技术资料

BACKGROUND AND OBJECTIVES: Chemokines are soluble mediators involved in angiogenesis,cellular growth control and immunomodulation. In the present study we investigated the effects of various chemokines on proliferation of acute myelogenous leukemia (AML) cells and constitutive chemokine release by primary AML cells. DESIGN AND METHODS: Native human AML cells derived from 68 consecutive patients were cultured in vitro. We investigated AML cell proliferation (3H-thymidine incorporation,colony formation),chemokine receptor expression,constitutive chemokine release and chemotaxis of normal peripheral blood mononuclear cells. RESULTS: Exogenous chemokines usually did not have any effect on AML blast proliferation in the absence of hematopoietic growth factors,but when investigating growth factor-dependent (interleukin 3 + granulocyte-macrophage colony-stimulating factor + stem cell factor) proliferation in suspension cultures the following patient subsets were identified: (i) patients whose cells showed chemokine-induced growth enhancement (8 patients); (ii) divergent effects on proliferation (15 patients); and (iii) no effect (most patients). These patient subsets did not differ in chemokine receptor expression,but,compared to CD34- AML cells,CD34+ cells showed higher expression of several receptors. Chemokines also increased the proliferation of clonogenic AML cells from the first subset of patients. Furthermore,a broad constitutive chemokine release profile was detected for most patients,and the following chemokine clusters could be identified: CCL2-4/CXCL1/8,CCL5/CXCL9-11 (possibly also CCL23) and CCL13/17/22/24/CXCL5 (possibly also CXCL6). Only the CCL2-4/CXCL1/8 cluster showed significant correlations between corresponding mRNA levels and NFkB levels/activation. The chemotaxis of normal immunocompetent cells for patients without constitutive chemokine release was observed to be decreased. INTERPRETATION AND CONCLUSIONS: Differences in chemokine responsiveness as well as chemokine release contribute to patient heterogeneity in AML. Patients with AML can be classified into distinct subsets according to their chemokine responsiveness and chemokine release profile. View Publication

Multiple myeloma is characterized by increased osteoclast activity that results in bone destruction and lytic lesions. With the prolonged overall patient survival achieved by new treatment modalities,additional drugs are required to inhibit bone destruction. We focused on a novel and more potent structural analog of the nonsteroidal anti-inflammatory drug etodolac,known as SDX-308,and its effects on osteoclastogenesis and multiple myeloma cells. SDX-101 is another structural analog of etodolac that is already used in clinical trials for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Compared with SDX-101,a 10-fold lower concentration of SDX-308 induced potent (60%-80%) inhibition of osteoclast formation,and a 10- to 100-fold lower concentration inhibited multiple myeloma cell proliferation. Bone resorption was completely inhibited by SDX-308,as determined in dentin-based bone resorption assays. SDX-308 decreased constitutive and RANKL-stimulated NF-kappaB activation and osteoclast formation in an osteoclast cellular model,RAW 264.7. SDX-308 effectively suppressed TNF-alpha-induced IKK-gamma and IkappaB-alpha phosphorylation and degradation and subsequent NF-kappaB activation in human multiple myeloma cells. These results indicate that SDX-308 effectively inhibits multiple myeloma cell proliferation and osteoclast activity,potentially by controlling NF-kappaB activation signaling. We propose that SDX-308 is a promising therapeutic candidate to inhibit multiple myeloma growth and osteoclast activity and that it should receive attention for further study. View Publication

IL-17A is a T cell-derived proinflammatory cytokine required for microbial host defense. In vivo expression profoundly stimulates granulopoiesis. At baseline,the hemopoietic system of IL-17R knockout mice (IL-17Ra(-/-)) is,with the exception of increased splenic progenitor numbers,indistinguishable from normal control mice. However,when challenged with gamma irradiation,hemopoietic toxicity is significantly more pronounced in IL-17Ra(-/-) animals,with the gamma irradiation-associated LD(50) being reduced by 150 rad. In spleen-derived T cells,gamma irradiation induces significant murine IL-17A expression in vivo but not in vitro. After sublethal radiation injury (500 rad),the infusion of purified CD4(+) T cells enhances hemopoietic recovery. This recovery is significantly impaired in IL-17Ra(-/-) animals or after in vivo blockade of IL-17Ra in normal mice,resulting in a reduction of hemopoietic precursors by 50% and of neutrophils by 43%. Following sublethal radiation-induced myelosuppression,in vivo overexpression of murine IL-17A in normal mice substantially enhanced granulopoietic restoration in mice with a 4-fold increase in neutrophils and splenic precursors on day 8 (CFU-granulocyte-macrophage/granulocyte-erythrocyte-megakaryocyte-monocyte,CFU-high proliferative potential),as well as 2- and 3-fold increases of bone marrow precursors,respectively. This establishes IL-17A as a hemopoietic response cytokine to radiation injury in mice and an inducible mechanism that is required for recovery of granulopoiesis after radiation injury. View Publication

