Hidetsugu Mitani

Activity of interleukin 6 in the differentiation of monocytes to macrophages and dendritic cells

Peripheral blood monocytes are common precursor cells of dendritic cells (DCs) and macrophages. We have searched for factors with the potential to regulate the differentiation of monocytes to DCs and macrophages. When CD14+ monocytes are cultured with granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL) 4, the CD14+CD1a− population, which consists of macrophages, was found in the serum‐containing cultures but not in the serum‐free cultures. Addition of IL‐6 receptor‐neutralizing monoclonal antibody (mAb) or gp130‐neutralizing mAb to the serum‐containing cultures resulted in a decreased population of CD14+CD1a− cells. An increase in the CD14+CD1a− population with reduction in CD14−CD1a+ DCs was observed with the addition of IL‐6 to cultures, whereas IL‐11, leukaemia inhibitory factor, oncostatin M or macrophage colony‐stimulating factor did not affect the differentiation of monocytes in the presence of GM‐CSF plus IL‐4. This effect of IL‐6 was blocked by tumour necrosis factor α (TNF‐α), lipopolysaccharide (LPS), IL‐1β, CD40 ligand (CD40L) and transforming growth factor β1 (TGF‐β1). Among these factors, TNF‐α was most potent in interfering with the action of IL‐6. These results suggest that IL‐6 inhibits the differentiation of monocytes to DCs by promoting their differentiation toward macrophages, which is modulated by factors such as TNF‐α, LPS, IL‐1β, CD40L and TGF‐β1.

The Soluble Notch Ligand, Jagged-1, Inhibits Proliferation of CD34 + Macrophage Progenitors

The Notch/Notch ligand system controls diverse cellular processes. The proteolytic cleavage generates transmembrane and soluble forms of Notch ligands.We examined the effect of a soluble Notch ligand, human Jagged-1, on human cord blood (CB) CD34+ cells, under serum-deprived conditions, using soluble human Jagged-1—immunoglobulin G1 chimera protein (hJagged-1). Soluble hJagged-1 inhibited myeloid colony formation but not erythroid-mix or erythroid colony formation, in the presence of stem cell factor (SCF), interleukin-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, thrombopoietin, and erythropoietin. Cytological analysis revealed that the decrease in myeloid colonies resulted mainly from the inhibition of macrophage colony formation. Furthermore, soluble hJagged-1 led to the inhibition of macrophage colony formation supported by M-CSF plus SCF and GM-CSF plus SCF. Delayed-addition experiments and the analysis of colony sizes demonstrated that soluble hJagged-1 inhibited the growth of macrophage progenitors by acting in the early stage of macrophage development. The direct action of hJagged-1 was confirmed by the enhanced expression of the HES-1 (hairy enhancer of the split-1) gene. These results suggest that soluble hJagged-1 may regulate human hematopoiesis in the monocyte/macrophage lineage.

Efficient ex vivo generation of dendritic cells from CD14+ blood monocytes in the presence of human serum albumin for use in clinical vaccine trials

Dendritic cells (DC) with the potential to induce anti‐tumour immunity represent one of the promising candidates for cancer vaccines. Efficiency of ex vivo DC generation depends on culture conditions, especially protein components in the plasma or serum used. Using human serum albumin (HSA), we devised a constant and reproducible culture method for DC generation from peripheral blood CD14+ cells. The number of DC obtained with 2% HSA‐supplemented cultures containing granulocyte‐macrophage colony‐stimulating factor and interleukin 4 were consistently higher than in cultures with various concentrations of autologous plasma or serum. The concentrations and time points tested for plasma or serum considerably affected the number of DC recovered. DC prepared with HSA acquired the ability to uptake dextran, and expressed high levels of major histocompatibility (MHC) and co‐stimulatory molecules similar to DC cultured with autologous plasma or serum. Although DC cultured with autologous plasma or serum consisted of CD1a+ and CD1a− populations, DC differentiated in the presence of HSA expressed CD1a. DC obtained with HSA primed and induced immunogenic peptide‐specific cytotoxic T lymphocytes against a tumour rejection antigen, HER2. These findings suggest that our method for preparation of DC with HSA should prove valuable in DC generation for immunotherapy.

