Biomedical subjects

K Sugamura

Publications and source records attributed to K Sugamura.

At least 37 records · Page 2Linked to original sources

PubMed2000

Cell-fate conversion of lymphoid-committed progenitors by instructive actions of cytokines.

The primary role of cytokines in haemato-lymphopoiesis is thought to be the regulation of cell growth and survival. But the instructive action of cytokines in haematopoiesis has not been well addressed. Here we show that a clonogenic common lymphoid progenitor, a bone marrow-resident cell that gives rise exclusively to lymphocytes (T, B and natural killer cells), can be redirected to the myeloid lineage by stimulation through exogenously expressed interleukin (IL)-2 and GM-CSF (granulocyte/macrophage colony-stimulating factor) receptors. Analysis of mutants of the beta-chain of the IL-2 receptor revealed that the granulocyte- and monocyte-differentiation signals are triggered by different cytoplasmic domains, showing that the signalling pathway(s) responsible for these unique developmental outcomes are separable. Finally, we show that the endogenous myelomonocytic cytokine receptors for GM-CSF and macrophage colony-stimulating factor (M-CSF) are expressed at low to moderate levels on the more primitive haematopoietic stem cells, are absent on common lymphoid progenitors, and are upregulated after myeloid lineage induction by IL-2. We conclude that cytokine signalling can regulate cell-fate decisions and propose that a critical step in lymphoid commitment is downregulation of cytokine receptors that drive myeloid cell development.

Animals↗

PubMed2000

In vivo competitive studies between normal and common gamma chain-defective bone marrow cells: implications for gene therapy.

Corrective gene transfer into hematopoietic stem cells (HSCs) is being investigated as therapy for X-linked severe combined immunodeficiency (XSCID) and it is hoped that selective advantage of gene-corrected HSCs will help in achieving full immune reconstitution after treatment. Lines of evidence from the results of allogeneic bone marrow transplantation in patients with XSCID support this hypothesis that, however, has not been rigorously tested in an experimental system. We studied the competition kinetics between normal and XSCID bone marrow (BM) cells using a murine bone marrow transplantation (BMT) model. For easy chimerism determination, we used genetic marking with retrovirus-mediated expression of the enhanced green fluorescent protein (EGFP). We found that XSCID BM cells were able to compete with normal BM cells for engraftment of myeloid lineages in a dose-dependent manner, whereas we observed selective repopulation of T, B, and NK cells deriving from normal BM cells. This was true despite the evidence of competitive engraftment of XSCID lineage marker-negative/c-Kit-positive (Lin-/c-Kit+) cells in the bone marrow of treated animals. From these results we extrapolate that genetic correction of XSCID HSCs will result in selective advantage of gene-corrected lymphoid lineages with consequent restoration of lymphocyte populations and high probability of clinical benefit.

Animals↗

PubMed2000

Involvement of LAT, Gads, and Grb2 in compartmentation of SLP-76 to the plasma membrane.

B cell linker protein (BLNK) and Src homology 2 domain-containing leukocyte protein of 76 kD (SLP-76) are adaptor proteins required for B cell receptor (BCR) and T cell receptor function, respectively. Here, we show that expression of SLP-76 cannot reconstitute BCR function in Zap-70(+)BLNK(-) B cells. This could be attributable to inability of SLP-76 to be recruited into glycolipid-enriched microdomains (GEMs) after antigen receptor cross-linking. Supporting this idea, the BCR function was restored when a membrane-associated SLP-76 chimera was enforcedly localized to GEMs. Moreover, we demonstrate that addition of both linker for activation of T cells (LAT) and Grb2-related adaptor downstream of Shc (Gads) to SLP-76 allow SLP-76 to be recruited into GEMs, whereby the BCR function is reconstituted. The Gads function was able to be replaced by overexpression of Grb2. In contrast to SLP-76, BLNK did not require Grb2 families for its recruitment to GEMs. Hence, these data suggest a functional overlap between BLNK and SLP-76, while emphasizing the difference in requirement for additional adaptor molecules in their targeting to GEMs.

Adaptor Proteins, Signal Transducing↗

PubMed2000

STAM2, a new member of the STAM family, binding to the Janus kinases.

