Skip Navigation


International Immunology Advance Access originally published online on February 16, 2007
International Immunology 2007 19(4):411-426; doi:10.1093/intimm/dxm006
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
19/4/411    most recent
dxm006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (1)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Jung, I. D.
Right arrow Articles by Park, Y.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jung, I. D.
Right arrow Articles by Park, Y.-M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?


© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Sphingosine kinase inhibitor suppresses a Th1 polarization via the inhibition of immunostimulatory activity in murine bone marrow-derived dendritic cells

In Duk Jung1, Jun Sik Lee2, Yong Joo Kim1, Young-Il Jeong3, Chang-Min Lee1, Thomas Baumruker4, Andreas Billlich4, Yoshiko Banno5, Min Goo Lee6, Soon-Choel Ahn1, Won Sun Park7, Jin Han7 and Yeong-Min Park1

1 Department of Microbiology and Immunology, National Research Laboratory of Dendritic Cell Differentiation and Regulation, Medical Research Institute, College of Medicine, Pusan National University, Ami-dong 1-10, Seo-gu, Busan 602-739, Korea
2 Department of Pharmacy, College of Pharmacy, Pusan National University, Geumjeong-gu, Busan 602-735, Korea
3 Department of Microbiology, College of Natural Science, Pusan National University, Geumjeong-gu, Busan 609-735, Korea
4 Novartis Institutes for BioMedical Research, Brunnerstrasse 59, Vienna A-1235, Austria
5 Department of Cell Signaling, Graduate School of Medicine, Gifu University, Japan
6 Department of Physiology, College of Medicine, Korea University, Seoul 136-705, Korea
7 Mitochondria Signaling Laboratory, FIRST project group, Department of Physiology and Biophysics, College of Medicine, Inje University, Gaegeum-dong, Busnajin-Gu, Busan 614-735, Korea

Correspondence to: Y.-M. Park; E-mail: immunpym{at}pusan.ac.kr


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Sphingosine kinase (Sphk) has been shown to be activated by growth factor and survival factors, and one of its products, sphingosine-1-phosphate, plays an important role in the regulation of various cellular responses. However, the effect of Sphk on the maturation and immunostimulatory function of dendritic cells (DCs) still remains largely unknown. In this study, we examined whether sphingosine kinase inhibitor (SKI) can influence co-stimulatory molecules (CD40, CD80, CD86 and MHC class II) and cytokine production (IL-12 and IL-10) in murine bone marrow-derived DCs. SKI significantly inhibited co-stimulatory molecules in DCs. SKI suppressed IL-12 production by DCs and IFN-{gamma} production by T cells. In addition, SKI-inhibited LPS induced the translocation of nuclear factor-{kappa}B, whereas it did not affect the degradation of IL-1 receptor-associated kinase-1 by LPS. These novel findings provide new insight into the immunopharmacological role of SKI in terms of its effects on DCs. These findings open a possibility for further understanding of the immunopharmacological functions of SKI, as well as therapeutic adjuvants for the treatment of DC-related acute and chronic diseases.

Keywords: dendritic cell, sphingosine kinase, sphingosine-1-phosphate, Th1 polarization


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
A considerable number of investigations are presently focused on the biology of bone marrow-derived dendritic cells (BMDCs) in terms of their possible clinical application as cellular therapies of chronic infections and cancers. BMDCs, as antigen-presenting cells, have a unique ability to enhance T and B cell responses as well as immune tolerance (1, 2). Under steady state, DCs exist in peripheral tissue at an immature state where they exert a guard function for incoming antigens (1, 2). Immature DCs do not enhance primary immune responses because they do not express the requisite co-stimulatory molecules and the antigenic peptides as stable complexes with MHC molecules. During pathogen invasion or after exposure to inflammatory cytokines, DCs undergo phenotypic change and functional maturation (3). Upon maturation, DCs in peripheral tissues migrate into the afferent lymphatics and move to T cell area of draining lymph nodes through the signals of both MHC molecules presenting antigenic peptides and co-stimulatory molecules, and subsequently initiate adaptive immune responses (3, 4).

Sphingosine-1-phosphate (S1P) has been shown to be associated with signals for calcium mobilization, cytoskeletal re-organization, chemotaxis and proliferation in immune cells (5–8). The level of S1P is tightly regulated by the balance between synthesis through sphingosine kinase (Sphk), irreversible cleavage by S1P lyase and reversible dephosphorylation to sphingosine by S1Ps. Until now, the biological activity of S1P in the immune system has been well understood. Recently, it is shown that cell migration and survival are enhanced by S1P (9–11), whereas the S1P precursor ceramide can induce apoptosis (12) in DCs. Furthermore, S1P in maturing DCs promotes their capacity to induced Th type-2 immune response (9) and inhibit T cell proliferation (5). However, the cellular targets of Sphk in DCs are not yet known, leaving the question of its global function in the maturation and immunoregulatory activities of DCs open.

Despite much effort in investigating sphingolipid signaling, there are very few established inhibitors of the enzymes of this pathway. In particular, the field suffers from a lack of potent and selective inhibitors of Sphk. Pharmacological studies to date have used sphingosine analogues, especially N.N-dimethylsohingosine (DMS) however these lipids are well-known to inhibit several protein kinases (13–16). Very recently, a few natural product inhibitors (compounds I–V) of Sphk have been isolated. It has been reported that compound II among the natural product inhibitors of Sphk is the most selective Sphk inhibitor and so may be the most attractive candidate for additional medicinal chemistry efforts (17). In this study, we used compound II as sphingosine kinase inhibitor, namely SKI, to investigate the effect of a non-cytotoxic concentration of SKI on the maturation and function in BMDCs. SKI inhibits LPS-induced co-stimulatory molecules expression and allostimulatory function, indicating that SKI is a putative inhibitor of DC maturation. These findings provide new insight into the immunopharmacology of SKI and Sphk as a potential target. Taken together, these data suggest that SKI may have critical implications for the manipulation of the functions of DCs for potential therapeutic application.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
Male 8- to 12-weeks-old C57BL/6 (H-2Kb and I-Ab) and BALB/c (H-3Kd and I-Ad) mice were purchased from the Korean Institute of Chemistry Technology (Daejeon, Korea). They were housed in a specific pathogen-free environment within our animal facility for at least 1 week before use.