Possible involvement of bcl-2 in regulation of cell-cycle progression of haemopoietic cells by transforming growth factor-β1

Transforming growth factor‐β1 (TGF‐β1) acts directly on haemopoietic progenitor cells to regulate their growth. To investigate a possible link between the action of TGF‐β1 and cell death regulators such as bcl‐2, we utilized Ba/F3 cells, the interleukin‐3 (IL‐3)‐dependent growth of which could be modulated by TGF‐β1, as well as haemopoietic progenitor cells. We demonstrate here that up‐regulation of bcl‐2 protein (Bcl‐2) as well as that of an inhibitor of cyclin/cyclin‐dependent kinase complex, p27, was associated with TGF‐β1‐induced deceleration of the cell‐cycling of haemopoietic progenitor cells and Ba/F3 cells. The data from cell‐cycle analysis of Ba/F3 cells showed that TGF‐β1 retarded the G1 to S phase transition. Analysis of cells with the potential to express Bcl‐2 in an inducible manner indicated that up‐regulation of Bcl‐2 was sufficient for not only an increase in the level of p27 but also to inhibit the cell growth. Using c‐kit‐overexpressing cells, we observed that the potential of TGF‐β1 to up‐regulate the expression of Bcl‐2 and p27 could be counteracted by the c‐kit ligand, stem cell factor. These results demonstrate that Bcl‐2 exerts an essential function in the regulation of G1 to S phase transition of haemopoietic cells by TGF‐β1.

Granulocyte colony-stimulating factor and its receptor in acute promyelocytic leukemia

Expression of granulocyte colony‐stimulating factor (G‐CSF) receptor (G‐CSFR) and in vitro proliferative response to G‐CSF were investigated by quantitative immunofluorescence and [3H] thymidine uptake, respectively, in a series of acute myeloid leukemias (AML). The results indicated that G‐CSFR was detected at high levels in acute promyelocytic leukemia (APL) cells, in comparison with other types of AML. Moreover, APL cells were also seen to predominantly proliferate in response to G‐CSF. Based on these observations, we administered recombinant human G‐CSF to a patient with APL in the third relapse that was resistant to both cytotoxic agents and all trans retinoic acid, in an attempt to sensitize the leukemic cells to cell‐cycle‐dependent agents. Complete remission was achieved. The finding that APL cells are exquisitely responsive to G‐CSF supports the view that G‐CSF is useful for augmentation of their vulnerability to cell‐cycle specific agents. Am. J. Hematol. 58:31–35, 1998.

Two Independent Clones in Myelodysplastic Syndrome Following Treatment of Acute Myeloid Leukemia

We describe a 55-year-old Japanese woman with therapy-related myelodysplastic syndrome (t-MDS) with 2 independent clones, t(1;2)(p36;p21) and t(11;12)(p15;q13). She was diagnosed with acute myeloid leukemia (AML) with cytological features of the bone marrow and peripheral blood. Cytogenetic evaluation revealed a 46,XX karyotype. She received chemotherapy and achieved complete remission (CR). Despite maintenance chemotherapy, she suffered a relapse. Chromosomal analysis showed t(1;2)(p36;p21) in 2 of 20 metaphases. At second CR, this clone transiently disappeared. Nine months later, t(1;2) (p36;p21) was detected again in 3 of 20 metaphases while the patient remained in CR. Six months later, bone marrow examination disclosed trilineage dysplasia without an excess of blasts, suggesting MDS. t(1;2)(p36;p21) was observed in 16 of 20 metaphases. The clinical course and serial cytogenetic findings were diagnostic of t-MDS. The duration of t-MDS was 6 years. During this period, persistent t(1;2)(p36;p21) and transient t(11;12)(p15;q13) were found.When t-MDS evolved to AML, cytogenetic evaluation revealed 46,XX,t(1;2)(p36;p21),del(7)(q22),add(19)(p13).

Efficient Ex Vivo Generation of Human Dendritic Cells from Mobilized CD34p+ Peripheral Blood Progenitors

We tried to efficiently generate human dendritic cells (DCs) from CD34p+ peripheral blood hematopoietic progenitor cells mobilized by high-dose chemotherapy and subsequent administration of granulocyte colony-stimulating factor, using a liquid suspension culture system. Among various combinations, the combination ofc-kit ligand,flt-3 ligand,c-mpl ligand (TPO), and interleukin (IL)-4 most potently generated the number of CD1ap+CD14p- DCs in cultures containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor α (TNF-α). The delayed addition of IL-4 on day 6 of culture gave rise to an additional increase in the yield of CD1ap+CD14p- DCs that were characterized by the expression of HLA-ABC, HLA-DR, CD80, CD86, and CD83. The majority of the sorted CD1ap-CD14p+ cells derived from 6-day culture of CD34p+cells gave rise to CD1ap+CD14p- DCs and CD1a-CD14p+ macrophages on day 12 of culture in the presence and absence of IL-4, respectively. These findings suggest that IL-4 promotes the differentiation of CD1ap-CD14p+ cells derived from mobilized CD34p+ peripheral blood hematopoietic progenitors to CD1ap+CD14p- DCs. The majority of these DCs expressed CD68 but not the Langerhans-associated granule antigen, a finding that suggests they emerge through the monocyte differentiation pathway. The addition of TPO and IL-4 to cultures did not affect the potential of DCs to stimulate the primary allogeneic T-cell response. These findings demonstrated that the combination ofc-kit ligand plusflt-3 ligand plus TPO with GM-CSF plus TNF-α, followed by IL-4, is useful for ex vivo generation of human DCs from mobilized CD34p+ peripheral blood progenitors.

