provided by Kirin Brewery (Takasaki, Japan). Polyclonal anti-LPA1 antibody was from Millipore (Billerica, MA), and polyclonal anti-LPA2 and LPA3 antibodies were from Exalpha Biologicals (Maynard, MA). Polyclonal anti-PPAR{gamma} antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell culture and LPA stimulation. The EAhy926 cell line is a hybridoma produced from HUVEC fused with cells of the carcinoma cell line A549 (generous gift from Dr Cora-Jean Edgell, University of North Carolina). These cells were maintained in Dulbecco's modified Eagle's high glucose (4,000 mg/l) medium (DHG) (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (GIBCO, Paisley, UK). The human acute monocytic leukemia cell line THP-1 was purchased from the ATCC (American Type Culture Collection, Manassas, VA) and cultured in RPMI-1640 medium (Invitrogen) supplemented with 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/l glucose, 1.5 g/l bicarbonate, 0.05 mM 2-mercaptoethanol, and 10% FBS. All cultures were grown at 37°C in a humidified atmosphere containing 5% CO2 in 75-cm2 culture flasks (Heraus, Hassau, Germany). For all experiments, EAhy926 cells were cultured at about 70%~80% confluence and starved in serum-free medium with 0.1% fatty acid-free BSA (as an LPA carrier) 1 h before LPA stimulation. THP-1 cells were cultured at 2,000.000 cells per T25 in RPMI and 0.1% fatty acid-free BSA 1 h before LPA stimulation. For each experiment, negative controls (= untreated cells = Ctl) were realized for each time, corresponding to cells treated the same way but in the absence of LPA. For the chemotaxis assay, THP-1 cells were prepared at 300,000 cells/500 µl/chamber in DHG + 0.1% fatty acid-free BSA.

Cell viability and mycoplasma detection. To estimate cell viability, directly after the incubations with the different chemical inhibitors, antagonists, or other factors, the medium was removed and cells were stained with a solution containing 10 µg/ml ethidium bromide and 3 µg/ml acridine orange in PBS. Viable cells appear in green while permeabilized dead cells in appear in orange when observed by fluorescence microscopy. Cell viability was also assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) by quantifying living metabolically MTT to purple formazan dye. Cytotoxicity was calculated as the percentage of formazan formation in cells treated or not. Tests to rule out the presence of mycoplasma contamination (MycoAlert Mycoplasma detection Kit, Lonza) were regularly performed on monocytes and endothelial cell line and were all negative.

Western blot analysis for LPA1-3. Total cell extracts were prepared from untreated EAhy926 cells grown in T25 flasks (1,000,000 cells seeded per T25 flask 36 h before the experiment). Cells were washed with cold PBS and lysed using lysis buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 10% glycerol, 1% NP-40, 0.1% SDS, and 0.5% deoxycholate) containing a protease inhibitor mixture ("complete" from Roche, Indianapolis, IN) and phosphatase inhibitors (1 mM NaVO3, 10 mM p-nitrophenylphosphate, 10 mM β-glycerophosphate, and 5 mM NaF). The lysate was then kept on ice and mixed every 10 min and centrifuged 10 min at 13,000 rpm (5415R centrifuge, Eppendorf, Germany) at 4°C, and the supernatants were collected. Protein concentrations were determined using the Bradford protein assay (Bio-Rad, Hercules, CA). Equal amounts of protein (20 µg) were then separated by SDS-PAGE on 10% acrylamide gels (NuPage) and transferred to a polyvinyldifluoride membrane (GE Healthcare, Little Chalfont, UK). After being blocked in TBS containing 0.1% Tween and 2% milk (GE Healthcare), the blots were probed with polyclonal anti-LPA1, anti-LPA2, and anti-LPA3 antibodies (diluted 1:1,000; the secondary antibody was a donkey anti-rabbit IgG antibody, diluted 1:100,000) (GE Healthcare). {alpha}-Tubulin revealed with a monoclonal anti-{alpha}-tubulin antibody (diluted 1:30,000); secondary antibody was a sheep anti-mouse IgG antibody, diluted 1:250,000 (GE Healthcare) was used as loading control. Chemiluminescent detection was performed using horseradish peroxidase conjugated to the secondary antibodies. Finally, the membranes were revealed with ECL reagent (GE Healthcare).

