Kaoru Tohyama

Flow cytometric method for enumeration and classification of reactive immature granulocyte populations.

We developed a flow cytometric method for the enumeration and classification of nonmalignant immature granulocytes (IG). In this study, IG are defined as most immature (IG stage 1: promyelocytes and myelocytes) and as more mature (IG stage 2: metamyelocytes). Blood specimens from 46 patients with documented infectious or inflammatory disease and known presence of IG (by routine manual microscopy) were analyzed. For a reference manual differential count, we used a 400 white blood cell (WBC) differential and separated granulocytes into promyelocytes and myelocytes combined, metamyelocytes, and included band cells in the mature, segmented neutrophil population. The flow cytometric method is based on three-color staining of whole, anticoagulated blood with CD45-PerCP, CD16-FITC, and CD11b-PE-labeled monoclonal antibodies and a three-step gating procedure. The flow cytometric results were confirmed by cell sorting and microscopic evaluation of the sorted cells. A total of 10,000 events, excluding debris, were recorded per specimen and IG stage 1 (CD16-/CD11b-), IG stage 2 (CD16-/CD11b+), and mature neutrophils (CD16+/CD11b+) were categorized. Regression and correlation between flow cytometric IG and the manual differential showed y = 1.34x + 0.95, r(2) = 0.86 for IG stages 1 and 2 combined versus promyelocytes, myelocytes, and metamyelocytes. For IG stage 1 versus microscopic counts of promyelocytes and myelocytes, the results were y = 1.53x + 1.24, r(2) = 0.76; for IG stage 2 versus manual metamyelocyte count, y = 0.77x + 0.21, r(2) = 0.58. Reproducibility of the flow cytometric method showed a coefficient of variation (CV) of 6.8% for all IG combined compared with a CV of 50.2% for manual differential IG count (based on a routine 100 WBC count). Samples were found stable at least 12 h at 25 degrees C and at least 48 h at 4 degrees C for flow cytometry. After staining and lysing, the sample was stable for at least 120 min at room temperature. We analyzed samples from patients with myelodysplastic and myeloproliferative disease separately. We found that CD16- mature neutrophils falsely elevated the flow cytometric IG count. Similar results were obtained in blood from patients treated with granulocyte-colony stimulating factor (G-CSF). Although this restricts the use of the method somewhat, we believe that this flow cytometric method is useful for enumerating reactive IG, as well as for evaluating automated methods for IG identification by hematology analyzers.

Plasma thrombopoietin (TPO) levels and expression of TPO receptor on platelets in patients with myelodysplastic syndromes

Data on endogenous thrombopoietin (TPO) levels and their regulation in myelodysplastic syndromes (MDS) are sparse. We examined the plasma TPO level of 85 MDS patients by a sensitive enzyme immunoassay and the platelet expression of TPO receptor (TPO‐R) protein, which metabolizes endogenous TPO, in 19 MDS patients with an equilibrium binding assay using 125I‐TPO. The MDS patients had higher plasma TPO levels (7.0 ± 9.3 fmol/ml) than 52 normal subjects (P < 0.0001). Refractory anaemia (RA) patients (n = 39) had higher plasma TPO levels than patients (n = 28) with RA with excess blasts (RAEB) or RAEB in transformation (RAEB‐t) (P = 0.0002), irrespective of similar platelet counts in these groups. The plasma TPO level correlated inversely with the platelet count in RA patients (P = 0.0027) but not in RAEB and RAEB‐t patients (P = 0.7865). These data suggest that the physiological pathway for TPO production and metabolism is conserved, at least partially, in RA, but deranged in RAEB/RAEB‐t. The number of TPO‐R per platelet was significantly smaller in 19 MDS patients (17.5 ± 13.3) than in normals (P = 0.0014), but similar between RA patients and patients with RAEB and RAEB‐t. Further, the bone marrow megakaryocyte count, determined in 31 MDS patients, was quite similar between RA patients and patients with RAEB or RAEB‐t. Thus, in addition to thrombocytopenia, a reduced platelet TPO‐R number may contribute to elevated plasma TPO levels in MDS, and a regulatory pathway for circulating TPO other than platelet TPO‐R and marrow megakaryocytes, such as blasts expressing TPO‐R, may operate in RAEB/RAEB‐t.

