Miya Yoshino

Potential of dental mesenchymal cells in developing teeth

The tooth, composed of dentin and enamel, develops through epithelium‐mesenchyme interactions. Neural crest (NC) cells contribute to the dental mesenchyme in the developing tooth and differentiate into dentin‐secreting odontoblasts. NC cells are known to differentiate into chondrocytes and osteoblasts in the craniofacial region. However, it is not clear whether the dental mesenchymal cells in the developing tooth possess the potential to differentiate into a lineage(s) other than the odontoblast lineage. In this study, we prepared mesenchymal cells from E13.5 tooth germ cells and assessed their potential for differentiation in culture. They differentiated into odontoblasts, chondrocyte‐like cells, and osteoblast‐like cells. Their derivation was confirmed by tracing NC‐derived cells as LacZ+ cells using P0‐Cre/Rosa26R mice. Using the flow cytometry‐fluorescent di‐β‐d‐galactosidase system, which makes it possible to detect LacZ+ cells as living cells, cell surface molecules of dental mesenchymal cells were characterized. Large number of LacZ+ NC‐derived cells expressed platelet‐derived growth factor receptor α and integrins. Taken together, these results suggest that NC‐derived cells with the potential to differentiate into chondrocyte‐like and osteoblast‐like cells are present in the developing tooth, and these cells may contribute to tooth organogenesis.

A Notch Ligand, Delta-Like 1 Functions As an Adhesion Molecule for Mast Cells

Mast cells (MCs) accumulate in chronic inflammatory sites; however, it is not clear which adhesion molecules are involved in this process. Recently, the expression of Notch ligands was reported to be upregulated in inflammatory sites. Although Notch receptors are known as signaling molecules that can activate integrins, their contributions to the adhesion of MCs have not been studied. In this study, we demonstrated that mouse MCs efficiently adhered to stromal cells forced to express a Notch ligand, Delta-like 1 (Dll1). Surprisingly, the adhesion was a consequence of direct cell–cell interaction between MCs and Dll1-expressing stromal cells rather than activation of downstream effectors of Notch receptor(s)-Dll1. The adhesion of MCs to Dll1-expressing stromal cells remained even when the cell metabolism was arrested. The recognition was blocked only by inhibition of Notch receptor(s)–Dll1 interaction by addition of soluble DLL1, or mAbs against Dll1 or Notch2. Taken together, these results indicate that Notch receptor(s) and Dll1 directly promote the adhesion of MCs to stromal cells by acting as adhesion molecules. This appreciation that Notch receptor–ligand interactions have an adhesion function will provide an important clue to molecular basis of accumulation of MCs to inflammatory sites.

Reduction of Osteoclasts in a Critical Embryonic Period Is Essential for Inhibition of Mouse Tooth Eruption

Alveolar bone resorption by osteoclasts is essential for tooth eruption. Osteoclast‐deficient Csfmop homozygous (op/op) mice, which lack functional macrophage colony‐stimulating factor (M‐CSF), suffer from osteopetrosis and completely lack tooth eruption. Although osteoclasts appear, and osteopetrosis is cured with age in op/op mice, tooth eruption is never seen. This fact suggests that there is a critical period when osteoclasts are required for tooth eruption. In this study, to detect the critical period, we administered an antagonistic antibody directed against c‐Fms, a receptor for M‐CSF, to inbred C57BL/6 mice for various periods. Administration of this antibody decreased tartrate‐resistant acid phosphatase‐positive (TRAP) osteoclasts, and incisor eruption was completely inhibited by continual administration of this antibody from embryonic day 15.5 (E15.5) until postnatal day 12.5 (D12.5). A 1‐day delay of this administration abolished the inhibition of incisor eruption. The number of TRAP‐positive osteoclasts was significantly reduced between E16.5 and E18.5 in the mice treated with antibody from E15.5 compared with those treated from E16.5. These results indicate that this period, during which the number of osteoclasts decreases significantly, is critical for inhibiting incisor eruption in C57BL/6 mice.