Ikaros is expressed in early hematopoietic progenitors and is required for lymphoid differentiation. In the absence of Ikaros,there is a lack of markers defining fate restriction along lympho-myeloid pathways,but it is unclear whether formation of specific progenitors or expression of their markers is affected. Here we use a reporter based on Ikaros regulatory elements to separate early progenitors in wild-type and Ikaros-null mice. We found previously undetected Ikaros-null lympho-myeloid progenitors lacking the receptor tyrosine kinase Flt3 that were capable of myeloid but not lymphoid differentiation. In contrast,lack of Ikaros in the common myeloid progenitor resulted in increased formation of erythro-megakaryocytes at the expense of myeloid progenitors. Using this approach,we identify previously unknown pivotal functions for Ikaros in distinct fate 'decisions' in the early hematopoietic hierarchy. View Publication

Menin is the product of the tumor suppressor gene Men1 that is mutated in the inherited tumor syndrome multiple endocrine neoplasia type 1 (MEN1). Menin has been shown to interact with SET-1 domain-containing histone 3 lysine 4 (H3K4) methyltransferases including mixed lineage leukemia proteins to regulate homeobox (Hox) gene expression in vitro. Using conditional Men1 knockout mice,we have investigated the requirement for menin in hematopoiesis and myeloid transformation. Men1 excision causes reduction of Hoxa9 expression,colony formation by hematopoietic progenitors,and the peripheral white blood cell count. Menin directly activates Hoxa9 expression,at least in part,by binding to the Hoxa9 locus,facilitating methylation of H3K4,and recruiting the methylated H3K4 binding protein chd1 to the locus. Consistent with signaling downstream of menin,ectopic expression of both Hoxa9 and Meis1 rescues colony formation defects in Men1-excised bone marrow. Moreover,Men1 excision also suppresses proliferation of leukemogenic mixed lineage leukemia-AF9 fusion-protein-transformed myeloid cells and Hoxa9 expression. These studies uncover an important role for menin in both normal hematopoiesis and myeloid transformation and provide a mechanistic understanding of menin's function in these processes that may be used for therapy. View Publication

Immunomodulatory derivative (IMiD) CC-4047,a new analog of thalidomide,directly inhibits growth of B-cell malignancies in vivo and in vitro and exhibits stronger antiangiogenic activity than thalidomide. However,there is little information on whether CC-4047 affects normal hematopoiesis. Here we investigated the effect of CC-4047 on lineage commitment and differentiation of hematopoietic stem cells. We found that CC-4047 effectively inhibits erythroid cell colony formation from CD34+ cells and increases the frequency of myeloid colonies. We also demonstrate that development of both erythropoietin-independent and erythropoietin-dependent red cell progenitors was strongly inhibited by CC-4047,while terminal red cell differentiation was unaffected. DNA microarray analysis revealed that red cell transcription factors,including GATA-1,GATA-2,erythroid Kruppel-like factor (EKLF),and growth factor independence-1B (Gfi-1b),were down-regulated in CC-4047-treated CD34+ cells,while myeloid transcription factors such as CCAAT/enhancer binding protein-alpha (C/EBPalpha),C/EBPdelta,and C/EBPepsilon were induced. Analysis of cytokine secretion indicated that CC-4047 induced secretion of cytokines that enhance myelopoiesis and inhibit erythropoiesis. In conclusion,these data indicate that CC-4047 might directly influence lineage commitment of hematopoietic cells by increasing the propensity of stem and/or progenitor cells to undergo myeloid cell development and concomitantly inhibiting red cell development. Therefore,CC-4047 provides a valuable tool to study the mechanisms underlying lineage commitment. View Publication

