Search PubMed⌕ Search

Biomedical subjects

S Koyasu

Publications and source records attributed to S Koyasu.

At least 37 records · Page 2Linked to original sources
PubMed2000

Development of chimeric molecules for recognition and targeting of antigen-specific B cells in pemphigus vulgaris.

Pemphigus vulgaris (PV) is an autoimmune blistering disease characterized by circulating pathogenic IgG antibodies against desmoglein 3 (Dsg3). The purpose of this study was to develop chimeric molecules for specific recognition and elimination of autoimmune B cells in PV. Mouse hybridoma cell lines producing anti-Dsg3 antibody (5H10, 12A2) were developed as an in vitro model system for targeting B cells. Dsg3-GFP, a baculoprotein containing the entire extracellular domain of Dsg3 fused with green fluorescence protein, recognized and targeted the hybridoma cells through their surface immunoglobulin receptors in an antigen-specific way. The epitopes of these monoclonal antibodies were mapped on the amino terminal EC1 and part of EC2, a region considered functionally important in cadherins. Chimeric toxin molecules containing the mapped region (Dsg3deltaN1) and modified Pseudomonas exotoxin were produced in bacteria (Dsg3deltaN1-PE40-KDEL, PE3 7-Dsg3deltaN1-KDEL) and tested in vitro on hybridoma cell lines. The chimeric toxins, but not Dsg3deltaN1 alone, showed dose-dependent toxic activity with a reduction in hybridoma cell number to 40-60% of toxin-negative control cultures, compared with little or no effect on anti-Dsg3-negative hybridoma cells. Furthermore, these toxins showed toxic effects on anti-Dsg3 IgG-producing B cells from Dsg3deltaN1-immunized mice, with a 60% reduction in cell number compared with Dsg3deltaN1 alone. Thus, specific recognition and targeting of antigen-specific B cells in PV was demonstrated; this strategy may hold promise as a future therapeutic option for PV and other autoimmune diseases.

ADP Ribose Transferases↗
PubMed2000

Two distinct action mechanisms of immunophilin-ligand complexes for the blockade of T-cell activation.

The immunosuppressive effects of cyclosporin A (CsA) and FK506 are mediated through binding to immunophilins. Here we show that FK506-FKBP complex suppresses the activation of JNK and p38 pathways at a level upstream of mitogen-activated protein kinase (MAPK) kinase kinase (MAPKK-K) besides the calcineurin-NFAT pathway. A238L, a viral gene product that binds to immunophilin, also blocks activation of both pathways. In contrast, direct inhibitors of calcineurin, Cabin 1 and FR901725, suppress the activation of NFAT but not the JNK or p38 pathway. We further demonstrate that co-expression of a constitutively active NFAT and a constitutively active MEKK1 renders the interleukin-2 promoter in Jurkat T lymphocytes resistant to CsA and FK506, whereas Jurkat cells expressing a constitutively active NFAT alone are still sensitive to CsA or FK506. Therefore, CsA and FK506 exert their immunosuppressive effects through targeting both the calcineurin-dependent NFAT pathway and calcineurin-independent activation pathway for JNK and p38.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Use of autoantigen-knockout mice in developing an active autoimmune disease model for pemphigus.

The development of experimental models of active autoimmune diseases can be difficult due to tolerance of autoantigens, but knockout mice, which fail to acquire tolerance to the defective gene product, provide a useful tool for this purpose. Using knockout mice lacking desmoglein 3 (Dsg3), the target antigen of pemphigus vulgaris (PV), we have generated an active disease model for this autoantibody-mediated disease. Dsg3(-/-) mice, but not Dsg3(+/-) littermates, produced anti-Dsg3 IgG that binds native Dsg3, when immunized with recombinant mouse Dsg3. Splenocytes from the immunized Dsg3(-/-) mice were then adoptively transferred into Rag-2(-/-) immunodeficient mice expressing Dsg3. Anti-Dsg3 IgG was stably produced in the recipient mice for more than 6 months without further boosting. This IgG bound to Dsg3 in vivo and disrupted the cell-cell adhesion of keratinocytes. Consequently, the recipient mice developed erosions in their oral mucous membranes with typical histologic findings of PV. In addition, the recipient mice showed telogen hair loss, as found in Dsg3(-/-) mice. Collectively, the recipient mice developed the phenotype of PV due to the pathogenic anti-Dsg3 IgG. This model will be valuable for developing novel therapeutic strategies. Furthermore, our approach can be applied broadly for the development of various autoimmune disease models.

