Published online before print June 12, 2006
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,1
,2,¶,3
* Departments of Anatomy and
¶ Physiology, University of Oslo, Norway;
Department of Pneumology, University of Freiburg, Germany;
Department of Dermatology, University of Jena, Germany; and
Bio-Quant, Inc., San Diego, California
2Correspondence: Department of Dermatology, University of Jena, Erfurterstrasse 35 D-07743, Jena, Germany. E-mail: johannes.norgauer{at}med.uni-jena.de
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: LPA • S1P • secretion • ERK • PLD • Rho • PLC
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, interleukin (IL)-10, and IL-12 [2
, 3
]. Sphingosine 1-phosphate (S1P) and a related lysophospholipid, lysophosphatidic acid (LPA), are released by platelets and constitute a major part of serum and plasma [4 , 5 ]. These lysophospholipids (LPLs) are also secreted by inflammatory cells. For example, S1P is secreted by mast cells upon ligation of their Fc receptor 1 for immunoglobulin E (IgE) and is elevated in asthmatic lungs after antigenic challenge [6 , 7 ]. In addition to inflammation, LPA promotes cell growth, survival, differentiation, and motility. For example, it regulates keratinocyte differentiation, induces smooth muscle contraction, and triggers chemotaxis of epithelial cells and leukocytes [8 ]. Furthermore, LPA is a growth factor for B cells and stimulates adhesion of monocytes and neutrophils to endothelial cells [9 ]. The receptors for LPLs have been cloned; those that bind S1P are known as S1P1, S1P2, S1P3, S1P4, and S1P5, whereas those that bind LPA are known as LPA1, LPA2, LPA3, and LPA4 [10 ].
The biochemical, cellular signaling events linking LPA to its pleomorphic activities are complex, as these receptors couple to different pertussis toxin (PTX)-sensitive and -insensitive G-proteins. Depending on the cell type, Gi as well as Gq/11/12 couple LPA1–3 to LPA, trigger tyrosine phosphorylation, and regulate Rho-dependent actin reorganization [11 ]. The bioactive sphingolipid S1P has also been implicated as a mediator of cellular functions. It has multiple actions and regulates many processes such as proliferation, differentiation, apoptosis, tumor cell invasion, cell migration, and angiogenesis [4 , 12 ]. S1P also increases intracellular Ca2+ levels via activation of phospholipase C (PLC) and inositol 1,4,5-trisphosphate-dependent Ca2+ release from the endoplasmic reticulum [13 ]. Depending on the subtype, S1P receptors are coupled via Gi, Gq, and/or G12/13 to multiple effector systems, such as Rho, adenylate cyclase, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), phospholipases, phosphatidylinositol-3 kinase, and nonreceptor tyrosine kinases [4 , 12 , 14 , 15 ].
IL-6 is a pleiotropic cytokine produced by several cell types including macrophages, DCs, and B cells. It has a wide range of biological activities in immune regulation, hematopoiesis, inflammation, and oncogenesis and is involved in the acute-phase response, B cell maturation, and macrophage differentiation [16 ]. IL-8 is produced by various cells upon stimulation, and it influences a variety of leukocyte functions [17 , 18 ].
S1P has been shown to stimulate IL-8 secretion in human bronchial epithelial cells [19 ]. However, the signaling mechanisms responsible for the induction of IL-6 and IL-8 secretion by LPLs have not been fully identified. As there is evidence that LPA and S1P might regulate cytokine production of DCs [20 , 21 ], we investigated the effects of LPA and S1P on IL-6 and IL-8 secretion on human maturing DCs and the participation of MAPK, PLD, PKC, and Rho in LPA- and S1P-mediated signal transduction pathways.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of human DCs
These cells were prepared as described previously [20
21
22
]. Briefly, CD14+ mononuclear cells were isolated from buffy coats by Ficoll centrifugation and MicroBeads using magnetic cell sorter single-use separation columns from Miltenyi Biotec (Bergisch Gladbach, Germany). Obtained CD14+ cells were resuspended in RPMI 1640 containing 10% fetal calf serum, 1% glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin, 1000 U/ml IL-4, and 200 ng/ml granulocyte macrophage-colony stimulating factor (GM-CSF; Promocell, Heidelberg, Germany) and incubated at 37°C for 5 days. Further differentiation into mature DCs was induced by treatment with 3 µg/ml lipopolysaccharide (LPS; Sigma-Aldrich) for 24–48 h as described previously by us [20
21
22
]. Immature DCs (5 days IL-4- and GM-CSF-differentiated cells) had low expression of the costimulatory molecules CD80 or CD83 and intermediate expression of CD86, whereas mature DCs (7 days IL-4- and GM-CSF-differentiated cells with additional exposure toward LPS at Day 5 for 48 h) up-regulated the expression of CD80, CD83, and CD86 (data not shown). Using immunoblot and flow cytometric analyses, we observed that mature DCs express CCR7 but not CCR5 (data not shown).