Enforced expression of PU.1 rescues osteoclastogenesis from embryonic stem cells lacking Tal-1

Transcription factor T‐cell acute lymphocytic leukemia 1 (Tal‐1) is essential for the specification of hematopoietic development. Mice lacking Tal1 fail to generate any hematopoietic precursors. Using our co‐culture system with stromal cells, we demonstrate that enforced expression of the transcription factor PU.1 under tetracycline control in Tal1‐null embryonic stem (ES) cells rescues the development of osteoclasts and macrophage–like phagocytes. It was low efficiency compared with wild–type ES cells; other hematopoietic lineage cells of granulocytes, B cells, mast cells, megakaryocytes, and erythroid cells were not generated. Osteoclasts developed in this culture were multinucleated and competent for bone resorption. Their development depended on macrophage colony‐stimulating factor and receptor activator of nuclear factor κB ligand. The majority of cells with the potential to differentiate into osteoclasts expressed fetal liver kinase 1 (Flk‐1) and could be isolated using anti–Flk‐1 antibody. These results suggest that the expression of PU.1 is a critical event for osteoclastogenesis and that Tal‐1 may lie upstream of PU.1 in a regulatory hierarchy during osteoclastogenesis.

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.

CCR7‐independent transport of skin antigens occurs in the dermis

Under homeostatic conditions, skin DCs migrate to regional LNs transporting self‐antigens (self‐Ags). The transport of self‐Ags is considered to be critical for maintaining peripheral tolerance. Although the chemokine receptor CCR7 potently induces the migration of skin DCs to regional LNs, Ccr7−/− (Ccr7‐KO) mice do not show skin auto‐immune diseases. To resolve this inconsistency, we examined Ccr7‐KO epidermis‐ or dermis‐hyperpigmented transgenic (Tg) mice, in which the transport of skin self‐Ags is traceable by melanin granules (MGs). Under CCR7‐deficient conditions, the transport of epidermal MGs to regional LNs was impaired at 7 weeks of age. However, epidermal MGs could be transported when they had accumulated in the dermis. Ccr7‐KO‐dermis‐pigmented Tg mice confirmed the presence of CCR7‐independent transport from the dermis. Compared with WT‐dermis‐pigmented Tg mice, the amount of transported melanin and number of MG‐laden CD11c+ cells were both approximately 40% of the WT levels, while the number of MG‐laden CD205+ or CD207+ cells decreased to about 10% in skin regional LNs of Ccr7‐KO‐dermis‐pigmented Tg mice. Cell sorting highlighted the involvement of CD11c+ cells in the CCR7‐independent transport. Here, we show that CCR7‐independent transport of skin self‐Ags occurs in the dermis. This system might contribute to the continuous transport of self‐Ags, and maintain peripheral tolerance.

A checkpoint in B-lymphopoiesis related to Notch resistance.

While murine B- and T-lymphopoiesis require overlapping molecules, they occur in separate organs: the bone marrow (BM) and the thymus, respectively. The BM microenvironment is incapable of supporting T-lymphopoiesis because of insufficient interactions of Notch1 with delta-like ligand (Dll). Notch1/Dll interactions also play a role in the suppression of B-lymphopoiesis in the thymus. However, it is still unclear whether the Notch1/Dll interaction alone explains why the thymus does not support B-lymphopoiesis. In this study, we compared the precursor population colonizing the thymus with that in the BM by culturing them on stromal cells expressing abundant Dll1. We demonstrated that Flt3(+) Il7r(+) B220(+) Cd19(+) BM cells gave rise to B cells under this condition. We defined them as resistant to Dll1. In the thymus, Dll1-resistant cells were undetectable. This suggested that the absence of Dll1-resistant cells might explain the absence of B-lymphopoiesis in the thymus.

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.

Characterization of Innate and Adaptive Immune Responses in PYNOD-Deficient Mice

PYNOD (also called NLRP10) is a member of the nucleotide-binding domain and leucine-rich repeat containing family. Many members of this family play important roles in the activation and/or regulation of immune and inflammatory responses. We previously showed that PYNOD inhibits the IL-1β secretion in response to microbial infection in PYNOD-transgenic mice. In this study, we generated PYNOD-knockout (KO) mice and further investigated PYNOD’s role in the innate and adaptive immune responses. Similar to wild-type macrophages, PYNOD-KO macrophages produced IL-1β and induced pyroptosis, a caspase-1–dependent programmed cell death, in response to various inflammasome activators and microbial infection. In addition, the PYNOD deficiency did not significantly affect the proliferation or cytokine production of T cells, the delayed-type hypersensitivity responses, the anti-tumor immunity, the Ag-specific Ab production, the cytotoxicity of NK cells, or the maturation, Ag-presenting capacity, or elicited migration of dendritic cells. Furthermore, the steady-state skin self-antigen transport to regional lymph nodes was not impaired in PYNOD-KO mice, suggesting that PYNOD is dispensable for steady-state dendritic cell migration. These results suggested that PYNOD is dispensable for the regulation of innate and adaptive immune responses in mice, unless PYNOD’s expression is highly induced under certain conditions.

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.