RNA extraction and cDNA synthesis. Real-time reverse transcriptase-polymerase chain-reaction (RT-PCR) was used to quantify MCP-1, IL-8, lipid phosphate phosphatase (LPP), and GAPDH transcript abundance. Briefly, RNA was extracted with TRIzol reagent (GIBCO) for THP-1 and with the "RNAgent total RNA isolation system" kit (Promega, Madison, WI) for EAhy926 cells, according to the manufacturer's instructions. Total RNA, in both cases, was reverse transcribed with the Superscript II transcriptase kit (Invitrogen) according to the manufacturer's instructions and using oligo-dT primers. Real-time RT-PCR was performed using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). A standard curve from several dilutions of a sample of total RNA was established to calculate the amplification efficiency for each gene. LPP-1, -2, -3, MCP-1, and IL-8 mRNA abundance was quantified using the threshold concentration method. Values were then normalized to the relative amounts of the mRNA of the housekeeping gene GAPDH determined from a similar standard curve.

Chemotaxis bioassay. The trans-well migration of human THP-1 cells toward LPA or endothelial cell-conditioned media (supernatants of EAhy926 treated or not with LPA or TNF{alpha}) was studied in a 24-well microchamber covered by a 8-µm pore polycarbonate membrane (Nunc) (VWR). Different concentrations of LPA or conditioned media (500 µl) were placed in the lower wells of the chamber or in both the lower and upper chambers for chemokinesis studies, and THP-1 cells (300,000 cells/500 µl/well) were loaded in the upper wells for 4 h or 24 h at 37°C in humidified air with 5% CO2. At the end of the incubation period, the cells having migrated into the lower part of the chamber were stained with Calcein-AM (Molecular Probes, Eugene, OR), and the number of migrating monocytes was estimated by measuring the corresponding fluorescence relatively to the fluorescence emitted by 300,000 THP-1 cells (excitation at 485 nm, emission at 520 nm) (Fluoroskan Ascent).

Transfection experiments and reporter gene assay. EAhy926 cells were seeded at 120,000 cells/well in 24-well plates (Corning, Valencia, CA) the day before transfection. Transfections were performed with the Superfect transfection reagent (Qiagen) according to the manufacturer's protocol. After 3 h transfection, cells were rinsed once with DHG medium alone before being stimulated during 18 h with LPA. To assay the transcriptional activity of PPAR{gamma}, we used a specific firefly luciferase reporter vector containing an artificial promoter with one TATA-box and six peroxisome proliferator response elements (PPRE) cis-elements, upstream of the firefly luciferase gene. A plasmid, which contains the renilla firefly gene downstream of the cytomegalovirus promoter, was also used to normalize the firefly luciferase activity. Luciferase assays were performed using the Dual-Luciferase Assay System (Promega) according to the manufacturer's protocol. Measurements were obtained using a luminometer (Luminoskan Ascent).

Statistical analysis. All experiments were performed at least three times independently, and data are expressed as means ± SD. Statistical analysis was achieved by the "Sigmastat" software using Student's t-test and one- or two-way ANOVA followed with post hoc corrections (Holm-Sidak or Tukey test). The differences were considered as significant if P values were <0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression of LPA receptors in EAhy926 cells. The immortal endothelial EAhy926 cell line is frequently used in vitro in the context of cardiovascular diseases. However, to our knowledge, there are no data available on the LPA receptors expressed in this model. That is why we first demonstrated the expression of LPA1, LPA2, and LPA3 receptors by Western blot analysis on total cellular extracts from untreated cells (data not shown). For LPA4, since no antibody was available, the abundance of mRNA was estimated by real-time RT-PCR, confirming the expression of LPA4 at least at the mRNA level in EAhy926 cells (data not shown). The expression of LPA5, described recently (16), was not yet investigated. Finally, by using a luciferase reporter vector containing six PPRE cis elements, we highlighted that LPA (25 µM) was able to induce the PPRE-driven expression of luciferase, even in the presence of Ki16425, a specific LPA1 and LPA3 receptor antagonist (25), which suggests the activation of the PPAR{gamma} transcription factor by LPA, bypassing the membrane G protein-coupled LPA receptors (Fig. 1). This effect was concentration dependent (from 1 to 25 µM); the activation of PPAR{gamma} being already significant at 1 µM (data not shown). Furthermore, when cells were preincubated with 1 µM GW9662, a specific antagonist of PPAR{gamma}, the LPA-induced PPAR{gamma} activation was almost completely abolished (Fig. 1). Taken together, these data confirmed the expression of at least four transmembrane LPA receptors and showed, for the first time, that LPA is able to increase the transactivating activity of PPAR{gamma} in the endothelial EAhy926 cells.


Figure 1
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  		Fig. 1. Effects of lysophosphatidic acid (LPA) on the transactivating activity of peroxisome proliferator-activator receptor (PPAR{gamma}). EAhy926 cells were transfected with a firefly luciferase reporter plasmid containing 6 peroxisome proliferator response elements (PPRE) cis-elements and with a renilla luciferase reporter plasmid for normalization. The firefly luciferase activity was assayed and normalized by the renilla luciferase activity. Results are means ± SD (n = 3). ***P < 0.001. Ctl, untreated cells; Ki + LPA and GW + LPA, cells preincubated 1 h with 10 µM Ki16425 or 1 µM GW9662 before 18 h stimulation with 25 µM LPA.