Reagents and antibodies
Materials were obtained from the following sources: recombinant mouse (rm) granulocyte macrophage colony-stimulating factor (GM-CSF) and rmIL-4 were purchased from R&D Systems. [{gamma}-32P] ATP was purchased from Amersham Biosciences, SIP and sphingosine were obtained from Biomol (Plymouth Meeting, PA, USA), VPC23019 from Avanti (Alabaster, AL, USA) and SKI was purchased from Calbiochem (San Diego, CA, USA). Dextran–FITC (molecular mass, 40 000), mitomycin C and LPS (from Escherichia coli 055:B5) were obtained from Sigma (St. Louis, MO, USA); FITC- or PE-conjugated mAbs of CD11c (HL3), CD80 (16-10A1), CD86 (GL1), I-Ab ß-chain (AF-120.1) or IL-12 p40/p70 (C15.6) and IL-10 (JESS-16E3), by flow cytometry; as well as isotype-matched control mAbs and biotinlyated anti-CD11c (N418) mAbs were purchased from eBioscience (San Diego, CA, USA). Mouse anti-rabbit Sphk1 antibody was prepared as described previously (18). Anti-IL-1 receptor-associated kinase (IRAK)-1, anti-p65 and anti-{alpha}-tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Isolation and culture of DCs
DCs were generated from murine BM cells according to the procedure of Inaba et al. (19) with minor modification. Briefly, BM was flushed from the tibiae and femurs of 6–8 weeks male C57BL/6 and depleted of RBCs with Red Blood Cell Lysing buffer (Sigma). The cells were plated in six-well culture plates (1 x 106 cells ml–1; 2 ml per well) in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U ml–1 penicillin, 100 mg ml–1 streptomycin, 20 ng ml–1 rmGM-CSF and 10 ng ml–1 rmIL-4 at 37°C, 5% CO2. On day 3 and 5 of the culture, floating cells were gently removed and fresh medium was added. On day 6 of the culture, non-adherent cells and loosely adherent proliferating DC aggregates were harvested for analysis or stimulation, or, in some experiments, re-plated in 60-mm dishes (1 x 106 cells ml–1; 5 ml per dishes). On day 7, 80% or more of the non-adherent cells expressed CD11c. In certain experiments, to obtain highly purified populations for subsequent analysis, the DCs were labeled with bead-conjugated anti-CD11c mAb (Miltenyi Biotec, Gladbach, Germany) followed by positive selection through paramagnetic columns (LS columns; Miltenyi Biotec) according to the manufacturer's instruction. The purity of the selected cell fraction was >90%.

Expression vectors of Sphk
The expression constructs for human Sphk1 and for the dominant-negative mutant G82D of human Sphk1 cloned into vector pEGFP-C2 have been described previously (20).

Determination of DC viability
SKI was added to cultures of isolated DCs in six-well plates (1 x 106 cells ml–1; 2 ml per well). For the determination of cell viability, DCs were stimulated with LPS or left without any stimuli, and viability was analyzed by double staining with 5 µg ml–1 propidium iodide (PI) and annexin-V, and then analyzed by flow cytometry.

Flow cytometry
On day 7, DCs were harvested, washed with PBS and re-suspended in FACS washing buffer (2% FBS and 0.1% sodium azide in PBS). Cells were first blocked with 10% (v/v) normal goat serum for 15 min at 4°C and stained with PE-conjugated mouse mAb against CD40, CD80, CD86 and MHC class II with FITC-conjugated CD11c for 30 min at 4°C, and then cells were analyzed on a FACSCalibur cytometer (Becton Dickenson). To calculate the percentage of positive cells, a proportion of 1% false-positive events were accepted in the negative control samples throughout.

Mixed leukocyte reaction assay
Responder T cells, which participate in allogeneic T cell reactions, were isolated when passed through mononuclear cells from BALB/c mice in a MACS column (Miltenyi Biotec). Staining with FITC-conjugated anti-CD3 antibodies (BD PharMingen, San Diego, CA, USA) revealed that they consisted mainly of CD3+ cells (>95%). The lymphocyte population (95% of CD3+ cells) was then washed twice in PBS and labeled with 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE), as previously described (21). The cells were re-suspended in 5 µM CFSE in PBS. After being shaken for 8 min at room temperature, the cells were washed once in pure FBS and twice in PBS with 10% FBS. DCs (1 x 104) or DCs exposed to SKI (2.5 µM) or LPS (200 ng ml–1) for 24 h were co-cultured with 1 x 105 allogeneic CFSE-labeled T lymphocytes in 96-well, Ubottom plates (Nunc). A negative control (CD3+ lymphocytes alone) and a positive control (CD3+ lymphocytes in 5 µg of Con A) were created for each experiment. After 4 days, the cells were harvested and washed in PBS. CFSE dilution optically gated lymphocytes were assessed by flow cytometry.

Cytokines measurement
Culture supernatants were analyzed by ELISA. The OD450 of triplicate sample was determined and corrected by a microplate reader, with reading at 570 nm. The minimum detections were as follows: IL-4, 7.8 pg ml–1, and IFN-{gamma}, 31.3 pg ml–1.

In vitro Sphk assay
The assay was performed as described by Olivera et al. (22) with minor modifications. Briefly, the cells were washed with ice-cold PBS and harvested by lysis buffer (20 mM Tris–Cl pH 7.4, 20% (v/v) glycerol, 1 mM mercaptoethanol, 1 mM EDTA, 1 mM Na3VO4, 15 mM NaF, 10 µg ml–1 leupeptin and aprotinin, 1 mM phenylmethylsulfonyl fluoride and 0.5 mM 4-deoxypyridoxine). Cells were lysed by freeze thawing three times, and cytosolic fractions were prepared by centrifugation at 13 000 x g for 20 min. Equal amounts of protein were then incubated with 20 µl of kinase buffer containing 5 µM sphingosine, 1 mM ATP, 10 µci [{gamma}-32P] ATP and 200 mM MgCl2 in a total incubation volume of 50 µl for 30 min at 30°C. Reactions were stopped with 188 µl of chloroform:methanol:HCl (100:200:1, v/v) and 125 µl of mixtures of chloroform:0.1 N HCl (1:1), and then centrifuged at 3000 x g for 5 min. The supernatant was aspirated, and the labeled lipids in the organic phase were then resolved on silica thin-layer chromatography plates (Merck) using 1-butanol/ethanol/acetic acid/water (80:20:10:20) as the solvent system. Labeled S1P was visualized by autoradiography and quantified by phosphoimager or scraped and counted in a scintillation counter.

Transient transfection by electroporation
On day 5, DCs (2.0 x 106 cells per 100 µl of RPMI 1640 without serum and antibiotics) were mixed with DNA (5 µg per 400 µl of RPMI 1640 without serum and antibiotics). Electroporation was carried out using Electro Cell Manipulator 2001 (BTX), which delivers a square wave pulse. Immediately after electroporation, cuvettes were incubated on ice for an additional 10 min and then washed three times with cold PBS. After transfection, cells were seeded in six-well plates and incubated for 24 h prior to each experiments.

siRNA studies
DCs were transfected with 100 nM siRNA specific to hSphk1 (Santa Cruz Biotechnology) and a negative control siRNA according to the manufacturer's protocol (Santa Cruz Biotechnology). After a 24-h incubation, the cells were rinsed with PBS and used for further analysis as described above.

Western blot
DCs were washed twice with Tris-buffered saline and lysed by the addition of ice-cold lysis buffer, containing 0.5% Triton X-100, 50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, 5 mM NaF, 1 mM Na3VO4, 10 µg ml–1 aprotinin and pepstatin A and 5 µg ml–1 leupeptin. Lysates were left on ice for 20 min and centrifuged for 30 min at 12 000 x g in a microcentrifuge at 4°C to remove nuclei. To examine the effect of SKI on nuclear factor-{kappa}B (NF-{kappa}B) nuclear translocation, nuclear proteins were extracted using NE-PERTM nuclear reagents (Pierce). Proteins were separated on 10% SDS–polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% non-fat dried milk in T-PBS (0.2% Tween 20 in PBS) and incubated with antibody against Sphk1, IRAK-1, NF-{kappa}B p65 or {alpha}-tubulin for overnight. Membranes were subsequently washed and incubated for 1 h with secondary antibody conjugated to HRP. Immunolabeling was detected using an enhanced chemiluminescence detection system (Millipore Corporation, Billerica, MA, USA).