We here cloned a cDNA encoding STAM2, a new member of the STAM family, which contains an SH3 domain and ITAM. STAM2 like STAM1 is associated with Jak2 and Jak3, and involved in the signaling for DNA synthesis and c-myc induction mediated by IL-2 and GM-CSF. Co-expression of the SH3 deletion mutants of STAM1 and STAM2 induces an additive effect on suppressing DNA synthesis upon stimulation with IL-2 and GM-CSF, suggesting that STAM1 and STAM2 exhibit compensatory effects on the signaling pathways downstream of Jak2 and Jak3 upon stimulation with GM-SCF and IL-2, respectively.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Cutting edge: differential production of prostaglandin D2 by human helper T cell subsets.

Several effector molecules, including cytokines, are differentially produced by Th1 and Th2 cells. We used a gene expression screen method to identify a gene encoding hematopoietic PG D synthase (hPGDS) which was preferentially expressed in human Th2 but not Th1 clones. Studies with anti-hPGDS mAbs confirmed the Th2-dominated expression of hPGDS protein. Upon stimulation with anti-CD3 plus anti-CD28 mAbs, coordinated cyclooxygenase-2 expression and PGD2 production were induced in Th2 lines. hPGDS expression was also observed in a small population (<1.0%) of peripheral blood CD4+ lymphocytes from healthy adults. Most hPGDS-expressing CD4+ lymphocytes showed a typical Th2-type cytokine pattern. Our results suggest that, at the sites of Ag presentation, at least part of the Th2 cell population produces PGD2, which may be involved in various aspects of Th2-related immune responses similar to mast cells.

Adult↗

PubMed2000

Impairment of antigen-presenting cell function in mice lacking expression of OX40 ligand.

OX40 expressed on activated T cells is known to be an important costimulatory molecule on T cell activation in vitro. However, the in vivo functional significance of the interaction between OX40 and its ligand, OX40L, is still unclear. To investigate the role of OX40L during in vivo immune responses, we generated OX40L-deficient mice and a blocking anti-OX40L monoclonal antibody, MGP34. OX40L expression was demonstrated on splenic B cells after CD40 and anti-immunoglobulin (Ig)M stimulation, while only CD40 ligation was capable of inducing OX40L on dendritic cells. OX40L-deficient and MGP34-treated mice engendered apparent suppression of the recall reaction of T cells primed with both protein antigens and alloantigens and a significant reduction in keyhole limpet hemocyanin-specific IgG production. The impaired T cell priming was also accompanied by a concomitant reduction of both T helper type 1 (Th1) and Th2 cytokines. Furthermore, antigen-presenting cells (APCs) derived from the mutant mice revealed an impaired intrinsic APC function, demonstrating the importance of OX40L in both the priming and effector phases of T cell activation. Collectively, these results provide convincing evidence that OX40L, expressed on APCs, plays a critical role in antigen-specific T cell responses in vivo.

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↗

PubMed2000

Expression of OX40 and OX40 ligand (gp34) in the normal and myasthenic thymus.

OBJECTIVES: To examine the expression of OX40, an activated memory T-cell marker, and its ligand (OX40L), a set of molecules for T-cell-B-cell interaction, and other lymphocyte activation markers in the thymuses of myasthenia gravis (MG) and controls. MATERIAL AND METHODS: We studied the expression of OX40, OX40L, IL-2Ralpha and HLA-DR in the thymic tissues of MG and controls using immunocytochemistry and flowcytometry. RESULTS: In both hyperplastic thymus of MG and control thymus, OX40+ cells were scattered mainly in the medulla with much fewer OX40L+ cells being distributed in the corticomedullary junctions. IL-2Ralpha and HLA-DR were expressed in the medulla at higher frequencies as compared with OX40 in controls as well as MG. In contrast, the numbers of OX40+ cells around the germinal centers (GC) were significantly greater than those of control thymuses, and some mononuclear cells in GC were OX40L+. A considerable number of OX40+ cells were seen in the thymic tissues adjacent to thymomas. OX40+ cells were CD4+ CD8- or CD4+ CD8+ and were mostly HLA-DR-. (The coexpression of OX40 and IL-2Ralpha on activated CD4+ T cells was previously reported.) CONCLUSION: OX40, expressed in a fraction of activated CD4+ T cells, may be upregulated in thymic tissues adjacent to GC and thymoma in MG, and OX40 may interact with OX40L in GC to enhance anti-acetylcholine receptor antibody production in MG.

