Nobukuni Sawai

Anakinra improves sensory deafness in a Japanese patient with Muckle-Wells syndrome, possibly by inhibiting the cryopyrin inflammasome.

Muckle-Wells syndrome (MWS) is a dominantly inherited autoinflammatory syndrome. Patients with MWS have a mutation in CIAS1, the gene encoding cryopyrin, a component of the inflammasome that regulates the processing of interleukin-1beta (IL-1beta). In this report we describe an 8-year-old Japanese girl with MWS who had symptoms of periodic fever, urticarial rash, conjunctivitis, arthropathy, and sensory deafness. Laboratory analysis of the patients serum showed abnormally high concentrations of C-reactive protein, serum amyloid A, and IL-1beta, and she had a heterozygous mutation in the CIAS1 gene, with C-to-T transversion at nucleotide position 778, encoding an arginine-to-tryptophan mutation at position 260 (R260W). Mononuclear cells (MNCs) isolated from the patient secreted large amounts of IL-1beta, without stimulation, and were highly sensitive to muramyldipeptide and lipopolysaccharide. After treatment with anakinra, laboratory results normalized, and clinical symptoms, including sensory deafness, disappeared, while MNCs appeared to remain activated. Thus, our case suggests that anakinra possibly affects the cryopyrin inflammasome and markedly improves the clinical and laboratory manifestations of MWS.

Establishment of a GM‐CSF‐dependent megakaryoblastic cell line with the potential to differentiate into an eosinophilic lineage in response to retinoic acids

We recently established a human granulocyte‐macrophage colony‐stimulating factor (GM‐CSF)‐dependent cell line (HML) from colony‐constituent cells grown by peripheral blood cells of a patient with acute megakaryoblastic leukaemia. The HML cells possessed megakaryocytic features, as determined by cytochemical, electron microscopic and flow cytometric analysis. In the present study we examined the effects of retinoic acid (RA) on the development of HML cells. All‐trans‐RA, 13‐cis‐RA and 9‐cis‐RA at 10−8 mol/l to 10−5 mol/l inhibited the GM‐CSF‐dependent cell growth. Some of the RA‐treated cells contained prominent azurophilic granules and were positive for peroxidase. They also reacted with Biebrich scarlet, Luxol fast blue and a monoclonal antibody against eosinophil peroxidase. In addition, exposure to RA increased the frequency and the intensity of major basic protein‐positive cells. However, eosinophil‐derived neurotoxin and eosinophil cationic protein were not detected or were only detected at a low level in the lysates of the HML cells treated with RA. Although IL‐5 alone could not stimulate cell growth, the addition of IL‐5 to the cultures containing stem cell factor + all‐trans‐RA was required for the expression of the eosinophilic phenotype. These results suggest that the HML cell line is a megakaryoblastic cell line with the potential to differentiate into the eosinophilic lineage. HML cells may be a useful model for elucidating the eosinophilic differentiation programme.

Megakaryocytes derived from CD34‐positive cord blood cells produce interleukin‐8

In a serum‐free liquid culture, thrombopoietin (TPO) selectively stimulated the growth of megakaryocytic cells from CD34‐positive cord blood cells. Using these cultured cells, we investigated cytokine production by human megakaryocytes. Day 10 megakaryocytes (2 × 105) secreted > 1000 pg/ml of interleukin (IL)‐8, in contrast to small amounts of IL‐1β and IL‐6. A time‐course study showed that the IL‐8 production of megakaryocytes occurred at the late phase of the culture period. The megakaryocyte‐conditioned medium had the chemotactic potential of polymorphonuclear leucocytes, which was abrogated by the addition of anti‐IL‐8 antibody, suggesting the secretion of biologically active IL‐8. The combination of TPO and IL‐1α was required for a significant augmentation of the IL‐8 secretion. Direct evidence for IL‐8 synthesis in megakaryocytes was provided by reverse transcription–polymerase chain reaction on purified CD41b+ cells and by the detection of intracellular IL‐8 in CD41b+ cells. These results suggest that TPO stimulates not only the proliferation and differentiation of the progenitors capable of megakaryocytic lineage expression but also IL‐8 release by the megakaryocytic cells with the aid of IL‐1.

