技术资料

There is evidence that neutrophil production is a balance between the proliferative action of granulocyte-colony-stimulating factor (G-CSF) and a negative feedback from mature neutrophils (the chalone). Two neutrophil serine proteases have been implicated in granulopoietic regulation: pro-proteinase 3 inhibits granulocyte macrophage-colony-forming unit (CFU-GM) growth,and elastase mutations cause cyclic and congenital neutropenia. We further studied the action of the neutrophil serine proteases (proteinase 3,elastase,azurocidin,and cathepsin G) on granulopoiesis in vitro. Elastase inhibited CFU-GM in methylcellulose culture. In serum-free suspension cultures of CD34+ cells,elastase completely abrogated the proliferation induced by G-CSF but not that of GM-CSF or stem cell factor (SCF). The blocking effect of elastase was prevented by inhibition of its enzymatic activity with phenylmethylsulfonyl fluoride (PMSF) or heat treatment. When exposed to enzymatically active elastase,G-CSF,but not GM-CSF or SCF,was rapidly cleaved and rendered inactive. These results support a role for neutrophil elastase in providing negative feedback to granulopoiesis by direct antagonism of G-CSF. View Publication

The differentiation of eosinophils from hematopoietic precursors and their subsequent maturation,chemotaxis,and activation is primarily regulated by interleukin-5 (IL-5). To examine the effect of chronic IL-5 exposure on hematopoiesis,IL-5 transgenic (IL-5trg) mice and wild-type BALB/c (WT) mice were examined. In comparison to WT mice,a significant alteration in bone marrow hematopoiesis was observed in IL-5trg mice. Although the total number of myeloid progenitors in the bone marrow of IL-5trg mice was not significantly altered,the number of long-term culture-initiating cells (LTC-ICs) was 1.5-fold lower than that observed in WT mice. Furthermore,IL-5trg mice failed to demonstrate hematopoietic activity in long-term bone marrow cultures,which correlated with a significant decrease in the number of bone marrow mesenchymal/stromal progenitor (MSP) cells in these mice. In comparison to WT mice,a 10-fold decrease was observed in the number of fibroblast colony-forming units (CFU-Fs) in IL-5trg bone marrow. Hematopoietic activity of IL-5trg bone marrow cells was rescued by cultivation on preestablished layers of bone marrow-derived stromal cells. However,in contrast to bone marrow,increased hematopoietic activity was observed in the spleen and peripheral blood of IL-5trg mice. Likewise,the numbers of LTC-ICs and granulocyte-macrophage,macrophage,eosinophil,B-lymphocyte progenitors in the peripheral blood and spleen of IL-5trg mice were approximately 20-fold higher than in WT mice. A significant increase in CFU-F numbers was also observed in the spleens of IL-5trg mice compared with WT mice. Overall,our results suggest that constitutive overexpression of IL-5 can potentially induce colonization of spleen with MSP cells,which provides the necessary microenvironment for establishment of hematopoiesis in extramedullary sites. View Publication

Here we describe the in vitro generation of a novel adherent cell fraction derived from highly enriched,mobilized CD133(+) peripheral blood cells after their culture with Flt3/Flk2 ligand and interleukin-6 for 3 to 5 weeks. These cells lack markers of hematopoietic stem cells,endothelial cells,mesenchymal cells,dendritic cells,and stromal fibroblasts. However,all adherent cells expressed the adhesion molecules VE-cadherin,CD54,and CD44. They were also positive for CD164 and CD172a (signal regulatory protein-alpha) and for a stem cell antigen defined by the recently described antibody W7C5. Adherent cells can either spontaneously or upon stimulation with stem cell factor give rise to a transplantable,nonadherent CD133(+)CD34(-) stem cell subset. These cells do not generate in vitro hematopoietic colonies. However,their transplantation into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice induced substantially higher long-term multilineage engraftment compared with that of freshly isolated CD34(+) cells,suggesting that these cells are highly enriched in SCID-repopulating cells. In addition to cells of the myeloid lineage,nonadherent CD34(-) cells were able to give rise to human cells with B-,T-,and natural killer-cell phenotype. Hence,these cells possess a distinct in vivo differentiation potential compared with that of CD34(+) stem cells and may therefore provide an alternative to CD34(+) progenitor cells for transplantation. View Publication