Statistical analysis
Experiments were repeated at least three times with consistent results. Unless otherwise stated, data are expressed as the mean ± SEM. Analysis of variance was used to compare experimental groups with control values. While comparisons between multiple groups were done using Tukey's multiple comparison test. Statistical significance was determined as P value < 0.05.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
LPS stimulates Sphk activity in BMDCs
In order to determine whether endogenous Sphk is expressed in immature or mature DCs, we measured Sphk1 expression by western blot analysis using anti-rabbit Sphk1 antibody. To obtain the lysates for immature DCs, cells were harvested on day 6 after culture with GM-CSF and IL-4. For the lysates for mature DCs, cells were harvested after treatment with 200 ng ml–1 LPS for 24 h on day 6. Expression of Sphk1 (49 kDa) was detected in both immature and mature DCs and LPS maturation did not cause any substantial change in the level of Sphk1 expression compared with immature DCs (Fig. 1A). Next, to investigate whether LPS affected Sphk activity in immature DCs, we analyzed Sphk activity after LPS stimulation in immature DCs. We found that LPS stimulated Sphk activity in a time-dependent manner. LPS-induced Sphk activation reached a maximum value (1.87-fold induction) at 60 min after LPS stimulation and decreased slightly thereafter (90–120 min) (Fig. 1B). An induction of Sphk in the same order of magnitude (~2-fold induction) was recently described by Wu et al. (23) in LPS-activated macrophages, and is also generally seen in at the same level in some other cell types when induced a variety of different stimuli such as platelet-derived growth factor (24) and phorbol myristate acetate (25).


Figure 1
View larger version (50K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. Sphk expression and activity in murine BMDCs. BMDCs were generated as described in Methods. (A) Sphk expression was measured by western blot analysis using with mouse anti-rabbit Sphk1 antibody in both immature DCs (iDC), harvested cells on day 6 after culture with GM-CSF and IL-4, and mature DCs (mDC), harvested cells with LPS stimulation (200 ng ml–1) for 24 h on day 6. (B) DCs were treated with 200 ng ml–1 LPS for the indicated amounts of time. Sphk activity was measured as described in Methods. (C) DCs were treated with indicated concentrations of SKI for 3 h and further incubated with or without LPS (200 ng ml–1) for 24 h. Then, DCs were stained with annexin-V and PI. The percentage within each positive cell represents the incidence of annexin-V+PI+. (D) DCs were pre-incubated with SKI for 3 h and then further incubated with 200 ng ml–1 LPS for 1 h. Sphk activity was measured as described in Methods. Values indicate mean ± SEM obtained from at least three separate experiments (B and D). The results are from one representative experiment of three performed (A and C). The asterisks in panel (B) indicate significant increases compared with that of 0 min at *P < 0.05 and ***P < 0.001 and for panel (D), asterisks indicate significant increases compared with that of LPS-treated DC at ***P < 0.001.

 
To exclude the possibility that impairment of DC function mediated by SKI is due to a reduction of DC numbers through apoptosis, we tested apoptotic sensitivity of DCs to SKI. On day 6 of DC cultures, SKI at a various concentration (0, 1, 2.5 and 5 µM) was pre-incubated for 3 h and then incubated with or without 200 ng ml–1 of LPS for a further 24 h. Because concentrations of SKI >5 µM were found to be somewhat cytotoxic to DCs, SKI was used at concentration ≤2.5 µM. There were no marked differences observed until reaching a concentration of 2.5 µM in the percentage of dead cells according to the CD11c+ cells and annexin-V–PI staining (Fig. 1C). Recently, it has been demonstrated that SKI acts as a highly specific inhibitor of Sphk (Inhibitory concentration at 50% = 0.5 µM for GST-human sohingosine kinase) and does not affect the kinase activity of hERK2, hPI3K or PKC{alpha} even at concentrations at high as 60 µM (17). We tested whether SKI could inhibit LPS-stimulated Sphk activity in DCs. When DCs were pre-treated with 2.5 µM SKI, LPS-induced Sphk activation was blocked (2.06-fold reduction) (Fig. 1D).

SKI inhibits the expression of co-stimulatory molecules and IL-12 production but not IL-10 in LPS-stimulated DCs
To determine whether SKI would affect LPS-induced co-stimulatory molecules, the levels of co-stimulatory molecules were measured in LPS-treated DCs with or without SKI. As shown in Fig. 2(A), stimulation of cells with LPS for 24 h from day 6 resulted in the up-regulation of co-stimulatory molecules (Fig. 2A-b, f, j and n). It was found that 2.5 µM SKI was sufficient to inhibit the expression of co-stimulatory molecules on CD11c+ cells in LPS-treated DCs (Fig. 2A-d, h, l and p). These data suggest that SKI-treated DCs were at least partially resistant to phenotype maturation. Specific cytokine patterns, IL-12 and IL-10, are known to select Th1 and Th2 dominant states of T cell-mediated immune responses, respectively (26). Matured DCs treated with LPS produced IL-12 to regulate the Th1 response (27). IL-23, a more recently discovered member of the IL-12 family, also promotes Th1 response, but has distinct functions from IL-12. IL-23 is a heterodimeric cytokine composed of the p40 and p19 subunits. While the p19 subunit is unique for IL-23, p40 is shared with IL-12 (28). The receptor used by IL-23 is formed by association of IL-12Rß1 and IL-23R (28, 29). IL-23 is required for the generation of effector memory T cells (28). IL-23 is also needed for the generation of IL-17-producing T cells which play a significant role in the inflammatory response (30). Through these activities, IL-23 plays a critical role in chronic inflammatory diseases. To assess whether SKI may modulate LPS-induced production of IL-12 or IL-10 in DCs, the respective productions of IL-12 and IL-10 were detected by flow cytometry. FITC-labeled anti-CD11c+ DCs with PE-labeled anti-IL-12 p40/p70 or PE-labeled anti-IL-10 mAbs indicated that LPS-treated DCs in the presence of SKI expressed a low level of IL-12 p40/p70 (Fig. 2B-d) compared with those of LPS-treated DCs in the absence of SKI (Fig. 2B-b), whereas IL-10 was not detected (Fig. 2B-f and h). These findings suggest that SKI attenuated the capability of DC to enhance high levels of IL-12p40p70. Taken together, these phenomena demonstrate that SKI suppresses the maturation function of DCs stimulated with LPS.


Figure 2
View larger version (55K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. SKI suppresses co-stimulatory molecules and IL-12 production during DC maturation. BMDCs were generated as described in Methods. (A) DCs were pre-incubated with SKI for 3 h and then further incubated with 200 ng ml–1 LPS for 24 h. Co-stimulatory molecules were then analyzed by flow cytometry. The cells were gated on CD11c+. The mean fluorescence intensity values were shown for each panel. (B) DCs were pre-incubated with SKI for 3 h and then further incubated with 200 ng ml–1 LPS for 24 h. The analysis of IL-12 p40/p70 and IL-10 in CD11c+ DCs was measured by flow cytometry. The numbers indicate the percentages of CD11c+ cells expressing IL-12 or IL-10. Values are mean ± SEM obtained from at least three separate experiments. The results are from one representative experiment of three performed.

