Tatsuya Kinoshita

IL-9 Enhances the Growth of Human Mast Cell Progenitors Under Stimulation with Stem Cell Factor

We examined the effects of IL-9 on human mast cell development from CD34+ cord blood (CB) and peripheral blood cells in serum-deprived cultures. IL-9 apparently enhanced cell production under stimulation with stem cell factor (SCF) from CD34+ CB cells. A great majority of the cultured cells grown with SCF + IL-9 became positive for tryptase at 4 wk. In methylcellulose cultures of CD34+ CB cells, IL-9 increased both the number and size of mast cell colonies grown with SCF. Furthermore, SCF + IL-9 caused an exclusive expansion of mast cell colony-forming cells in a 2-wk liquid culture of CD34+ CB cells, at a level markedly greater than for SCF alone. Clonal cell cultures and RT-PCR analysis showed that the targets of SCF + IL-9 were the CD34+CD38+ CB cells rather than the CD34+CD38− CB cells. IL-9 neither augmented the SCF-dependent generation of progeny nor supported the survival of 6-wk-cultured mast cells. Moreover, there was no difference in the appearance of tryptase+ cells and histamine content in the cultured cells between SCF and SCF + IL-9. The addition of IL-9 increased numbers of mast cell colonies grown with SCF from CD34+ peripheral blood cells in children with or without asthma. It is of interest that mast cell progenitors of asthmatic patients responded to SCF + IL-9 to a greater extent than those of normal controls. Taken together, IL-9 appears to act as a potent enhancer for the SCF-dependent growth of mast cell progenitors in humans, particularly asthmatic patients.

Regulation of Highly Cytokinergic IgE-Induced Mast Cell Adhesion by Src, Syk, Tec, and Protein Kinase C Family Kinases

Mast cells play a critical role in IgE-dependent immediate hypersensitivity. Recent studies have shown that, contrary to the traditional view, binding of monomeric IgE to FcεRI results in a number of biological outcomes in mast cells, including survival. However, IgE molecules display heterogeneity in inducing cytokine production; highly cytokinergic (HC) IgEs cause extensive FcεRI aggregation, which leads to potent enhancement of survival and other activation events, whereas poorly cytokinergic (PC) IgEs can do so inefficiently. The present study demonstrates that HC, but not PC, IgEs can efficiently induce adhesion and spreading of mouse mast cells on fibronectin-coated plates in slow and sustained kinetics. HC IgE-induced adhesion through β1 and β7 integrins promotes survival, IL-6 production, and DNA synthesis. Importantly, we have identified Lyn and Syk as requisite tyrosine kinases and Hck, Btk, and protein kinase C θ as contributory kinases in HC IgE-induced adhesion and spreading, whereas protein kinase C ε plays a negative role. Consistent with these results, Lyn, Syk, and Btk are activated in HC IgE-stimulated cells in a slower but more sustained manner, compared with cells stimulated with IgE and Ag. Thus, binding of HC IgEs to FcεRI induces adhesion of mast cells to fibronectin by modulating cellular activation signals in a unique fashion.

An Increase in Circulating Mast Cell Colony-Forming Cells in Asthma

We compared a potential to generate mast cells among various sources of CD34+ peripheral blood (PB) cells in the presence of stem cell factor (SCF) with or without thrombopoietin (TPO), using a serum-deprived liquid culture system. From the time course of relative numbers of tryptase-positive and chymase-positive cells in the cultured cells grown by CD34+ PB cells of nonasthmatic healthy individuals treated with G-CSF, TPO appears to potentiate the SCF-dependent growth of mast cells without influencing the differentiation into mast cell lineage. CD34+ PB cells from asthmatic patients in a stable condition generated significantly more mast cells under stimulation with SCF alone or SCF+TPO at 6 wk of culture than did steady-state CD34+ PB cells of normal controls. Single-cell culture studies showed a substantial difference in the number of SCF-responsive or SCF+TPO-responsive mast cell progenitors in CD34+ PB cells between the two groups. In the presence of TPO, CD34+ PB cells from asthmatic children could respond to a suboptimal concentration of SCF to a greater extent, compared with the values obtained by those of normal controls. Six-week cultured mast cells of asthmatic subjects had maturation properties (intracellular histamine content and tryptase/chymase enzymatic activities) similar to those derived from mobilized CD34+ PB cells of nonasthmatic subjects. An increase in a potential of circulating hemopoietic progenitors to differentiate into mast cell lineage may contribute to the recruitment of mast cells toward sites of asthmatic mucosal inflammation.

STI571 inhibits growth and adhesion of human mast cells in culture

Stem cell factor (SCF)/c‐kit system is critical for human mast cell development. We thus examined the effects of STI571, an inhibitor of the c‐kit tyrosine kinase receptor, on the proliferation and function of human mast cells. STI571 at concentrations of 10−6 M or higher almost completely abolished the SCF‐dependent progeny generation from cord blood‐derived cultured mast cells through an inhibition of the tyrosine phosphorylation of c‐kit. The compound also suppressed the early phase of mast cell development. The extinction of mast cell growth induced by STI571 may be due largely to apoptosis according to the flow cytometric analysis and gel electrophoresis. Two‐hour exposure to STI571 that failed to influence the total viable cell number suppressed adhesion of the cells to fibronectin in the presence of SCF without altering the expressions of integrin molecules. Our results may provide a fundamental insight for the clinical application of STI571 in allergic disorders.