Analysis of synergism between stem cell factor and granulocyte-macrophage colony-stimulating factor on human megakaryoblastic cells: An increase in tyrosine phosphorylation of 145 KDA subunit of c-kit in two-factor combination

In normal hematopoiesis, stem cell factor (SCF) stimulates survival, proliferation and differentiation of hematopoietic progenitors. Although SCF acts synergistically with a variety of cytokines, the mechanism of growth factor-cooperation remains to be determined. To analyze the synergism between SCF and granulocyte-macrophage colony-stimulating factor (GM-CSF), we established a new megakaryoblastic cell line, HML-2, by culture in the presence of both SCF and GM-CSF. While SCF alone or GM-CSF alone supported modest cell growth, SCF and GM-CSF together induced substantial growth of this cell line. SCF alone tyrosine-phosphorylated several bands including the 145 kDa subunit of c-kit. GM-CSF alone did not cause the tyrosine phosphorylation of the 145 kDa subunit, but markedly up-regulated the expression of the 145 kDa subunit of c-kit. The combination of SCF and GM-CSF resulted in a synergistic increase in tyrosine phosphorylation of the 145 kDa subunit of c-kit. Several proliferation inhibitors which removed the two-factor interaction on the growth of the HML-2 cells down-regulated the 145 kDa subunit of c-kit. Thus, a synergistic increase in tyrosine phosphorylation of the 145 kDa subunit of c-kit may be one possible mechanism underlying the cooperation of SCF and GM-CSF on the HML-2 cell growth.

Apoptosis of Erythroid Precursors under Stimulation with Thrombopoietin: Contribution to Megakaryocytic Lineage Choice

Although the effect of thrombopoietin (TPO) on megakaryocyte production is well established, its role in the commitment of multipotential hematopoietic progenitors to the megakaryocytic lineage remains to be determined. In the present study, we attempted to clarify the determination process of megakaryocytic lineage as a terminal differentiation pathway under stimulation with TPO. Day 7 cultured cells grown by TPO derived from cord blood CD34+ cells were divided into four subpopulations on the basis of CD34 and CD41 expression. The CD34−/CD41− cells showed the labeling pattern of anti‐CD42b and anti‐CD9 antibodies closer to that of the CD34+/CD41− cells than the CD34+/CD41+ cells. Replating experiments revealed that approximately 40% of the CD34−/CD41− cells proliferated in response to a combination of growth factors, and more than 80% of them were pure erythroid precursors. However, this subpopulation failed to grow/survive and fell into apoptosis in the presence of TPO alone. In contrast, the CD34+/CD41+ cells, which predominantly contained megakaryocytic precursors, exerted a low but significant proliferative potential in the presence of TPO. The insufficient response to TPO of the CD34−/CD41− cells may result from the apparently low expression of c‐Mpl, as determined by flow cytometric analysis and reverse transcription‐polymerase chain reaction analysis. Therefore, these results suggest that the apoptosis of hematopoietic precursors other than megakaryocytic precursors is related to the determination of the terminal differentiation under the influence of TPO.

Cytogenetic clonality analysis in monosomy 7 associated with juvenile myelomonocytic leukemia: clonality in B and NK cells, but not in T cells

It remains unclear which lymphoid lineages are involved in juvenile myelomonocytic leukemia (JMML). We report a JMML patient who acquired monosomy 7 after intensive chemotherapy. In this case, the expression of monosomy 7 was analyzed in T, B and natural killer (NK) cells highly purified from peripheral blood mononuclear cells of the patient. The fluorescence in situ hybridization method revealed the expression of monosomy 7 in B cells, but not T cells. Half of the NK cells expressed monosomy 7; when NK cells were divided into CD2- and CD2+ populations, this abnormality was positive in 91.1% of CD2- NK cells but in only 14.7% of CD2+ NK cells. These results suggest that, in this JMML patient who acquired monosomy 7 after intensive chemotherapy, B cells and half of NK cells, but not T cells, have monosomy 7.

Quantitative and qualitative differences in thrombopoietin-dependent hematopoietic progenitor development between cord blood and bone marrow.

BACKGROUND To assess the clinical application of thrombopoietin (TPO) for thrombocytopenia of patients receiving cord blood (CB) or bone marrow (BM) transplants, we examined whether various types of hematopoietic progenitors including megakaryocyte (MK) progenitors from CB and BM exerted different proliferative and differentiative potential in the presence of TPO. METHODS The development of MK, granulocyte-macrophage, and erythroid/mixed erythroid (E/Mix) progenitors in a serum-deprived liquid culture medium supplemented with TPO was compared between CD34+ CB and BM cells. RESULTS The CD34+ CB cells generated 30-fold more MKs than the CD34+ BM cells, but the CB-derived MKs were more immature. A single-cell culture study showed that CB CD34+CD38- cells as well as CD34+CD38+ cells proliferated in response to TPO, whereas the two subpopulations of CD34+ BM cells showed little multiplication. In short-term liquid cultures containing CD34+ CB or BM cells, TPO significantly increased the absolute numbers of various types of colony-forming cells, compared with the input values. In particular, MK progenitors and E/Mix progenitors in CB were amplified to a substantially greater extent than in BM. The superior response of CD34+ CB cells to TPO observed in this study may be due in part to the use of cryopreserved cells. CONCLUSIONS Our results suggest that TPO alone cannot only stimulate megakaryocytopoiesis but also increase the numbers of various types of hematopoietic progenitors, and that quantitative and qualitative differences in TPO-dependent hematopoietic progenitor development exist between CB and BM.