 
The expression of co-stimulatory molecules and IL-12 production were enhanced in DCs over-expressing Sphk1
To test directly the idea that Sphk, especially Sphk1, may have influence on LPS-induced expression of co-stimulatory molecules and cytokine production, cells were transfected with vectors expressing green fluorescent protein (GFP)-tagged human Sphk1, namely SPHK1, or dominant-negative Sphk1, namely SPHK1(G82D), in DCs. First, to confirm the transfection efficiency in our system, we measured the level of GFP expression analyzed by fluorescence microscopy. The expression of GFP was clearly visualized in DCs transfected with either vector, SPHK1 or SPHK1(G82D) (Fig. 3A). Furthermore, we measured percentage of survival of DCs over-expressing either vector, SPHK1 or SPHK1(G82D), after stimulation with or without LPS for 24 h. There were no marked differences in the percentage of dead cells according to the annexin-V–PI staining (Fig. 3B). Next, we sought to determine whether DCs over-expressing either vector, SPHK1 or SPHK1(G82D), influenced Sphk activity. Therefore, we measured Sphk activity in DCs over-expressing either vector, SPHK1 or SPHK1(G82D), in the presence and absence of SKI after LPS stimulation. As shown in Fig. 3(C), the activation of Sphk after LPS stimulation was about the same as those activated by the parental cells or vector-transfected cells (black bars in DCs and vector transfectant). However, LPS-induced Sphk activation was even higher in DCs over-expressing SPHK1 without changing the kinetics (white and black bars of SPHK1 transfectant). The fold increase in Sphk activity by LPS in DCs over-expressing SPHK1 (407.6 ± 60.4%) (black bar of SPHK1 transfectant) was almost twice of those in parental cells (223.3 ± 16.44%) (black bar of DCs) or vector-transfected cells (215 ± 7.63%) (black bar of vector transfectant), and LPS-induced Sphk activation was strongly inhibited in DCs over-expressing SPHK1(G82D) (70.3 ± 12.06%) (black bar of SPHK1(G82D) transfectant). Additionally, the induction of Sphk activity in parental cells or DCs over-expressing vector, SPHK1 or SPHK1(G82D), was completely diminished below the baseline by SKI treatment (all gray bars).


Figure 3
Figure 3
Figure 3
View larger version (101K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. Over-expression of Sphk1 enhances co-stimulatory molecules and IL-12 production during DC maturation. BMDCs were generated as described in Methods. On day 5, DCs were transfected with either vector, SPHK1 or SPHK1(G82D), as described in Methods. (A) DCs were seeded in six-well-containing glass cover slips for overnight. On day 6, cover slips were fixed with 4% PFA for 15 min and mounted with 4',6-diamidino-2-phenylindole-contained mounting medium to stain nucleus. (B) DCs over-expressing either vector, SPHK1 or SPHK1(G82D), were incubated with or without LPS (200 ng ml–1) for 24 h. Then, DCs were stained with annexin-V and PI. The percentage within each positive cell represents the incidence of annexin-V+PI+. (C) DCs over-expressing either vector, SPHK1 or SPHK1(G82D), were pre-incubated with 2.5 µM SKI for 3 h and then further incubated with or without LPS for 1 h. Sphk activity was measured as described in Methods. (D) DCs over-expressing either vector, SPHK1 or SPHK1(G82D), were incubated with or without LPS (200 ng ml–1) for 24 h. Then, co-stimulatory molecules were analyzed by flow cytometry after LPS stimulation (200 ng ml–1) for 24 h. The cells were gated on CD11c+. The means fluorescence intensity values were shown for each panel. (E) DCs over-expressing either vector, SPHK1 or SPHK1(G82D), were incubated with or without LPS (200 ng ml–1) for 24 h. The analysis of IL-12 p40/p70 and IL-10 in CD11c+ DCs was measured by flow cytometry. The numbers indicate the percentages of CD11c+ cells expressing IL-12 or IL-10. Values indicate mean ± SEM obtained from at least three separate experiments (C). The results are from one representative experiment of three performed (A, B, D and E). The asterisks in panel (C) indicate significant increases at ***P < 0.001 and P > 0.05 (n.s., not significant).

 
As shown in Fig. 3(D), the expression of co-stimulatory molecules was completely inhibited in DCs over-expressing SPHK1(G82D) (Fig. 3D-f, l, r and x) even after LPS stimulation, compared with the corresponding vector transfectant (Fig. 3D-b, h, n and t) or SPHK1 transfectant (Fig. 3D-d, j, p and v). Interestingly, co-stimulatory molecules were clearly enhanced in DCs over-expressing SPHK1 even after no LPS stimulation (Fig. 3D-c, i, o and u). The enhancement of maturation in DCs over-expressing SPHK1 even if no LPS stimulation was used is likely due to the increased production of S1P.

Furthermore, we found that the production of IL-12 p40/p70 was significantly enhanced in DCs over-expressing SPHK1 even without LPS stimulation (Fig. 3E-c) compared with the corresponding vector transfectant (Fig. 3E-a) or SPHK1(G82D) transfectant (Fig. 3E-e). However, the production of IL-12 p40/p70 was completely inhibited in LPS-treated DCs over-expressing SPHK1(G82D) (Fig. 3E-f) compared with the corresponding vector transfectant (Fig. 3E-b) or SPHK1 transfectant (Fig. 3E-d). Our finding suggested that Sphk1 may affect T cell-mediated responses by provoking DCs to release Th1-promoting cytokines, such as IL-12.

SKI impairs the allostimulatory capacity of DCs
CFSE, a fluorescein-based dye, has biochemical properties that render it particularly appropriate for this application. Specifically, CFSE dye is loaded into cells in vitro, and the CFSE in a given cell is monitored over time. The CFSE segregates equally between the daughter cells on division, such that the intensity of cellular fluorescence decreases 2-fold with each successive generation. This property of CFSE allows for the accurate tracking of the number of divisions that a given cell has undergone, either in vitro or following transfer in vivo (31). In order to determine whether SKI exerts detectable effects on allogeneic T cell stimulation, we subjected the DCs to 24 h of stimulation with SKI.

As shown in Fig. 4(A), the rate of proliferation of allogeneic T cells elicited by LPS-treated DCs (Fig. 4A-e) appeared to be faster than that of in untreated DCs (Fig. 4A-b), whereas SKI appeared to inhibit proliferation responses in the allogeneic T cells elicited by the LPS-stimulated DCs (Fig. 4A-f). These findings suggest that the maturation induced by LPS stimulation (24 h of 200 ng ml–1) profoundly promoted the allostimulatory capacity of the untreated DCs, whereas exposure to SKI significantly inhibited their allostimulatory capacity.


Figure 4
Figure 4
View larger version (90K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. SKI impaired the ability to induce the proliferation of allogeneic T cells and initiate Th1 responses in vitro. DCs were pre-incubated with SKI for 3 h and then further incubated with or without 200 ng ml–1 LPS for 24 h. The DCs were washed and co-cultured with T cells. (A) The treated DCs were harvested and thoroughly washed to remove all SKI. A mixed lymphocyte reaction was allowed to proceed for 4 days, as described in Methods. (B) Clustering was assessed after 64 h. (C) The cells were then examined for cytokine release after 48 h using ELISA. Medium represents the chemically untreated control group. (D) DCs were transfected with Sphk1 siRNA at a concentration of 25, 50 and 100 nM, no siRNA (Mock) or control siRNA (Con) for 24 h, and then cells were harvested. The level of Sphk1 and {alpha}-tubulin were determined by western blotting. (E) DCs were transfected with Sphk1 siRNA (100 nM), no siRNA (Mock) or control siRNA (Con) for 24 h, and then further incubated with LPS for 24 h. DCs were harvested and washed. A mixed lymphocyte reaction was allowed to proceed for 4 days, as described in Methods. (F) DCs were transfected with Sphk1 siRNA (100 nM), no siRNA (Mock) or control siRNA (Con) for 24 h, and then further incubated with LPS for 24 h. The cells were then examined for cytokine release after 48 h using ELISA. Values indicate mean ± SEM obtained from at least three separate experiments (C and F). The results were from one representative experiment of three performed (A, B, D and E). The asterisks in panel (C) and (F) indicate significant increases compared with that of LPS treatment at **P < 0.01, ***P < 0.001 and P > 0.05 (n.s., not significant).