Animals↗
PubMed2000

[Why T cells?].

Explore the source record for details and available documents.

Animals↗
PubMed1999

Temperature-sensitive ZAP70 mutants degrading through a proteasome-independent pathway. Restoration of a kinase domain mutant by Cdc37.

CD8 deficiency is an autosomal recessive form of severe combined immunodeficiency diseases characterized by the absence of CD8(+) T lymphocytes and impaired T cell functions. We identified two novel mis-sense mutations in the zap70 genes of a CD8-deficiency patient. One mutation (P80Q) affects a residue in an SH2 domain and another (M572L) in the kinase subdomain XI. Both mutations cause a degradation of ZAP70 protein in a temperature-sensitive manner through an ATP-dependent and proteasome-independent pathway. We further demonstrated that Cdc37, a protein kinase-specific chaperone, bound to M572L but not P80Q mutant and restored the expression of the M572L mutant when overexpressed. The restoration of M572L mutant by Cdc37 required the function of HSP90. These results indicate that Cdc37 in conjunction with HSP90 functions as a molecular chaperone for a temperature-sensitive kinase domain mutant of ZAP70.

Adenosine Triphosphate↗
PubMed1999

Interleukin 12-dependent interferon gamma production by CD8alpha+ lymphoid dendritic cells.

We investigated the role of antigen-presenting cells in early interferon (IFN)-gamma production in normal and recombinase activating gene 2-deficient (Rag-2(-/-)) mice in response to Listeria monocytogenes (LM) infection and interleukin (IL)-12 administration. Levels of serum IFN-gamma in Rag-2(-/-) mice were comparable to those of normal mice upon either LM infection or IL-12 injection. Depletion of natural killer (NK) cells by administration of anti-asialoGM1 antibodies had little effect on IFN-gamma levels in the sera of Rag-2(-/-) mice after LM infection or IL-12 injection. Incubation of splenocytes from NK cell-depleted Rag-2(-/-) mice with LM resulted in the production of IFN-gamma that was completely blocked by addition of anti-IL-12 antibodies. Both dendritic cells (DCs) and monocytes purified from splenocytes were capable of producing IFN-gamma when cultured in the presence of IL-12. Intracellular immunofluorescence analysis confirmed the IFN-gamma production from DCs. It was further shown that IFN-gamma was produced predominantly by CD8alpha+ lymphoid DCs rather than CD8alpha- myeloid DCs. Collectively, our data indicated that DCs are potent in producing IFN-gamma in response to IL-12 produced by bacterial infection and play an important role in innate immunity and subsequent T helper cell type 1 development in vivo.

Animals↗
PubMed1999

Cutting edge: LFA-1 is required for liver NK1.1+TCR alpha beta+ cell development: evidence that liver NK1.1+TCR alpha beta+ cells originate from multiple pathways.

Using mice deficient for LFA-1, CD44, and ICAM-1, we examined the role of these adhesion molecules in NK1.1+TCR alpha beta+ (NKT) cell development. Although no defect in NKT cell development was observed in CD44-/- and ICAM-1-/- mice, a dramatic reduction of liver NKT cells was observed in LFA-1-/- mice. Normal numbers of NKT cells were present in other lymphoid organs in LFA-1-/- mice. When LFA-1-/- splenocytes were injected i.v. into wild-type mice, the frequency of NKT cells among donor-derived cells in the recipient liver was normal. In contrast, when LFA-1-/- bone marrow (BM) cells were injected i.v. into irradiated wild-type mice, the frequency of liver NKT cells was significantly lower than that of mice injected with wild-type BM cells. Collectively, these data indicate that LFA-1 is required for the development of liver NKT cells, rather than the migration to and/or subsequent establishment of mature NKT cells in the liver.