 
LPA upregulates MCP-1 and IL-8 expression through LPA1-, LPA3-, Gi/o-, and PPAR{gamma}-dependent pathways. We next investigated whether LPA was able to modulate gene expression in EAhy926 cells. We showed that LPA upregulates IL-8 and MCP-1 at the mRNA and protein levels in a time-dependent (maximal effect at the mRNA and protein levels at 1 and 4 h, respectively, that declines thereafter) and concentration-dependent (from 1 to 25 µM) manner (Figs. 2 and 3, A and B). These results were also confirmed at the protein level in HUVEC cells (Fig. 3, A and B). Basal production of MCP-1 at 4 h was about 148.5 ± 2.12 and 387.07 ± 3.25 pg/ml in EAhy926 and HUVEC cells, respectively, and at 12 h, it was about 550.1 ± 3.04 and 1268.27 ± 1.98 pg/ml, respectively. As shown in Fig. 3, we observed a peak of secretion at 4 h corresponding to 327.5 ± 7.77 pg/ml for the EAhy926 cells and 795.03 ± 2.06 pg/ml for the HUVEC cells, and at 12 h it corresponded to 802.2 ± 2.3 and 1424.96 ± 2.08 pg/ml, respectively.


Figure 2
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  		Fig. 2. LPA upregulates monocyte chemoattractant receptor-1 (MCP-1) and IL-8 expression in EAhy926 cells in a time- and concentration-dependent manner. A: EAhy926 cells were stimulated during the indicated time with 25 µM LPA. B: EAhy926 cells were stimulated during 1 h, with different LPA concentrations, as indicated. MCP-1 and IL-8 mRNA abundance was monitored by real-time RT-PCR with specific primers for human MCP-1, IL-8, and GAPDH, chosen as a housekeeping gene. Results are expressed as fold induction of the appropriate controls, and data are means ± SD (n = 3). ***P < 0.001; **P < 0.01 vs. corresponding control.

 

Figure 3
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  		Fig. 3. LPA upregulates MCP-1 and IL-8 expression in both EAhy926 and human umbilical vein endothelial cells (HUVEC). A and B: endothelial cells were stimulated during the indicated time with 25 µM LPA. Supernatants of endothelial cells were collected at the indicated times, and MCP-1 (A) and IL-8 (B) secretion were assayed by ELISA and normalized by the total amount of proteins (per well). Results are expressed in fold induction relative to the appropriate controls, and data are means ± SD (n = 3). ***P < 0.001; **P < 0.01; *P < 0.05 vs. negative control. C: effects of various inhibitors on the LPA-induced overexpression of MCP-1 and IL-8. Cells were pretreated for 1 h with either 10 µM Ki, 1 µM GW, or 50 ng/ml pertussis toxin (PTX), before 1 h stimulation with 25 µM LPA. Real-time RT-PCR was performed, as previously described. Results are expressed as fold induction of the control cells, and data are means ± SD (n = 3). ***P < 0.001; **P < 0.01; *P < 0.05 vs. LPA.

 
Since LPA interacts with both transmembrane LPA receptors and the nuclear receptor PPAR{gamma}, we investigated the relative contribution of the membrane and nuclear LPA receptors in mediating LPA-induced IL-8 and MCP-1 expression (Fig. 3C). Pretreatment of EAhy926 cells during 1 h with 10 µM Ki16425 before treatment with 25 µM LPA for 1 h markedly diminished the LPA-induced chemokine expression at the mRNA level (by 72% for MCP-1 and 67% for IL-8). Similar results were obtained by preincubating EAhy926 cells with 25 µM Ki16425 (data not shown). In addition, preincubation with 50 ng/ml of PTX that blocks the signaling downstream of Gi/o protein-coupled receptors significantly inhibited the LPA-induced chemokine expression (by 55% for MCP-1 and 64% for IL-8). Finally, the stimulatory effect of 25 µM LPA (after 1 h) on IL-8 and MCP-1 expression was diminished (47% and 49%, respectively) in the presence of 1 µM GW9662, a PPAR{gamma} antagonist. We never observed any additive effect when these antagonists/inhibitors were combined (data not shown). These results suggest that the overexpression of IL-8 and MCP-1 induced by LPA is LPA1, LPA3, and Gi/o dependent. Moreover, we demonstrated that the LPA-induced upregulation of IL-8 and MCP-1 at the mRNA level is also partially (at about 50%) PPAR{gamma} dependent in the endothelial EAhy926 cells.