 
We also attempted to determine the potency of DCs in terms of their ability to adhere to T cells, and thus form clusters. The size of the DC/T cell clusters diminished with exposure to SKI as compared with what was observed in the LPS-treated cells (Fig. 4B). In the presence of SKI (Fig. 4B-d), the LPS-treated DCs formed clusters that were 60% of the size of the clusters formed by the LPS-stimulated DCs in the absence of SKI (Fig. 4B-b). Considering the inhibitory effects of SKI on the production of IL-12 (a Th1-inducing cytokine) in DCs, we attempted to characterize the quality of the primary T cell response in DCs that had matured in the presence of SKI. Naive allogeneic T cells primed with mature DCs were observed to differentiate into Th1 lymphocytes when they generated high levels of IFN-{gamma} and low levels of IL-4 (Fig. 4C). In contrast, T lymphocytes primed with DCs that had matured in the presence of SKI-inhibited LPS-induced IFN-{gamma} production.

To verify the allostimulatory capacity of DCs through Sphk1-dependent signaling, DCs were transfected with Sphk1-specific siRNA and then measured the rate of proliferation of allogeneic T cells (Fig. 4E) and the levels of IFN-{gamma} or IL-4 (Fig. 4F). Western blotting verified that the expression of Sphk1 was specifically and significantly reduced by the cognate Sphk1 siRNA duplex compared with mock transfections or transfections with a control siRNA (Fig. 4D). Importantly, compared with mock transfections (Fig. 4E-f) and control siRNA transfections (Fig. 4E-g), Sphk1 siRNA (Fig. 4E-h) significantly reduced proliferation responses in the allogeneic T cells elicited by the LPS-stimulated DCs and also inhibited LPS-induced IFN-{gamma} production (Fig. 4F). These results show that the majority of the effects of SKI on the T cell differentiating properties of DCs are a consequence of the inhibition of IL-12 production.

SKI inhibits the activation of NF-{kappa}B, but not the upstream signaling components of IRAK-1 including MyD88.
NF-{kappa}B activation is an important event underlying DCs maturation (31). Also, LPS stimulation has been shown to activate NF-{kappa}B signal pathways in DCs (32). To gain insight into Sphk-induced signaling, we determined whether NF-{kappa}B might be involved in Sphk-induced signaling pathways in DCs. To determine whether SKI decreases LPS-induced activation of NF-{kappa}B, nuclear translocation of the NF-{kappa}B p65 subunit was performed. Pre-treatment with SKI suppressed the NF-{kappa}B p65 nuclear translocation that was induced by LPS stimulation (Fig. 5A). The stimulation of toll-like receptor (TLR)-4 with agonists enhances the homodimerization of TLR4 leading to the recruitment of MyD88. MyD88 induces the phosphorylation of IRAK-4, which in turn phosphorylates IRAK-1 leading to the degradation of IRAK-1 and the activation of NF-{kappa}B signal pathways. Thus, IRAK-1 degradation was used as an additional read out for the activation of TLRs. SKI did not inhibit LPS-induced degradation of IRAK-1 (Fig. 5B). MyD88 is the upstream molecule which enhances the phosphorylation and degradation of IRAK-1. Therefore, our data suggest that SKI does not inhibit MyD88. These results suggest that the molecular target of SKI is not the upstream signaling components of IRAK-1 including MyD88, but is the upstream molecules of NF-{kappa}B signal pathways.


Figure 5
View larger version (76K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5. SKI suppresses the activation of NF-{kappa}B in MyD88-dependent signaling pathways. DCs were pre-incubated with SKI for 3 h and then further incubated with 200 ng ml–1 LPS for 30 min, and then cells were harvested. (A) Nuclear extracts were prepared and the nuclear-translocated NF-{kappa}B p65 was measured by western blot using anti-p65 antibody. (B) IRAK-1 and {alpha}-tubulin expression was measured by western blot using anti-IRAK-1 and anti-{alpha}-tubulin.

 
S1P produced by LPS acts intracellularly on DCs maturation
Recent evidence suggests that S1P regulates the production of cytokines in different cell types (33, 34). However, Idzko et al. (9) and Renkl et al. (35) reported that S1P did not effect basal cytokine production such as IL-12 and IL-10 from immature DC. To investigate whether S1P produced by Sphk1 can act extracellularly through its specific G-protein-coupled receptors binding or intracellularly as second messenger. First, we applied S1P extracellularly, and then measured co-stimulatory molecules and cytokine production in DCs. S1P at a concentration of 0.1 and 1 µM for 24 h had no influence on co-stimulatory molecules (Fig. 6A) and cytokine production (Fig. 6B) in DCs. Next, to explore the candidate receptors involved in S1P-mediated co-stimulatory molecules and cytokine production, we used VPC23019, an inhibitor of S1P1 and S1P3. VPC23019, a S1P-related synthetic compound, is a very useful agent for examining the involvement of S1P, since it acts as a competitive inhibitor of S1P1 and S1P3, but partly stimulates S1P4 and S1P5 (36). We measured co-stimulatory molecules and cytokine production by LPS in the presence and absence of VPC23019 (1 µM). LPS-induced co-stimulatory molecules and cytokine production were not significantly changed in DCs in the presence and absence of VPC23019 (Fig. 6C and D). These data suggest that S1P produced by LPS might act intracellularly on DCs maturation independent of S1PR.


Figure 6
Figure 6
View larger version (107K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6. LPS stimulates co-stimulatory molecules and IL-12 production in S1P receptor-independent pathway. BMDCs were generated as described in Methods. (A) DCs were incubated with S1P and LPS for 24 h. Co-stimulatory molecules were then analyzed by flow cytometry. The cells were gated on CD11c+. The mean fluorescence intensity values were shown for each panel. (B) DCs were incubated with S1P and LPS for 24 h. The analysis of IL-12 p40/p70 and IL-10 in CD11c+ DCs was measured by flow cytometry. The numbers indicate the percentages of CD11c+ cells expressing IL-12 or IL-10. (C) DCs were pre-incubated with VPC23019 (1 µM) for 2 h and then further incubated with or without 200 ng ml–1 LPS for 24 h. Co-stimulatory molecules were then analyzed by flow cytometry. The cells were gated on CD11c+. The mean fluorescence intensity values were shown for each panel. (D) DCs were pre-incubated with VPC23019 (1 µM) for 2 h and then further incubated with or without 200 ng ml–1 LPS for 24 h. The analysis of IL-12 p40/p70 and IL-10 in CD11c+ DCs was measured by flow cytometry. The numbers indicate the percentages of CD11c+ cells expressing IL-12 or IL-10. Values are mean ± SEM obtained from at least three separate experiments. The results are from one representative experiment of three performed.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
This is, to the best of our knowledge, the first description of the effects of SKI on the phenotypic and functional maturation of murine BMDCs. In addition, this is the first study in which SKI-exposed DCs have been analyzed with regard to their capacity to sensitize recipients for cell-mediated immune responses. Sphk has been generally considered a cytosolic protein that is constitutively active, thus generating basal levels of S1P within the cells. S1P produced by Sphk can regulate various cellular process including growth, survival, differentiation, cytoskeleton rearrangements, chemotaxis, angiogenesis and immunity (37–39). Although it is clear that SIP can modulate various physiological processes in the literature, it is not clear that the effects of Sphk1 on the maturation and immunological responses of DCs.

