Immunosuppressant drugs such as cyclosporin have allowed widespread organ transplantation, but their utility remains limited by toxicities, and they are ineffective in chronic management of autoimmune diseases such as multiple sclerosis. In contrast, the immune modulating drug FTY720 is efficacious in a variety of transplant and autoimmune models without inducing a generalized immunosuppressed state and is effective in human kidney transplantation. FTY720 elicits a lymphopenia resulting from a reversible redistribution of lymphocytes from circulation to secondary lymphoid tissues by unknown mechanisms. Using FTY720 and several analogs, we show now that FTY720 is phosphorylated by sphingosine kinase; the phosphorylated compound is a potent agonist at four sphingosine 1-phosphate receptors and represents the therapeutic principle in a rodent model of multiple sclerosis. Our results suggest that FTY720, after phosphorylation, acts through sphingosine 1-phosphate signaling pathways to modulate chemotactic responses and lymphocyte trafficking.FTY720 is derived from ISP-1 (myriocin), a fungal metabolite that is an eternal youth nostrum in traditional Chinese herbal medicine (1). The compound (2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol) is a novel, high potency immune modulating agent that is remarkably effective in a variety of autoimmune and transplant models including islet transplantation (2) and has recently proven to be effective in renal transplantation in man (3). Unlike the currently used immunosuppressive agents (e.g. the calcineurin inhibitors cyclosporin and tacrolimus), FTY720 does not inhibit T cell activation and proliferation and in rodent models does not impair immunity to systemic viral infection (4). If confirmed in man, the latter property provides a striking advantage over current immunosuppressive therapies. FTY720 apparently sequesters lymphocytes from circulation to secondary lymph tissue compartments (5) with concomitant reduction of specific effector T cells recirculating from the lymph nodes to inflamed peripheral tissues (4) and graft sites (6). FTY720 does not act via the lymphocytehoming chemokine receptor CCR-7 because FTY720 is active both in CCR-7-deficient mice and plt (paucity of lymph node T cells) mice, which lack CCR-7 ligands (CCL-19 and CCL-21) (7).FTY720-induced lymphocyte homing is sensitive to suppression by pertussis toxin (6 -8), which suggests that the molecular target of the drug is a G protein-coupled receptor (GPCR) 1 interacting with heterotrimeric G proteins of the ␣ i/o type. The affected GPCR(s) is on the lymphocyte since fluorescently labeled lymphocytes treated with pertussis toxin ex vivo and transferred to mice are not depleted by FTY720 in vivo (8). The structural similarity of FTY720 and sphingosine has prompted speculation that the drug might act via the sphingosine 1-phosphate (S1P) receptor S1P 4 (formerly 2 that is known to be expressed by lymphocytes (9). S1P is a pleiotropic lysophospholipid mediator; the prominent cellular responses to applied S...