Membrane preparation and immunoblot analysis using antibodies to receptors for S1P and LPA were as described [15 , 22 , 23 ]. A 1:100–1:250 dilution of various antibodies to LPL receptors was used.
Determination of the phosphorylation of ERK1/2
DCs were first starved in serum-free AIM-V medium (Invitrogen, Carlsbad, CA) and then stimulated with 1 µM S1P and LPA or with 200 ng/ml CCL19 for 0–120 min. Lysates were prepared from these cells, and immunoblot analysis was done as described [23
] using 1:5000 dilution of antibodies recognizing phosphorylated or nonphosphorylated ERK1/2.
Flow cytometric analysis
Surface labeling of DCs with FITC-conjugated anti-CD14, -CD80, -CD83, and -CD86 was done as described [22
]. Labeling of CCR7 was as described previously using anti-CCR7 antibodies [24
]. Staining of receptors for S1P or LPA in DCs was performed as described for other cells using antibodies to S1P1–5 or LPA1–3 [22
, 23
]. The specificity of these antibodies has been determined previously [22
, 23
, 25
].
Cell culture and cytokine assays
To investigate the influence on LPA- and S1P-mediated signal transduction pathways, immature DCs were pretreated in RPMI with or without PTX at a concentration of 4 µg/ml for 1 h, 1 µM BIM for 1 h, 1 µM Y27632 for 1 h, or 10 µM PD98059 for 1 h, prior to stimulation. The concentrations of these inhibitors were found to be optimal in these assays (data not shown). The media were removed, and the cells were stimulated in RPMI 1640 containing LPA or S1P at the concentrations indicated in Results at 37°C with 5% CO2 for an additional 1–2 days in the presence of 3 µg/ml LPS. For the experiments involving 1- or 3-butanol (0.05%), cells were pretreated for 15 min, and the alcohol incubations were continued during stimulation with LPA and S1P. After stimulation, cell supernatants were removed, centrifuged at 10,000 revolutions per minute for 5 min at 37°C, and frozen at –80°C for later analysis for IL-6 and IL-8 by enzyme-linked immunosorbent assay (ELISA), which was performed according to the manufacturer’s instructions using matched pairs of mAb from R&D Systems.
Statistical analysis
Unless otherwise stated, data are expressed as the mean ± SEM. ANOVA was used to compare experimental groups to control values. When the global test of differences was significant at the 5% level, pair-wise tests of differences between groups were applied (Tukey’s Multiple Comparison test).
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figure 1. (A–E) Immature and mature DCs express receptors for LPLs. Flow cytometric analysis of LPA receptor (LPA1–3) and S1P receptor (S1P1–5) expression in immature DCs (A), mature DCs (B), and ex vivo-isolated CD14+ cells (C). Numbers indicate the percentages of positive cells. Background controls with isotype-matched antibodies are shown in filled histograms, and percentages of positive cells are shown in open histograms. (D) The percentages of positive cells for S1P and LPA receptors from four different donors in immature DCs. Mean ± SEM (n=4). (E) The percentages of positive cells for S1P and LPA receptors from four different donors in mature DCs. Mean ± SEM (n=4).
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Figure 2. The expression of S1P and LPA receptors (S1P1, S1P2, S1P3, S1P4, S1P5, LPA1, LPA2, and LPA3) in immature DCs (iDC) and mature DCs (mDC) is determined by immunoblot analysis. Numbers to the left indicate the approximate molecular weights. Experiments were repeated three times with identical results.