Hematopoietic stem cells (HSC) are tightly regulated through,as yet,undefined mechanisms that balance self-renewal and differentiation. We have identified a role for the transcriptional coactivators CREB-binding protein (CBP) and p300 in such HSC fate decisions. A full dose of CBP,but not p300,is crucial for HSC self-renewal. Conversely,p300,but not CBP,is essential for proper hematopoietic differentiation. Furthermore,in chimeric mice,hematologic malignancies emerged from both CBP(-/-) and p300(-/-) cell populations. Thus,CBP and p300 play essential but distinct roles in maintaining normal hematopoiesis,and,in mice,both are required for preventing hematologic tumorigenesis. View Publication

The small cytokine-inducible SH2 domain-containing protein (CIS) has been implicated in the negative regulation of signaling through cytokine receptors. CIS reduces growth of erythropoietin receptor (EpoR)-dependent cell lines,but its role in proliferation,differentiation,and survival of erythroid progenitor cells has not been resolved. To dissect the function of CIS in cell lines and erythroid progenitor cells,we generated green fluorescent protein (GFP)-tagged versions of wild type CIS,a mutant harboring an inactivated SH2 domain (CIS R107K),and a mutant with a deletion of the SOCS Box (CISDeltaBox). Retroviral expression of the GFP fusion proteins in BaF3-EpoR cells revealed that both Tyr-401 in the EpoR and an intact SH2 domain within CIS are prerequisites for receptor recruitment. As a consequence,both are essential for the growth inhibitory effect of CIS,whereas the CIS SOCS box is dispensable. Accordingly,the retroviral expression of GFP-CIS but not GFP-CIS R107K impaired proliferation of erythroid progenitor cells in colony assays. Erythroid differentiation was unaffected by either protein. Interestingly,apoptosis of erythroid progenitor cells was increased upon GFP-CIS expression and this required the presence both of an intact SH2 domain and the SOCS box. Thus,CIS negatively regulates signaling at two levels,apoptosis and proliferation,and thereby sets a threshold for signal transduction. View Publication

The WT1 tumor-suppressor gene is expressed by many forms of acute myeloid leukemia. Inhibition of this expression can lead to the differentiation and reduced growth of leukemia cells and cell lines,suggesting that WT1 participates in regulating the proliferation of leukemic cells. However,the role of WT1 in normal hematopoiesis is not well understood. To investigate this question,we have used murine cells in which the WT1 gene has been inactivated by homologous recombination. We have found that cells lacking WT1 show deficits in hematopoietic stem cell function. Embryonic stem cells lacking WT1,although contributing efficiently to other organ systems,make only a minimal contribution to the hematopoietic system in chimeras,indicating that hematopoietic stem cells lacking WT1 compete poorly with healthy stem cells. In addition,fetal liver cells lacking WT1 have an approximately 75% reduction in erythroid blast-forming unit (BFU-E),erythroid colony-forming unit (CFU-E),and colony-forming unit-granulocyte macrophage-erythroid-megakaryocyte (CFU-GEMM). However,transplantation of fetal liver hematopoietic cells lacking WT1 will repopulate the hematopoietic system of an irradiated adult recipient in the absence of competition. We conclude that the absence of WT1 in hematopoietic cells leads to functional defects in growth potential that may be of consequence to leukemic cells that have alterations in the expression of WT1. View Publication