Abnormal differentiation of myeloid cells is one of the hallmarks of cancer. However,the molecular mechanisms of this process remain elusive. In this study,we investigated the effect of tumor-derived factors on Janus kinase (Jak)/STAT signaling in myeloid cells during their differentiation into dendritic cells. Tumor cell conditioned medium induced activation of Jak2 and STAT3,which was associated with an accumulation of immature myeloid cells. Jak2/STAT3 activity was localized primarily in these myeloid cells,which prevented the differentiation of immature myeloid cells into mature dendritic cells. This differentiation was restored after removal of tumor-derived factors. Inhibition of STAT3 abrogated the negative effects of these factors on myeloid cell differentiation,and overexpression of STAT3 reproduced the effects of tumor-derived factors. Thus,this is a first demonstration that tumor-derived factors may affect myeloid cell differentiation in cancer via constitutive activation of Jak2/STAT3. View Publication

We previously found that some myeloma cell lines express the heparin-binding epidermal growth factor-like growth factor (HB-EGF) gene. As the proteoglycan syndecan-1 is an HB-EGF coreceptor as well as a hallmark of plasma cell differentiation and a marker of myeloma cells,we studied the role of HB-EGF on myeloma cell growth. The HB-EGF gene was expressed by bone marrow mononuclear cells in 8 of 8 patients with myeloma,particularly by monocytes and stromal cells,but not by purified primary myeloma cells. Six of 9 myeloma cell lines and 9 of 9 purified primary myeloma cells expressed ErbB1 or ErbB4 genes coding for HB-EGF receptor. In the presence of a low interleukin-6 (IL-6) concentration,HB-EGF stimulated the proliferation of the 6 ErbB1+ or ErbB4+ cell lines,through the phosphatidylinositol 3-kinase/AKT (PI-3K/AKT) pathway. A pan-ErbB inhibitor blocked the myeloma cell growth factor activity and the signaling induced by HB-EGF. This inhibitor induced apoptosis of patients'myeloma cells cultured with their tumor environment. It also increased patients' myeloma cell apoptosis induced by an anti-IL-6 antibody or dexamethasone. The ErbB inhibitor had no effect on the interaction between multiple myeloma cells and stromal cells. It was not toxic for nonmyeloma cells present in patients' bone marrow cultures or for the growth of hematopoietic progenitors. Altogether,these data identify ErbB receptors as putative therapeutic targets in multiple myeloma. View Publication

Dysregulation of the JAK/STAT signaling pathway is associated with Multiple Sclerosis (MS) and its mouse model,Experimental Autoimmune Encephalomyelitis (EAE). Suppressors Of Cytokine Signaling (SOCS) negatively regulate the JAK/STAT pathway. We previously reported a severe,brain-targeted,atypical form of EAE in mice lacking Socs3 in myeloid cells (Socs3DeltaLysM),which is associated with cerebellar neutrophil infiltration. There is emerging evidence that neutrophils are detrimental in the pathology of MS/EAE,however,their exact function is unclear. Here we demonstrate that neutrophils from the cerebellum of Socs3DeltaLysM mice show a hyper-activated phenotype with excessive production of reactive oxygen species (ROS) at the peak of EAE. Neutralization of ROS in vivo delayed the onset and reduced severity of atypical EAE. Mechanistically,Socs3-deficient neutrophils exhibit enhanced STAT3 activation,a hyper-activated phenotype in response to G-CSF,and upon G-CSF priming,increased ROS production. Neutralization of G-CSF in vivo significantly reduced the incidence and severity of the atypical EAE phenotype. Overall,our work elucidates that hypersensitivity of G-CSF/STAT3 signaling in Socs3DeltaLysM mice leads to atypical EAE by enhanced neutrophil activation and increased oxidative stress,which may explain the detrimental role of G-CSF in MS patients. View Publication

