Kenji Muraoka

Defective response to thrombopoietin and impaired expression of c‐mpl mRNA of bone marrow cells in congenital amegakaryocytic thrombocytopenia

Congenital amegakaryocytic thrombocytopenia (CAMT) is an uncommon disorder in newborns and infants, characterized by isolated thrombocytopenia and megakaryocytopenia in the first year without physical anomalies. The defect of thrombopoiesis is not well understood. Recently, thrombopoietin (TPO), the ligand for the c‐mpl receptor, was cloned. Accumulating evidence from in vitro and in vivo studies indicate that TPO plays a key role in the regulation of megakaryocytopoiesis. In this study we examined the effect of TPO on megakaryocyte colony formation from a patient with CAMT using a plasma‐containing methylcellulose clonal culture. The in vitro results demonstrated a defective response to TPO in megakaryocyte colony formation from bone marrow mononuclear cells (MNC) of the patient, although interleukin‐3 (IL‐3) but not stem cell factor (SCF) induced only a small number of megakaryocyte colonies. These findings indicated that thrombocytopenia in CAMT could not be corrected by administration of TPO in vitro. Additionally, clonal cultures containing SCF, IL‐3, IL‐6 and erythropoietin showed decreased numbers of erythroid and myelocytic progenitors in the bone marrow of the patient. The serum TPO level measured by enzyme‐linked immunosorbent assay was significantly higher than that in healthy controls. By PCR, marrow MNC from healthy children and from a patient with essential thrombocytosis expressed c‐mpl mRNA, whereas no c‐mpl mRNA was detected in marrow MNC from the patient with CAMT. There was no difference in the CD34 expression and c‐kit mRNA between the CAMT patient and healthy children. The results of this study suggest that the pathophysiology in CAMT may be a defective response to TPO in haemopoietic cells through impaired expression of c‐mpl mRNA.

Thrombopoietin alone stimulates the early proliferation and survival of human erythroid, myeloid and multipotential progenitors in serum-free culture

We examined the effects of recombinant human thrombopoietin (TPO, c‐Mpl ligand) on the proliferation and differentiation of human haemopoietic progenitors other than megakaryocytic progenitors using serum‐free cultures. TPO alone supported the generation of not only megakaryocytic (MK) but also blast cell (blast) colonies from cord blood CD34+ cells. Delayed addition of a cytokine cocktail (cytokines; interleukin (IL)‐3, IL‐6, stem cell factor, erythropoietin, granulocyte‐macrophage colony‐stimulating factor, and TPO) to cultures with TPO alone on day 7 induced various colonies including granulocyte‐macrophage (GM) colonies, erythroid bursts (E), granulocyte‐erythrocyte‐macrophage‐megakaryocyte (GEMM) colonies. Replating experiments of blast colonies supported by TPO alone for culture with cytokines revealed that approximately 60% of the blast colonies contained various haemopoietic progenitors. Single cell cultures of clone‐sorted CD34+ cells indicated that TPO supported the early proliferation and/or survival of both primitive and committed haemopoietic progenitors. In serum‐free suspension cultures, TPO alone significantly stimulated the production of progenitors for MK, GM, E and GEMM colonies as well as long‐term culture‐initiating cells. These effects were completely abrogated by anti‐TPO antibody. These results suggest that TPO is an important cytokine in the early proliferation of human primitive as well as committed haemopoietic progenitors, and in the ex vivo manipulation of human haemopoietic progenitors.

Characterization of peripheral blood progenitor cells (PBPC) mobilized by filgrastim (rHuG-CSF) in normal volunteers: dose-effect relationship for filgrastim with the character of mobilized PBPC.

Filgrastim (rHuG‐CSF)‐mobilized peripheral blood progenitor cells (PBPC) in healthy Japanese volunteers were characterized in detail using two clonal cell culture systems and double‐colour flow cytometry to detect multilineage colony‐forming cells and subsets of CD34+ cells. The kinetics of PBPC during the administration of filgrastim was studied, and possible differences in the character of progenitor cells relative to given doses of filgrastim were investigated. Filgrastim was administered subcutaneously to normal volunteers for 7 d at doses of 100, 200 or 400 μg/m2 (10 per cohort). Treatment with 100 or 200 μg/m2 filgrastim was well tolerated; however, the 400 μg/m2 dose level was not completed because of bone pain and myalgia. The treatment strikingly mobilized various types of progenitor cells, including highly proliferative megakaryocytic colony‐forming cells. The number of progenitor cells peaked on days 5 and 6. The fold increase of circulating progenitor cells from the baseline value in the volunteers treated with 200 μg/m2 filgrastim was more pronounced than in those treated with 100 μg/m2. Treatment with 200 μg/m2 also released the less mature progenitor cells (i.e. mixed colony‐forming cells, CD34+/33− cells, and CD34+/HLA‐DR− cells) into circulation better than the 100 μg/m2 dose. These results suggest that daily subcutaneous injection with 200 μg/m2 filgrastim for 5 d will effectively mobilize, both qualitatively and quantitatively, PBPC in healthy donors.