Sphingosine 1-phosphate (S1P) is a lysophospholipid mediator that evokes a variety of cell and tissue responses via a set of cell surface receptors. The recent development of S1P receptor agonists, led by the immunomodulatory pro-drug FTY720, has revealed that S1P signaling is an important regulator of lymphocyte trafficking. With the twin goals of understanding structureactivity relationships of S1P ligands and developing tool compounds to explore S1P biology, we synthesized and tested numerous S1P analogs. We report herein that a subset of our aryl amide-containing compounds are antagonists at the S1P 1 and S1P 3 receptors. The lead compound in the series, VPC23019, was found in broken cell and whole cell assays to behave as a competitive antagonist at the S1P 1 and S1P 3 receptors. The structureactivity relationship of this series is steep; for example, a slight modification of the lead compound resulted in VPC25239, which was one log order more potent at the S1P 3 receptor. These new chemical entities will enable further understanding of S1P signaling and provide leads for further S1P receptor antagonist development. Sphingosine 1-phosphate (S1P)1 is a lysophospholipid mediator that evokes a variety of cellular responses by stimulation of five members of the endothelial cell differentiation gene receptor family. The endothelial cell differentiation gene receptors are G-protein coupled receptors that, upon stimulation, propagate second messenger signals via activation of heterotrimeric G-protein ␣ subunits and ␤-␥ dimers. Ultimately, this S1P-driven signaling results in cell survival, increased cell migration, and, often, mitogenesis. The recent development of agonists targeting S1P receptors has provided insight regarding the role of this signaling system in physiologic homeostasis. For example, the immunomodulator FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol), which is a pan S1P receptor agonist following phosphorylation, revealed that S1P tone influences lymphocyte trafficking (1-4). The utility of an S1P receptor agonist was unexpected; indeed, prior speculation focused on the potential (as yet unrealized) for S1P antagonists as anti-angiogenic agents.Recent findings also suggest a physiological influence for S1P in the vasculature.Although not yet explored in detail, it has been hypothesized that S1P may exert anti-inflammatory actions on endothelial cells through its release from high density lipoprotein (5). A recent study found that S1P inhibited tumor necrosis factor ␣-mediated monocyte-endothelial cell adhesion.2 Furthermore, an S1P 1 receptor antagonist described herein blocked the anti-inflammatory action of S1P, thereby providing evidence that this effect maps to the S1P 1 receptor. If verified, these results would expand the role of the S1P 1 receptor to include influencing monocyte extravasation and would further highlight how the development of S1P receptor-specific compounds is expanding our understanding of the biology of this important signaling system.To further characterize ...

Background The relevance of allergic sensitization, judged by titers of serum IgE antibodies, to the risk of an asthma exacerbation caused by rhinovirus is unclear. Objective To examine the prevalence of rhinovirus infections in relation to the atopic status of children treated for wheezing in Costa Rica, a country with an increased asthma burden. Methods The children enrolled (n=287) were 7 through 12 years old. They included 96 with acute wheezing, 65 with stable asthma, and 126 non-asthmatic controls. PCR methods, including gene sequencing to identify rhinovirus strains, were used to identify viral pathogens in nasal washes. Results were examined in relation to wheezing, total IgE, allergen-specific IgE antibody, and levels of expired nitric oxide (FENO). Results Sixty-four percent of wheezing children compared to 13% of children with stable asthma and 17% of the non-asthmatic controls tested positive for rhinovirus (p<0.001 for both comparisons). Among wheezing subjects, 75% of the rhinoviruses detected were Group C strains. High titers of IgE antibodies to dust mite allergen (especially Dermatophagoides sp) were common and correlated significantly with levels of total IgE and FENO. The greatest risk for wheezing was observed among children with titers of IgE antibodies to dust mite ≥17.5 IU/ml who tested positive for rhinovirus (odds ratio for wheezing: 31.5; 95% CI 8.3–108, p<0.001). Conclusions High titers of IgE antibody to dust mite allergen were common and significantly increased the risk for acute wheezing provoked by rhinovirus among asthmatic children.

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Sphingosine-1 Phosphate (S1P) helps mediate lymphocyte egress from lymph nodes, yet significant mechanistic questions remain. Here we show that B lymphocyte egress sites exist close to lymph node follicles. Recent B cell emigrants localize towards follicle centers, while longer-term residents tend towards cortical sinusoids. Exiting B lymphocytes squeeze through apparent portals in the lymphatic endothelium. Treatment with the S1P receptor agonist FTY720 empties the cortical sinusoids of lymphocytes, blocks lymphatic endothelial penetration, and displaces B lymphocytes into the T cell zone. S1P3−/− B cells, which lack chemoattractant responses to S1P, transit lymph nodes normally, while Gnai2−/− B cells, which have impaired responses to chemokines and S1P, transit more rapidly than do wild type cells. This study identifies a major site of B lymphocyte lymph node egress, shows that FTY720 treatment blocks passage through the cortical lymphatic endothelium, and argues against a functional role for S1P chemotaxis in B lymphocyte egress.