Abstract
The immunomodulator fingolimod (FTY720) induces lymphopenia by inhibiting lymphocyte egress from thymus and secondary lymphoid organs (SLOs). It is phosphorylated mainly by sphingosine kinase (SK) 2 in vivo. FTY720-phosphate (FTY-P) activated and rapidly internalized S1P1, which is the major sphingosine 1-phosphate (S1P) receptor for mediating lymphocyte egress. Although FTY-P is thought to be the active metabolite for triggering the onset of lymphopenia, nonphosphorylated FTY720 was much more potent in inhibiting cellular calcium flux and splenocyte chemotaxis via S1P1 than FTY-P after preincubation. Determination of both compounds by liquid chromatography coupled to mass spectrometry revealed efficient uptake and accumulation of FTY720 but not FTY-P by splenocytes. Coculture experiments of B and T cells with and without FTY720 pretreatment led to rapid cellular transfer and phosphorylation by mouse lymphocytes. The presence of FTY720 in lymphoid tissues of FTY720-treated SK2-deficient mice without onset of lymphopenia excluded a potential role of the nonphosphorylated compound for lymphocyte egress. Local concentrations of both phosphorylated and nonphosphorylated FTY720 were much higher in lymphoid tissues than in blood. Therefore, we conclude that cellular accumulation of FTY720 generates a reservoir in thymus and SLOs, leading to sustained FTY-P production and activation of S1P1 within tissues.
Footnotes
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This work was supported by the Emmy Noether Program and priority program 1267 of the German Research Foundation [Grants GR-1943/1-4, GR-1943/2-1].
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doi:10.1124/jpet.108.148163.
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ABBREVIATIONS: FTY720, fingolimod, 2-amino-2-(2-(4-octylphenyl)ethyl)propane-1,3-diol; SK, sphingosine kinase; SLO, secondary lymphoid organ; S1P, sphingosine 1-phosphate; FTY-P, FTY720-phosphate; 2-MEE, 2-(2-methoxyethoxy)ethanol; DMS, N,N-dimethylsphingosine; Sph, sphingosine; AAL(R/S), 2-amino-4-(4-heptyloxyphenyl)-2-methylbutanol; AFD(R/S), phosphate ester of AAL(R/S); C17-Sph, 1,3-dihydroxy-2-amino-4-heptadecene; FCS, fetal calf serum; HA, hemagglutinin; FACS, fluorescence-activated cell sorter; PBS, phosphate-buffered saline; LC/MS/MS, liquid chromatography-mass spectrometry; ESI, electrospray ionization.
- Received October 31, 2008.
- Accepted December 11, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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