Liver becomes the predominant site of hematopoiesis by 11.5 dpc (days after coitus) in the mouse and 15 gestational weeks in humans and stays so until the end of gestation. The reason the liver is the major hematopoietic site during fetal life is not clear. In this work,we tried to define which of the fetal liver microenvironmental cell populations would be associated with the development of hematopoiesis and found that a population of cells with mixed endodermal and mesodermal features corresponded to hematopoietic-supportive fetal liver stroma. Stromal cells generated from primary cultures or stromal lines from mouse or human fetal liver in the hematopoietic florid phase expressed both mesenchymal markers (vimentin,osteopontin,collagen I,alpha smooth muscle actin,thrombospondin-1,EDa fibronectin,calponin,Stro-1 antigens,myocyte-enhancer factor 2C) and epithelial (alpha-fetoprotein,cytokeratins 8 and 18,albumin,E-cadherin,hepatocyte nuclear factor 3 alpha) markers. Such a cell population fits with the description of cells in epithelial-to-mesenchymal transition (EMT),often observed during development,including that of the liver. The hematopoietic supportive capacity of EMT cells was lost after hepatocytic maturation,induced by oncostatin M in the cell line AFT024. EMT cells were observed in the fetal liver microenvironment during the hematopoietic phase but not in nonhematopoietic liver by the end of gestation and in the adult. EMT cells represent a novel stromal cell type that may be generated from hepatic endodermal or mesenchymal stem cells or even from circulating hematopoietic stem cells (HSCs) seeding the liver rudiment. View Publication

Natural killer (NK) cell differentiation from pluripotent CD34(+) human hematopoietic stem cells or oligopotent lymphoid progenitors has already been reported. In the present study,long-term cultures of the CD56(-)/CD34(-) myeloid-like adherent cell fraction (ACF) from umbilical cord blood (UCB),characterized by the expression of CD14(+) as well as other myeloid markers,were set up with flt3 ligand (FL) and interleukin-15 (IL-15). The UCB/ACF gradually expressed the CD56 marker,which reached fairly high levels (approximately 90% of the cells were CD56(+)) by day 15. FL plus IL-15-driven ACF/CD56(+) cells progressively expressed a mature NK functional program lysing both NK- and lymphokine-activate killer (LAK)-sensitive tumor targets and producing high levels of interferon-gamma (IFN-gamma),granulocyte-macrophage colony-stimulating factor,tumor necrosis factor alpha,and IL-10 upon stimulation with IL-12 and IL-18. Similar results were obtained when highly purified CD14(+) cells from UCB were cultured with FL and IL-15. In contrast,UCB/CD34(+) cells cultured under the same conditions showed a delayed expression of CD56 and behaved functionally differently in that they exhibited NK but not LAK cytotoxicity and produced significantly fewer cytokines. Kinetic studies on the phenotype of UCB/ACF or UCB/CD14(+) cells cultured in the presence of FL and IL-15 showed a rapid decrease in CD14 expression after day 5,which reached levels of zero by day 20. Approximately 60% of the CD56(+) derived from the UCB/ACF or the UCB/CD14(+) cells coexpressed CD14 by day 5. Taken together,our data support the role of CD14(+) myeloid-like cells within UCB as a novel progenitor for lymphoid NK cells. View Publication

Interleukin (IL)-13 has recently been shown to play important and unique roles in asthma,parasite immunity,and tumor recurrence. At least two distinct receptor components,IL-4 receptor (R)alpha and IL-13Ralpha1,mediate the diverse actions of IL-13. We have recently described an additional high affinity receptor for IL-13,IL-13Ralpha2,whose function in IL-13 signaling is unknown. To better appreciate the functional importance of IL-13Ralpha2,mice deficient in IL-13Ralpha2 were generated by gene targeting. Serum immunoglobulin E levels were increased in IL-13Ralpha2-/- mice despite the fact that serum IL-13 was absent and immune interferon gamma production increased compared with wild-type mice. IL-13Ralpha2-deficient mice display increased bone marrow macrophage progenitor frequency and decreased tissue macrophage nitric oxide and IL-12 production in response to lipopolysaccharide. These results are consistent with a phenotype of enhanced IL-13 responsiveness and demonstrate a role for endogenous IL-13 and IL-13Ralpha2 in regulating immune responses in wild-type mice. View Publication

