Dendritic cells (DCs) are the cutting edge in innate and adaptive immunity. The major functions of these antigen-presenting cells are the capture, endosomal processing and presentation of antigens, providing them an exclusive ability to provoke adaptive immune responses and to induce and control tolerance. Immature DCs capture and process antigens, migrate towards secondary lymphoid organs where they present antigens to naive T cells in a well-synchronized sequence of procedures referred to as maturation. Indeed, recent research indicated that sphingolipids are modulators of essential steps in DC homeostasis. It has been recognized that sphingolipids not only modulate the development of DC subtypes from precursor cells but also influence functional activities of DCs such as antigen capture, and cytokine profiling. Thus, it is not astonishing that sphingolipids and sphingolipid metabolism play a substantial role in inflammatory diseases that are modulated by DCs. Here we highlight the function of sphingosine 1-phosphate (S1P) on DC homeostasis and the role of S1P and S1P metabolism in inflammatory diseases.

Conventional cytostatic cancer treatments rarely result in the complete eradication of tumor cells. Therefore, new therapeutic strategies focus on antagonizing the immunosuppressive activity of established tumors. In particular, recent studies of antigen-loaded dendritic cells (DCs) eliciting a specific antitumor immune response has raised the hopes of achieving the complete elimination of tumor tissue. Genistein, fingolimod and betulin have already been described as active compounds in different types of cancer. Herein, we applied an integrated screening approach to characterize both their cytostatic and their immune-modulating properties side-by-side. As will be described in detail, our data confirmed that all three compounds exerted proapoptotic and antiproliferative activity in different B16 melanoma cell lines to a given extent, as revealed by an MTT assay, CFSE and DAPI staining. However, while genistein and fingolimod also affected the survival of primary bone marrow (BM) derived DCs of C57BL/6 mice, betulin exhibited a lower cytotoxicity for BMDCs in comparison to the melanoma cells. Moreover, we could show for the first time, that only betulin caused a simultaneous, highly specific immune-stimulating activity, as measured by the IL-12p70 release of Toll-like receptor 4-stimulated BMDCs by ELISA, which was due to increased IL-12p35 mRNA expression. Interestingly, the activation of DCs resulted in enhanced T lymphocyte stimulation, indicated by increased IL-2 and IFN-γ production of cytotoxic T cells in spleen cell co-culture assays which led to a decreased viability of B16 cells in an antigen specific model system. This may overcome the immunosuppressive environment of a tumor and destroy tumor cells more effectively in vivo if the immune response is specific targeted against the tumor tissue by antigen-loaded dendritic cells. In summary, cytostatic agents, such as betulin, that simultaneously exhibit immune stimulatory activity may serve as lead compounds and hold great promise as a novel approach for an integrated cancer therapy.

FTY720 (Fingolimod; Gilenya ® ) is an immunemodulatory prodrug which, after intracellular phosphorylation by sphingosine kinase 2 (SphK2) and export, mimics effects of the endogenous lipid mediator sphingosine-1-phosphate. Fingolimod has been introduced to treat relapsing-remitting multiple sclerosis. However, little has been published about the immune cell membrane penetration and subcellular distribution of FTY720 and FTY720-P. Thus, we applied a newly established LC-MS/MS method to analyze the subcellular distribution of FTY720 and FTY720-P in subcellular compartments of spleen cells of wild type, SphK1-and SphK2-deficient mice. These studies demonstrated that, when normalized to the original cell volume and calculated on molar basis, FTY720 and FTY720-P dramatically accumulated several hundredfold within immune cells reaching micromolar concentrations. The amount and distribution of FTY720 was differentially affected by SphK1-and SphK2-deficiency. On the background of recently described relevant intracellular FTY720 effects in the nanomolar range and the prolonged application in multiple sclerosis, this data showing a substantial intracellular accumulation of FTY720, has to be considered for benefit/risk ratio estimates.

Sphingosine-1-phosphate (S1P) is an immune modulator produced by sphingosine kinase (SphK)1 and SphK2 and dephosphorylated by two S1P phosphatases or irreversibly degraded by a lyase. We recently showed that Toll-like receptor (TLR)4-induced IL-12p70 is selectively counter regulated by SphK1 and extracellular S1P/S1PR1. Others demonstrated that specific SphK1-dependent binding of intracellular S1P to TRAF2 enhances TNF signaling. In an ongoing investigation to determine the influence of extra- and intracellular S1P on dendritic cell signaling our experiments revealed that activation of TLR4 by lipopolysaccharide dose and time dependently decreased S1P lyase mRNA expression of murine bone marrow-derived dendritic cells by up to 70%. This set of realtime PCR data was further confirmed by semi-quantitative RT-PCR using exon-specific primers for murine sgpl1. Systematic analysis with dose-optimized ligands of TLRs 1/2, 5, 2/6, 7/8 and TLR9 showed a differential pattern of S1P lyase down regulation with concomitant increase of intracellular S1P. Although S1P lyase defcient mice are immune compromised, we found that TLR-induced transient S1P lyase downregulation resulted in a TLR adaptor-dependent upregulation of IL-12p70, IL-23 and IL-6 expression on mRNA and protein level. Further experiments including cell compartment-specific quantification of S1P, hexadecenal and, possibly counter regulatory, sphingolipid enzymes are necessary to understand the role of sphingolipids in DC.

No abstract