A case of acute myeloblastic leukemia with a novel variant of t(8;21)(q22;q22)

We encountered a case of acute myeloblastic leukemia (AML), with extramedullary leukemia (EML) and a masked type of the variant translocation t(8;21)(q22;q22). Morphologically, the AML M2 subtype according to the French–American–British (FAB) classification was present. Phenotypically, leukemic cells were negative for CD19 and positive for CD56. Clinically, the case showed chemo-refractoriness and a poor outcome. The initial karyotypic interpretation was t(8;9)(q22;q34) on G-banding. Multiplex-fluorescence in situ hybridization (multiplex-FISH) analysis revealed a three-way translocation involving chromosomes 8, 9, and 21, and identified a masked type of variant t(8;21)q22;q22) translocation. The karyotype was finally determined as 45,X,-Y,der(8)t(8;21)(q22;q22), der(9)(8;9)(q22;q34), and der(21)t(9;21)(q34;q22). Results of FISH using the AML1/ETO probe and detection of the AML1/ETO fusion transcripts by reverse transcriptase-polymerase chain reaction (RT-PCR) support the karyotype as well as the sequence of the PCR product. Additionally, C-KIT mutation was detected.

Molecular analysis of PDGFRα/β genes in core binding factor leukemia with eosinophilia

Abstract:  Eosinophilia sometimes occurs in acute myeloid leukemia (AML), especially in core binding factor (CBF) leukemia. However, the pathogenesis of the differentiation from leukemic progenitors to eosinophils is not well understood in this type of leukemia. Recent reports showed that a novel fusion tyrosine kinase, Fip1‐like1 (FIP1L1) platelet‐derived growth factor receptor alpha (PDGFRα), is found in idiopathic hypereosinophilic syndrome. The involvement of another chimeric gene, PDGFRβ, was also reported in myeloproliferative disorder with eosinophilia. These chimeric genes cause constitutive activation of PDGFR tyrosine kinases. On the other hand, a two‐hit model for the pathogenesis of AML, which seems to be caused by inactivating mutations in transcription factors and genetic lesions in tyrosine kinase resulting in constitutive activation, has been proposed. On the basis of these findings, we screened for the expression of the FIP1L1‐PDGFRα fusion gene and for mutations in the juxtamembrane and tyrosine kinase domains of PDGFRα/β genes in 22 cases of CBF leukemia with eosinophilia. Among these cases, no FIP1L1‐PDGFRα fusion gene was found. Although cDNA sequencing also detected three types of single‐nucleotide alterations at kinase domains in PDGFRα/β genes, all of them were silent changes and polymorphisms. Therefore, PDGFRα/β genes do not appear to play a significant pathogenetic role in eosinophilia or leukemogenesis of CBF leukemia.

Bcl-2 in cell-cycle regulation of hematopoietic cells by transforming growth factor-β1

We reported that several growth factors regulate the doubling lime of hematopoietic progenitor cells by modulating the time required to pass through the GI phase. As recent studies revealed the link between cell death and cell-cycle progression. we asked if cell death regulators such ss Bel-2 play a role in regulating the cell-cycle of hematopoietic cells by growth factors. Among growth factors. transforming growth factor-β1 (TCF-β1). a negative regulator of hematopoietis, was chosen. When a large number of cells was required for analysis, we used IL-3-dependent Ba/F3 cells instead of primary hematopoietic progenitor cells because the response of Ba/F3 cells to TGF-β1 was similar to that of primary hematopoietic progenitor cells. TGF-β1 decelerated the cell-cycling of hematopoietic cells by inducing a delay in G1 to S phase transition, an event associated with increase in the level of Bel-2 as well as p27. a cyclin/cyclin-dependent kinase inhibitor. In experiments using Ba/F3 cells with the potential to produce Bel-2 in an inducible manner. Bel-2 apparently functions upstream of p27. The effects of TGF-β1 on Bel-2 and p27 expression as well as cell growth were abrogated by c-kit ligand. These findings suggest that Bel-2 plays a crucial role in regulating the cell-cycle of hematopoietic progenitor cells.