Adolescent↗

PubMed2000

Hgs (Hrs), a FYVE domain protein, is involved in Smad signaling through cooperation with SARA.

Smad proteins are effector molecules that transmit signals from the receptors for the transforming growth factor beta (TGF-beta) superfamily to the nucleus; of the Smad proteins, Smad2 and Smad4 are essential components for mouse early embryogenesis. We demonstrated that Hgs, a FYVE domain protein, binds to Smad2 in its C-terminal half and cooperates with another FYVE domain protein, the Smad anchor for receptor activation (SARA), to stimulate activin receptor-mediated signaling through efficient recruitment of Smad2 to the receptor. Furthermore, a LacZ knock-in allele of the C-terminal half-deletion mutant of mouse Hgs was created by gene targeting. The introduced mutation causes an embryonic lethality between embryonic days 8.5 and 10.5. Mutant cells showed significantly decreased responses to stimulation with activin and TGF-beta. These findings suggest that the two FYVE domain proteins, Hgs and SARA, are prerequisites for receptor-mediated activation of Smad2.

Activin Receptors↗

PubMed1999

CRTH2, an orphan receptor of T-helper-2-cells, is expressed on basophils and eosinophils and responds to mast cell-derived factor(s).

We have recently cloned a putative chemoattractant receptor, named CRTH2, which is preferentially expressed on human T-helper- (Th) 2 but not Th1 cells. In this study, we demonstrated that CRTH2 is also highly expressed on peripheral blood basophils and eosinophils. Our search for a CRTH2 ligand identified mast cells as the possible producers of a ligand. When stimulated with an anti-FcepsilonR1 antibody, cord blood-derived mast cells secreted factor(s) that induced Ca(2+) mobilization in CRTH2-expressing K562 cells but not in mock transfected cells. These findings implied the involvement of CRTH2 in mast cell-mediated immune responses such as allergic reactions.

Basophils↗

PubMed1999

Possible involvement of a novel STAM-associated molecule "AMSH" in intracellular signal transduction mediated by cytokines.

STAM containing an SH3 (Src homology 3) domain and an immunoreceptor tyrosine-based activation motif was previously revealed to be implicated in signaling pathways immediately downstream of Jak2 and Jak3 tyrosine kinases associated with cytokine receptors. We molecularly cloned a novel molecule interacting with the SH3 domain of STAM, which was named AMSH (associated molecule with the SH3 domain of STAM). AMSH contains a putative bipartite nuclear localization signal and a homologous region of a c-Jun activation domain-binding protein 1 (JAB1) subdomain in addition to a binding site for the SH3 domain of STAM. AMSH mutant deleted of the C-terminal half conferred dominant negative effects on signaling for DNA synthesis and c-myc induction mediated by interleukin 2 and granulocyte macrophage-colony-stimulating factor. These results suggest that AMSH plays a critical role in the cytokine-mediated intracellular signal transduction downstream of the Jak2/Jak3.STAM complex.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Grf40, A novel Grb2 family member, is involved in T cell signaling through interaction with SLP-76 and LAT.

We molecularly cloned a new Grb2 family member, named Grf40, containing the common SH3-SH2-SH3 motif. Expression of Grf40 is predominant in hematopoietic cells, particularly T cells. Grf40 binds to the SH2 domain-containing leukocyte protein of 76 kD (SLP-76) via its SH3 domain more tightly than Grb2. Incidentally, Grf40 binds to linker for activation of T cells (LAT) possibly via its SH2 domain. Overexpression of wild-type Grf40 in Jurkat cells induced a significant increase of SLP-76-dependent interleukin (IL)-2 promoter and nuclear factor of activated T cell (NF-AT) activation upon T cell receptor (TCR) stimulation, whereas the COOH-terminal SH3-deleted Grf40 mutant lacked any recognizable increase in IL-2 promoter activity. Furthermore, the SH2-deleted Grf40 mutant led to a marked inhibition of these regulatory activities, the effect of which is apparently stronger than that of the SH2-deleted Grb2 mutant. Our data suggest that Grf40 is an adaptor molecule involved in TCR-mediated signaling through a more efficient interaction than Grb2 with SLP-76 and LAT.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Molecular cloning of a glycosylphosphatidylinositol-anchored molecule CDw108.

