Blood lymphocyte numbers, essential for the development of efficient immune responses, are maintained by recirculation through secondary lymphoid organs. We show that lymphocyte trafficking is altered by the lysophospholipid sphingosine-1-phosphate (S1P) and by a phosphoryl metabolite of the immunosuppressive agent FTY720. Both species were high-affinity agonists of at least four of the five S1P receptors. These agonists produce lymphopenia in blood and thoracic duct lymph by sequestration of lymphocytes in lymph nodes, but not spleen. S1P receptor agonists induced emptying of lymphoid sinuses by retention of lymphocytes on the abluminal side of sinus-lining endothelium and inhibition of egress into lymph. Inhibition of lymphocyte recirculation by activation of S1P receptors may result in therapeutically useful immunosuppression.

Sphingosine-1-phosphate (S1P), a lipid signaling molecule that regulates many cellular functions, is synthesized from sphingosine and ATP by the action of sphingosine kinase. Two such kinases have been identified, SPHK1 and SPHK2. To begin to investigate the physiological functions of sphingosine kinase and S1P signaling, we generated mice deficient in SPHK1. Sphk1 null mice were viable, fertile, and without any obvious abnormalities. Total SPHK activity in most Sphk1؊/؊ tissues was substantially, but not completely, reduced indicating the presence of multiple sphingosine kinases. S1P levels in most tissues from the Sphk1؊/؊ mice were not markedly decreased. In serum, however, there was a significant decrease in the S1P level. Although S1P signaling regulates lymphocyte trafficking, lymphocyte distribution was unaffected in lymphoid organs of Sphk1؊/؊ mice. The immunosuppressant FTY720 was phosphorylated and elicited lymphopenia in the Sphk1 null mice showing that SPHK1 is not required for the functional activation of this sphingosine analogue prodrug. The results with these Sphk1 null mice reveal that some key physiologic processes that require S1P receptor signaling, such as vascular development and proper lymphocyte distribution, can occur in the absence of SPHK1.Sphingosine-1-phosphate (S1P) 1 is a signaling molecule that influences cellular functions including proliferation, survival, migration, adhesion molecule expression, and morphogenesis (1-4). S1P binds to members of the S1P receptor family (also known as EDG receptors) and, via G proteins, triggers multiple signaling pathways (5, 6). S1P has also been shown to function intracellularly mediating calcium homeostasis, cell growth, and suppression of apoptosis (7,8). In mammals, vascular development and lymphocyte trafficking are dependent on S1P receptor signaling (9 -13).Sphingosine kinase (SPHK) catalyzes the synthesis of S1P via the phosphorylation of sphingosine. SPHK activity is elevated by several stimuli, including platelet-derived growth factor, vascular endothelial growth factor, tumor necrosis factor-␣, and phorbol ester, which trigger an increase in cellular S1P levels (14). Sphk genes have been identified in mammals (15-18), insects (19), plants (20), yeast (21), worm (22), and slime mold (23, 24). Mammals carry two known SphK genes, which in mice are encoded by Sphk1 and Sphk2. The two enzymes contain five highly conserved regions (C1-C5) and an ATP binding site within a conserved lipid kinase catalytic domain (15, 16). SPHK1 has a predominantly cytoplasm localization but can be induced to localize to the inner leaflet of the plasma membrane. Interestingly in endothelial cells SPHK1 is secreted and is capable of producing S1P extracellularly (25). Sphk1 shows a tissue distribution and developmental expression pattern different from Sphk2, although both enzymes are widely expressed (16,26).The importance of S1P receptor signaling in lymphocyte trafficking was first illuminated by the activities of FTY720, a potent immunosuppressive agent. FT...

Sphingosine 1-phosphate (S1P) is a bioactive lysolipid with pleiotropic functions mediated through a family of G proteincoupled receptors, S1P 1,2,3,4,5 . Physiological effects of S1P receptor agonists include regulation of cardiovascular function and immunosuppression via redistribution of lymphocytes from blood to secondary lymphoid organs. The phosphorylated metabolite of the immunosuppressant agent FTY720 (2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol) and other phosphonate analogs with differential receptor selectivity were investigated. No significant species differences in compound potency or rank order of activity on receptors cloned from human, murine, and rat sources were observed. All synthetic analogs were high-affinity agonists on S1P 1 , with IC 50 values for ligand binding between 0.3 and 14 nM. The correlation between S1P 1 receptor activation and the ED 50 for lymphocyte reduction was highly significant (p Ͻ 0.001) and lower for the other receptors. In contrast to S1P 1 -mediated effects on lymphocyte recirculation, three lines of evidence link S1P 3 receptor activity with acute toxicity and cardiovascular regulation: compound potency on S1P 3 correlated with toxicity and bradycardia; the shift in potency of phosphorylated-FTY720 for inducing lymphopenia versus bradycardia and hypertension was consistent with affinity for S1P 1 relative to S1P 3 ; and toxicity, bradycardia, and hypertension were absent in S1P 3 Ϫ/Ϫ mice. Blood pressure effects of agonists in anesthetized rats were complex, whereas hypertension was the predominant effect in conscious rats and mice. Immunolocalization of S1P 3 in rodent heart revealed abundant expression on myocytes and perivascular smooth muscle cells consistent with regulation of bradycardia and hypertension, whereas S1P 1 expression was restricted to the vascular endothelium.

The increasing incidence of life-threatening fungal infections has driven the search for new, broad-spectrum fungicidal agents that can be used for treatment and prophylaxis in immunocompromised patients. Naturalproduct inhibitors of cell wall (

Rustmicin is a 14-membered macrolide previously identified as an inhibitor of plant pathogenic fungi by a mechanism that was not defined. We discovered that rustmicin inhibits inositol phosphoceramide synthase, resulting in the accumulation of ceramide and the loss of all of the complex sphingolipids. Rustmicin has potent fungicidal activity against clinically important human pathogens that is correlated with its sphingolipid inhibition. It is especially potent against Cryptococcus neoformans, where it inhibits growth and sphingolipid synthesis at concentrations <1 ng/ml and inhibits the enzyme with an IC 50 of 70 pM. This inhibition of the membrane-bound enzyme is reversible; moreover, rustmicin is nearly equipotent against the solubilized enzyme. Rustmicin was efficacious in a mouse model for cryptococcosis, but it was less active than predicted from its in vitro potency against this pathogen. Stability and drug efflux were identified as two factors limiting rustmicin's activity. In the presence of serum, rustmicin rapidly epimerizes at the C-2 position and is converted to a ␥-lactone, a product that is devoid of activity. Rustmicin was also found to be a remarkably good substrate for the Saccharomyces cerevisiae multidrug efflux pump encoded by PDR5.