Sphingosine-1-phosphate receptor 1 (S1P(1)) was recently shown to be required for lymphocyte egress from lymphoid organs. Here we have examined the relationship between S1P(1) abundance on the cell and egress efficiency. Using an integrin neutralization approach to separate the processes of entry and exit, we show that pertussis toxin treatment reduces lymphocyte egress from lymph nodes. Retrovirally mediated S1P(1) overexpression is sufficient to reduce B cell accumulation in the splenic white pulp and to promote egress of activated T cells from lymph nodes, whereas S1P(1)(+/-) cells have reduced lymph node exit efficiency. Furthermore, lymphocyte S1P(1) is down-regulated in the blood, up-regulated in lymphoid organs, and down-regulated again in the lymph. We propose that cyclical ligand-induced modulation of S1P(1) on circulating lymphocytes contributes to establishing their lymphoid organ transit time.

Activation of the GPCR sphingosine-1-phosphate receptor 1 (S1P1) by sphingosine-1-phosphate (S1P) regulates key physiological processes. S1P1 activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S1P1 activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S1P1 activation. These mice, known as S1P1 GFP signaling mice, produce a S1P1 fusion protein containing a transcription factor linked by a protease cleavage site at the C terminus as well as a β-arrestin/protease fusion protein. Activated S1P1 recruits the β-arrestin/protease, resulting in the release of the transcription factor, which stimulates the expression of a GFP reporter gene. Under normal conditions, S1P1 was activated in endothelial cells of lymphoid tissues and in cells in the marginal zone of the spleen, while administration of an S1P1 agonist promoted S1P1 activation in endothelial cells and hepatocytes. In S1P1 GFP signaling mice, LPS-mediated systemic inflammation activated S1P1 in endothelial cells and hepatocytes via hematopoietically derived S1P. These data demonstrate that S1P1 GFP signaling mice can be used to evaluate S1P1 activation and S1P1-active compounds in vivo. Furthermore, this strategy could be potentially applied to any GPCR to identify sites of receptor activation during normal physiology and disease.

After induction in secondary lymphoid organs, a subset of antibody-secreting cells (ASCs) homes to the bone marrow (BM) and contributes to long-term antibody production. The factors determining secondary lymphoid organ residence versus BM tropism have been unclear. Here we demonstrate that in mice treated with FTY720 or that lack sphingosine-1-phosphate (S1P) receptor-1 (S1P1) in B cells, IgG ASCs are induced and localize normally in secondary lymphoid organs but they are reduced in numbers in blood and BM. Many IgG ASCs home to BM on day 3 of the secondary response and day 3 splenic ASCs exhibit S1P responsiveness, whereas the cells remaining at day 5 are unable to respond. S1P1 mRNA abundance is higher in ASCs isolated from blood compared to spleen, whereas CXCR4 expression is lower. Blood ASCs also express higher amounts of Kruppel-like factor (KLF)2, a regulator of S1P1 gene expression. These findings establish an essential role for S1P1 in IgG plasma cell homing and they suggest that differential regulation of S1P1 expression in differentiating plasma cells may determine whether they remain in secondary lymphoid organs or home to BM.

Hematologic measures such as hematocrit and white blood cell (WBC) count are heritable and clinically relevant. We analyzed erythrocyte and WBC phenotypes in 52,531 individuals (37,775 of European ancestry, 11,589 African Americans, and 3,167 Hispanic Americans) from 16 population-based cohorts with Illumina HumanExome BeadChip genotypes. We then performed replication analyses of new discoveries in 18,018 European-American women and 5,261 Han Chinese. We identified and replicated four new erythrocyte trait-locus associations (CEP89, SHROOM3, FADS2, and APOE) and six new WBC loci for neutrophil count (S1PR4), monocyte count (BTBD8, NLRP12, and IL17RA), eosinophil count (IRF1), and total WBC count (MYB). The association of a rare missense variant in S1PR4 supports the role of sphingosine-1-phosphate signaling in leukocyte trafficking and circulating neutrophil counts. Loss-of-function experiments for S1pr4 in mouse and s1pr4 in zebrafish demonstrated phenotypes consistent with the association observed in humans and altered kinetics of neutrophil recruitment and resolution in response to tissue injury.