Effects of LPA on monocyte chemotaxis. Since MCP-1 and IL-8 overexpression induced by LPA was also observed at the protein level, we wondered whether these elevated protein levels could lead to an increased biological activity, with a particular focus on chemotactic activity. But, as mentioned in the introduction, LPA by itself, according to the cellular model and the conditions used, can exert variable effects on cellular migration. So, we first studied the capacity of LPA, over a large concentration range (from 100 pM to 100 µM) to favor or not favor THP-1 migration, using a modified Boyden's chamber. By adding LPA to the lower chamber, we observed a typical bell-shaped dose-response curve since LPA enhanced migration from 100 pM to 100 nM with a maximum effect observed at 10 nM, whereas inhibiting this transmigration from 10 to 100 µM (Fig. 4A). No effect was observed for 1 µM LPA.


Figure 4
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  		Fig. 4. Concentration-dependent effect of LPA on monocyte migration in an in vitro chemotaxis assay. A: different concentrations of LPA diluted in Dulbecco's high glucose medium (DHG) + 0.1% fatty acid-free BSA were tested on the migration of THP-1 monocytes during 4 h. The number of migrating monocytes was estimated as described in MATERIALS AND METHODS. Results are means ± SD (n = 3), and data are expressed as a percentage of control (Ctl = DHG + 0.1% fatty acid-free BSA). In the absence of LPA, the number of migrating monocytes was about 1,500 cells. ***P < 0.001; **P < 0.01 vs. Ctl. B: LPA receptors involved in LPA-induced monocyte migration. THP-1 cells were preincubated 1 h with 10 µM Ki, 1 µM GW, or 50 ng/ml PTX before being added to the upper chamber to migrate for 4 h toward the lower chamber containing 10 nM or 100 µM LPA. Results are expressed as number of migrating monocytes estimated as described in MATERIALS AND METHODS. Results are means ± SD (n = 3). ***P < 0.001; **P < 0.01; *P < 0.05 vs. LPA.

 
Next, to confirm the chemotactic effect of LPA and exclude any chemokinesis (increased random migration), we repeated the experiments by adding equal LPA concentrations in both lower and upper chambers. In these conditions, in the range of 100 pM to 1 µM, LPA was unable to stimulate monocyte migration (data not shown). This confirms that LPA indeed induces monocyte chemotaxis. At higher concentrations of LPA (from 10 to 100 µM), we observed an inhibition of monocyte migration both without gradient (data not shown) or with a LPA gradient as already mentioned (see Fig. 4A). To identify some of the LPA receptors involved in LPA-induced THP-1 migration, we tested several antagonists/inhibitors, as previously used for the EAhy926 cells. As shown in Fig. 4B, PTX, GW9662, and Ki16425 significantly inhibited 10 nM LPA-induced THP-1 cell migration. Moreover, these inhibitors also significantly reduced the inhibitory effect of 100 µM LPA on monocyte migration (Fig. 4B). Taken together, these findings indicated that LPA induces THP-1 monocyte chemotaxis at concentrations ranging from 100 pM to 100 nM as well as an inhibition of THP-1 migration from 10 to 100 µM, with the participation of LPA1, LPA3, Gi/o, and PPAR{gamma} receptors.

Effects of conditioned media from LPA-treated endothelial cells on THP-1 migration. Since LPA at 1 µM had no effect on monocyte migration, we first tested conditioned media from endothelial EAhy926 cells treated with 1 µM LPA for 4 h on THP-1 monocyte migration (Fig. 5A). The number of migrating THP-1 cells was estimated after 4 h migration by using a modified Boyden's chamber. Since these conditioned media contain MCP-1 and IL-8 (Fig. 3), neutralizing antibodies against IL-8 and MCP-1 were added, combined or not, to the conditioned media of LPA-treated EAhy926 cells (4 h at 1 µM). As shown in Fig. 5A, these antibodies abrogate the chemotactic activity of the endothelial cell supernatants, but only partially. When combined, they almost completely abrogate the LPA-induced chemotactic activity. Normal mouse IgG (1 µg/ml) were unable to reproduce the effects of the anti-IL-8/anti-MCP-1 antibodies. These results suggest that the LPA enhancement of the chemotactic activity in the supernatant of endothelial cells is largely mediated through secreted IL-8 and MCP-1. We could also exclude that the presence of LPA by itself was required for inducing monocyte migration, by performing a chase experiment: after an incubation in the presence of LPA launching the LPA-dependent signaling, the cells were rinsed to remove the residual LPA and incubated for a further period of 2 h; the chemotactic activity in the supernatant devoided of LPA was then evaluated. As shown by Gustin et al. (13), this LPA chase did not affect the chemotactic activity in the supernatant. Second, since LPA upregulates MCP-1 and IL-8 with a maximal response at 25 µM (Fig. 2B), we also tested the effects of conditioned media from 25 µM LPA-treated EAhy926 cells, using the same migration assay during 4 h (Fig. 5B). In these conditions, we observed that the conditioned media reduced monocyte migration compared with the control conditioned medium (cells incubated during 4 h without LPA treatment). This result is consistent with the inhibition of THP-1 migration observed for LPA concentrations higher than 1 µM (Fig. 4A). Taken together, these results suggest that 1 µM LPA triggers the release by endothelial EAhy926 cells of chemotactic factors such as IL-8 and MCP-1, which induce monocyte chemotaxis. In the presence of higher concentrations of LPA (25 µM), although the amount of chemotactic factors released by endothelial cells is much more important (Fig. 2B), LPA by itself inhibits monocyte migration, counteracting the chemotactic effect of endothelial cells-secreted IL-8 and MCP-1.


