Maintenance of hematopoietic stem cells (HSCs) depends on interaction with their niche. Here we show that the long-term (LT)-HSCs expressing the thrombopoietin (THPO) receptor, MPL, are a quiescent population in adult bone marrow (BM) and are closely associated with THPO-producing osteoblastic cells. THPO/MPL signaling upregulated beta1-integrin and cyclin-dependent kinase inhibitors in HSCs. Furthermore, inhibition and stimulation of THPO/MPL pathway by treatments with anti-MPL neutralizing antibody, AMM2, and with THPO showed reciprocal regulation of quiescence of LT-HSC. AMM2 treatment reduced the number of quiescent LT-HSCs and allowed exogenous HSC engraftment without irradiation. By contrast, exogenous THPO transiently increased quiescent HSC population and subsequently induced HSC proliferation in vivo. Altogether, these observations suggest that THPO/MPL signaling plays a critical role of LT-HSC regulation in the osteoblastic niche.

SHPS-1 is a receptor-type glycoprotein that binds and activates the protein-tyrosine phosphatases SHP-1 and SHP-2, and thereby negatively modulates intracellular signaling initiated by various cell surface receptors coupled to tyrosine kinases. SHPS-1 also regulates intercellular communication in the neural and immune systems through its association with CD47 (integrin-associated protein) on adjacent cells. Furthermore, recent studies with fibroblasts derived from mice expressing an SHPS-1 mutant that lacks most of the cytoplasmic region suggested that the intact protein contributes to cytoskeletal function. Mice homozygous for this SHPS-1 mutation have now been shown to manifest thrombocytopenia. These animals did not exhibit a defect in megakaryocytopoiesis or in platelet production. However, platelets were cleared from the bloodstream more rapidly in the mutant mice than in wild-type animals. Furthermore, peritoneal macrophages from the mutant mice phagocytosed red blood cells more effectively than did those from wild-type mice; in addition, they exhibited an increase both in the rate of cell spreading and in the formation of filopodia-like structures at the cell periphery. These results indicate that SHPS-1 both contributes to the survival of circulating platelets and down-regulates the macrophage phagocytic response.SHPS-1 is a transmembrane glycoprotein that is abundant in neural and myeloid tissues (1-6). This molecule is also known as SIRP␣1 (7), BIT (8), MFR (9), and p84 neural adhesion molecule (10). The cytoplasmic region of SHPS-1 contains two immunoreceptor tyrosine-based inhibitory motifs, which recruit and activate the Src homology 2 domain-containing protein-tyrosine phosphatases SHP-1 and SHP-2 in a phosphorylation-dependent manner (1, 7, 11). The putative extracellular region of this protein comprises three immunoglobulin (Ig)-like domains, of which the most amino-terminal, IgV-like domain associates with the ligand CD47, also known as integrin-associated protein (6,12,13).Tyrosine phosphorylation of SHPS-1 is induced by soluble growth factors (1,7,14,15), integrin-mediated cell adhesion (16 -18), or cross-linking of Fc␥ receptors (19). Overexpression of SHPS-1 inhibits the activation of extracellular signal-regulated kinases induced by growth factors such as insulin, epidermal growth factor, and platelet-derived growth factor (7); it also inhibits promotion of the motility and survival of glioblastoma cells by epidermal growth factor (20). Furthermore, SHPS-1 inhibits IgE-induced mediator secretion and cytokine synthesis by mast cells (21). These observations suggest that SHPS-1, presumably by recruiting SHP-1 or SHP-2, negatively modulates a wide range of cellular activation signals initiated by tyrosine kinase-coupled receptors. However, the physiological significance of these observations remains unclear.Recent studies have suggested that SHPS-1, through its association with CD47, contributes to cellular functions that depend on intercellular communication, including T cell activation (13),...

