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T Heike

Publications and source records attributed to T Heike.

At least 19 recordsLinked to original sources
PubMed2001

[Stem cell and self-renewal].

Stem cells are defined as cells with the ability for self-renewal and differentiation. Hematopoietic stem cells are well known, and their application is useful for the treatment of various kinds of diseases. Recently, neural stem cells have been identified even in the adult brain, which has up to now been considered to be a tissue with no regenerative capacity. In addition, it has emerged that tissue stem cells can differentiate into various kinds of cells beyond their original characteristics. Here, we discuss the self-renewal mechanisms of embryonic stem (ES) cells, hematopoietic stem cells and neural stem cells.

Animals↗
PubMed2000

Characterization of the gammac chain among 27 unrelated Japanese patients with X-linked severe combined immunodeficiency (X-SCID).

X-linked severe combined immunodeficiency (X-SCID) is a rare fatal disease that is caused by mutations in the gene encoding the gammac chain. In this study, 27 unrelated Japanese patients with X-SCID were examined in terms of their genetic mutations and surface expression of the gammac chain. Among 25 patients examined, excluding two patients with large deletions, 23 different mutations were identified in the IL2RG gene, including 10 novel mutations. One patient bearing an extracellular mutation and all three of the patients bearing intracellular mutations after exon 7 expressed the gammac chain on the cell surface. Overall, 84% of patients lacked surface expression of the gammac chain leading to a diagnosis of X-SCID.

Antibodies, Monoclonal↗
PubMed1999

Activation of Stat3 by cytokine receptor gp130 ventralizes Xenopus embryos independent of BMP-4.

Stat3 is one of the main signaling components of cytokine receptors, including gp130. Here we show that activation of cytokine receptor gp130 resulted in a dramatic ventralization of Xenopus embryos and that the ventralization correlated well with Stat3 activation potential of the receptor. This finding led to identification of Xenopus Stat3 (Xstat3), which showed a 95% homology to its murine and human counterparts, at the amino acid level, and was expressed from the one-cell stage throughout development. The mechanism of gp130/XStat3-mediated ventralization proved to be independent of BMP-4. gp130/Xstat3 stimulation inhibited Smad2-induced ectopic axis formation in embryos and Smad2-dependent luciferase activity. A dominant-negative Stat3, in contrast, dorsalized Xenopus embryos, resulting in ectopic axis formation. We propose that Stat3-mediated signaling has the capacity to modify dorsoventral patterning in the early development of Xenopus.

Animals↗
PubMed1999

Hypomethylation of the proximal and intronic regulatory regions of the IFN-gamma gene is not essential for its transcription by naive CD4+ T cells cultured with IL-4.

Recently, long-term preculture with IL-4 or IL-7 has been reported to induce IFN-gamma-producing ability in naive CD4+ T cells without stimulation via TCR. The mechanism of IFN-gamma-transcription in naive CD4+ T cells precultured with IL-4 was analyzed and compared with that in typical Th1 cells by focusing on the TATA proximal and first intronic regulatory regions of the IFN-gamma gene. Both regulatory regions in these IL-4-primed naive CD4+ T cells, which produce a large amount of IFN-gamma upon stimulation with PMA and ionomycin, were completely methylated in contrast to the same hypomethylated regions in Th1 cells. DNase I hypersensitive site analysis suggested that both regulatory regions in IL-4-primed naive CD4+ T cells were not active for IFN-gamma-expression. Moreover, we demonstrated that the composition of transcriptional factors that can bind to the proximal regulatory region is different between IL-4-primed naive CD4+ T cells and Th1 cells. These results indicated that the transcriptional machinery involved in the expression of the IFN-gamma gene by CD4+ T cells varied depending on their modes of differentiation in both the responsive regulatory regions and the specific nuclear factors.

CD4-Positive T-Lymphocytes↗
PubMed1999

STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells.