Animals↗
PubMed1999

Differential activation of c-Jun NH2-terminal kinase and p38 pathways during FTY720-induced apoptosis of T lymphocytes that is suppressed by the extracellular signal-regulated kinase pathway.

FTY720 is a novel immunosuppressive drug derived from a metabolite from Isaria sinclairii that is known to induce apoptosis of rat splenic T cells. In this study, we examined the intracellular signaling pathway triggered by FTY720. Treatment of human Jurkat T lymphocytes with FTY720-induced apoptosis characterized by DNA fragmentation. The same treatment induced activation of protein kinases such as c-Jun NH2-terminal kinase (JNK), p38/CSBP (CSAID-binding protein), and a novel 36-kDa myelin basic protein (MBP) kinase, but not extracellular signal-regulated kinase (ERK). Pretreatment of Jurkat cells with DEVD-CHO blocked FTY720-induced DNA fragmentation as well as the activation of p38/CSBP. However, DEVD-CHO treatment failed to inhibit FTY720-induced activation of JNK and the 36-kDa MBP kinase. We have also demonstrated that activation of the ERK signaling pathway completely suppressed the FTY720-induced apoptotic process including activation of caspase 3 and activation of JNK and the 36-kDa MBP kinase. Furthermore, transient expression of constitutively active mitogen-activated protein kinase/ERK kinase (MEK) protected the cells from FTY720-induced cell death. The effect of MEK was canceled by coexpression of a mitogen-activated protein kinase phosphatase, CL100. These results indicate that JNK and p38 pathways are differentially regulated during FTY720-induced apoptosis and that activation of ERK pathway alone is sufficient to cancel the FTY720-induced death signal.

Apoptosis↗
PubMed1999

Xid-like immunodeficiency in mice with disruption of the p85alpha subunit of phosphoinositide 3-kinase.

Mice with a targeted gene disruption of p85alpha, a regulatory subunit of phosphoinositide 3-kinase, had impaired B cell development at the pro-B cell stage, reduced numbers of mature B cells and peritoneal CD5+ Ly-1 B cells, reduced B cell proliferative responses, and no T cell-independent antibody production. These phenotypes are nearly identical to those of Btk-/- or xid (X-linked immunodeficiency) mice. These results provide evidence that p85alpha is functionally linked to the Btk pathway in antigen receptor-mediated signal transduction and is pivotal in B cell development and functions.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1999

Increased insulin sensitivity and hypoglycaemia in mice lacking the p85 alpha subunit of phosphoinositide 3-kinase.

The hallmark of type 2 diabetes, the most common metabolic disorder, is a defect in insulin-stimulated glucose transport in peripheral tissues. Although a role for phosphoinositide-3-kinase (PI3K) activity in insulin-stimulated glucose transport and glucose transporter isoform 4 (Glut4) translocation has been suggested in vitro, its role in vivo and the molecular link between activation of PI3K and translocation has not yet been elucidated. To determine the role of PI3K in glucose homeostasis, we generated mice with a targeted disruption of the gene encoding the p85alpha regulatory subunit of PI3K (Pik3r1; refs 3-5). Pik3r1-/- mice showed increased insulin sensitivity and hypoglycaemia due to increased glucose transport in skeletal muscle and adipocytes. Insulin-stimulated PI3K activity associated with insulin receptor substrates (IRSs) was mediated via full-length p85 alpha in wild-type mice, but via the p50 alpha alternative splicing isoform of the same gene in Pik3r1-/- mice. This isoform switch was associated with an increase in insulin-induced generation of phosphatidylinositol(3,4,5)triphosphate (PtdIns(3,4,5)P3) in Pik3r1-/- adipocytes and facilitation of Glut4 translocation from the low-density microsome (LDM) fraction to the plasma membrane (PM). This mechanism seems to be responsible for the phenotype of Pik3r1-/- mice, namely increased glucose transport and hypoglycaemia. Our work provides the first direct evidence that PI3K and its regulatory subunit have a role in glucose homeostasis in vivo.