SUMMARYWe examined the effects of interferon-g (IFN-g) on 100% pure human mast cells generated in suspension cultures of umbilical cord blood mononuclear cells in the presence of stem cell factor (SCF) and interleukin-6 (IL-6). When mast cells were suspended in serum-free medium without any cytokine after the withdrawal of SCF and IL-6, they died over a period of 5 days because of apoptosis. IFN-g in the cultures suppressed apoptosis and prolonged their survival in a dose-dependent manner. This survival-promoting effect of IFN-g was blocked by neutralizing antibodies to IFN-g or to IFN-g receptor (IFN-gR). When mast cells were incubated with IFN-g in serum-free medium for more than 4 hr during sensitization, immunoglobulin E (IgE)/anti-IgE antibody-induced histamine release was effectively enhanced. Polymerase chain reaction (PCR) amplification of the ␣-chain of IFN-gR (IFN-gR␣) yielded products of the correct size predicted from the sequence of the receptor. In addition, flow cytometry using anti-IFN-gR monoclonal antibodies (mAbs) indicated that these mast cells bear IFN-gR on their surface. These findings suggested that IFN-g activates human mast cells via specific receptors in certain aspects of inflammatory reactions.

UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on interleukin-3, granulocyte-macrophage colony-stimulating factor, or erythropoietin (EPO) for growth and survival. We investigated the effect of thrombopoietin (TPO), the ligand for the receptor encoded by c-mpl proto-oncogene, on the proliferation and differentiation of UT-7 and its sublines. We found that UT-7/GM, which is a subline of UT-7, but neither UT-7 nor UT-7/EPO, can proliferate in response to TPO. The subline, UT-7/TPO, was established from UT-7/GM by culture at lower concentrations of TPO. UT-7/TPO cells had morphologically mature megakaryocytic characteristics such as developed demarcation membrane in the cytoplasm and multinucleated appearance. This was also confirmed by the high expression of platelet factor-4 and glycoprotein IIb at the mRNA levels and by the high level of DNA content. UT-7/TPO can be maintained by TPO alone, with a doubling time of 24 hours in log growth phase. In the absence of TPO, the majority of the cells died within a few days. Thus, UT-7/TPO has an absolute dependence on TPO for growth and survival and has mature megakaryocytic features. The mRNA for c-mpl was detected in UT-7/TPO and, to a lesser degree, in UT-7/GM. The mRNA level of NF- E2 p45, reported to be an erythroid-specific transcription factor, was upregulated in UT-7/TPO, whereas it was down-regulated in the erythroid subline, UT-7/EPO. There were no significant differences in GATA-1 and GATA-2 mRNA levels among UT-7 and its sublines. Not only EPO but also TPO induced the tyrosine phosphorylation of JAK2 tyrosine kinase and STAT5-related protein. These findings indicate that UT-7/TPO would be a useful model with which to analyze the gene regulation of megakaryocytic maturation- associated proteins and to study the specific actions of TPO.

BackgroundPatients with Alzheimer’s disease (AD) often present with apathy symptoms resembling the decreased motivation observed in depressed patients. Therefore, differentiating the initial phase of AD from late life depression may be difficult in some cases. Near-infrared spectroscopy (NIRS) is a functional neuroimaging modality that uses near-infrared light to measure changes in hemoglobin concentration on the cortical surface during activation tasks. The objective of this study was to investigate differences in brain activation associated with late life depression and with AD by means of NIRS.MethodsNIRS was performed in 30 patients with depression, 28 patients with AD, and 33 healthy controls, all aged 60 years or older. During two tasks, a verbal fluency task and a visuospatial task, changes in oxygenated hemoglobin concentration in the frontal and parietal cortices were investigated.ResultsIn the visuospatial task, cortical activation was lower in the depressed group than in the AD group, and significant differences were observed in the parietal cortex.ConclusionsNIRS can detect differences in brain activation between patients with late life depression and those with AD. NIRS is a promising tool for the differential diagnosis of late life depression and AD.