Figure 5
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  		Fig. 5. Effect of EC-conditioned media on the migration of THP-1 monocytes. A: EAhy926 cells were or were not treated for 4 h with 1 µM LPA, and the corresponding conditioned media were tested on the migration of THP-1 monocytes. The lower chamber contained either conditioned media of untreated EAhy926 cells (= control conditioned media or Ctl) or conditioned media of LPA 1 µM-treated cells (LPA) supplemented or not with the specific IL-8/MCP-1 neutralizing antibody, and control conditioned media or conditioned media of LPA 1 µM-treated cells supplemented with normal mouse IgG. Cells migrating into the lower chamber were estimated as described in Fig. 4. {ddagger}{ddagger}{ddagger}P < 0.001; {ddagger}{ddagger}P < 0.01; {ddagger}P < 0.05 vs. Ctl. ***P < 0.001. B: EAhy926 cells were or were not treated for 4 h in the presence of LPA (25 µM), and the corresponding conditioned media were tested on the migration of THP-1 monocytes preincubated or not with Ki16425. The lower chamber contained either control conditioned media (Ctl) or conditioned media of 25 µM LPA-treated EC (LPA). ***P < 0.001; **P < 0.01. C: EAhy926 cells were or were not treated (Ctl) for 8 h in the presence of TNF{alpha} (10 ng/ml) and the corresponding conditioned media were tested on the migration of THP-1 monocytes. The lower chamber contained either control conditioned media (Ctl), conditioned media of TNF-{alpha}-treated cells, or the latter combined with freshly added LPA (25 µM). Cells migrating into the lower chamber were estimated as described in Fig. 4. Results are means ± SD (n = 3). ***P < 0.001; **P < 0.01.

 
To confirm this hypothesis, we first used an LPA receptor antagonist to limit the THP-1 response to LPA. Since THP-1 cells are known to express the three membrane LPA1, LPA2 and LPA3 receptors (7), we preincubated THP-1 cells with the LPA1 and LPA3 receptor antagonist (Ki16425) 30 min before the chemotaxis assay (Fig. 5B). This preincubation had no effect on the basal chemotactic activity of the endothelial cells supernatants; however, the reduction in monocyte migration observed with the conditioned media from LPA-treated cells was not observed anymore. This set of data is in agreement with the results shown in Fig. 4B, confirming that the inhibitory activity of LPA at high concentrations on monocyte migration involves LPA membrane receptors (Figs. 4B and 5B) and PPAR{gamma} (Fig. 4B). Second, to confirm the inhibitory effect of LPA on the monocytes, we evaluated the effect of LPA on a well-described chemotactic model, totally independent of LPA. We used conditioned media of EAhy926 stimulated with the pro-inflammatory cytokine TNF-{alpha} during 8 h, and this cytokine is well known to enhance the secretion of chemokines such as IL-8 and MCP-1. As expected, a significant increase in monocyte migration was observed (Fig. 5C). But, when we combined the TNF-{alpha}-conditioned media with 25 µM fresh LPA during the migration assay, the TNF-{alpha}-induced monocyte migration was also significantly reduced.

Altogether, the results presented in Fig. 5 suggest opposing effects of different concentrations of LPA (in the range of 100 pM to 100 µM); the lower concentrations favoring at the one side the secretion of chemotactic factors (such as MCP-1 and IL-8 by endothelial cells) and being chemoattractive for THP-1 cells, but the higher concentrations inhibiting monocyte migration at the other side, through the same receptors expressed on the monocytes, despite a significant induction of chemokines secretion by the endothelial cells. In this context, it is conceivable that if LPA would be progressively degraded in the medium, the resulting effect of conditioned media from endothelial cells treated with LPA concentration >1 µM could again progressively become chemoattractive to monocytes for longer incubation times.