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Figure 3. (A–D) Time course and concentration response of LPA- and S1P-induced IL-6 and IL-8 secretion. Immature DCs (5 days IL-4- and GM-CSF-differentiated cells) were incubated with 10–6 M LPA or S1P in the presence of LPS for the indicated times (0–24 h). IL-6 (A) and IL-8 (B) secretion was quantified by ELISA. Immature DCs were treated for 24 h with the indicated concentrations of LPA or S1P in the presence of LPS. IL-6 (C) and IL-8 (D) secretion was quantified by ELISA. Data are means ± SEM (n=3). *, **, and ***, P values less than 0.05, 0.01, and 0.001, respectively, as compared with the controls.
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Figure 4. (A and B) Effect of LPA on secretion of IL-6 and IL-8 in different maturation stages of DCs. First (iDC LPA/LPS –/–) and second (iDC LPA/LPS +/–) columns show immature DCs stimulated without and with 10–6 M LPA in the absence of LPS for 24 h. Third [iDC LPA/LPS –/+(s)] and fourth [iDC LPA/LPS +/+(s)] columns represent data from immature DCs stimulated without and with 10–6 M LPA exposed simultaneously (s) to 3 µg LPS for 24 h. Fifth [mDC LPA/LPS –/+(2d)] and sixth [mDC LPA/LPS +/+(2d)] columns are mature cells [7 days IL-4- and GM-CSF-differentiated cells with additional exposure toward LPS at Day 5 for 48 h (2d)] stimulated without and with 10–6 M LPA for 24 h. IL-6 (A) and IL-8 (B) levels were quantified by ELISA. Data are means ± SEM (n=3). ***, P values less than 0.001, as compared with the control (cells incubated in the absence of LPA).
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Figure 5. (A–D) Time-kinetic study showing the effects of LPA and S1P in maturing DCs. Immature DCs (5 days IL-4- and GM-CSF-differentiated cells) were incubated with 3 µg LPS for 48 h. In addition, DC were exposed to 10–6 M LPA (A and C) or S1P (B and D) simultaneously with LPS (Time 0), or 3, 6, 12, or 24 h after the induction of maturation by LPS. The supernatants were harvested, and IL-6 (A, B) or IL-8 (C, D) levels were analyzed by ELISA. Data are mean ± SEM (n=3). *, **, and ***, P values less than 0.05, 0.01, and 0.001, respectively, as compared with the response of immature DCs incubated with LPA or S1P in the presence of LPA at Time 0.
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ß-methyladenosine-5-triphosphate and 2'-3'-(4-benzoyl) benzoyladenosine-5-triphosphate, and Ca2+ transients were followed. This response was not influenced by PTX (data not shown).
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Figure 6. Effect of Gi/o or MAPK inhibition on LPA- and S1P-induced IL-6 and IL-8 secretion. Immature DCs were pretreated with 4 µg/ml PTX or left untreated. IL-6 (A) and IL-8 (B) secretion was quantitated from these cells 24 h after incubation with LPS in the presence of 10–6 M LPA or 10–6 M S1P. Immature DCs were pretreated with 10 µM PD98059 or left untreated. IL-6 (C) and IL-8 (D) secretion into the medium was quantified by ELISA after stimulating these cells with LPS in the presence of 10–6 M LPA or 10–6 M S1P for 24 h. Data are means ± SEM (n=3). * and **, P values less than 0.05 and 0.01, respectively, as compared with untreated (control) cells.
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To correlate these functional assays with biochemical analysis, we observed that LPA or S1P induced the phosphorylation of ERK2 in immature DCs (Fig. 7A ). Lysates from a control Jurkat cell line (JUR) expressing phosphorylated ERK1/2 confirmed that the antibody used in this assay recognizes phosphorylated ERK1/2. In contrast, neither LPA nor S1P induced the phosphorylation of ERK2 in mature DCs. This effect was seen even when DCs were stimulated with LPLs for up to 2 h (Fig. 7B) . Immature and mature DCs expressed nonphosphorylated ERK using pan-ERK antibody (lower panels in Fig. 7A and 7B , respectively), ruling out the possibility that the differential effects of these lipids may be a result of the lack of ERK in mature DCs. To ascertain that mature DCs have no intrinsic disability to phosphorylate ERK2 after ligand binding, we stimulated these cells with CCL19, the ligand of CCR7. This chemokine induced the phosphorylation of ERK1/2 in mature DCs 5 min after stimulation (Fig. 7C) . Collectively, these findings indicate that ERKs play important roles in mediating LPL-induced release of inflammatory cytokines and that the effect of ERK inhibitor PD98059 is exerted on the level of immature or maturing DCs but not on fully mature DCs.
