Biomedical subjects

Hiroshi Watarai

Publications and source records attributed to Hiroshi Watarai.

11 recordsLinked to original sources

PubMed2006

IL-21-induced Bepsilon cell apoptosis mediated by natural killer T cells suppresses IgE responses.

Epidemiological studies have suggested that the recent increase in the incidence and severity of immunoglobulin (Ig)E-mediated allergic disorders is inversely correlated with Mycobacterium bovis bacillus Calmette Guerin (BCG) vaccination; however, the underlying mechanisms remain uncertain. Here, we demonstrate that natural killer T (NKT) cells in mice and humans play a crucial role in the BCG-induced suppression of IgE responses. BCG-activated murine Valpha14 NKT cells, but not conventional CD4 T cells, selectively express high levels of interleukin (IL)-21, which preferentially induces apoptosis in Bepsilon cells. Signaling from the IL-21 receptor increases the formation of a complex between Bcl-2 and the proapoptotic molecule Bcl-2-modifying factor, resulting in Bepsilon cell apoptosis. Similarly, BCG vaccination induces IL-21 expression by human peripheral blood mononuclear cells (PBMCs) in a partially NKT cell-dependent fashion. BCG-activated PBMCs significantly reduce IgE production by human B cells. These findings provide new insight into the therapeutic effect of BCG in allergic diseases.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Coupling of STIM1 to store-operated Ca2+ entry through its constitutive and inducible movement in the endoplasmic reticulum.

Depletion of intracellular calcium (Ca(2+)) stores induces store-operated Ca(2+) (SOC) entry across the plasma membrane (PM). STIM1, a putative Ca(2+) sensor in the endoplasmic reticulum (ER), has been recently shown to be necessary for SOC channel activation. Here we show that STIM1 dynamically moves in tubulovesicular shape on the ER and its subcompartment in resting living cells, whereas, upon Ca(2+) store depletion, it is rapidly redistributed into discrete puncta that are located underneath, but not inserted into the PM. Normal constitutive movement of STIM1 is mediated through the coiled-coil and Ser/Thr-rich C-terminal domains in the cytoplasmic region of STIM1, whereas subsequent inducible puncta formation further requires the sterile alpha motif domain protruding into the ER lumen. Each of these three domains (coiled-coil, Ser/Thr-rich, and sterile alpha motif) was essential for activating SOC channels. Hence, our findings based on structure-function experiments suggest that constitutive dynamic movement of STIM1 in the ER and its subcompartment is obligatory for subsequent depletion-dependent redistribution of STIM1 into puncta underneath the PM and activation of SOC channels.

Animals↗

PubMed2006

The importance of CD25+ CD4+ regulatory T cells in mouse hepatic allograft tolerance.

In mouse liver transplantation, tolerance is readily inducible. Recent studies have revealed that CD25+ CD4+ regulatory T cells play an important role in regulating various immune responses, including transplant tolerance. However, the contribution of these cells to tolerance in mouse liver transplantation has not been elucidated. We showed here that depletion of CD25+ CD4+ T cells increased proliferative response of CD4+ T cells and cytotoxic T lymphocyte induction of CD8+ T cells. Depletion of these cells in the recipient but not in the donor before liver transplantation caused rejection. Furthermore, the number of CD25+ CD4+ population and forkhead/winged helix transcription factor expression in liver mononuclear lymphocytes derived from tolerant mice were higher than those from grafts undergoing rejection. In conclusion, these results indicate that CD25+ CD4+ regulatory T cells in the recipient but not in the donor of liver transplantation are important for the tolerance induction.

Animals↗

PubMed2005

PKC beta regulates BCR-mediated IKK activation by facilitating the interaction between TAK1 and CARMA1.