Embryonic stem (ES) cells can be maintained in an undifferentiated state in the presence of leukemia inhibitory factor (LIF). LIF acts through a receptor complex composed of a low affinity LIF receptor (LIFRbeta) and gp130. We reported that the intracellular domain of gp130 plays an important role in self-renewal of ES cells. In the present study, we examined the signaling pathway through which gp130 contributes to the self-renewal of ES cells. Mutational analysis of the cytoplasmic domain of gp130 revealed that the tyrosine residue of gp130 responsible for STAT3 activation is necessary for self-renewal of ES cells, while that required for SHP2 and MAP kinase activation was dispensable. Next, we constructed a fusion protein composed of the entire coding region of STAT3 and the ligand binding domain of the estrogen receptor. This construction (STAT3ER) induced expression of junB (one of the targets of STAT3) in ES cells in the presence of the synthetic ligand 4-hydroxytamoxifen (4HT), thereby indicating that STAT3ER is a conditionally active form. ES cells transfected with STAT3ER cultured in the presence of 4HT maintained an undifferentiated state. Taken together, these results strongly suggest that STAT3 activation is required and sufficient to maintain the undifferentiated state of ES cells.

Animals↗
PubMed1999

Cytoplasmic domains of the leukemia inhibitory factor receptor required for STAT3 activation, differentiation, and growth arrest of myeloid leukemic cells.

Leukemia inhibitory factor (LIF) induces growth arrest and macrophage differentiation of mouse myeloid leukemic cells through the functional LIF receptor (LIFR), which comprises a heterodimeric complex of the LIFR subunit and gp130. To identify the regions within the cytoplasmic domain of LIFR that generate the signals for growth arrest, macrophage differentiation, and STAT3 activation independently of gp130, we constructed chimeric receptors by linking the transmembrane and intracellular regions of mouse LIFR to the extracellular domains of the human granulocyte macrophage colony-stimulating factor receptor (hGM-CSFR) alpha and betac chains. Using the full-length cytoplasmic domain and mutants with progressive C-terminal truncations or point mutations, we show that the two membrane-distal tyrosines with the YXXQ motif of LIFR are critical not only for STAT3 activation, but also for growth arrest and differentiation of WEHI-3B D+ cells. A truncated STAT3, which acts in a dominant negative manner was introduced into WEHI-3B D+ cells expressing GM-CSFRalpha-LIFR and GM-CSFRbetac-LIFR. These cells were not induced to differentiate by hGM-CSF. The results indicate that STAT3 plays essential roles in the signals for growth arrest and differentiation mediated through LIFR.

Animals↗
PubMed1999

Chimeric cytokine receptor can transduce expansion signals in interleukin 6 receptor alpha (IL-6Ralpha)-, IL-11Ralpha-, and gp130-low to -negative primitive hematopoietic progenitors.

We generated transgenic mice expressing chimeric receptors, which comprise extracellular domains of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor and transmembrane and cytoplasmic domains of the mouse leukemia inhibitory factor receptor. In suspension cultures of lineage-negative (Lin(-)), 5-fluorouracil-resistant bone marrow cells of the transgenic mice, a combination of hGM-CSF and stem cell factor (SCF) induced exponential expansions of mixed colony-forming unit. The combination of hGM-CSF and SCF was effective on enriched, Lin(-)Sca-1(+)c-kit(+) progenitors and increased either mixed colony-forming unit or cobblestone area-forming cells. In case of stimulation with hGM-CSF and SCF, interleukin-6 (IL-6) and SCF, or IL-11 and SCF, the most efficient expansion was achieved with hGM-CSF and SCF. When Lin(-)Sca-1(+)c-kit(+)CD34(-) further enriched progenitors were clone sorted and individually incubated in the presence of SCF, hGM-CSF stimulated a larger number of cells than did IL-6, IL-6 and soluble IL-6 receptor (IL-6R), or IL-11. These data suggest the presence of IL-6Ralpha-, IL-11Ralpha-, and gp130-low to -negative primitive hematopoietic progenitors. Such primitive progenitors are equipped with signal transduction molecules and can expand when these chimeric receptors are genetically introduced into the cells and stimulated with hGM-CSF in the presence of SCF.

Animals↗
PubMed1998

A selective switch-on system for self-renewal of embryonic stem cells using chimeric cytokine receptors.