Establishment of an interleukin-5-dependent subclone from an interleukin-3-dependent murine hemopoietic progenitor cell line, LyD9, and its malignant transformation by autocrine secretion of interleukin-5.

An interleukin‐5 (IL‐5)‐dependent subclone, K‐5, was established from an IL‐3‐dependent murine hemopoietic progenitor cell line by co‐culturing with bone marrow stroma cells. K‐5 cells were induced to differentiate into myeloid lineage cells by co‐culturing with cloned PA6 stroma cells. By co‐culturing with another cloned stroma cell (ST‐2s10), K‐5 cells gave rise to a factor‐independent transformant cell line LT‐5 which proliferated in an autocrine manner by secretion of IL‐5 and produced tumors in nude mice. Molecular cloning of the IL‐5 gene of LT‐5 cells and the nucleotide sequencing of its 5′ flanking region indicate that a transposition of an intracisternal A‐particle (IAP) element to the 5′ flanking region of the IL‐5 gene is responsible for the constitutive expression of IL‐5 mRNA of an aberrant size in LT‐5 cells.

Myelodysplastic syndrome (MDS)-associated inhibitory activity on haemopoietic progenitor cells.

We studied MDS‐associated inhibitory activity, which inhibited colony formation in vitro of granulocyte‐macrophage progenitors (CFU‐GM). Macrophages obtained from MDS bone marrow mononuclear cells (BM‐MNC) when pretreated with granulocyte‐macrophage colony stimulating factor (GM‐CSF) suppressed the growth of normal CFU‐GM. These macrophages were designated as ‘MDS‐derived inhibitory macrophages’. Media conditioned by MDS‐derived inhibitory macrophages (MDS‐CM) also suppressed the growth of normal CFU‐GM. In the MDS‐CM, high levels of prostaglandin E2 (PGE2) and ferritin were found. However, MDS‐CM did not contain detectable levels of tumour necrosis factor (TNF) or gamma‐interferon (γ‐IFN). Antiserum against human placental ferritin and/or against PGE2 blocked the haemopoietic inhibitory activity to some extent.

Detection of Granulocyte Colony‐stimulating Factor Produced by a Newly Established Human Hepatoma Cell Line Using a Simple Bioassay System

A colony‐stimulating factor(CSF)‐producing tumor cell line (KX‐87) was established from a patient with hepatocellular carcinoma and marked granuloeytosis. This cell line formed tumors on nude mice in high frequency and the mice revealed marked granuloeytosis. In clonogenic assays of human bone marrow cells, KX‐87 conditioned medium (CM) supported the formation of colonies mainly consisting of neutrophilic granulocytes but had no burst‐promoting activity. The molecular weight of the colony‐stimulating activity (CSA) in KX‐87CM was estimated about 25,000 daltons by gel filtration and a new bioassay system. In principle, a subline of murine hemopoietic cell line NFS‐60 was cloned which was dependent on KX‐87CM. Then the growth of this subline was examined by a rapid and sensitive colorimetric tetrazolium assay. From these results, it was concluded that the CSA which KX‐87 cell line produced was G‐CSF.

Retrovirus-mediated gene transfer of granulocyte colony-stimulating factor receptor (G-CSFR) cDNA into MDS cells and induction of their differentiation by G-CSF

Myelodysplastic syndromes (MDS) are clonal disorders in which the proper differentiation of hematopoietic stem cells is impaired. There is no effective treatment for this stem cell disorder at present. In an attempt to find a new strategy that promotes the differentiation of MDS blast cells, we tried retroviral transduction of granulocyte colony-stimulating factor receptor (G-CSFR) into an interleukin-3-dependent MDS cell line, MDS-L, since expression of G-CSFR is known to be essential for the differentiation of myeloid progenitor cells and this expression is impaired in most MDS cells. Ectopic expression of human G-CSFR cDNA in MDS-L cells gave rise to granulocytic differentiation by G-CSF stimulation. G-CSF caused the transformants expressing G-CSFR to display a morphological characteristic of mature granulocytes, upregulated CD11b on the cell surface, and improved NBT reduction activity. These results demonstrate that MDS-L cells ecopically expressing G-CSFR are induced to granulocytic differentiation upon exposure to G-CSF, and shed light on the molecular mechanisms of maturation arrest in MDS cells.