We tested the latter hypothesis by extending the migration time during the migration assay. Conditioned media of LPA 25 µM-treated EAhy926 cells were tested on THP-1 cells for 24 h in the chemotaxis assay. Interestingly, in this case compared with Fig. 5A, the conditioned media again enhanced monocyte migration compared with the control conditioned media (Fig. 6A). Once again, as shown previously, this migration was significantly reduced after preincubation with 1 µg/ml of neutralizing antibodies against human IL-8 and MCP-1. Similar results were obtained with conditioned media of 25 µM LPA-treated HUVEC cells (Fig. 6B). These data suggests that LPA might be degraded during the 24-h period of the assay, reaching concentrations too low to exert its inhibitory effect on the monocyte transmigration. To verify this hypothesis, we studied in a preliminary study the expression of three phosphatases well known to be involved in the degradation of LPA: the lipid phosphate phosphatases-1, -2 and -3 (LPP-1, -2, -3) (31). THP-1 cells were stimulated with 25 µM LPA during the period of time indicated, and LPP expression was monitored by measuring the abundance of the corresponding mRNA by real-time RT-PCR. As shown in Fig. 7, LPA induces a significant LPP-2 overexpression (180%, 150%, and 130% of the appropriate negative controls) after 16, 20, and 24 h stimulation, respectively. LPP-1 and LPP-3 were detected, but no variation of expression was observed after LPA treatment (data not shown). Based on this LPP-2 overexpression, we hypothesize a possible negative feedback loop, since LPP-2 is known to inactivate exceeding LPA.


Figure 6
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  		Fig. 6. Conditioned media from EAhy926 and HUVEC cells treated with 25 µM LPA favor the chemotactic activity of THP-1 cells measured after 24 h of cell migration through the secretion of IL-8 and MCP-1. EAhy926 (A) and HUVEC (B) cells were or were not treated for 4 h in the presence of 25 µM LPA, and the conditioned media were tested on the migration of THP-1 monocytes for 24 h toward the lower chamber, which contained either control conditioned media (Ctl) or conditioned media of LPA-treated cells (LPA) supplemented or not with the specific IL-8/MCP-1 neutralizing antibody. Results are expressed as described in Fig. 4. Data are means ± SD (n = 3). ***P < 0.001; **P < 0.01; *P < 0.05.

 

Figure 7
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  		Fig. 7. LPA upregulates lipid phosphate phosphatase (LPP-2) expression at the mRNA level in THP-1 cells. Cells were or were not treated during the indicated time with LPA (25 µM) and after RNA extraction, real-time RT-PCR was performed. Results are expressed as fold induction of the appropriate control and are means ± SD (n = 4). ***P < 0.001; **P < 0.01 vs. corresponding Ctl.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Endothelial cells respond to local injury by overexpressing adhesion molecules and actively secreting pro-inflammatory cytokines and chemokines, leading to monocyte chemotaxis followed by their transmigration into the vascular wall. This sequence of cellular processes relying on a complex interplay between, among others, endothelial cells and monocytes, has been claimed to play a critical role in the onset of early atherosclerotic lesions and is then amplified by LDL accumulation and oxidation in the intima. In this context, the effects of LPA, which is released by activated platelets and is mainly found as a bioactive component of oxLDL (35, 36), were investigated both in endothelial cells and monocytes, with a particular focus on endothelium-induced monocyte transmigration.

First, we characterized the LPA receptors expressed in the EAhy926 cells, used as an in vitro model for endothelial cells. Not only do these cells express the LPA1 to LPA4 receptors, but we also highlighted the capacity of LPA to specifically induce the PPRE-driven expression of luciferase in EAhy926 cells, independently of the LPA1 and LPA3 membrane receptors. Our results are in agreement with the data of McIntyre et al. (19) on murine RAW 264.7 macrophages, suggesting that LPA is a high-affinity lipid ligand for PPAR{gamma}, at least in macrophages and endothelial cells. This observation again supports the ambiguous role of LPA, since PPAR{gamma} is expressed in cells of the vascular wall, such as monocytes/macrophages, endothelial cells, and smooth muscle cells (for a review, see Ref. 4), but it is now generally accepted that PPAR{gamma} activators in this context exert antioxidant and anti-inflammatory actions by transrepression of pro-inflammatory genes (39).