DCs are considered to play an important role in deciding the establishment of immunity and tolerance. (40, 41). Thus, we performed a series of functional assays to ascertain the phenotype and function of SKI in murine BMDCs. To confirm that the observed effects of SKI could be attributed to DCs and not to contaminating cells persisting in the BMDC cultures, DCs were purified (>95%) prior to analysis in each of the conducted assays. Our findings indicated that SKI is a potent inhibitor of LPS-induced DC maturation. LPS-induced DC maturation was inhibited by SKI. The acute suppressive effects of SKI on DC maturation can be attributed to a non-specific inhibitory effect. Therefore, we also measured that the level of co-stimulatory molecules present after LPS stimulation in DC over-expressing either vector, SPHK1 or SPHK1(G82D), to determine the effect of Sphk1 on the maturation of DCs. The level of co-stimulatory molecule stimulated by LPS in DC over-expressing SPHK1(G82D) was found to be significantly attenuated, whereas that in DCs over-expressing SPHK1 was strongly enhanced even if no LPS stimulation occurred, thereby suggesting that Sphk1 regulated both the phenotypic and functional maturation of DCs.

Recently collected evidence suggests that the production of cytokines by DCs is dependent on either the specific type of DC, or on the stimuli received by DC (42). IL-12, in particular, exerts multiple immunoregulatory functions, inducing the activation of the Th1 subset, which performs a critical role in the induction of inflammation (43, 44). The results obtained in this report indicated that Sphk1 caused CD11c+ DC to generate IL-12 in the presence of LPS, and also confirmed that SKI or DCs over-expressing SPHK1(G82D) exert inhibitory effects on the expression of intracellular IL-12 p40/p70.

In addition, this study shows that LPS-stimulated CD11c+ DCs skewed naive T cells toward developing into IFN-{gamma}-producing T cells. SKI was determined to significantly impair the capacity of these cells to proliferate and initiate Th1 responses in vitro. Naive T cells stimulated with SKI-treated DCs generated lower IFN-{gamma} levels, but did not exhibit significantly altered abilities to generate IL-4. Collectively, our data suggest that SKI affects, at least in part, the function of DCs to polarize the immune response toward an increased Th1 response. In the point of view in Th1-skewed immunity, many lines of evidence have been accumulated from murine models and human pre-clinical studies that hold up potent CD8+ T cell responses for tumor and viral immunity (45–47). Considering the practical development for inducing of Th1 responses such as ex vivo manipulation of DCs and cytokine therapy for adaptive transfer, our newly discovered resullts suggest that Th1-polarizing roles of Sphk1 may provide rationale for another target of DC based-therapies.

Because Th1 cells exert functionally immunogenic or defensive effects against invading pathogens, the inhibition of DC-mediated Th1 polarization might constitute a SKI-associated immunosuppressive mechanism. However, the inhibition of Th1 development exerts negative regulatory roles on a wide variety of immune cells (48). Consequently, SKI-mediated inhibition of IL-12 generation in LPS-stimulated CD11c+ DCs may also contribute to the induction of an immunosuppressive state.

Involvement of NF-{kappa}B in tumor necrosis factor {alpha} signaling via the Sphk pathway had been previously suggested in endothelial cells, based on the suppression of NF-{kappa}B activation by DMS (49) and on the activation of NF-{kappa}B by S1P (49, 50). More recently, Wu et al. (23) reported that NF-{kappa}B activation by LPS in murine macrophages, as measured by transcriptional activity in a reporter gene assay, was inhibited by over-expression of a dominant-negative mutant of SPHK1. Our results suggest that NF-{kappa}B may be the target of inhibition by SK1 in DC maturation that results from impairing the translocation of NF-{kappa}B by SKI.

TLRs induce innate immune responses that are essential for host defense against invading microbial pathogens (51). In general, TLR activation triggers the activation of two downstream signaling pathways, MyD88-dependent and MyD88-independent pathways (52). TLR4 stimulates both MyD88- and TIR domain-containing adaptor-inducing IFN-ß (TRIF)-dependent pathways while TLR2 and TLR3 stimulate MyD88- and TRIF-dependent signaling pathway, respectively. MyD88 recruits IRAK-4 and induces IRAK-4 phosphorylation. The phosphorylated IRAK-4 induces the phosphorylation of IRAK-1 leading to the degradation of IRAK-1 and the activation of NF-{kappa}B signal pathways. The activation of NF-{kappa}B leads to the induction of inflammatory gene products including cytokines. If SKI impairs the interaction of LPS with the receptor, it should inhibit the dimerization of TLR4 and the degradation of IRAK-1. LPS-induced degradation of IRAK-1 was not inhibited by SKI in DCs (Fig. 5B). Our results suggest that SKI does not inhibit the interaction of LPS with TLR. In this context, our study shows for the first time that Sphk activation is not the component in the upstream of IRAK-1, including TLR4 or MyD88, but the upstream molecule such as NF-{kappa}B.

S1P which is present in high concentration in serum and is also secreted by inflammatory cells has been known to play roles in allergic reactions and tumor pathogenesis (53, 54). Biological activities and signaling pathways of S1P are mediated in a S1P receptor-dependent manners in DCs, T cells and NK cells in various lymphoid tissues (55). Interestingly, in comparison to those reports, we observed that extracellular treatment with S1P on DCs did not affect the expression of co-stimulatory molecules or IL-12 production of immature and mature DCs, and inhibition of S1P receptors by VPC23019 (competitive antagonist to S1P1 and S1P3 receptors) also had no effect. Furthermore, although there have been raised a massive discrepancy regarding the function of intracellular and extracellular S1P, our findings in this issue strongly support that, at least in the DCs, by the LPS-induced high-level induction of intracellular Sphk and its metabolite, S1P, DCs can be directed to maturation in the end.

In this study, we demonstrated that the effect of SKI inhibit the maturation of mouse BM-derived myeloid DCs. At non-toxic concentration, SKI proved to be a potent inhibitor of DC maturation. This inhibitory effect of SKI on DC maturation is associated with suppressed translocation of NF-{kappa}B as potential target.

In conclusion, we have characterized a variety of effects exerted by SKI on BMDCs. SKI was demonstrated to inhibit phenotypic maturation and modulate cytokine production in these DCs, resulting in a significant inhibition of Th1 development. Moreover, our experimental data showed that SKI suppressed LPS-induced co-stimulatory molecules and IFN-{gamma}. These findings illustrate the immunopharmacological functions of SKI. Moreover, exposure to this readily available drug may constitute a non-toxic and highly effective means for the modulation of DC immunostimulatory capacity.