CDw108, also known as the John-Milton-Hagen human blood group Ag, is an 80-kDa glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that is preferentially expressed on activated lymphocytes and E. The molecular characteristics and biological function of the CDw108 were not clarified previously. In this manuscript, we identify the cDNA clone containing the entire coding sequence of the CDw108 gene and report its molecular characteristics. The 1998-base pairs of the open reading frame of the cloned cDNA encoded a protein of 666 amino acids (aa), including the 46 aa of the signal peptide and the 19 aa of the GPI-anchor motif. Thus, the membrane-anchoring form of CDw108 was the 602 aa, and the estimated molecular mass of the unglycosylated form was 68 kDa. The RGD (Arg-Gly-Asp) cell attachment sequence and the five potential N-linked glycosylation sites were located on the membrane-anchoring form. Flow cytometric and immunoprecipitation analyses of the CDw108 cDNA transfectants confirmed that the cloned cDNA encoded the native form of CDw108. The CDw108 mRNA was expressed in activated PBMCs as well as in the spleen, thymus, testis, placenta, and brain, but was not expressed in any other tissues tested. Radiation hybrid mapping indicated that the CDw108 gene was located in the middle of the long arm of chromosome 15 (15q23-24). This molecular information will be critical for understanding the biological function of the CDw108 Ag.

Amino Acid Sequence↗

PubMed1999

Molecular cloning, chromosome mapping and characterization of the mouse CRTH2 gene, a putative member of the leukocyte chemoattractant receptor family.

We have cloned the mouse CRTH2 (chemoattractant receptor-homologous molecule expressed on TH2 cells) gene encoding a putative leukocyte chemoattractant receptor, of which human homologue is expressed selectively in Th2 but not in Th1 clones among T cell clones. The deduced amino-acid sequence of mouse CRTH2 bears 77% identity with its human homologue. Phylogenetic analysis suggested that both mouse and human CRTH2 are closely related to the N-formyl peptide receptor and the C5a receptor among leukocyte chemoattractant receptors. The mouse CRTH2 gene was mapped on chromosome 19c with FISH, where no other genes for leukocyte chemoattractant receptors are mapped. RT-PCR analysis revealed that mouse CRTH2 mRNA is expressed in various cell lineages, including both hematopoietic and non-hematopoietic cell lines. Expression was also observed in liver, lung, kidney, brain, heart, thymus, and spleen. These results suggest that mouse CRTH2 functions in a variety of cells, making the effects of CRTH2 pleiotropic.

Amino Acid Sequence↗

PubMed1999

Selective expression of a novel surface molecule by human Th2 cells in vivo.

The search for reliable marker molecules discriminating between human Th1 and Th2 cells identified a gene encoding a novel member of the G protein-coupled leukocyte chemoattractant receptor family, which is selectively expressed in Th2 but not Th1 lineage cells, thereby named CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells). Studies with anti-CRTH2 mAbs demonstrated that CRTH2 was expressed in a small population (0.4-6.5%) of CD4+ T cells in fresh PBMCs of healthy adults, but no remarkable expression was seen in B cells and NK cells. In some cases, CD8+ T cells ( approximately 3.5%) expressed CRTH2. Phenotypes of CD4+ T cells expressing CRTH2 were CD45RA-, CD45RO+, and CD25+, similar to those of Ag-activated effector/memory T cells. Freshly isolated CRTH2+ CD4+ T cells produced Th2- but little or no Th1-type cytokines upon stimulation with PMA and ionomycin. In addition, an allergen-induced proliferative response in fresh PBMCs was significantly and selectively reduced by subtracting CRTH2+ cells. Together, these results indicate that CRTH2 is selectively expressed in vivo in an activated state of Th2 cells including allergen-responsive Th2 cells, suggesting its pivotal roles in ongoing Th2-type immune reactions.

Adult↗

PubMed1999

Functional role of interleukin-4 (IL-4) and IL-7 in the development of X-linked severe combined immunodeficiency.

