The bioactive lysophospholipid mediator sphingosine-1-phosphate (S1P) promotes the egress of newly formed T cells from the thymus and the release of immature B cells from the bone marrow. It has remained unclear, however, where and how S1P is released. Here, we show that in mice, the S1P transporter spinster homolog 2 (Spns2) is responsible for the egress of mature T cells and immature B cells from the thymus and bone marrow, respectively. Global Spns2-KO mice exhibited marked accumulation of mature T cells in thymi and decreased numbers of peripheral T cells in blood and secondary lymphoid organs. Mature recirculating B cells were reduced in frequency in the bone marrow as well as in blood and secondary lymphoid organs. Bone marrow reconstitution studies revealed that Spns2 was not involved in S1P release from blood cells and suggested a role for Spns2 in other cells. Consistent with these data, endothelia-specific deletion of Spns2 resulted in defects of lymphocyte egress similar to those observed in the global Spns2-KO mice. These data suggest that Spns2 functions in ECs to establish the S1P gradient required for T and B cells to egress from their respective primary lymphoid organs. Furthermore, Spns2 could be a therapeutic target for a broad array of inflammatory and autoimmune diseases.

The migration and positioning of osteoclast precursor monocytes are controlled by the blood-enriched lipid mediator sphingosine-1-phosphate (S1P) and have recently been shown to be critical points of control in osteoclastogenesis and bone homeostasis. Here, we show that calcitriol, which is the hormonally active form of vitamin D, and its therapeutically used analog, eldecalcitol, inhibit bone resorption by modulating this mechanism. Vitamin D analogs have been used clinically for treating osteoporosis, although the mode of its pharmacologic action remains to be fully elucidated. In this study, we found that active vitamin D reduced the expression of S1PR2, a chemorepulsive receptor for blood S1P, on circulating osteoclast precursor monocytes both in vitro and in vivo. Calcitriol-or eldecalcitol-treated monocytoid RAW264.7 cells, which display osteoclast precursor-like properties, migrated readily to S1P. Concordantly, the mobility of circulating CX 3 CR1 + osteoclast precursor monocytes was significantly increased on systemic administration of active vitamin D. These results show a mechanism for active vitamin D in controlling the migratory behavior of circulating osteoclast precursors, and this action should be conducive to limiting osteoclastic bone resorption in vivo.B one is a highly dynamic organ, and it is continuously remodeled cooperatively by bone-resorbing osteoclasts and bone-replenishing osteoblasts (1). Osteoclasts, which have bone-resorbing capacity, are a unique cell type differentiated from monocyte/ macrophage lineage hematopoietic precursor cells termed osteoclast precursors. Previous studies have identified key molecular signals, such as mediated by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), that regulate osteoclastic differentiation and function (2, 3). Unlike osteoblasts, which are of mesenchymal origin and essentially reside in bone tissues, osteoclasts and their precursor monocytes are highly dynamic. Their migratory mechanisms in systemic circulation and homing into bone spaces have recently emerged as critical points of control for osteoclastogenesis and thus, bone homeostasis. We have recently used intravital two-photon microscopy to visualize the bone tissues of live mice and found that sphingosine-1-phosphate (S1P), a lysophospholipid mediator enriched in blood, plays a vital role in regulating the migration and positioning of osteoclast precursors on the bone surface (4, 5).Osteoclast precursor monocytes express S1PR1 (formerly designated as S1P 1 or Edg-1), a cognate receptor for S1P, and can use this receptor to migrate from bone tissues to blood that contains S1P. The deletion of S1PR1 in monocytoid cells leads to an accumulation of osteoclast precursors and a resultant increase in bone resorption, which suggests that the S1P-S1PR1 interaction is essential for the recirculation of osteoclast precursors from bone to blood (4). The expression of S1PR1 was suppressed on stimulation with RANKL, representing a reasonable mechanism wher...

Monocytes give rise to macrophages and dendritic cells (DCs) under steady-state and inflammatory conditions, thereby contributing to host defense and tissue pathology. A common monocyte progenitor (cMoP) that is strictly committed to the monocyte lineage has been recently identified in mice. Here, we identified human cMoPs as a CLEC12ACD64 subpopulation of conventional granulocyte-monocyte progenitors (cGMPs) in umbilical cord blood and in bone marrow. Human cMoPs gave rise to monocyte subsets without showing any potential for differentiating into myeloid or lymphoid cells. Within the cGMP population, we also identified revised GMPs that completely lacked DC and lymphoid potential. Collectively, our findings expand and revise the current understanding of human myeloid cell differentiation pathways.

On the basis of sequence comparison of thermophilic and mesophilic DNA binding protein HUs, Bacillus stearothermophilus DNA binding protein HU (BstHU) seems to gain thermostability with a change in amino acid residues present on the molecular surface. To evaluate the contribution of exchange of each amino acid to the thermostability of BstHU, we constructed three mutants, BstHU-T13A (Thr13 to Ala), BstHU-G15E (Gly15 to Glu), and BstHU-T33L (Thr33 to Leu), in which the amino acids in BstHU were changed to the corresponding ones in Bacillus subtilis DNA binding protein HU (BsuHU). Stability of the mutant proteins was determined from thermal-denaturation curves. Replacement of Gly15 located in the turn region between alpha 1 and alpha 2 helices (HTH motif), with Glu (BstHU-G15E), resulted in a decrease in thermostability, and the Tm value was 54.0 degrees C compared to the Tm value of 63.9 degrees C for BstHU. The mutants, BstHU-T13A and BstHU-T33L, were, by contrast, slightly more stable (Tm values of 67.0 and 65.6 degrees C for BstHU-T13A and BstHU-T33L, respectively) than the wild type. We then generated the BsuHU mutant protein BsuHU-E15G, where Glu15 in BsuHU was in turn replaced by Gly, and we analyzed the thermostability. This substitution clearly enhanced the melting temperature by 11.8 degrees C (Tm value: 60.4 degrees C for BsuHU-E15G) compared to the value for BsuHU (Tm: 48.6 degrees C). Thus, Gly15 in the HTH motif of BstHU has an important role in the thermostability of BstHU. Characterization of the structure of the BstHU-G15E by 1H-NMR analysis showed that solvent accessibility of amide proton of Ala21 in the mutant was significantly increased compared with that of wild type, which means that the structure of the HTH motif in the N-terminal region in the mutant was changed to a more open conformation, thereby avoiding the interaction of Ala21 with either Ser17 by hydrogen bond or Ala11 by hydrophobic interaction.