The
sphingosine 1-phosphate receptor type 1 (S1P(1)) is important for the maintenance of lymphocyte circulation. S1P(1) receptor surface expression on lymphocytes is critical for their egress from thymus and lymph nodes. Premature activation-induced internalization of the S1P(1) receptor in lymphoid organs, mediated either by pharmacological agonists or by inhibition of the S1P degrading
enzyme S1P-lyase, blocks lymphocyte egress and induces
lymphopenia in blood and lymph. Regulation of S1P(1) receptor surface expression is therefore a promising way to control adaptive immunity. Hence, we analyzed potential cellular targets for their ability to alter S1P(1) receptor surface expression without stimulation. The initial observation that preincubation of mouse splenocytes with its natural analog
sphingosine was sufficient to block Transwell chemotaxis to S1P directed subsequent investigations to the underlying mechanism.
Sphingosine is known to inhibit
protein kinase C (PKC), and PKC inhibition with nanomolar concentrations of
staurosporine,
calphostin C, and
GF109203X down-regulated surface expression of S1P(1) but not S1P(4) in transfected rat
hepatoma HTC(4) cells. The PKC activator
phorbol 12-myristate 13-acetate partially rescued FTY720-induced down-regulation of the S1P(1) receptor, linking PKC activation with S1P(1) receptor surface expression.
FTY720, but not
FTY720 phosphate, efficiently inhibited PKC. Cell-based efficacy was obvious with 10 nm
FTY720, and in vivo treatment of mice with 0.3-3 mg/kg/day
FTY720 showed increasing concentration-dependent effectiveness. PKC inhibition therefore may contribute to
lymphopenia by down-regulating S1P(1)
receptor cell surface expression independently from its activation.