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Figure 7. (A–C) LPLs induce ERK1/2 phosphorylation in immature DCs but not mature DCs. Lysates were prepared from immature DCs after stimulation with 10–6 M LPA or S1P for 0–20 min. Jurkat (JUR) cell line was used as a positive control. Phosphorylation of ERK1/2 was done using antibodies to phospho-ERK1/2 (A). In the lower lane, total ERK was determined using nonphosphorylated antipan ERK1/2. (B) Similar to A, except that mature DCs were used instead of immature DCs. (C) Phosphorylation of ERK1/2 induced by 200 ng/ml CCL19 in mature DCs. Representative of three experiments performed. ELC, EBI1-ligand chemokine.
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Figure 8. (A–D) Effect of PLD inhibition on LPA- and S1P-induced IL-6 and IL-8 secretion. Immature DCs were pretreated with 1-butanol (0.05%) or 3-butanol (0.05%). IL-6 secretion into the medium was quantified by ELISA after stimulation with LPS in the presence of 10–6 M LPA (A) or S1P (B) for 24 h. DCs were pretreated with 1-butanol (0.05%) or 3-butanol (0.05%), respectively. IL-8 secretion into the medium was quantified by ELISA after stimulation with LPS in the presence of 10–6 M LPA (C) or S1P (D) for 24 h. Data are means ± SEM (n=3). * and **, P values less than 0.05 and 0.01, respectively, as compared with untreated (control) cells.
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Figure 9. (A and B) Effect of BIM and Y27632 on LPA- and S1P-induced IL-6 and IL-8 secretion. (C and D) Immature DCs were pretreated with 1 µM BIM. IL-6 (A) and IL-8 (B) secretion into the medium was quantified by ELISA after stimulation these cells with LPS in the presence of 10–6 M LPA or S1P for 24 h. Immature DCs were pretreated with 1 µM Y27632. IL-6 (C) and IL-8 (D) secretion into the medium was quantified by ELISA after stimulating the cells with 10–6 M LPA or S1P in the presence of LPS for 24 h. Data are means ± SEM (n=3). * and **, P values less than 0.05 and 0.01, respectively, as compared with untreated (control) cells.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We further characterized the influence of LPLs on cytokine secretion and attempted to identify the signaling pathways involved in their activity. Thereby, we were able to show that LPA enhances the secretion of IL-6 and IL-8 in human DCs. In this context, it might be of interest that LPA has been implicated in the pathogenesis of allergic diseases such as asthma and rhinitis allergic [27 ]. Several studies have shown that challenge by allergens in humans as well as in animal models causes an IL-8-mediated increase of neutrophils in the lungs [28 ]. Moreover, asthma is characterized by mucus hypersecretion, which might increase the obstruction of the airways and the impairment of gas exchange. One of the main components of mucus secretion is the mucin protein. In this context, it is interesting that expression of the mucin genes is mediated through an IL-6-dependent autocrine/paracrine loop [29 ]. Therefore, it can be suggested that in patients with allergic diseases such as asthma, LPL-activated DCs release IL-8 to cause the infiltration of neutrophils. Therefore, enhanced secretion of IL-6 and IL-8 may add to the exacerbation of the proasthmatic changes.
Cellular recognition of LPA and S1P is mediated by G-protein-coupled receptors [4
, 5
, 12
, 14
]. To characterize the involvement of heterotrimeric G-proteins in signaling, PTX has been widely used. This toxin catalyzes the adenosine 5'-diphosphate-ribosylation of specific G-protein subunits of the Gi family, and this modification prevents the occurrence of the receptor-G-protein interaction. PTX pretreatment nearly abolished LPA- and S1P-induced IL-6 and IL-8 secretion by mature DCs, suggesting that signaling, at least to a large extent, follows the binding of Gi-protein-coupled receptors. As there is no evidence that PTX completely reduces IL-6 and IL-8 secretion of mature DCs, one can speculate that LPA and S1P receptors could be differentially coupled to different G-protein subunits in DCs.
