Movement towards or away from a given stimulus guides the directional migration of prokaryotes, simple eukaryotes and neurons. As bi-directional cues may influence entry and exit of immune effector cells from tissue sites, we evaluated the migratory responses of T-cell subsets to varying concentrations of the chemokine stromal cell derived factor-1 (SDF-1). There was selective repulsion of subpopulations of T cells at high concentrations of recombinant SDF-1 or naturally occurring bone marrow-derived SDF-1, which could be inhibited by pertussis toxin and antibody against the chemokine receptor CXCR4. Distinct sensitivity profiles to genistein, herbimycin and 8-Br-cAMP biochemically distinguished movement of cells towards or away from an SDF-1 gradient. In vivo, antigen-induced T-cell recruitment into the peritoneal cavity was reversed by high but not low concentrations of SDF-1. The phenomenon of movement away from a chemokine represents a previously unknown mechanism regulating the localization of mature T cells. It adds to the functional repertoire of chemokines that may participate in immune physiology and may be applied therapeutically to alter the immune response.

No abstract

Hematopoiesis in mammals undergoes a developmental shift in location from fetal liver to bone marrow accompanied by a gradual transition from highly proliferative to deeply quiescent stem cell populations. P2Y receptors are G-protein-coupled nucleotide receptors participating in vascular and immune responses to injury. We identified a P2Y-like receptor for UDP-conjugated sugars, GPR105 (P2Y 14 ), with restricted expression on primitive cells in the hematopoietic lineage. Anti-GPR105 antibody selectively isolated a subset of hematopoietic cells within the fetal bone marrow, but not in the fetal liver, that was enriched for G0 cell cycle status and for in vitro stem-cell-like multipotential long-term culture capability. Conditioned media from bone marrow stroma induced receptor activation and chemotaxis that was sensitive to G␣i and anti-receptor antibody inhibition. GPR105 is a G-protein-coupled receptor identifying a quiescent, primitive population of hematopoietic cells restricted to bone marrow. It mediates primitive cell responses to specific hematopoietic microenvironments and extends the known immune system functions of P2Y receptors to the stem cell level. These data suggest a new class of receptors participating in the regulation of the stem cell compartment. G-protein-coupled receptors (GPRs) have a broad repertoire of activating ligands ranging from photons to chemokines and induce an array of cellular events in virtually every physiologic system. Yet there remains a large proportion of GPR without known ligands or with limited known functions. A group of GPRs responsive to nucleotides (termed P2Y receptors) has been defined mediating cell-cell communication in the nervous system and in modulating vascular tone . A well-defined effect of nucleotides on platelet activation and vascular smooth muscle migration and growth has suggested participation of P2Y receptors in the response to injury. More recently, these receptors have been noted to affect cellular constituents of the innate immune system altering functional characteristics of monocytes, eosinophils, and dendritic cells and to play a critical role in terminating the inflammatory response in vivo (Mutini et al. 1999;Ferrari et al. 2000;Idzko et al. 2001;Santiago-Perez et al. 2001;Warny et al. 2001;Wilkin et al. 2001). The P2Y receptor specificity originally thought to be restricted to purine (adenine) nucleotides has been extended to pyrimidine nucleotides (uridine) and more recently to a receptor with specificity for UDP, but only when conjugated to glucose or related sugars (Chambers et al. 2000). This receptor, GPR105 (recently designated P2Y 14 ; Abbracchio et al. 2003), was originally noted to be expressed in rat brain tegmentum, but has no known function apart from being the presumed basis for UDP-glucose to induce diaphragmatic contraction or neural action potentials (Pastoris et al. 1979(Pastoris et al. , 1981. We provide evidence that this receptor participates in regulation of hematopoietic cells with stem cell characteristics.

The chemokine stroma-derived factor (SDF)-1, and its receptor, CXCR-4, have been shown to be essential for the translocation of hemopoietic stem cells from the fetal liver to the bone marrow (BM). We hypothesized that if CXCR-4 plays a crucial role in the localization of human hemopoiesis, stem cells from distinct tissue sources should demonstrate distinct CXCR-4 expression or signaling profiles. CD34+ cells from BM were compared with blood: either mobilized peripheral blood or umbilical cord blood. Unexpectedly, significantly higher levels of CXCR-4 surface expression on CD34+ cells from blood sources, mobilized peripheral blood, or cord blood were observed compared with BM (p = 0.0005 and p = 0.002, respectively). However, despite lower levels of CXCR-4, responsiveness of the cells to SDF-1 as measured by either calcium flux or transmigration was proportionally greatest in cells derived from BM. Further, internalization of CXCR-4 in response to ligand, associated with receptor desensitization, was significantly lower on BM-derived cells. Therefore, preserved chemokine receptor signaling was highly associated with marrow rather than blood localization. To test the functional effects of perturbing CXCR-4 signaling, adult mice were exposed to the methionine-SDF-1β analog that induces prolonged down-regulation/desensitization of CXCR-4 and observed mobilization of Lin−, Sca-1+, Thy-1low, and c-kit+ hemopoietic progenitor cells to the peripheral blood with a >30-fold increase compared with PBS control (p = 0.0007 day 1 and p = 0.004 day 2). These data demonstrate that CXCR-4 expression and function can be dissociated in progenitor cells and that desensitization of CXCR-4 induces stem cell entry into the circulation.