Propagation of embryonic stem (ES) cells with an undifferentiated pluripotential phenotype depends on leukemia inhibitory factor (LIF). The LIF receptor complex is composed of a heterodimer of LIF receptor alpha (LIFR alpha) and gp130. To activate LIFR signaling pathways independently from endogenous ones, we constructed chimeric receptors by linking the extracellular domain of human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha or beta (hGMR alpha or beta) to the transmembrane and cytoplasmic regions of either mouse LIFR alpha or gp130. hGMR alpha-mLIFR/hGMR beta-mgp130 or hGMR alpha-mgp130/hGMR beta-mgp130, but not hGMR alpha-mLIFR/hGMR beta-mLIFR, preserved the self-renewal activity in A3 ES cells. All of these chimeric receptors were phosphorylated after hGM-CSF stimulation without phosphorylation of endogenous gp130. Phosphorylation of the signal transducer and activator of transcription 3 through chimeric receptors correlated with the undifferentiated phenotype. Therefore, these chimeric receptors prove useful to analyze mechanisms of the self-renewal of ES cells.

Animals↗
PubMed1998

IL-4 and prostaglandin E2 inhibit hypomethylation of the 5' regulatory region of IFN-gamma gene during differentiation of naive CD4+ T cells.

We have previously shown that prostaglandin E2 (PGE2) and IL-4 inhibit the priming of IFN-gamma-production during the differentiation of naive CD4+ T cells from human cord blood by different signal-transducing mechanisms. To compare and analyse the molecular mechanisms by which PGE2 and IL-4 inhibit the priming of IFN-gamma production, we investigated the effects of PGE2 and IL-4 on the methylation of the IFN-gamma gene during the in vitro differentiation of naive CD4+ T cells. In human naive CD4+ T cells, which produce primarily IL-2 and a little amount of IFN-gamma, the IFN-gamma gene was methylated. After stimulation via TCR, CD4+ T cells produced IFN-gamma and the CpG dinucleotide contained within the TATA proximal regulatory element of the IFN-gamma gene was partially hypomethylated. Both IL-4 and PGE2 inhibited the hypomethylation of this site and the acquisition of IFN-gamma-producing ability. In contrast to the SnaBI site in the TATA proximal regulatory element, the HpalI site in the first intron of the IFN-gamma gene of the CD4+ T cells from cord blood was completely methylated even after stimulation via TCR. 5-azacytidine restored the IFN-gamma-producing ability of these cells treated with IL-4 and PGE2. These findings suggest that, although the signal transduction that inhibits the priming of IFN-gamma-production is different for each reagent, the protection from hypomethylation of the regulatory region of the IFN-gamma gene is involved in the molecular mechanisms by which these reagents inhibit the priming of IFN-gamma-production during the differentiation of human naive CD4+ T cells.

Azacitidine↗
PubMed1997

X-linked severe combined immunodeficiency with gamma delta T cells.

A patient with X-linked severe combined immunodeficiency (X-SCID) was found to have a deletion mutation of a four base pair in the transmembrane domain of the IL-2 receptor gamma chain gene, a subunit shared by the receptors for IL-4, IL-7, IL-9, and IL-15 (common gamma chain; gamma c). He had very few alpha beta T cells but had a considerable number of gamma delta T cells in his peripheral blood. Fluorescence in situ hybridization (FISH) analysis showed that the gamma delta T cells in his peripheral blood were not of maternal origin. He had received a Bacillus Calmette-Guerin (BCG) vaccination before recognition of the disease, and the BCG infection remained quiescent with no reaction for 19 months. After successful bone marrow transplantation, the site of the BCG vaccination showed a reaction, and live BCG were detected. It is useful to consider the relationship between the existence of gamma delta T cells and BCG in this case, and it is suggested that gamma delta T cells may be, in a given situation, less dependent on the gamma c chain than are alpha beta T cells.

Gene Deletion↗
PubMed1996

Signal transduction pathway of interleukin-4 and interleukin-13 in human B cells derived from X-linked severe combined immunodeficiency patients.