MicroRNAs (miRNAs) are small,noncoding RNAs that regulate gene expression by sequence-specific targeting of multiple mRNAs. Although lineage-,maturation-,and disease-specific miRNA expression has been described,miRNA-dependent phenotypes and miRNA-regulated signaling in hematopoietic cells are largely unknown. Combining functional genomics,biochemical analysis,and unbiased and hypothesis-driven miRNA target prediction,we show that lentivirally over-expressed miR-125b blocks G-CSF-induced granulocytic differentiation and enables G-CSF-dependent proliferation of murine 32D cells. In primary lineage-negative cells,miR-125b over-expression enhances colony-formation in vitro and promotes myelopoiesis in mouse bone marrow chimeras. We identified Stat3 and confirmed Bak1 as miR-125b target genes with approximately 30% and 50% reduction in protein expression,respectively. However,gene-specific RNAi reveals that this reduction,alone and in combination,is not sufficient to block G-CSF-dependent differentiation. STAT3 protein expression,DNA-binding,and transcriptional activity but not induction of tyrosine-phosphorylation and nuclear translocation are reduced upon enforced miR-125b expression,indicating miR-125b-mediated reduction of one or more STAT3 cofactors. Indeed,we identified c-Jun and Jund as potential miR-125b targets and demonstrated reduced protein expression in 32D/miR-125b cells. Interestingly,gene-specific silencing of JUND but not c-JUN partially mimics the miR-125b over-expression phenotype. These data demonstrate coordinated regulation of several signaling pathways by miR-125b linked to distinct phenotypes in myeloid cells. View Publication

Because lymphoid progenitors can give rise to natural killer (NK) cells,NK ontogeny has been considered to be exclusively lymphoid. Here,we show that rare human CD34(+) hematopoietic progenitors develop into NK cells in vitro in the presence of cytokines (interleukin-7,interleukin-15,stem cell factor,and fms-like tyrosine kinase-3 ligand). Adding hydrocortisone and stromal cells greatly increases the frequency of progenitor cells that give rise to NK cells through the recruitment of myeloid precursors,including common myeloid progenitors and granulocytic-monocytic precursors to the NK-cell lineage. WNT signaling was involved in this effect. Cells at more advanced stages of myeloid differentiation (with increasing expression of CD13 and macrophage colony-stimulating factor receptor [M-CSFR]) could also differentiate into NK cells in the presence of cytokines,stroma,and hydrocortisone. NK cells derived from myeloid precursors (CD56(-)CD117(+)M-CSFR(+)) showed more expression of killer immunoglobulin-like receptors,a fraction of killer immunoglobulin-like receptor-positive-expressing cells that lacked NKG2A,a higher cytotoxicity compared with CD56(-)CD117(+)M-CSFR(-) precursor-derived NK cells and thus resemble the CD56(dim) subset of NK cells. Collectively,these studies show that NK cells can be derived from the myeloid lineage. View Publication

Although microbial infections can alter steady-state hematopoiesis,the mechanisms that drive such changes are not well understood. We addressed a role for IFN-γ signaling in infection-induced bone marrow suppression and anemia in a murine model of human monocytic ehrlichiosis,an emerging tick-borne disease. Within the bone marrow of Ehrlichia muris-infected C57BL/6 mice,we observed a reduction in myeloid progenitor cells,as defined both phenotypically and functionally. Infected mice exhibited a concomitant increase in developing myeloid cells within the bone marrow,an increase in the frequency of circulating monocytes,and an increase in splenic myeloid cells. The infection-induced changes in progenitor cell phenotype were critically dependent on IFN-γ,but not IFN-α,signaling. In mice deficient in the IFN-γ signaling pathway,we observed an increase in myeloid progenitor cells and CDllb(lo)Gr1(lo) promyelocytic cells within the bone marrow,as well as reduced frequencies of mature granulocytes and monocytes. Furthermore,E. muris-infected IFN-γR-deficient mice did not exhibit anemia or an increase in circulating monocytes,and they succumbed to infection. Gene transcription studies revealed that IFN-γR-deficient CDllb(lo)Gr1(lo) promyelocytes from E. muris-infected mice exhibited significantly reduced expression of irf-1 and irf-8,both key transcription factors that regulate the differentiation of granulocytes and monocytes. Finally,using mixed bone marrow chimeric mice,we show that IFN-γ-dependent infection-induced myelopoiesis occurs via the direct effect of the cytokine on developing myeloid cells. We propose that,in addition to its many other known roles,IFN-γ acts to control infection by directly promoting the differentiation of myeloid cells that contribute to host defense. View Publication