   Acknowledgements
 
This work was supported by grants from the Korea Science and Engineering Foundation through National Research Laboratory Program grant M106-0000000805J000000810.


   Abbreviations
 
BMDC, bone marrrow-derived DC
CFSE, carboxylfluorescein diacetate succinimidyl ester
DC, dendritic cell
DMS, N,N-dimethylsophingosine
FBS, fetal bovine serum
GFP, green fluorescent protein
GM-CSF, granulocyte macrophage colony-stimulating factor
IRAK, IL-1 receptor-associated kinase
NF-{kappa}B, nuclear factor-{kappa}B
rm, recombinant mouse
PI, propidium iodide
Sphk, sphingosine kinase
SKI, sphingosine kinase inhibitor
S1P, sphingosine-1-phosphate
TLR, toll-like receptor
TRIF, TIR domain-containing adaptor-inducing IFN-ß

   Notes
 
Transmitting editor: K. Inaba

Received 30 November 2006, accepted 16 January 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Steinman RM, Hawiger D, Nussenzweig MC. (2003) Tolerogenic dendritic cells. Annu. Rev. Immunol. 21:685.[CrossRef][Web of Science][Medline]
  2. Banchereau J, Briere F, Caux C, et al. (2000) Immunobiology of dendritic cells. Annu. Rev. Immunol. 18:767.[CrossRef][Web of Science][Medline]
  3. Banchereau J and Steinman RM. (1998) Dendritic cells and the control of immunity. Nature 392:245.[CrossRef][Medline]
  4. Randolph GJ, Angeli V, Swartz MA. (2005) Dendritic-cell trafficking to lymph nodes through lymphatic vessels. Nat. Rev. Immunol. 5:617.[CrossRef][Web of Science][Medline]
  5. Jin Y, Knudsen E, Wang L, et al. (2003) Sphingosine 1-phosphate is a novel inhibitor of T-cell proliferation. Blood 101:4909.[Abstract/Free Full Text]
  6. Baumruker T and Prieschl EE. (2002) Sphingolipids and the regulation of the immune response. Semin. Immunol. 14:57.[CrossRef][Web of Science][Medline]
  7. Spiegel S. (1999) Sphingosine 1-phosphate: a prototype of a new class of second messengers. J. Leukoc. Biol. 65:341.[Abstract]
  8. Ott VL and Cambier JC. (2002) Introduction: multifaceted roles of lipids and their catabolites in immune cell signaling. Semin. Immunol. 14:1.[CrossRef][Web of Science][Medline]
  9. Idzko M, Panther E, Corinti S, et al. (2002) Sphingosine 1-phosphate induces chemotaxis of immature and modulates cytokine-release in mature human dendritic cells for emergence of Th2 immune responses. FASEB J. 16:625.[Free Full Text]
  10. Czeloth N, Bernhardt G, Hofmann F, Genth H, Forster R. (2005) Sphingosine-1-phosphate mediates migration of mature dendritic cells. J. Immunol. 175:2960.[Abstract/Free Full Text]
  11. Eigenbrod S, Derwand R, Jakl V, Endres S, Eigler A. (2006) Sphingosine kinase and sphingosine-1-phosphate regulate migration, endocytosis and apoptosis of dendritic cells. Immunol. Invest. 35:149.[CrossRef][Web of Science][Medline]
  12. Franchi L, Malisan F, Tomassini B, Testi R. (2006) Ceramide catabolism critically controls survival of human dendritic cells. J. Leukoc. Biol. 79:166.[Abstract/Free Full Text]
  13. Igarashi Y, Hakomori S, Toyokuni T, et al. (1989) Effect of chemically well-defined sphingosine and its N-methyl derivatives on protein kinase C and src kinase activities. Biochemistry 28:6796.[CrossRef][Medline]
  14. Megidish T, White T, Takio K, Titani K, Igarashi Y, Hakomori S. (1995) The signal modulator protein 14-3-3 is a target of sphingosine- or N, N-dimethylsphingosine-dependent kinase in 3T3(A31) cells. Biochem. Biophys. Res. Commun. 216:739.[CrossRef][Web of Science][Medline]
  15. King CC, Zenke FT, Dawson PE, et al. (2000) Sphingosine is a novel activator of 3-phosphoinositide-dependent kinase 1. J. Biol. Chem. 275:18108.[Abstract/Free Full Text]
  16. McDonald OB, Hannun YA, Reynolds CH, Sahyoun N. (1991) Activation of casein kinase II by sphingosine. J. Biol. Chem. 266:21773.[Abstract/Free Full Text]
  17. French KJ, Schrecengost RS, Lee BD, et al. (2003) Discovery and evaluation of inhibitors of human sphingosine kinase. Cancer Res. 63:5962.[Abstract/Free Full Text]
  18. Murate T and Banno YK, et al. (2001) Cell type-specific localization of sphingosine kinase 1a in human tissues. J. Histochem. Cytochem. 49:845.[Abstract/Free Full Text]
  19. Inaba K, Inaba M, Romani N, et al. (1992) Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 176:1693.[Abstract/Free Full Text]
  20. Billich A, Bornancin F, Mechtcheriakova D, Natt F, Huesken D, Baumruker T. (2005) Basal and induced sphingosine kinase 1 activity in A549 carcinoma cells: function in cell survival and IL-1beta and TNF-alpha induced production of inflammatory mediators. Cell. Signal. 17:1203.[CrossRef][Web of Science][Medline]
  21. Lyons AB. (2000) Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution. J. Immunol. Methods 243:147.[CrossRef][Web of Science][Medline]
  22. Olivera A, Kohama T, Tu Z, Milstien S, Spiegel S. (1998) Purification and characterization of rat kidney sphingosine kinase. J. Biol. Chem. 273:12576.[Abstract/Free Full Text]
  23. Wu W, Mosteller RD, Broek D. (2004) Sphingosine kinase protects lipopolysaccharide-activated macrophages from apoptosis. Mol. Cell. Biol. 24:7359.[Abstract/Free Full Text]
  24. Rosenfeldt HM, Hobson JP, Maceyka M, et al. (2001) EDG-1 links the PDGF receptor to Src and focal adhesion kinase activation leading to lamellipodia formation and cell migration. FASEB J. 15:2649.[Abstract/Free Full Text]
  25. Johnson KR, Becker KP, Facchinetti MM, Hannun YA, Obeid LM. (2002) PKC-dependent activation of sphingosine kinase 1 and translocation to the plasma membrane. Extracellular release of sphingosine-1-phosphate induced by phorbol 12-myristate 13-acetate (PMA). J. Biol. Chem. 277:35257.[Abstract/Free Full Text]
  26. de Jong EC, Smits HH, Kapsenberg ML. (2005) Dendritic cell-mediated T cell polarization. Springer Semin. Immunopathol. 26:289.[CrossRef][Web of Science][Medline]
  27. Langrish CL, McKenzie BS, Wilson NJ, de Waal Malefyt R, Kastelein RA, Cua DJ. (2004) IL-12 and IL-23: master regulators of innate and adaptive immunity. Immunol. Rev. 202:96.[CrossRef][Web of Science][Medline]
  28. Oppmann B, Lesley R, Blom B, et al. (2000) Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13:715.[CrossRef][Web of Science][Medline]
  29. Parham C, Chirica M, Timans J, et al. (2002) A receptor for the heterodimeric cytokine IL-23 is composed of IL-12Rbeta1 and a novel cytokine receptor subunit, IL-23R. J. Immunol. 168:5699.[Abstract/Free Full Text]