Interleukin-4 (IL-4) and IL-13 are functionally similar cytokines. The functional IL-4 receptor (IL-4R) consists of the IL-4R alpha chain (IL-4R alpha) and the IL-2R gamma chain (gamma c), which is shared by the IL-2, IL-7, IL-9, and IL-15 receptors. The functional IL-13R is thought to involve the IL-4R alpha but not gamma c. In this study, we have analyzed activation of members of the Janus tyrosine kinase (Jak) family and signal transducers and activators of transcription (STAT) 6 induced by IL-4 and IL-13 in Epstein-Barr virus-transformed B cells derived from two patients of X-linked severe combined immunodeficiency, who have mutations of the gamma c gene in the extracellular and intracellular domains. In these B cells, IL-4 failed to induce tyrosine phosphorylation of Jak3 and activation of STAT6, or activation of these molecules was significantly decreased compared with Epstein-Barr virus-transformed normal B cells. In contrast, IL-13 activated STAT6 in these cells as well as normal B cells. However, Jak3 was not activated by IL-13, even in normal B cells. These results clearly indicated that gamma c is essential for activation of Jak3 and STAT6 in the signal transduction pathway of IL-4 in human B cells and that IL-13 does not utilize gamma c but activates STAT6 through an alternative pathway, which is not impaired in B cells of X-linked severe combined immunodeficiency patients.

B-Lymphocytes↗
PubMed1996

Negative signaling in B cells by surface immunoglobulins.

Cross-linking of surface immunoglobulins generates negative signals that cause B-cell death unless appropriate rescue signals are provided. Surface IgM is the main transducer of the negative signaling, but surface IgD and IgG may also transduce negative signaling when cross-linked intensively. In the surface IgM+, IgD+ human malignant B lymphoma cell lines B104 and DND-39, cross-linking of surface IgM by anti-IgM antibodies induced cell death. Anti-IgM antibody-induced B104 cell death was inhibited by stimulation with alpha- and beta-interferons but not stimulation with anti-CD40 antibody or IL-4, whereas anti-IgM antibody-induced DND-39 cell death was inhibited by stimulation with anti-CD40 antibody but not stimulation with alpha- and beta-interferons. Anti-IgM antibody-stimulated B104 cells had morphologic features compatible with necrosis, whereas anti-IgM antibody-stimulated DND-39 cells showed morphologic features of apoptosis. CD11a/CD54-dependent cell adhesion induced by stimulation with anti-CD40 antibody was involved in anti-CD40 antibody-mediated inhibition of anti-IgM antibody-induced DND-39 cells. In normal human mature B cells, cross-linking of surface IgM induced different signaling consequences, including DNA synthesis or cell division (positive signaling) or cell cycle arrest or death (negative signaling). In this system, too, CD40-transduced signal inhibited anti-IgM antibody-induced negative signaling, and CD11a/CD54-dependent cell adhesion played a role in the rescue process. It is suggested that quantitatively different intensities of surface IgM cross-linking induce qualitatively different signaling consequences; relatively weak cross-linking may induce DNA synthesis; moderate cross-linking may induce DNA synthesis with cell cycle arrest at the G2/M interphase; and intense cross-linking may induce apoptotic cell death. The reasons for this difference are not yet known. Further elucidation of the molecular mechanisms responsible for surface IgM-mediated negative signaling and its rescue signaling may contribute toward development of therapy for allergic disorders by artificial modulation of specific immunoglobulin production.

Animals↗
PubMed1995

Pyrrolidine dithiocarbamate inhibits intercellular adhesion molecule-1 biosynthesis induced by cytokines in human fibroblasts.