Recruitment of circulating monocytes into the subendothelial space due to secretion of chemoattractant factors such as IL-8 and MCP-1 by endothelial cells is one of the early steps in lesion progression (42). Hence, we wondered whether LPA could stimulate endothelial EAhy926 cells to express these two chemokines. As already demonstrated by Lin et al. (17) on primary HUVEC cells, we showed that LPA induces MCP-1 and IL-8 at the mRNA and protein levels in a concentration- and time-dependent manner in EAhy926 cells, a model more and more characterized in several in vitro studies related to the context of cardiovascular diseases (14, 18, 30). We also observed similar results on primary HUVEC cells. To further unravel the relative contribution of the membrane LPA-receptors and of PPAR{gamma} in this response, we used specific inhibitors/antagonists of these receptors. We demonstrated that LPA induced IL-8 and MCP-1 expression through at least two membrane receptors but also through the intracellular nuclear receptor PPAR{gamma} in EAhy926 cells. The PPAR{gamma} antagonist GW9662 reduced the chemokine secretion to about 50%. It is well documented that MCP-1 and IL-8 are regulated by AP-1 and NF{kappa}B, two major transcription factors known to be involved in the inflammatory response, in ovarian cancer cells (11), bronchial epithelial cells (44), or endothelial cells (17). The participation of PPAR{gamma} in the overexpression of MCP-1 and IL-8 is surprising since PPAR{gamma} has been claimed to exert anti-inflammatory actions by transrepressing pro-inflammatory genes. However, Dwarakanth and colleagues (8) recently showed that 13-hydroperoxyoctadecadienoic acid (13-HPODE) (an oxidized metabolite of 12/15-lipoxygenase), which could activate PPAR{gamma} (22), was able to induce by itself the overexpression of MCP-1 in vascular smooth muscle cells. But in the presence of a pro-inflammatory cytokine such as TNF-{alpha}, oxidized lipids (13-hydroxyoctadecadienoic acid, 13-oxooctadecadienoic acid, and 15-HETE) recognized by PPAR{gamma} inhibit TNF-{alpha}-induced chemokine production (1). In other words, it appears that some oxidized lipids recognized by PPAR{gamma}, when used alone are rather pro-inflammatory, while they exert anti-inflammatory actions by transrepression, when combined with strong activators of NF{kappa}B (as TNF-{alpha}). Moreover, despite the anti-inflammatory actions of PPAR{gamma} ligands, Zhang et al. (43) have demonstrated that activation of PPAR{gamma} by LPA in vivo on a rat carotid artery model is both necessary (at 100%) and sufficient for neointimal lesion formation, which again suggests the complexity of the effects of LPA and PPAR{gamma} in the context of atherosclerotic lesions.

Since diapedesis is a highly space- and time-regulated process, we also investigated the effects of LPA, as well as of conditioned media obtained from LPA-treated EAhy926 cells, on monocyte migration, which is poorly documented in the literature, compared with other cell types (as mentioned in the introduction). First, our data clearly showed that LPA directly modulates monocyte chemotaxis, exerting opposing effects according to its concentration: monocyte migration is enhanced in the presence of LPA 100 pM to 100 nM with a maximum effect at 10 nM, while it is inhibited at higher concentrations (10–100 µM). No effect was observed at 1 µM LPA. We also confirmed that LPA induced monocyte chemotaxis and not chemokinesis. The bell-shaped response curve obtained for monocyte chemotaxis in response to LPA is in agreement with the recent data obtained on breast cancer cells. Indeed, Chen and colleagues (6), demonstrated that LPA1-expressing cells show a peak of migration at 100 nM LPA that declines at 1 µM LPA, whereas LPA2-expressing cells show a peak of migration between 1 and 10 µM LPA, through the activation of RhoA. Interestingly, they also showed the chemotaxis of cells expressing both LPA1 and LPA2 over a wider range of LPA concentrations (from 10 nM to 10 µM), suggesting a cooperative rather than a competitive effect. However, the molecular mechanisms underlying the effects of LPA on monocyte chemotaxis are totally unknown. In general, cell migration is driven by signaling pathways controlled by the three Rho GTPases, RhoA, Rac1, and Cdc42, modulating in a coordinate way the actin cytoskeleton (23). These Rho GTPases might be activated dowstream of LPA1 and LPA2 receptors that signal mainly via Gi/o and G12/13 heterotrimeric proteins. Van Leeuwen et al. (40) have shown that the LPA1 receptor, in addition to transiently activating RhoA, mediates prolonged activation of Rac via a Gi-phosphatidylinositol 3 kinase pathway leading to lamellipodia formation, cell spreading, and migration in fibroblasts. Moreover, they observed that Rac activation through the guanine nucleotide exchange factor Tiam1 inhibits RhoA activation. On the other hand, Sugimoto and co-workers (38) have also demonstrated that the LPA1 receptor (when Gi is inactivated by PTX) mediates a strong inhibitory signal for the migration of Chinese hamster ovary cells through a G13- and RhoA-dependent activation, regulating negatively Rac. Therefore, the Rac-RhoA activity balance in a given cellular context is critical in determining whether LPA receptor stimulation leads either to lamellipodia formation (Rac dependent), leading to cell spreading and migration or to actin stress fiber formation (Rho dependent) hampering cell migration. To characterize the LPA receptors (membrane and nuclear) involved in LPA-regulated THP-1 migration, we observed, by using specific LPA-receptors antagonists (Ki16425, GW9662, PTX), that LPA modulates THP-1 chemotaxis through a LPA1-, LPA3-, Gi/o-, and PPAR{gamma}-dependent pathways. However, further studies on THP-1 monocytes are needed to unravel at the molecular level the downstream signaling resulting in the bell-shaped curve obtained for LPA-induced monocyte migration.

