SHARAQ Project: Progress in 2009

On March 23, 2009, the first beam was successfullytransported to the final focal plane of the SHARAQspectrometer. We investigated detector responses toheavy-ion beams and the ion optical properties ofthe SHARAQ spectrometer1) and the high-resolutionbeam line2) in the subsequent commissioning runs andfound that the system as a whole worked almost as perits design. The first physics run with the spectrometerwas performed in November 2009. In this article, wereview the progress in the SHARAQ project in 2009

Kit-independent mast cell adhesion mediated by Notch

Kit/CD117 plays a crucial role in the cell–cell and cell–matrix adhesion of mammalian mast cells (MCs); however, it is unclear whether other adhesion molecule(s) perform important roles in the adhesion of MCs. In the present study, we show a novel Kit-independent adhesion mechanism of mouse cultured MCs mediated by Notch family members. On stromal cells transduced with each Notch ligand gene, Kit and its signaling become dispensable for the entire adhesion process of MCs from tethering to spreading. The Notch-mediated spreading of adherent MCs involves the activation of signaling via phosphatidylinositol 3-kinases and mitogen-activated protein kinases, similar to Kit-mediated spreading. Despite the activation of the same signaling pathways, while Kit supports the adhesion and survival of MCs, Notch only supports adhesion. Thus, Notch family members are specialized adhesion molecules for MCs that effectively replace the adhesion function of Kit in order to support the interaction of MCs with the surrounding cellular microenvironments

Correlation between cell aggregation and antibody production in IgE-producing plasma cells

Allergic conditions result in the increase of immunoglobulin (Ig)E-producing plasma cells (IgE-PCs); however, it is unclear how IgE production is qualitatively controlled. In this study, we found that IgE-PCs in spleen of immunized mice formed homotypic cell aggregates. By employing IgE-producing hybridomas (IgE-hybridomas) as a model of IgE-PCs, we showed that these cells formed aggregates in the presence of specific antigens (Ags). The formation of the Ag-induced cell aggregation involved secreted IgE and Fcγ receptor (FcγR)II/FcγRIII, but not FcεRs. Ag-induced cell aggregation plus lipopolysaccharide signaling resulted in an enhancement of IgE production in aggregated IgE-hybridomas. Furthermore, the administration of anti-FcγRII/FcγRIII antagonistic monoclonal antibody to immunized mice tended to reduce the splenic IgE-PC aggregation as well as the serum IgE levels. Taken together, our results suggested that Ag-IgE complexes induced IgE-PCs aggregation via FcγRII/FcγRIII, leading to the enhancement of IgE production. These findings suggest the presence of a novel mechanism for regulation of IgE production

An evolutionary-conserved function of mammalian notch family members as cell adhesion molecules.

Notch family members were first identified as cell adhesion molecules by cell aggregation assays in Drosophila studies. However, they are generally recognized as signaling molecules, and it was unclear if their adhesion function was restricted to Drosophila. We previously demonstrated that a mouse Notch ligand, Delta-like 1 (Dll1) functioned as a cell adhesion molecule. We here investigated whether this adhesion function was conserved in the diversified mammalian Notch ligands consisted of two families, Delta-like (Dll1, Dll3 and Dll4) and Jagged (Jag1 and Jag2). The forced expression of mouse Dll1, Dll4, Jag1, and Jag2, but not Dll3, on stromal cells induced the rapid and enhanced adhesion of cultured mast cells (MCs). This was attributed to the binding of Notch1 and Notch2 on MCs to each Notch ligand on the stromal cells themselves, and not the activation of Notch signaling. Notch receptor-ligand binding strongly supported the tethering of MCs to stromal cells, the first step of cell adhesion. However, the Jag2-mediated adhesion of MCs was weaker and unlike other ligands appeared to require additional factor(s) in addition to the receptor-ligand binding. Taken together, these results demonstrated that the function of cell adhesion was conserved in mammalian as well as Drosophila Notch family members. Since Notch receptor-ligand interaction plays important roles in a broad spectrum of biological processes ranging from embryogenesis to disorders, our finding will provide a new perspective on these issues from the aspect of cell adhesion

MCs efficiently adhered to OP9-Dll1, -Dll4, -Jag1, and -Jag2, but not OP9-Dll3, than to OP9-Ctrl.