Intercellular adhesion molecule-1 (ICAM-1), the ligand of lymphocyte function-associated antigen-1, plays an important role in the interactions of a variety of hemopoietic and nonhemopoietic cells, including leukocytes, fibroblasts, and endothelial cells. ICAM-1 is known to be involved in the onset of several diseases such as inflammation, allograft rejection, and so on. In this report, we investigated the effects of dexamethasone, cyclosporin A, FK506, and pyrrolidine dithiocarbamate (PDTC) on the induction of the ICAM-1 gene by cytokines in fibroblasts. PDTC, a potent inhibitor of NF-kappa B, was shown by ELISA and FACS analysis to prevent dramatically the expression of the ICAM-1 gene stimulated by IL-1 alpha, IFN-gamma, and PMA, although the other reagents inhibited it only slightly. Ribonuclease protection assay revealed that PDTC blocked the expression of the ICAM-1 gene at the mRNA level. To elucidate the mechanism of this inhibition, we constructed a series of ICAM-1 promoter deletion mutants linked to the chloramphenicol acetyl transferase gene and analyzed the effect of PDTC on their activities. Transient transfection analysis indicated that the critical region for inhibition by PDTC is an NF-kappa B binding site-like motif (GGGAGGATTCC, ICAM-1 kappa B) that is located at position-540. Electrophoresis mobility shift assay revealed that PDTC actually inhibits the binding of NF-kappa B (or NF-kappa B-like) protein to the ICAM-1 kappa B site. These findings suggest that PDTC inhibits ICAM-1 gene expression by inhibiting the association of NF-kappa B (or NF-kappa B-like) protein with the ICAM-1 kappa B site.

Base Sequence↗
PubMed1995

Cis-acting DNA elements of mouse granulocyte-macrophage colony-stimulating factor gene responsive to Fc epsilon receptor cross-linking stimulation in the mouse mast cell line MC/9.

Mouse mast cells produce many kinds of cytokines in response to cross-linking of high affinity Fc epsilon receptor (Fc epsilon RI). Among these cytokines, granulocyte-macrophage CSF (GM-CSF) gene induction in mouse mast cells has been reported to be regulated at both the transcriptional level and the post-transcriptional level. We analyzed the mechanism of the transcriptional regulation of GM-CSF gene induction through Fc epsilon RI cross-linking stimulation in the mouse mast cell line MC/9. In MC/9, the GM-CSF gene was activated transcriptionally by Fc epsilon RI cross-linking stimulation. The 5' deletion analysis of GM-CSF gene promoter indicated that the 5' boundary of the responsive promoter region lay between positions -113 and -95. When the deletion was extended to positions -72 or -60, the stimulatory effect was significantly diminished. We then examined 3' deletion of pmGMCAT -113 from position -60. This analysis indicated that the 3' boundary lay between positions -84 and -72. No subfragments of the region spanning positions -113 to -72 could cover the full induction level. A site-directed mutagenesis experiment revealed that the sequence spanning positions -108 to -72 was needed for full activation. These data indicate that GM-CSF gene in mast cells is activated mainly through the sequence spanning positions -108 to -72.

Animals↗
PubMed1995

Role of LFA-1/ICAM-1-dependent cell adhesion in CD40-mediated inhibition of anti-IgM antibody-induced B-cell death.

Cross-linking of surface IgM by anti-IgM antibody caused activation-induced cell death of a surface IgM+, IgD+ human B lymphoma cell line, B104. The dying B104 cells did not show the morphology of apoptosis but did show that of necrosis. However, anti-IgM antibody caused apoptosis of another surface IgM+, IgD+ human B lymphoma cell line, DND-39. The influx of extracellular Ca2+ was necessary for the cell deaths of B104 and DND-39 caused by anti-IgM antibody. Their cell deaths were inhibited by cyclosporine. The anti-IgM antibody-induced cell death of DND-39, but not that of B104, was prevented by costimulation with anti-CD40 antibody. In human peripheral blood B-cells, anti-IgM antibody inhibited cell cycle transition induced by Staphylococcus aureus Cowan I at the G2/M interphase without inhibition of DNA synthesis. In this system, too, anti-CD40 antibody canceled the inhibitory signal transduced through surface IgM and increased the number of M phase cells. Blocking antibodies against the leukocyte function-associated antigen-I/intercellular adhesion molecule-1 system decreased the rescue effect of anti-CD40 antibody in both DND-39 cells and peripheral B-cells, which shows that leukocyte function-associated antigen-1/intercellular adhesion molecule-1-dependent cell adhesion plays an important role in the CD40-mediated inhibition of surface IgM-mediated negative signals.

