Effekte von Sphingosin-1-Phosphat und Analoga auf die Entzündungsreaktion und epidermale Hyperproliferation in Modellen der Psoriasis

Sphingosine-1-phosphate (S1P) is a biological mediator, which has been implicated in many physiological cell functions. In the present study the influence of S1P, S1P receptor agonists (FTY720, FTY720P, SEW2871) and antagonists (W146, JTE-013) on the cytokine profile of murine dendritic cells and murine keratinocytes was investigated. Furthermore, the in vivo efficacy of locally administered S1P and FTY720 was tested in the imiqumiod-induced psoriasis model, as well as in the mouse-tail-assay, since the strategy of targeting the migratory response of Langerhans cells with locally acting S1P, which also inhibits keratinocyte proliferation, represents a new option for the treatment of psoriasis vulgaris. The in vitro experiments were carried out with bone marrow derived dendritic cells from BALB/c mice and murine keratinocytes (MSC-P5). Initially, the presence of the S1P-receptor 1, 2 and 3 was examined by means of PCR. As expected, dendritic cells expressed the mRNA of S1P-receptors 1, 2 and 3 while only the S1P-receptors 1 and 2 were detected in murine keratinocytes. In cell culture experiments dendritic cells were incubated with S1P, FTY720 and FTY720P and subsequently stimulated with toll like receptor agonist LPS. In the supernatants the concentration of IL-12, -23 and -27 were determined by means of ELISA. Pre-treatment of DC with S1P markedly reduced the LPS-mediated secretion of IL-12 and IL-23 in a dose dependent manner, whereas it partially enhanced the secretion of IL-27. For FTY720 and FTY720P there were only slight effects on cytokine production. To evaluate which receptor subtype is responsible for this pattern of cytokine modulation, experiments using the S1PR1-agonist SEW2871, the S1PR1-antagonist W146, as well as the S1PR2-antagonist JTE-013 were performed. SEW2871 reduced the cytokine secretion to the same extend as S1P. Pre-treatment of DC with the S1PR1-antagonist W146 blocked this inhibition, while treatment with the S1PR2-antagonist JTE-013 had no effect. So, it is likely that S1PR1 is the main receptor mediating the modulation of cytokine production. It was further tested, if S1P had any influence on the cross talk between dendritic cells and keratinocytes, since these two cell types are crucial for the initiation and maintenance of psoriasis. Again, in this setting S1P reduced cytokine production in DC, which were activated by the stimulated keratinocytes. In contrast the preincubation of keratinocytes with S1P showed no effect. In a combined in-vivo/ex-vivo study the impact of S1P on the antigen uptake by dendritic cells was investigated, because antigen uptake is a key event in immunological processes and has recently been discussed also as an important parameter in the pathogenesis of psoriasis. This experiment showed a markedly reduced uptake of FITC-dextran by Langerhans cells in the epidermal layer of mice ears, which were pre-treated with S1P. The in vivo influence of S1P and FTY720 was tested in the imiquimod (IMQ)-induced psoriasis model. The psoriasis area and severity index (PASI), the mouse-earswelling-test, the local lymph node reaction (weight and total cell number), the IL-17 and IL-23 production in ear homogenate, as well as histological analysis of the epidermal back and ear skin were evaluated as parameters. Application of IMQ creme to mouse back skin and ear skin induced inflammation, characterized by erythema, scaling and lichenification of the back skin, as well as a marked swelling of the ear. While both, S1P and FTY720 had no impact on the phenotypical and histological changes at the back skin of mice, S1P significantly reduced the ear swelling. Furthermore, S1P and FTY720 decreased weight and total cell number of the draining auricular lymph node. The epidermal hyperproliferation of the ear was also attenuated by topically administered S1P. Whereas the IL-23 secretion in the ear was too low for quantification, FTY720 elicited a strong decrease in IL-17 production. S1P showed no effect on this parameter. The second in-vivo study, the mouse tail assay, offers the opportunity to examine the effect of test agents on the differentiation of keratinocytes. In contrast the positive control calcipotriol, S1P as well as FTY720 exhibited no effect. In summary, this study implicates, that S1P has an anti-inflammatory impact on various immunological reactions. S1P modulates the cytokine profile of dendritic cells, with a reduced TH1 dominated response. In addition to the inhibition of antigen uptake and the general anti-inflammatory effect in vivo S1P also reduces epidermal proliferation. This suggests that the topical use of S1P might represent a novel option for the treatment of psoriasis vulgaris.