  30. Aggarwal S, Ghilardi N, Xie MH, de Sauvage FJ, Gurney AL. (2003) Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17. J. Biol. Chem. 278:1910.[Abstract/Free Full Text]
  31. Rescigno M, Martino M, Sutherland CL, Gold MR, Ricciardi-Castagnoli P. (1998) Dendritic cell survival and maturation are regulated by different signaling pathways. J. Exp. Med. 188:2175.[Abstract/Free Full Text]
  32. An H, Yu Y, Zhang M, et al. (2002) Involvement of ERK, p38 and NF-kappaB signal transduction in regulation of TLR2, TLR4 and TLR9 gene expression induced by lipopolysaccharide in mouse dendritic cells. Immunology 106:38.[CrossRef][Web of Science][Medline]
  33. Lin CI, Chen CN, Chen JH, Lee H. (2006) Lysophospholipids increase IL-8 and MCP-1 expressions in human umbilical cord vein endothelial cells through an IL-1-dependent mechanism. J. Cell. Biochem. 99:1216.[CrossRef][Web of Science][Medline]
  34. Oz-Arslan D, Ruscher W, Myrtek D, et al. (2006) IL-6 and IL-8 release is mediated via multiple signaling pathways after stimulating dendritic cells with lysophospholipids. J. Leukoc. Biol. 80:287.[Abstract/Free Full Text]
  35. Renkl A, Berod L, Mockenhaupt M, et al. (2004) Distinct effects of sphingosine-1-phosphate, lysophosphatidic acid and histamine in human and mouse dendritic cells. Int. J. Mol. Med. 13:203.[Web of Science][Medline]
  36. Davis MD, Clemens JJ, Macdonald TL, Lynch KR. (2005) Sphingosine 1-phosphate analogs as receptor antagonists. J. Biol. Chem. 280:9833.[Abstract/Free Full Text]
  37. Spiegel S and Milstien S. (2003) Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat. Rev. Mol. Cell. Biol. 4:397.[CrossRef][Web of Science][Medline]
  38. Saba JD and Hla T. (2004) Point-counterpoint of sphingosine 1-phosphate metabolism. Circ. Res. 94:724.[Abstract/Free Full Text]
  39. Anliker B and Chun J. (2004) Lysophospholipid G protein-coupled receptors. J. Biol. Chem. 279:20555.[Abstract/Free Full Text]
  40. Arrighi JF, Rebsamen M, Rousset F, Kindler V, Hauser C. (2001) A critical role for p38 mitogen-activated protein kinase in the maturation of human blood-derived dendritic cells induced by lipopolysaccharide, TNF-alpha, and contact sensitizers. J. Immunol. 166:3837.[Abstract/Free Full Text]
  41. Shlomchik WD, Couzens MS, Tang CB, et al. (1999) Prevention of graft versus host disease by inactivation of host antigen-presenting cells. Science 285:412.[Abstract/Free Full Text]
  42. Kadowaki N, Antonenko S, Ho S, et al. (2001) Distinct cytokine profiles of neonatal natural killer T cells after expansion with subsets of dendritic cells. J. Exp. Med. 193:1221.[Abstract/Free Full Text]
  43. Sallusto F, Cella M, Danieli C, Lanzavecchia A. (1995) Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J. Exp. Med. 182:389.[Abstract/Free Full Text]
  44. Triantaphyllopoulos KA, Williams RO, Tailor H, Chernajovsky Y. (1999) Amelioration of collagen-induced arthritis and suppression of interferon-gamma, interleukin-12, and tumor necrosis factor alpha production by interferon-beta gene therapy. Arthritis Rheum. 42:90.[CrossRef][Web of Science][Medline]
  45. Antony PA, Piccirillo CA, Akpinarli A, et al. (2005) CD8+ T cell immunity against a tumor/self-antigen is augmented by CD4+ T helper cells and hindered by naturally occurring T regulatory cells. J. Immunol. 174:2591.[Abstract/Free Full Text]
  46. Rocha B and Tanchot C. (2004) Towards a cellular definition of CD8+ T-cell memory: the role of CD4+ T-cell help in CD8+ T-cell responses. Curr. Opin. Immunol. 16:259.[CrossRef][Web of Science][Medline]
  47. Smith CM, Wilson NS, Waithman J, et al. (2004) Cognate CD4(+) T cell licensing of dendritic cells in CD8(+) T cell immunity. Nat. Immunol. 5:1143.[CrossRef][Web of Science][Medline]
  48. Murphy CA, Langrish CL, Chen Y, et al. (2003) Divergent pro- and antiinflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation. J. Exp. Med. 198:1951.[Abstract/Free Full Text]
  49. Xia P, Gamble JR, Rye KA, et al. (1998) Tumor necrosis factor-alpha induces adhesion molecule expression through the sphingosine kinase pathway. Proc. Natl Acad. Sci. USA 95:14196.[Abstract/Free Full Text]
  50. Shatrov VA, Lehmann V, Chouaib S. (1997) Sphingosine-1-phosphate mobilizes intracellular calcium and activates transcription factor NF-kappa B in U937 cells. Biochem. Biophys. Res. Commun. 234:121.[CrossRef][Web of Science][Medline]
  51. Medzhitov R. (2001) Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1:135.[CrossRef][Medline]
  52. O'Neill LA. (2006) How Toll-like receptors signal: what we know and what we don't know. Curr. Opin. Immunol. 18:3.[CrossRef][Web of Science][Medline]
  53. Murata N, Sato K, Kon J, et al. (2000) Interaction of sphingosine 1-phosphate with plasma components, including lipoproteins, regulates the lipid receptor-mediated actions. Biochem. J. 352:Pt 3809.[CrossRef][Web of Science][Medline]
  54. Yatomi Y, Ohmori T, Rile G, et al. (2000) Sphingosine 1-phosphate as a major bioactive lysophospholipid that is released from platelets and interacts with endothelial cells. Blood 96:3431.[Abstract/Free Full Text]
  55. Windh RT, Lee MJ, Hla T, An S, Barr AJ, Manning DR. (1999) Differential coupling of the sphingosine 1-phosphate receptors Edg-1, Edg-3, and H218/Edg-5 to the G(i), G(q), and G(12) families of heterotrimeric G proteins. J. Biol. Chem. 274:27351.[Abstract/Free Full Text]

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


This article has been cited by other articles:


Home page
 Pharmacol. Rev.Home page
K. Takabe, S. W. Paugh, S. Milstien, and S. Spiegel
"Inside-Out" Signaling of Sphingosine-1-Phosphate: Therapeutic Targets
Pharmacol. Rev., June 1, 2008; 60(2): 181 - 195.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
K. Itagaki, J. K. Yun, J. A. Hengst, A. Yatani, C. J. Hauser, Z. Spolarics, and E. A. Deitch
Sphingosine 1-Phosphate Has Dual Functions in the Regulation of Endothelial Cell Permeability and Ca2+ Metabolism
J. Pharmacol. Exp. Ther., October 1, 2007; 323(1): 186 - 191.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
19/4/411    most recent
dxm006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (1)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Jung, I. D.
Right arrow Articles by Park, Y.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jung, I. D.
Right arrow Articles by Park, Y.-M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?