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.

Serum inhibitors for human mast cell growth: possible role of retinol

Background: In vitro culture systems have been used to study the physiological and pathological characteristics of human mast cells. However, there are some differences in proliferation and maturation of mast cells between fetal bovine serum (FBS)‐containing and serum‐deprived cultures. Accordingly, we attempted to identify circulating factor(s) affecting the development of human mast cells.

One-month relative dose intensity of not less than 50% predicts favourable progression-free survival in sorafenib therapy for advanced renal cell carcinoma in Japanese patients

BACKGROUND Sorafenib is a multikinase inhibitor used as a second-line treatment for metastatic renal cell carcinoma (mRCC). However, it is very difficult to estimate sorafenib dosage because it is difficult to maintain stable administration and dosage intervals due to several side-effects. We examined the correlation between relative dose intensity (RDI) and clinical outcome of sorafenib therapy in a multi-institutional study. METHODS A study population of 70 first-line therapy-refractory patients with pathologically confirmed RCC was eligible for this investigation. Clinical outcomes were evaluated according to clinicopathological features and RDI for 1 month (1M-RDI). RESULTS There was significant difference in progression-free survival (PFS) time but not overall survival (OS) time when the 1M-RDI cut-off value was ≥ 50%. In 15 patients (21.4%) with 1M-RDI of <50%, median PFS time was 4.1 months (95% I collagen (95% CI): 2.0-6.2), whereas it was 10.5 months (95% CI: 7.6-13.4) in the patients with 1M-RDI of ⩾50% (P=0.022). Multivariate analysis showed 1M-RDI status to be significantly associated with PFS (HR: 3.838, 95% CI: 1.658-8.883, P=0.002) but not OS (P=0.328). CONCLUSION Although this study was retrospective, a 1M-RDI cut-off value of ≥ 50% for sorafenib may be the first factor to predict PFS but not OS in cytokine pretreated mRCC patients. The data indicate that a dose of 400mg/day of sorafenib administered successively for the first one month was necessary to prolong disease stabilisation and could be tolerated by Japanese patients.

Prostacyclin Inhibits the Production of MMP-9 Induced by Phorbol Ester through Protein Kinase A Activation, but Does Not Affect the Production of MMP-2 in Human Cultured Mesangial Cells

Background/Aims: The imbalance between degradation and synthesis of the glomerular extracellular matrix (ECM) causes glomerular sclerosis in various types of glomerulonephritis. We investigated the effect of prostacyclin, which is an inflammatory mediator, on the production of matrix metalloproteinase (MMP)-9 and MMP-2 in human cultured mesangial cells. The synthesis of Jun proteins and Ets-1 proteins, which are related with MMP-9 gene, was also studied. Methods: The production of MMP-9 and MMP-2 was investigated by gelatin zymography. Western blotting was undertaken to analyze the protein synthesis of Jun and Ets-1. Results: Prostacyclin inhibited the production of MMP-9 induced by phorbol ester. The inhibitory effect by prostacyclin was reversed in part by the pretreatment with an inhibitor of protein kinase A, such as H-89. Forskolin also inhibited the production of MMP-9. The production of MMP-2 was constitutionally seen and was not influenced by prostacyclin and forskolin. The synthesis of Jun protein augmented by phorbol ester was suppressed by prostacyclin. Ets-1 protein was constitutionally synthesized and was not affected by phorbol ester and prostacyclin. Conclusion: Prostacyclin plays an important role in inflammatory glomerular disorders by regulating the metabolism of ECM. The production of MMP-9 and MMP-2 may be under the different control pathways.

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.

Neurologically normal development of a patient with severe methionine adenosyltransferase I/III deficiency after continuing dietary methionine restriction.

BACKGROUND There is not much information on established standard therapy for patients with severe methionine adenosyltransferase (MAT) I/III deficiency. CASE PRESENTATION We report a boy with MAT I/III deficiency, in whom plasma methionine and total homocysteine, and urinary homocystine were elevated. Molecular genetic studies showed him to have novel compound heterozygous mutations of the MAT1A gene: c.191T>A (p.M64K) and c.589delC (p.P197LfsX26). A low methionine milk diet was started at 31 days of age, and during continuing dietary methionine restriction plasma methionine levels have been maintained at less than 750 μmol/L. He is now 5 years old, and has had entirely normal physical growth and psychomotor development. CONCLUSIONS Although some severely MAT I/III deficient patients have developed neurologic abnormalities, we report here the case of a boy who has remained neurologically and otherwise normal for 5 years during methionine restriction, suggesting that perhaps such management, started in early infancy, may help prevent neurological complications.