X-linked severe combined immunodeficiency (X-SCID) is characterized by an absent or diminished number of T cells and natural-killer (NK) cells with a normal or elevated number of B cells, and results from mutations of the gammac chain. The gammac chain is shared by interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15 receptors. Recently, a survival signal through the IL-7 receptor alpha (IL-7Ralpha) chain was shown to be important for T-cell development in mice and was suggested to contribute to the X-SCID phenotype. In the present study, we examined function of a mutant gammac chain (A156V) isolated from an X-SCID patient and found that T cells expressing the mutant gammac chain were selectively impaired in their responses to IL-4 or IL-7 compared with the wild-type gammac chain expressing cells although responses to IL-2 or IL-15 were relatively maintained. The result shows that IL-4- and/or IL-7-induced signaling through the gammac chain is critical for T-cell development and plays an important role in the development of the X-SCID phenotype.

Genetic Linkage↗

PubMed1999

Serologic study of human parvovirus B19 infection in pregnancy in Japan.

OBJECTIVES: To clarify the relationship between hydrops fetalis and parvovirus outbreaks in the community, seroprevalence of B19 antibody among women of childbearing age, and adverse effects of intrauterine B19 infection. METHODS: Sera were collected from 168 cases of hydrops fetalis which were diagnosed between 1987 and 1997 in Miyagi prefecture, Japan, from 232 healthy pregnant women in 1987 and 277 healthy pregnant women in 1997 in Miyagi, and from 48 women infected with B19 during pregnancy. The sera were examined for B19 IgG and IgM antibodies by enzyme-linked immunosorbent assay and for B19 DNA by polymerase chain reaction. The number of cases of erythema infectiosum in Miyagi had been monitored each month. RESULTS: Thirteen of the 168 cases of hydrops fetalis were found to be caused by intrauterine B19 infection and 12 of the 13 cases clustered in two periods of outbreaks of erythema infectiosum in the community. The positive rates of B19 IgG antibody between 1987 and 1997 were significantly different: 33% in 1987 and 46% in 1997. Nine of the 48 women infected during pregnancy showed adverse effects of the fetus: eight hydrops fetalis and one early abortion with positive B19 DNA. The fetal death rate (>12 weeks of gestation) among them was 15% (7/48), far higher than the calculated 1% among the general population. The nine mothers with adverse fetal outcomes had contact with the infectious source at the 16 weeks of gestation or earlier. CONCLUSIONS: These data clearly showed a relationship between hydrops fetalis and parvovirus outbreaks in the community, and it may be important to follow the seroprevalence for an extrapolated period time to predict occurrence of hydrops fetalis caused by B19. Also the data indicated that the gestational week infection occurred is the most important determinant of an adverse effect to the fetus as described previously.

Adolescent↗

PubMed1999

Parvovirus B19 infection induces apoptosis of erythroid cells in vitro and in vivo.

OBJECTIVE: intrauterine parvovirus B19 infection is related to non-immune hydrops fetalis, the pathogenesis of which is based on the strict tropism of B19 for erythroid precursor cells and the massive destruction of the infected erythroid cells, although the mechanism of beta19-induced cytotoxicity has not been studied in detail. The purpose of this study is to provide empirical evidence that beta19 induces apoptosis of erythroid cells both in vitro and ill vivo. METHODS: we analysed culture cells infected in vitro by B19 and tissues of nine cases of hydrops fetalis caused by B19 intrauterine infection by histological and biological methods. RESULTS: cells infected iil vitro by B19 showed nuclear changes characteristic of apoptosis by light microscopic examination and DNA extracted from the infected cells was fragmented. Electron microscopic examination showed the nuclei of infected cells contained crescent-shaped clumps of heterochromatin with increased density and double staining with anti-B1 9 antibody and terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling (TUNEL) confirmed apoptosis of individual cells. Tissues of cases of hydrops fetalis caused by B19 contained erythroid cells with nuclear inclusions and characteristic nuclear changes of apoptosis by light microscopy. The double-staining confirmed apoptosis of erythroid cells in the tissues. Immunohistochemical analysis with antibodies against cellular factors involved in apoptosis showed that caspase3, p53 and p21 were positive in infected cells.

Apoptosis↗