Animals↗
PubMed1999

Existence of activated and memory CD4+ T cells in peripheral blood and their skin infiltration in CD8 deficiency.

CD8 deficiency is a rare primary immunodeficiency caused by the defect of a tyrosine kinase, ZAP-70, which transduces signals from the T cell receptor. We report here a case of CD8 deficiency, having CD4+ T cells with a unique phenotype. The patient's T cells did not respond to anti-CD3 stimulation in vitro, suggesting that they were naive. However, many CD4+ T cells with activated and memory phenotypes, which expressed CD45RO+, HLA-DR+ and CD25+, were present in the peripheral blood, and these cells accumulated in the perivascular area of his infiltrative erythematous skin lesions. The patient's T cells could be activated by a high concentration of phytohaemagglutinin (PHA), indicating the presence of an alternate signalling pathway which bypasses ZAP-70 and activates CD4+ T cells in vivo. The origin and role of activated CD4+ T cells in the pathogenesis involved in the skin lesions are discussed.

CD4-Positive T-Lymphocytes↗
PubMed1999

[Regulatory mechanisms of NK cell functions].

Natural Killer (NK) cells discriminate self from non-self in a manner distinct from T cells. NK cells exhibit cytotoxicity against "missing-self" by killing any cells in principle except normal self-cells. Cells expressing low levels of self MHC class I molecules such as tumor cells and foreign cells are killed, whereas normal self cells are neglected by NK cells. Although identities of activation receptors triggering NK activity are still unclear, recent studies have revealed molecules inhibiting cytotoxicity against normal self cells. In this review, we summarize current understanding of molecular basis for missing-self hypothesis and control mechanisms of NK cell activation.

Animals↗
PubMed1998

T lymphocyte activation signals for interleukin-2 production involve activation of MKK6-p38 and MKK7-SAPK/JNK signaling pathways sensitive to cyclosporin A.

p38/CSBP, a subgroup member of mitogen-activated protein kinase (MAPK) superfamily molecules, is known to be activated by proinflammatory cytokines and environmental stresses. We report here that p38 is specifically activated by signals that lead to interleukin-2 (IL-2) production in T lymphocytes. A p38 activator MKK6 was also markedly activated by the same stimulation. Pretreatment of cells with SB203580, a specific inhibitor of p38, as well as expression of a dominant-negative mutant of MKK6, suppressed the transcriptional activation of the IL-2 promoter. We also demonstrated that MKK7, a recently described MAPK kinase family member, plays a major role in the activation of stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) in T lymphocytes. Moreover, a dominant-negative mutant of MKK7 abrogated the transcriptional activation of the distal nuclear factor of activated T cells response element in the IL-2 promoter. Cyclosporin A, a potent immunosuppressant, inhibited activation of both p38 and SAPK/JNK pathways but not the MAPK/extracellular signal-regulated kinase (ERK) pathway. Our results indicate that both MKK6 to p38 and MKK7 to SAPK/JNK signaling pathways are activated in a cyclosporin A-sensitive manner and contribute to IL-2 gene expression in T lymphocytes.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1998

Positive selection of CD4+ T cells by TCR-specific antibodies requires low valency TCR cross-linking: implications for repertoire selection in the thymus.

