The immunomodulator FTY720 is phosphorylated and released from platelets
Introduction
The sphingolipid metabolite sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that regulates several cellular processes (proliferation, regulation of cell migration, actin cytoskeletal rearrangement, and adherens junction assembly) through binding to its cell surface receptors (Kihara et al., 2007, Sanchez and Hla, 2004). To date, five mammalian S1P receptors (S1P1/Edg1, S1P2/Edg5, S1P3/Edg3, S1P4/Edg6, S1P5/Edg8) have been identified.
S1P is abundant in plasma and is physiologically important, especially in the vascular and immune systems and through its binding to S1P1. The importance of S1P in the immune system has been demonstrated using a novel immunomodulating agent, FTY720 (fingolimod; 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol). FTY720, an effective agent against transplantation rejection and autoimmune diseases, does not impair lymphocyte activation, proliferation, or effector function, but rather reduces circulating lymphocyte numbers (especially those of mature T cells) (Budde et al., 2006). Treatment causes accumulation of lymphocytes in secondary lymphoid organs such as lymph nodes and Peyer's patches (Chiba et al., 1998). FTY720 is phosphorylated in vivo by sphingosine kinase and binds to all S1P receptors except S1P2 (Brinkmann et al., 2002, Mandala et al., 2002).
S1P1 is highly expressed in mature T cells and endothelial cells, and is the most important S1P receptor in the immune system. S1P and the S1P1 receptor have pivotal functions in the egress of lymphocytes from secondary lymphoid organs and thymus (Allende et al., 2004, Matloubian et al., 2004). Two models have been proposed for the molecular mechanism of FTY720-induced peripheral lymphopenia (Rosen and Goetzl, 2005). In the first, phosphorylated FTY720 (FTY720-P) causes prolonged internalization of S1P1 on lymphocytes. Since S1P is a potent chemoattractant for lymphocytes, loss of cell surface S1P1 results in reduced responsiveness to the S1P gradient between lymphoid organs and plasma. In the second model, FTY720-P binds to S1P1 on endothelial cells of the lymphoid organs and stimulates adherens junction assembly. As a result, lymphocytes become unable to pass through the cell junctions, and accumulate in the lymphoid organs.
Two major blood cells, platelets and erythrocytes, provide plasma S1P (Hänel et al., 2007, Ito et al., 2007, Yatomi et al., 1997a, Yatomi et al., 1997b). Most, but not all, of the S1P release from platelets is dependent on stimuli such as thrombin and protein kinase C activation (Yatomi et al., 2000, Yatomi et al., 1997b), whereas erythrocytes release S1P constitutively (Ito et al., 2007, Yang et al., 1999). In contrast, it has been unclear where FTY720-P is produced. Therefore, we tested the involvement of plasma and related cells (endothelial cells, platelets, and erythrocytes) in the production of FTY720-P. We found that only platelets can produce, store, and release FTY720-P into plasma. This release was largely independent of stimuli, in contrast to S1P release.
Section snippets
Preparation of erythrocytes, platelets, and plasma
Wistar ST rats (7–14 weeks old, male) were anesthetized with diethyl ether and pentobarbital (50 mg/kg rat weight), and whole blood was collected from their hearts. This study was carried out in accordance with the Declaration of Helsinki and/or with the guide for the Committee on the Care and Use of Laboratory Animals of Hokkaido University.
Erythrocytes, platelets, and plasma were prepared as described elsewhere (Ito et al., 2007, Yang et al., 1999). Blood (∼ 10 ml) was collected in 2 ml
Platelets produce and release FTY720-P
We first investigated the ability of two major blood cells, platelets and erythrocytes, to produce and release FTY720-P. After platelets and erythrocytes were incubated with FTY720, cells and medium were separated by centrifugation. FTY720 and FTY720-P from each fraction were measured by HPLC. Almost no FTY720-P was detected in the erythrocyte fraction or its incubation medium (Fig. 1A). In contrast, FTY720-P was detected both in the platelet fraction and the incubation medium. About 60% of the
Discussion
FTY720 is a prodrug, and its conversion to FTY720-P is necessary for its immunomodulating effects. Conversion occurs not only at the time of administration but also constitutively during its circulation, since FTY720 exhibits a cycle of phosphorylation/dephosphorylation (Brinkmann et al., 2002, Kharel et al., 2005). However, it has been unclear where FTY720-P is produced. We have demonstrated here that platelets have the ability to produce FTY720-P and supply it to the plasma.
FTY720-P is
Acknowledgements
We thank Dr. F. Nara (Sankyo Co., Tokyo, Japan) for providing FTY720 and Dr. E.A. Sweeney for scientific editing of the manuscript. A.K. is supported by a Grant-in-Aid for Young Scientists (A) (17687011) from the Ministry of Education, Culture, Sports, Sciences and Technology of Japan.
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