Hoffmann Franziska S, Hofereiter Johann, Rübsamen Heike, Melms Johannes, Schwarz Sigrid, Faber Hans, Weber Peter, Pütz Benno, Loleit Verena, Weber Frank, Hohlfeld Reinhard, Meinl Edgar, Krumbholz Markus
Institute of Clinical Neuroimmunology, Ludwig Maximilian University, 81377, Munich, Germany.
German Center for Neurodegenerative Diseases (DZNE) and Technical University, 81377, Munich, Germany.
J Neuroinflammation. 2015 Sep 30;12:184. doi: 10.1186/s12974-015-0393-6.
Fingolimod (FTY720) is the first sphingosine-1-phosphate (S1P) receptor modulator approved for the treatment of multiple sclerosis. The phosphorylated active metabolite FTY720-phosphate (FTY-P) interferes with lymphocyte trafficking. In addition, it accumulates in the CNS and reduces brain atrophy in multiple sclerosis (MS), and neuroprotective effects are hypothesized.
Human primary astrocytes as well as human astrocytoma cells were stimulated with FTY-P or S1P. We analyzed gene expression by a genome-wide microarray and validated induced candidate genes by quantitative PCR (qPCR) and ELISA. To identify the S1P-receptor subtypes involved, we applied a membrane-impermeable S1P analog (dihydro-S1P), receptor subtype specific agonists and antagonists, as well as RNAi silencing.
FTY-P induced leukemia inhibitory factor (LIF), interleukin 11 (IL11), and heparin-binding EGF-like growth factor (HBEGF) mRNA, as well as secretion of LIF and IL11 protein. In order to mimic an inflammatory milieu as observed in active MS lesions, we combined FTY-P application with tumor necrosis factor (TNF). In the presence of this key inflammatory cytokine, FTY-P synergistically induced LIF, HBEGF, and IL11 mRNA, as well as secretion of LIF and IL11 protein. TNF itself induced inflammatory, B-cell promoting, and antiviral factors (CXCL10, BAFF, MX1, and OAS2). Their induction was blocked by FTY-P. After continuous exposure of cells to FTY-P or S1P for up to 7 days, the extent of induction of neurotrophic factors and the suppression of TNF-induced inflammatory genes declined but was still detectable. The induction of neurotrophic factors was mediated via surface S1P receptors 1 (S1PR1) and 3 (S1PR3).
We identified effects of FTY-P on astrocytes, namely induction of neurotrophic mediators (LIF, HBEGF, and IL11) and inhibition of TNF-induced inflammatory genes (CXCL10, BAFF, MX1, and OAS2). This supports the view that a part of the effects of fingolimod may be mediated via astrocytes.
芬戈莫德（FTY720）是首个被批准用于治疗多发性硬化症的鞘氨醇-1-磷酸（S1P）受体调节剂。磷酸化的活性代谢物FTY720-磷酸（FTY-P）会干扰淋巴细胞运输。此外，它在中枢神经系统中蓄积并减少多发性硬化症（MS）患者的脑萎缩，因此推测其具有神经保护作用。
用FTY-P或S1P刺激原代人星形胶质细胞以及人星形细胞瘤细胞。我们通过全基因组微阵列分析基因表达，并通过定量PCR（qPCR）和酶联免疫吸附测定（ELISA）验证诱导的候选基因。为了确定所涉及的S1P受体亚型，我们应用了一种膜不可渗透的S1P类似物（二氢-S1P）、受体亚型特异性激动剂和拮抗剂以及RNA干扰沉默技术。
FTY-P诱导白血病抑制因子（LIF）、白细胞介素11（IL11）和肝素结合表皮生长因子样生长因子（HBEGF）的mRNA表达，以及LIF和IL11蛋白的分泌。为了模拟在活动性MS病变中观察到 的炎性环境，我们将FTY-P的应用与肿瘤坏死因子（TNF）相结合。在这种关键炎性细胞因子存在的情况下，FTY-P协同诱导LIF、HBEGF和IL11的mRNA表达，以及LIF和IL11蛋白的分泌。TNF本身诱导炎性、促进B细胞和抗病毒因子（CXCL10、BAFF、MX1和OAS2）。它们的诱导被FTY-P阻断。细胞连续暴露于FTY-P或S1P长达7天后，神经营养因子的诱导程度和对TNF诱导的炎性基因的抑制作用有所下降，但仍可检测到。神经营养因子的诱导是通过表面S1P受体1（S1PR1）和3（S1PR3）介导的。
我们确定了FTY-P对星形胶质细胞的作用，即诱导神经营养介质（LIF、HBEGF和IL11）以及抑制TNF诱导的炎性基因（CXCL10、BAFF、MX1和OAS2）。这支持了芬戈莫德的部分作用可能通过星形胶质细胞介导的观点。