Ligation of LPA to its G-protein-coupled cell surface receptors results in a rapid activation of PLD [30 ]. S1P has also been shown to activate PLD in a number of different cell types [31 ]. PLD catalyzes the hydrolysis of phosphatidylcholine, the most abundant membrane phospholipid, and generates phosphatidic acid (PA) and choline. To study the role of PLD in LPA- and S1P-induced IL-6 and IL-8 secretion by mature DCs, we used the PLD antagonist 1-butanol, which reduces the generation of phosphatidate in cells by serving as a substrate for PLD. Our data show a significant decrease in LPA- and S1P-induced IL-6 and IL-8 secretion in the presence of low concentrations of 1-butanol, suggesting that PLD-catalyzed PA generation is an important signaling regulator in this pathway. These findings were strengthened further by using the tertiary alcohol 3-butanol as a negative control, which shows a lack of inhibition in IL-6 and IL-8 secretion of DCs. Our findings are consistent with studies on human bronchial epithelial cells, where an involvement of PLD-derived PA in S1P signaling resulting in IL-8 secretion has been demonstrated [19 ].
Similar to PTX-induced inhibition of Gi, the inhibition of PLD by 1-butanol did not result in total attenuation of LPA- and S1P-mediated IL-6 and IL-8 secretion. These results indicate that other signal transduction intermediates also play a role in LPA- and S1P-induced IL-6 and IL-8 secretion by DCs. Many agonists including LPA and S1P, whose receptors are linked to heterotrimeric G-proteins, regulate PLD via activation of PKC and/or small G-proteins of the Rho family. Our studies with the PKC inhibitor BIM and with Y27632, a Rho kinase inhibitor, indicate that LPA- and S1P-mediated IL-6 and IL-8 secretion is regulated via PKC and G-proteins of the Rho family.
It has been reported that LPL-activated signaling pathways involve MAPKs and their activators such as MEK [4 , 5 , 12 ]. Similar to PTX, the MEK inhibitor PD98059 did not influence the baseline secretion of IL-6 and IL-8 in DCs but abrogated the modulatory effect of LPLs. In addition, we observed that LPA and S1P robustly induced the phosphorylation of ERK2 in immature DCs but were unable to stimulate this signaling pathway in mature DCs. These findings suggest that the signal pathways coupling LPLs to ERK2 are rapid and occur before DCs are matured. Once DCs are matured by bacterial products, signal coupling from LPLs to ERK2 is disrupted, despite the expression of ERK2 in mature DCs, which can be phosphorylated by other ligands such as CCL19, the ligand of CCR7. The results also suggest that the ability of PD98059 to inhibit IL-6 and IL-8 secretion upon stimulation with LPLs is related to inhibiting ERK kinase activation in immature or maturing DCs but not in mature DCs.
During maturation, expression of several chemokine receptors, such as CCR1, CCR2, and CCR5, is down-regulated to facilitate the migration of DCs toward the lymph nodes [32 ]. It is interesting that insensitivity of mature DCs toward LPA-induced ERK2 activation is not caused by down-regulation or disappearance of receptors for LPLs from the cell membranes. Therefore, our results suggest that LPS apparently uncouples these receptors from ERK2 activation, and consequently, the response can no longer be modulated by the addition of LPLs. Moreover, these "frozen" LPL receptors, retained on the surface of LPS-treated cells, are also unable to elicit migration, actin reorganization, and Ca2+transients [20 , 21 ]. In conclusion, we have uncovered a novel pathway regarding how LPLs regulate the secretion of inflammatory cytokines by DCs. According to this model, LPS uncouples LPL receptors from the signaling pathway in mature DCs. Consequently, LPLs are no longer capable of phosphorylating ERK and hence, cannot modulate or increase cellular responses from mature DCs beyond the maximum threshold. Because of the lack of ERK phosphorylation by LPLs in mature DCs, other signaling pathways via the activation of PLD, PKC, and Rho kinase may take over and may mediate the release of these inflammatory cytokines from these cells.
3 Correspondence: Department of Physiology, University of Oslo, Oslo, Norway. E-mail: azzam.maghazachi{at}medisin.uio.no
Received December 21, 2005; revised March 27, 2006; accepted April 21, 2006.
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