The B cell antigen receptor (BCR)-mediated activation of IkappaB kinase (IKK) and nuclear factor-kappaB require protein kinase C (PKC)beta; however, the mechanism by which PKCbeta regulates IKK is unclear. Here, we demonstrate that another protein kinase, TGFbeta-activated kinase (TAK)1, is essential for IKK activation in response to BCR stimulation. TAK1 interacts with the phosphorylated CARMA1 (also known as caspase recruitment domain [CARD]11, Bimp3) and this interaction is mediated by PKCbeta. IKK is also recruited to the CARMA1-Bcl10-mucosal-associated lymphoid tissue 1 adaptor complex in a PKCbeta-dependent manner. Hence, our data suggest that phosphorylation of CARMA1, mediated by PKCbeta, brings two key protein kinases, TAK1 and IKK, into close proximity, thereby allowing TAK1 to phosphorylate IKK.

Amino Acid Sequence↗

PubMed2005

Induction of regulatory properties in dendritic cells by Valpha14 NKT cells.

Valpha14 NKT cells exhibit various immune regulatory properties in vivo, but their precise mechanisms remain to be solved. In this study, we demonstrate the mechanisms of generation of regulatory dendritic cells (DCs) by stimulation of Valpha14 NKT cells in vivo. After repeated injection of alpha-galactosylceramide (alpha-GalCer) into mice, splenic DCs acquired properties of regulatory DCs in IL-10-dependent fashion, such as nonmatured phenotypes and increased IL-10 but reduced IL-12 production. The unique cytokine profile in these DCs appears to be regulated by ERK1/2 and IkappaB(NS). These DCs also showed an ability to suppress the development of experimental allergic encephalomyelitis by generating IL-10-producing regulatory CD4 T cells in vivo. These findings contribute to explaining how Valpha14 NKT cells regulate the immune responses in vivo.

Animals↗

PubMed2005

Cutting edge: critical role of CXCL16/CXCR6 in NKT cell trafficking in allograft tolerance.

It is well-documented that certain chemokines or their receptors play important roles in the graft rejection. However, the roles of chemokines and their receptors in the maintenance of transplantation tolerance remain unclear. In this study, we demonstrate that blocking of the interaction between the chemokine receptor, CXCR6, highly expressed on V alpha14+ NKT cells and its ligand, CXCL16, resulted in the failure to maintain graft tolerance and thus in the induction of acceleration of graft rejection. In a mouse transplant tolerance model, the expression of CXCL16 was up-regulated in the tolerated allografts, and anti-CXCL16 mAb inhibited intragraft accumulation of NKT cells. In vitro experiments further showed that blocking of CXCL16/CXCR6 interaction significantly affected not only chemotaxis but also cell adhesion of NKT cells. These results demonstrate the unique role of CXCL16 and CXCR6 molecules in the maintenance of cardiac allograft tolerance mediated by NKT cells.

Animals↗

PubMed2005

Regulation of T helper type 2 cell differentiation by murine Schnurri-2.

Schnurri (Shn) is a large zinc finger protein implicated in cell growth, signal transduction, and lymphocyte development. Vertebrates possess at least three Shn orthologues (Shn-1, Shn-2, and Shn-3), which appear to act within the bone morphogenetic protein, transforming growth factor beta, and activin signaling pathways. However, the physiological functions of the Shn proteins remain largely unknown. In Shn-2-deficient mice, mature peripheral T cells exhibited normal anti-T cell receptor-induced proliferation, although there was dramatic enhancement in the differentiation into T helper type (Th)2 cells and a marginal effect on Th1 cell differentiation. Shn-2-deficient developing Th2 cells showed constitutive activation of nuclear factor kappaB (NF-kappaB) and enhanced GATA3 induction. Shn-2 was able to compete with p50 NF-kappaB for binding to a consensus NF-kappaB motif and inhibit NF-kappaB-driven promoter activity. Thus, Shn-2 plays a crucial role in the control of Th2 cell differentiation by regulating NF-kappaB function.

Animals↗

PubMed2005

Plasma membrane-focused proteomics: dramatic changes in surface expression during the maturation of human dendritic cells.

The differential expression of surface molecules on dendritic cells (DC) reflects their functional differences as immature and mature subsets. It is difficult, however, to characterize differences in surface expression by standard proteomic approaches, due mainly to the hydrophobic nature and low abundance of the individual proteins in question. We have established a method for obtaining high-yield plasmalemma preparations which contain surface molecules enriched more than 200-fold by coating cells with beads conjugated with antibody against a cell type-specific cell-surface molecule, followed by nitrogen cavitated disruption, magnetic separation, and density gradient ultracentrifugation. We identified and quantified 339 human monocyte-derived DC transmembrane proteins, including 33 previously uncharacterized molecules. Whereas 106 proteins were selectively expressed in immature cells or down-regulated after maturation, 191 proteins were selectively expressed in mature cells or up-regulated after maturation.