Prognostic implication of sequential bone marrow cultures in the myelodysplastic syndromes

Twenty-five patients with newly-diagnosed myelodysplastic syndromes were studied by the clonal culture method at least three times during the clinical course. Clinical outcomes of the patients were classified into: a stable disease (ten patients); subsequent leukemic transformation (eight patients) and nonleukemic death (seven patients). The growth of the marrow granulocyte-macrophage progenitors (CFU-GM) at the time of diagnosis was significantly related to the survival. In addition, sequential changes in the CFU-GM growth patterns correlated with the different clinical outcome of myelodysplastic syndromes patients.

Effects of the Tyrosine Kinase Inhibitor Imatinib Mesylate on a Bcr-Abl-Positive Cell Line: Suppression of Autonomous Cell Growth but No Effect on Decreased Adhesive Property and Morphological Changes

Expression of the Bcr-Abl oncoprotein alters various aspects of hematopoietic cells. We investigated the effects of a Bcr-Abl tyrosine kinase inhibitor, imatinib mesylate, on the proliferation, adhesive properties, and morphology of a Bcr-Abl-transferred cell line, TF-1 Bcr-Abl, in comparison with parental TF-1. First, the factor-independent growth of TF-1 Bcr-Abl was inhibited in the presence of imatinib mesylate, but this inhibition was overcome by addition of exogenous granulocyte-macrophage colony-stimulating factor. Imatinib mesylate remarkably reduced tyrosine phosphorylation of Bcr-Abl, Cbl, and Crkl in a time-dependent manner, and their complex formation also was affected. Imatinib mesylate inhibited activation of Stat5 rather than the MEK-ERK1/2 pathway. TF-1 Bcr-Abl cells exhibited a round shape, unlike TF-1, and the adhesive property to fibronectin was much lower than that of TF-1. Although the Bcr-Abl oncoprotein may be involved negatively in cell adhesion, the decreased adhesion and altered morphology of TF-1 Bcr-Abl cells were minimally affected by imatinib mesylate and seemed independent of Bcr-Abl kinase activity. The present data indicated that the Bcr-Abl-specific kinase inhibitor cannot control Bcr-Abl-induced cell alterations other than autonomous growth.Int J Hematol. 2003;78:233-240.

Effects of recombinant G‐CSF and GM‐CSF on in vitro differentiation of the blast cells of RAEB and RAEB‐T

To evaluate the effects of recombinant G‐CSF and GM‐CSF on RAEB and RAEB‐T cells, blast cells from 6 patients were incubated in liquid culture systems with these CSFs for 7 days, and their numerical, morphological and functional changes were assessed. Both CSFs stimulated cell growth, but decreased the proportion of blast cells in 5 of the 6 cases. Karyotypic abnormalities persisted during cultivation in some cases. The CSFs also stimualted the expression of part of the esterase activities, and a positive interaction of both CSFs was seen in part. Although CSFs had no significant effects on the ability of cells to reduce NBT or to phagocytize latex particles, the results indicated that they induce partial differentiation of blast cells. It appears that such pathological cells still retain the capacity to respond to growth factors.

Reversible Acceleration of Disease Progression Following Cyclosporin A Treatment in a Patient With Myelodysplastic Syndrome

A 38-year-old Japanese man with myelodysplastic syndrome (MDS), whose bone marrow smears demonstrated hypercellularity, was treated with oral cyclosporin A (CsA) therapy. During the course of this therapy, the numbers of peripheral blood and bone marrow blasts increased and the level of serum lactate dehydrogenase increased.After discontinuation of CsA treatment, all of these levels rapidly decreased. We consider that CsA might accelerate disease progression in certain MDS cases.