<p>(A and B) Flow cytometric analysis of the expression of (A) Dll1, Dll3, Dll4, Jag1, and Jag2 on OP9 stromal cells transduced with each Notch ligand gene, and (B) Notch receptors on MCs after staining with specific mAbs (open histograms) or isotype-matched control mAbs (filled histograms). (C) Total RNA was analyzed by RT-PCR for the expression of Notch receptors in MCs and OP9-Ctrl cells. (D) Relative expression levels of <i>Notch1</i> and <i>Notch2</i> to <i>Gapdh</i> in MCs were analyzed by quantitative RT-PCR. Data represent the mean ± SEM of 3 independent experiments. (E and F) An adhesion assay for MCs on each OP9 cell (E) in a 48-well plate for 60 min and (F) in 96-well plates with serial incubation times of 5, 15, 30, 60, and 120 min. Data represent the percentages of non-adherent MCs (mean ± SEM of triplicate cultures) (*p<0.05 significantly different from OP9-Ctrl at each time point, the Student’s <i>t-</i>test).</p

Enhanced adhesion of MCs by Notch ligands involved both Notch1 and Notch2 on MCs.

<p>An adhesion assay (60 min) for MCs on each OP9 cell in a 96-well plate (A and B) with or without 10 µg/ml of the indicated polyclonal Ab (pAb) and (C) with control pAb (20 µg/ml), anti-Notch2 pAb (10 µg/ml) plus control pAb (10 µg/ml) or anti-Notch2 pAb (10 µg/ml) plus anti-Notch1 pAb (10 µg/ml). Data represent the percentages of non-adherent MCs (mean ± SEM of triplicate cultures) (*p<0.05, the Student’s <i>t-</i>test). Cultures with pAbs contained (A and B) 1.0% PBS (vol/vol) and 38.5 µM NaN₃ and (C) 2.0% PBS (vol/vol) and 76.9 µM NaN₃, which had no effect on the adhesion of MCs.</p

Notch signaling in stromal cells or MCs did not account for the enhanced adhesion.

<p>(A) An adipocyte differentiation assay of OP9-Ctrl cells stimulated with immobilized Notch ligands or human IgG1 (control) for 5 days in the presence of DAPT (10 µM) or the same volume of DMSO (control, 0.1% vol/vol). Data represent the numbers of adipocytes in a field (magnification; x200) in the center of the wells (mean ± SEM of triplicate cultures) (*p<0.05 significantly different from IgG1, the Student’s <i>t-</i>test). ND: not detected. (B) OP9-Ctrl cells were stimulated with each immobilized Notch ligand or human IgG1 (control) for 2 days in a 48-well plate, and an adhesion assay (60 min) for MCs was then performed. (C) An adhesion assay (60 min) for MCs on each OP9 cell with DAPT (10 µM) or the same volume of DMSO (control, 0.1% vol/vol). (B and C) Data represent the percentages of non-adherent MCs (mean ± SEM of triplicate cultures). (B) *p<0.05 significantly different from IgG1 and (C) no significant differences between the responses with DMSO and DAPT on the same OP9 cells (the Student’s <i>t-</i>test).</p

Reduction in Notch2 on MCs did not have marked effects on the enhanced adhesion.

<p>MCs were analyzed 48 hours after transfection with siRNA against Notch2 or control siRNA. (A) Relative expression levels of <i>Notch1</i> and <i>Notch2</i> to <i>Gapdh</i> in MCs transfected with each siRNA were analyzed by quantitative RT-PCR. Data represent the mean ± SEM of three independent experiments. (*p<0.05 significantly different from the control siRNA treatment, the Student’s <i>t-</i>test) (B) Flow cytometric analysis of the expression of Notch2 and Kit on MCs transfected with each siRNA after staining with specific mAbs (open histograms) or isotype-matched control mAbs (filled histograms). Representative histograms from one of three independent experiments are shown. Numbers indicate the relative mean fluorescence intensities (MFIs) of specific mAbs relative to that of the control siRNA treatment (MFIs of specific mAbs were normalized by the MFIs of control mAbs) (mean ± SEM of three independent experiments). (C) An adhesion assay (60 min) for MCs transfected with each siRNA on each OP9 cell in a 96-well plate. Data represent the percentages of non-adherent MCs (mean ± SEM of triplicate cultures) (*p<0.05, the Student’s <i>t-</i>test).</p