Animals↗
PubMed1995

IgM-mediated B cell apoptosis.

Cross-linking of surface immunoglobulin M (sIgM) on normal mature B cells induces different signaling consequences, including DNA synthesis (positive signaling) and cell cycle arrest and/or death by apoptosis (negative signaling). Presumably, the difference depends on the intensity of sIgM cross-linking: relatively weak cross-linking induces DNA synthesis, moderate cross-linking induces DNA synthesis with cell cycle arrest at the G2/M interphase, and intense cross-linking induces apoptosis. In vivo experiments with transgenic mice have shown that relatively weak cross-linking of sIgM by soluble antigens induces anergy in autoreactive B cells, whereas intense sIgM cross-linking by membrane-bound forms of antigens induces deletion of them. However, it is still unknown whether the different intensities of sIgM cross-linking generate qualitatively different signals responsible for DNA synthesis or cell death or whether they generate qualitatively the same but quantitatively different signals, and the quantitative difference is responsible for the induction of positive or negative signaling. The sIgM-mediated negative signaling presumably plays an important role in the induction and maintenance of B cell tolerance, and sIgD and sIgG also possess the machinery necessary for negative signaling. Negative signaling through sIgM is dependent on tyrosine kinase(s) and Ca2+ influx and is sensitive to cyclosporin A in certain types of B cells but not in all B cells. It has been suggested that there are different intracellular signaling pathways that transduce negative signaling via sIgM, and that activation-induced B cell death by sIgM cross-linking does not necessarily show DNA fragmentation and the morphology of apoptosis. On the other hand, sIgM-mediated B cell death may be inhibited in the presence of appropriate co-stimulators such as IL-4, alpha-, and beta-interferons and CD40-mediated signaling. The CD40-mediated signaling effectively inhibits sIgM-mediated B cell apoptosis in many but not all experimental systems. Although homotypic cell adhesion through the LFA-1/ICAM-1 dependent pathway was shown to be involved in certain types of CD40-mediated inhibition of sIgM-mediated negative signaling, it is still not known how the cytokines and CD40-mediated signaling inhibit sIgM-mediated B cell death. The molecular mechanisms responsible for sIgM-mediated negative signaling and for the inhibitory signaling against sIgM-mediated negative signaling need further elucidation.

Animals↗
PubMed1994

Involvement of LFA-1/intracellular adhesion molecule-1-dependent cell adhesion in CD40-mediated inhibition of human B lymphoma cell death induced by surface IgM crosslinking.

B cells have been shown to receive negative signals for their growth through crosslinking of surface IgM (sIgM), and it has been demonstrated that anti-IgM Abs induce B cell death. Proliferation of B cells in response to Ag stimulation in vivo may thus require additional signals that inhibit the sIgM-transduced negative signals. Signaling through CD40 has been proposed as a candidate for such costimulatory signals. To investigate the role of CD40-transduced signals in sIgM-mediated B cell death, we used a human B cell line (DND-39) that expresses sIgM, sIgD, and CD40. Crosslinking of sIgM, but not sIgD, by Abs induced DND-39 cell death. The dying cells showed the morphology of apoptosis and DNA fragmentation. Anti-CD40 Abs induced homotypic adhesion of DND-39 cells and rescued them from anti-IgM Ab-induced cell death. Anti-CD40 Abs inhibited anti-IgM Ab-induced cell death when added within 3 h after stimulation with anti-IgM Ab. Treatment with Abs against CD11a, CD18, or CD54 inhibited not only the homotypic adhesion but also the inhibition of anti-IgM Ab-induced apoptosis by anti-CD40 Ab. CD11a antisense decreased the surface CD11a expression, the anti-CD40 Ab-induced homotypic adhesion, and the inhibitory effect of anti-CD40 Ab on anti-IgM Ab-induced apoptosis. The data show that LFA-1/ICAM-1-dependent cell adhesion induced by signaling through CD40 plays an important role in the inhibition of anti-IgM Ab-induced apoptosis of DND-39 cells.

Antigens, CD↗