OBJECTIVE: We examined if cellular elements or adhesive ligands were able to alter asymmetric divisions of CD34(+)/CD38(-) cells in contrast to soluble factors at a single cell level. MATERIALS AND METHODS: After single cell deposition onto 96-well plates,cells were cocultured for 10 days with the stem cell supporting cell line AFT024,fibronectin (FN),or bovine serum albumin (BSA). The divisional history was monitored with time-lapse microscopy. Subsequent function for the most primitive cells was assessed using the myeloid-lymphoid-initiating cell (ML-IC) assay. Committed progenitors were measured using colony-forming cells (CFC). RESULTS: Only contact with AFT024 recruited significant numbers of CD34(+)/CD38(-) cells into cell cycle and increased asymmetric divisions. Although most ML-IC were still identified among cells that have divided fewer than 3 times,a significant number of ML-IC shifted into the fast-dividing fraction after exposure to AFT024. The increase in ML-IC frequency was predominantly due to recruitment of quiescent and slow-dividing cells from the starting population. Increase in CFC activity induced by AFT024 was found only among rapidly dividing cells. CONCLUSIONS: For the first time,we have demonstrated that asymmetric divisions can be altered upon exposure with a stem cell-supporting microenvironment. For the primitive subset of cells (ML-IC),this was predominantly due to recruitment into cell cycle and increased rounds of cycling without loss of function. Exposure to AFT024 cells also increased proliferation and asymmetric divisions of committed CFC. Hence direct communication between hematopoietic progenitors with stroma cells is required for maintaining self-renewal potential. View Publication

Oncogenic Kit mutations are found in somatic gastrointestinal (GI) stromal tumors (GISTs) and mastocytosis. A mouse model for the study of constitutive activation of Kit in oncogenesis has been produced by a knock-in strategy introducing a Kit exon 11-activating mutation into the mouse genome based on a mutation found in a case of human familial GIST syndrome. Heterozygous mutant KitV558Delta/+ mice develop symptoms of disease and eventually die from pathology in the GI tract. Patchy hyperplasia of Kit-positive cells is evident within the myenteric plexus of the entire GI tract. Neoplastic lesions indistinguishable from human GISTs were observed in the cecum of the mutant mice with high penetrance. In addition,mast cell numbers in the dorsal skin were increased. Therefore KitV558Delta/+ mice reproduce human familial GISTs,and they may be used as a model for the study of the role and mechanisms of Kit in neoplasia. Importantly,these results demonstrate that constitutive Kit signaling is critical and sufficient for induction of GIST and hyperplasia of interstitial cells of Cajal. View Publication

A battery of clonal assays for myeloid progenitor cells (HPP-CFC,CFU-gemm,CFU-gm,CFU-g) was utilized to evaluate the myelotoxicity of a series of alkylating agents representing the spectrum of clinical times to nadir. Bone marrow aspirates from normal volunteers were incubated with mechlorethamine,busulfan,melphalan,carmustine or lomustine for 1 h and then cultured in methylcellulose with 30% serum and cytokines. There was a concentration-dependent inhibition of colony formation and often a differential toxicity to the myeloid progenitors with the alkylators tested. On a molar basis,mechlorethamine and melphalan were the most toxic of the alkylator drugs to the myeloid precursors. The most sensitive progenitor was CFU-gemm with the lowest inhibitory concentration IC(70) concentrations for mechlorethamine,melphalan,carmustine and lomustine. Generally,there was great similarity for drug effects between CFU-g and CFU-gm with overlapping inhibition curves. HPP-CFC proved to be the least sensitive of the progenitors to the toxic actions of the drugs. While there was no correlation between the time to clinical neutropenic nadir and the most sensitive progenitor in the clonal assays,the CFU-gm assay remains a suitable method for determining the myelotoxic potential of cytotoxic agents. View Publication