Cell Membrane↗

PubMed2004

Structure of the receptor-binding domain of human thrombopoietin determined by complexation with a neutralizing antibody fragment.

The cytokine thrombopoietin (TPO), the ligand for the hematopoietic receptor c-Mpl, acts as a primary regulator of megakaryocytopoiesis and platelet production. We have determined the crystal structure of the receptor-binding domain of human TPO (hTPO(163)) to a 2.5-A resolution by complexation with a neutralizing Fab fragment. The backbone structure of hTPO(163) has an antiparallel four-helix bundle fold. The neutralizing Fab mainly recognizes the C-D crossover loop containing the species invariant residue Q111. Titration calorimetric experiments show that hTPO(163) interacts with soluble c-Mpl containing the extracellular cytokine receptor homology domains with 1:2 stoichiometry with the binding constants of 3.3 x 10(9) M(-1) and 1.1 x 10(6) M(-1). The presence of the neutralizing Fab did not inhibit binding of hTPO(163) to soluble c-Mpl fragments, but the lower-affinity binding disappeared. Together with prior genetic data, these define the structure-function relationships in TPO and the activation scheme of c-Mpl.

Amino Acid Sequence↗

PubMed2004

Classification for dimethylarsenate-decomposing bacteria using a restrict fragment length polymorphism analysis of 16S rRNA genes.

A new monitoring system for bacterial communities involving dimethylarsinic acid (DMAA) decomposition was provided by combining the MPN (Most Probable Number) method and RFLP (restriction-fragment-length polymorphism analysis). The abundance of DMAA decomposing bacteria was estimated by the MPN method using a bacterial culture medium, which included DMAA as the sole carbon source, indicating bacterial cell densities of 1700 cells/ml in Lake Kahokugata and 330 cells/ml in Lake Kibagata. After isolating the dominant bacteria using agar plates, the isolates were classified into some genotype groups by RFLP analysis using 16S rDNA sequences. Classification of the RFLP analysis indicated that 14 isolates of Lake Kahokugata were classified into 6 types, which included 2 dominant types related to genus Pseudomonas, while 8 isolates of Lake Kibagata displayed 6 types including one or two isolates. Moreover, the RFLP types were unique for each lake, suggesting that DMAA decomposing bacteria were specific for the aquatic environment related to the arsenic cycle. The activities of DMAA decomposition mostly matched with the RFLP type category of the isolates. Accordingly, combining the MPN method with the RFLP analysis will play an important role in elucidating the distributions and dynamics of the DMAA-decomposing bacterial community.

Bacteria↗

PubMed2002

Crystallization of the functional domain of human thrombopoietin using an antigen-binding fragment derived from neutralizing monoclonal antibody.

Thrombopoietin (TPO) is a cytokine which primarily stimulates megakaryocytopoiesis and thrombopoiesis. The functional domain of TPO (TPO(163)) consisting of the N-terminal 163 amino acids was prepared and crystallized. Since the crystallization of TPO(163) was unsuccessful using the standard screening methods, a Fab fragment derived from a neutralizing monoclonal antibody was used for crystallization. It was found that the TPO(163)-Fab complex crystallized reproducibly in 0.1 M potassium phosphate buffer pH 6.0 containing 20-25% polyethylene glycol 4000. Thin crystals (0.2 x 0.2 x 0.02 mm) grew in two space groups: P2(1), with unit-cell parameters a = 133.20, b = 46.71, c = 191.47 A, beta = 90.24 degrees, and C2, with unit-cell parameters a = 131.71, b = 46.48, c = 184.63 A, beta = 90.42 degrees. The results of a molecular-replacement analysis indicate that the Fab molecules interact with each other and provide a suitable interface for crystallization.

Antibodies, Monoclonal↗