The developmental fate of immature CD4+ 8+ thymocytes is determined by intrathymic signals transduced by surface TCR complexes. In particular, TCR signals are required for immature CD4+ 8+ thymocytes to further differentiate into CD4+ 8- or CD4- 8+ T cells, a process referred to as positive selection. It is generally thought that positive selection results from low affinity TCR interactions with self antigens which engage the relatively few surface TCR complexes that are on immature CD4+ 8+ thymocytes. However, we now demonstrate with TCR-specific antibodies that positive selection of CD4+ T cells requires low valency cross-linking of surface TCR complexes on immature thymocytes. That is, positive selection signals are only generated within a narrow range of TCR cross-linking: cross-linking either too few or too many surface TCR complexes fails to signal positive selection. We interpret these results as indicating that positive selection of CD4+ T cells is not signaled by low affinity TCR interactions per se, but rather can be signaled by any combination of TCR affinity and ligand density that induces low valency TCR cross-linking on immature thymocytes.

Animals↗
PubMed1998

Energy of adhesion of human T cells to adsorption layers of monoclonal antibodies measured by a film trapping technique.

A novel method for studying the interaction of biological cells with interfaces (e.g., adsorption monolayers of antibodies) is developed. The method is called the film trapping technique because the cell is trapped within an aqueous film of equilibrium thickness smaller than the cell diameter. A liquid film of uneven thickness is formed around the trapped cell. When observed in reflected monochromatic light, this film exhibits an interference pattern of concentric bright and dark fringes. From the radii of the fringes one can restore the shape of interfaces and the cell. Furthermore, one can calculate the adhesive energy between the cell membrane and the aqueous film surface (which is covered by a layer of adsorbed proteins and/or specific ligands), as well as the disjoining pressure, representing the force of interaction per unit area of the latter film. The method is applied to two human T cell lines: Jurkat and its T cell receptor negative (TCR-) derivative. The interaction of these cells with monolayers of three different monoclonal antibodies adsorbed at a water-air interface is studied. The results show that the adhesive energy is considerable (above 0.5 mJ/m2) when the adsorption monolayer contains antibodies acting as specific ligands for the receptors expressed on the cell surface. In contrast, the adhesive energy is close to zero in the absence of such a specific ligand-receptor interaction. In principle, the method can be applied to the study of the interaction of a variety of biological cells (B cells, natural killer cells, red blood cells, etc.) with adsorption monolayers of various biologically active molecules. In particular, film trapping provides a tool for the gentle micromanipulation of cells and for monitoring of processes (say the activation of a T lymphocyte) occurring at the single-cell level.

Adsorption↗
PubMed1997

Functional analysis of immunoreceptor tyrosine-based activation motif (ITAM)-mediated signal transduction: the two YxxL segments within a single CD3zeta-ITAM are functionally distinct.

Functional analysis of the immunoreceptor tyrosine-based activation motif (ITAM) derived from the membrane-proximal ITAM of CD3zeta demonstrates that mutations at either the tyrosine or leucine residues in the N-terminal YxxL segment of the ITAM abolish all signal transduction functions of this ITAM. In contrast, mutations at the tyrosine or leucine residues in the C-terminal YxxL segment abrogate signals for interleukin (IL)-2 production but do not prevent tyrosine phosphorylation of the N-terminal tyrosine of the ITAM, lck association with the ITAM, activation of phospholipase C-gamma1 or calcium mobilization. Cross-linking of chimeric receptors containing a C-terminal YxxL leucine mutation induces tyrosine phosphorylation of ZAP70 but without stable binding to the phosphorylated ITAM. These results indicate that the two YxxL segments in an ITAM are functionally distinct and that both are essential for ZAP70 binding and IL-2 production. Furthermore, tyrosine phosphorylation of ZAP70 per se is not sufficient to trigger the downstream events leading to IL-2 production. Substitution of an alanine for the bulky side chain at the Y+1 position of the N-terminal YxxL segment reduces the receptor cross-linking requirement necessary to achieve cellular activation and the absolute dependence on lck in this process. Our results reveal that both the number of ITAM as well as the specific amino acid residues within a single ITAM determine the extent of chimeric receptor cross-linking required to trigger tyrosine phosphorylation-dependent signaling events.

Amino Acid Sequence↗