Thrombopoietin-independent effect of interferon-γ on the proliferation of human megakaryocyte progenitors

Flow cytometric study revealed that almost all CD34+ cells in human umbilical cord blood expressed interferon‐γ receptor (IFN‐γR). To clarify the precise functional roles of IFN‐γR in human CD34+ cells, we examined the effect of IFN‐γ alone and in combination with various cytokines on the growth of haemopoietic progenitor cells in CD34+ cells using a serum‐free clonal culture. Surprisingly, IFN‐γ alone supported only megakaryocyte (MK) colonies in a dose‐dependent manner with a plateau level at 1000 U/ml of IFN‐γ. IFN‐γ at 1000 U/ml induced 10 ± 1.2 MK colonies from 1 × 103 CD34+ cells, whereas thrombopoietin (TPO), interleukin (IL)‐3, stem cell factor (SCF) or IL‐6 alone induced 22 ± 4.0, 22 ± 4.2, 4 ± 0.6 and 0 MK colonies, respectively. The addition of anti‐IFN‐γ monoclonal antibody (mAb) to the IFN‐γ culture completely abrogated MK colony formation, whereas the mAb had no effect on TPO‐dependent production of MK colonies. In contrast, although anti‐TPO polyclonal Ab almost completely blocked TPO‐dependent MK colony formation, it failed to inhibit the generation of MK colonies induced by IFN‐γ, suggesting that the observed effect of IFN‐γ on the proliferation of human MK progenitor cells is independent of TPO. The addition of IFN‐γ to culture with TPO or SCF significantly augmented the development of MK colonies, whereas it did not affect IL‐3‐dependent MK colony formation. Additionally, IFN‐γ induced the increase of DNA content of cultured glycoprotein IIb/IIIa‐positive megakaryocytes. These results suggest that IFN‐γ may have regulatory roles in human megakaryocytopoiesis.

Role of glycoprotein 130 and c-Kit signaling in proliferation and differentiation of human hematopoietic progenitor cells

Abstract Glycoprotein (gp) 130, a receptor component for interleukin 6 (IL-6), can associate with a soluble IL-6 receptor (sIL-6R) – IL-6 complex. To examine the role of gp130 signaling in human hematopoietic progenitor-cell proliferation and differentiation, we studied the effects of the sIL-6R – IL-6 complex in combination with other cytokines on human CD34+ cells in clonal and suspension cultures. The sIL-6R – IL-6 complex, but not sIL-6R or IL-6 alone, in the presence of stem-cell factor (SCF) produced dramatic increases in the populations of various cell lineages, including erythroid cells and various hematopoietic progenitors, in suspension culture. Significant numbers of colonies of (particularly) multilineage and blast cells were generated in methylcellulose culture supplemented with a combination of sIL-6R – IL-6 complex and SCF. Addition of anti-gp130 monoclonal antibodies (MAbs) and anti-IL-6R MAbs to the above-mentioned cultures dose-dependently inhibited the generation of cells of various lineages and of progenitor cells in suspension culture and completely blocked multilineage colony production in methylcellulose culture; an anti-erythropoietin antibody did not cause inhibition. These findings demonstrate that both proliferation and differentiation of hematopoietic progenitor cells can be induced through gp130 and c-Kit signaling, indicating that progenitor cells are responsive to the sIL-6R – IL-6 complex, even though they do not express IL-6R. Together with previous studies showing that detectable levels of sIL-6R, IL-6, and SCF are present in human serum, these results suggest that gp130 signaling may play an important role in human hematopoiesis in vivo.

INTERFERON-GAMMA AND HUMAN MEGAKARYOPOIESIS

Interferon-gamma (IFN-gamma) exhibits various properties including antigrowth activity in neoplastic and normal cells and regulatory roles in immune responses and hematopoiesis, but studies of IFN-gamma effects on human megakaryopoiesis have been inconclusive. Recently we have used serum-free culture of purified CD34+ cells to demonstrate that IFN-gamma stimulates the proliferation of relatively mature megakaryocytic progenitors independently of thrombopoietin. It has been also shown that IFN-gamma stimulates the maturation of megakaryocytes, and has a significant synergism with stem cell factor in human megakaryopoiesis. Further studies are needed to clarify the in vivo effect of IFN-gamma on human megakaryopoiesis and the clinical relevance of IFN-gamma.