The immunosuppressant drug FTY720 inhibits cytosolic phospholipase A2 independently of sphingosine-1-phosphate receptors

Abstract

FTY720 is a potent immunomodulator drug that inhibits the egress of lymphocytes from secondary lymphoid tissues and thymus. FTY720 is phosphorylated in vivo by sphingosine kinase 2 to FTY720-phosphate, which acts as a potent sphingosine-1-phosphate (S1P) receptor agonist. However, in contrast to S1P, FTY720 has no effect on mast-cell degranulation, yet significantly reduces antigen-induced secretion of PGD 2 and cysteinyl-leukotriene. Unexpectedly, this effect of FTY720 was independent of its phosphorylation and S1P receptor functions. The rate-limiting step in the biosynthesis of all eicosanoids is the phospholipase A 2 (PLA 2 )-mediated release of arachidonic acid from glycerol phospholipids. Although FTY720 also reduced arachidonic acid release in response to antigen, it had no effect on translocation of cPLA 2 or ERK1/2 activation, suggesting that it does not interfere with Fc⑀RI-mediated events leading to cPLA 2 activation. Remarkably, however, FTY720 drastically inhibited recombinant cPLA 2 ␣ activity, whereas FTY720-phosphate, sphingosine, or S1P had no effect. This study has uncovered a unique action of FTY720 as an inhibitor of cPLA 2 ␣ and hence on production of all eicosanoids. Our results have important implications for the potential therapeutic mechanism of action of FTY720 in eicosanoid-driven inflammatory disorders such as asthma and multiple sclerosis. IntroductionFTY720 is a novel immunosuppressive agent that was derived from myriocin, a sphingosine-like fungal metabolite. 1 FTY720 potently inhibits a variety of experimental autoimmune disorders, such as type I diabetes 2 and arthritis, 3 but clinically, its greatest potential for humans appears to be in the prevention of renal graft rejection and treatment of multiple sclerosis (MS) where it is currently in phase 3 clinical trials. 4,5 The immunosuppressive properties of FTY720 stem from its ability to prevent T cells' egress from secondary lymphoid organs and back into circulation, sequestering them away from the transplanted graft, [6][7][8] or preventing their entry into the central nervous system. 9 FTY720 is phosphorylated by sphingosine kinase 2 (SphK2), [10][11][12] and a large body of evidence suggests that the phosphorylated drug (FTY720-P), an analog of sphingosine-1-phosphate (S1P), is the biologically active form. FTY720-P can bind to all of the known S1P receptors, except S1P 2 , 6,13,14 and has been shown to regulate S1P 1 actions that are crucial for lymphocyte migration and trafficking. [15][16][17][18] Nevertheless, the exact mechanism of action of this prodrug is still a matter of debate. [19][20][21] S1P is also an important chemoattractant in the immune system, modulating T-cell responses to chemokines (reviewed in Cyster 13 and Rosen and Goetzl 18 ).FTY720 inhibits airway inflammation, induction of bronchial hyperresponsiveness, and lymphocyte and eosinophil infiltration in an actively Ag-sensitized murine asthma model, 22 suggesting that it might have therapeutic benefits in asthma.Given that S1P and its receptors may pla...

Subcellular distribution of FTY720 and FTY720-phosphate in immune cells – another aspect of Fingolimod action relevant for therapeutic application

“…It is however very possible that the significant intracellular accumulation of this substance described by our data, may explain part of the surprising therapeutic efficacy of even low dosage levels in MS as compared to its formerly applied dosage in transplantation (Kappos et al, 2010). Therefore, by interpreting our findings in a positive direction, Fingolimod may affect immune cell-relevant intracellular targets, such as phospholipase A2 (PLA2) and protein phosphatase 2A (PP2A) (Payne et al, 2007;Saddoughi et al, 2013;Arlt et al, 2014). These results have to be further confirmed by an in vivo model, e.g.…”

FTY720 Ameliorates Th1-Mediated Colitis in Mice by Directly Affecting the Functional Activity of CD4+CD25+ Regulatory T Cells

“…Whereas several studies support its effects on lymphocyte trafficking, more recent investigations indicate that the FTY720 action distinct from S1P might not only be due to its interaction with other S1PR subtypes but may be based on direct intracellular effects on e.g., the S1P lyase or the cytosolic PlA 2 (33,43). For S1P it has recently been documented that the S1P-S1P 1 axis mediates many of the T cell-associated activities.…”

“…Furthermore, the definition of T lymphocytes as the prime and major target of FTY720/S1P has been challenged by several publications showing significant effects on other immune competent cells comprising monocytes and dendritic cells (DC) (26 -28, 31-33). These investigations raise the possibility that not only migratory but rather differentiating effects are involved early during an immune response, which include e.g., a direct intracellular blockade of cPLA2 that is independent of FTY720 phosphorylation and significantly contributes to its therapeutic activity in experimental autoimmune encephalomyelitis (33).…”

Distinct roles of sphingosine kinases 1 and 2 in human mast-cell functions

“…4 In agreement, FTY720-phosphate, an agonist of all S1P receptors except S1P 2 , did not induce degranulation. 19 Moreover, quantitative real-time PCR (QPCR) revealed that like rodent MCs, LAD2 cells as well as cord blood-derived human MCs and human skin MCs express similar levels of S1P 1 and S1P 2 , but do not express S1P 3-5 receptors (data not shown). S1P at 1 M was nearly as effective as Ag or ionomycin at stimulating secretion of TNF-␣ and IL-6 ( Figure 1B,C For personal use only.…”