We next wanted to challenge the monocytes with endothelial cells in the presence of LPA, as it occurs in vivo. Therefore, we tested conditioned media of LPA-treated EAhy926 cells on monocyte migration during 4 h, and we also observed two opposing effects comparable to the previous results obtained on the monocytes: 1) conditioned media of 1 µM LPA-treated cells significantly enhanced monocyte migration, mainly because of the enhancement of IL-8 and MCP-1 secretion and 2) conditioned media of 25 µM LPA-treated cells significantly inhibited monocyte migration through a LPA1- and LPA3-dependent way. When the monocytes were treated with Ki16425, this inhibition disappears, but the chemotactic activity remained comparable to the control. This may be explained by the fact that Ki16425 does not antagonize PPAR{gamma} nor LPA2 that may still activate the signaling pathway leading to the inhibition of THP-1 migration. Moreover, we demonstrated that this inhibition with conditioned media of 25 µM LPA-treated endothelial cells (EAhy926 and HUVEC) disappears by increasing the migration time to 24 h. Therefore, we considered the possibility that LPA might be degraded during the 24 h of the assay, reaching concentrations too low to affect monocyte migration. In a preliminary experiment, we showed that monocytes respond to high concentrations of LPA by overexpressing LPP-2 at the mRNA level after 16 h of LPA treatment, which is in agreement with the hypothesis of LPA degradation. Indeed, LPP-2 is one the LPP isoforms involved in LPA degradation, but the overexpression was not evaluated at the protein level. The induction of LPPs was also evaluated in the EAhy926 cells (data not shown), indicating an overexpression at the mRNA level of LPP-3 of about 1, 4, and 2 times after 4 and 8 h of LPA stimulation, respectively. This preliminary observation could explain the progressive decrease of chemokine secretion by the endothelial cells as a function of time due to a progressive LPA degradation, mediated among others by the LPPs (see Fig. 2).

Although we are aware that the in vitro model we used is far away of the in vivo conditions where shear stress, for instance, may have locally an impact on the monocyte transmigration into the vascular wall, our results clearly indicate that LPA is an important factor favoring endothelial dysfunction as well as monocyte-endothelium interactions, both involved in the progression of atherosclerosis. All together our data fits into the following model: in a local inflammatory context, LPA accumulates, produced from activated platelets, blood cells, oxLDL..., reaching concentrations sufficient to activate endothelial cells to secrete several pro-inflammatory proteins like IL-8 and MCP-1, but also pentraxin-3, a pro-inflammatory protein belonging to the same family as C-reactive protein (CRP), but that contrary to CRP, is expressed ubiquitously and in particular by activated endothelial cells (13). At 1 µM LPA and for short periods of incubation (4 h), both cell types respond cooperatively, favoring the monocyte potential of transmigrating across the endothelial barrier. At higher LPA concentration (25 µM), the migratory response varies as a function of time. After 4 h, the inhibitory effect of LPA on monocytes counteracts the chemotactic activity of MCP-1 and IL-8 secreted by the endothelial cells. For longer periods of time (24 h), the concentration of available bioactive LPA could decrease, probably due to the overexpression of LPP-2 in response to LPA by the monocytes, LPP-2 being known to specifically inactivate exceeding LPA.

In conclusion, our data, for the first time, highlight the very complex facets of bioactive LPA affecting both endothelial cells and monocytes, as well as the interplay between both cell types, that is fine tuned and regulated in a time- and LPA concentration-dependent way, taking into account the multiple LPA receptors expressed in both endothelial cells and monocytes.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Cindy Gustin is a recipient of the Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture doctoral fellowship. We thank the Fonds de la Recherche Fondamentale Collective for financial support. The scientific responsibility is assumed by the authors.


   ACKNOWLEDGMENTS
 
This paper presents research results of the Belgian Programme on Interuniversity Poles of Attraction (PAI 6/30) initiated by the Belgian State, Prime Minister's Office, Science Policy Programming.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Gustin, Laboratory of Biochemistry and Cellular Biology, URBC, Univ. of Namur (FUNDP), 61, rue de Bruxelles, 5000 Namur, Belgium (e-mail: cindy.gustin{at}fundp.ac.be)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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