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-1ß 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-1ß 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.

Blood supply--susceptible formation of melanin pigment in hair bulb melanocytes of mice.

BACKGROUND: Allogeneic skin grafts onto C57BL/6 mice are rejected, and the rejected skin is replaced by surrounding skin with black hair. In contrast, syngeneic skin grafts are tolerated, and gray hair grows on the grafts. METHODS: To explore the mechanism of gray hair growing on the tolerated skin grafts, we prepared full-thickness skin (2-cm square) autografts, 2 (2 cm + 2 cm) horizontal or vertical parallel incisions, and U-shaped (2 cm × 2 cm × 2 cm) flaps with or without pedicle vessels. The grafts, incisions, and flaps were fixed by suturing with string and protected by a transparent bandage. On day 14 after the operation, the bandages were removed to observe the color of the hair growing on the skin. RESULTS: Skin autografts from wild-type or hepatocyte growth factor-transgenic (Tg) C57BL/6 mice survived with gray hair, whereas those from steel factor (Kitl)-Tg C57BL/6 mice survived with black hair. In addition, U-shaped flaps lacking both of the 2 main feeding vessels of wild-type mice had gray hair at the tip of the flaps. Light microscopy after staining with hematoxylin and eosin or dihydroxyphenylalanine showed that the formation of melanin pigment in the follicles, but not in the interadnexal skin, was susceptible to the blood supply. CONCLUSIONS: Melanin pigment formation in the hair bulb melanocytes appeared to be susceptible to the blood supply, and melanocytosis was promoted in the follicles and in the epidermis of Kitl-Tg C57BL/6 mice.

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.

Stochastic differentiation into an osteoclast lineage from cloned macrophage-like cells.

Differentiation into osteoclasts is induced by a macrophage colony-stimulating factor and receptor activator of nuclear-factor κB ligand. The macrophage-like cell line, C7 has the potential to differentiate into osteoclasts when it is cultured with both factors for 6 days. Although C7 is an established cell line, the frequency of differentiation into this lineage was less than 10%, and the ratio was maintained at a constant level, even after repeated cloning. In this study, to increase the differentiation of C7 cells to osteoclasts, C7 derivative treatments with several activators and/or inhibitors were performed for 3 days prior to setting osteoclast induction analysis; however, a reagent to significantly up-regulate the frequency of differentiation was not found. Only extended cultures for osteoclastogenesis exponentially increased the frequency of osteoclast precursors. It is likely that C7 cell differentiation into committed osteoclast precursors is on 'autopilot' rather than requiring specific signals to drive this process.

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.

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.

Analysis of capturing skin antigens in the steady state using milk fat globule EGF factor 8-deficient skin-hyperpigmented mice.

Dendritic cells (DCs) can capture apoptotic cells and present them to immune competent cells as self-antigens (Ags). Langerhans cells (LCs), DCs in the epidermis, are capable of presenting tissue-associated Ags in the steady state, suggesting that LCs may also capture apoptotic cells and transport them to skin regional lymph nodes (LNs). However, to what extent LCs utilize apoptotic cells as self-Ags in vivo is still unclear. To clarify this point, we examined the contribution of milk fat globule EGF factor 8 (MFG-E8), a secreted glycoprotein, to capturing skin Ags. MFG-E8 is expressed in several subsets of macrophages (M phi s) and DCs, including LCs, and crucial for recognizing and engulfing apoptotic cells. Using a skin-hyperpigmented KRT14-Kitl-Tg (Kitl-Tg) mouse system, we measured the accumulation of melanin granules (MGs), a marker of skin Ags, transported from the skin to regional LNs in Mfge8-deficient mice. Unexpectedly, their accumulation in Mfge8-deficient Kitl-Tg mice was comparable to that in Mfge8-heterozygous littermates. Mfge8-deficient DCs engulfed skin-derived MGs efficiently in vitro. The results indicate that MFG-E8 does not contribute critically or functions redundantly to capturing and trafficking of skin Ags in the steady state, and suggest a possibility that LCs may capture skin Ags in forms other than apoptotic cells in vivo.

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-beta-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 alpha 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.

Constant rate of steady-state self-antigen trafficking from skin to regional lymph nodes.

It is suggested that dendritic cells (DCs) capture and present both foreign antigens such as components of pathogens as well as endogenous self-antigens. However, the magnitude of self-antigen trafficking to secondary lymphoid organs is still unclear. Here we show constitutive trafficking of self-antigens from skin to regional lymph nodes (LNs) quantitatively using a KRT14-Kitl transgenic mouse. This mouse model expresses the Kit ligand in keratinocytes, shows hyperpigmentation of the epidermis and exhibits constitutive accumulation of melanin granules (MGs) in skin regional LNs transported by Langerhans cells. Using an MG-solubilization technique, we revealed that 128 microg per week of MGs, a marker of self-antigens, accumulated in skin regional LNs and that the rate of accumulation was constant from 3 to 50 weeks. Activation markers such as CD40, CD54 and CD86 did not increase in the LNs, and abrogation of CD40 signaling did not affect the accumulation. Additionally, the total amount of MGs did not increase significantly following stimulation with intravenous LPS injections. These results suggest that the accumulation is not caused by inflammatory stimuli, and the steady-state trafficking of self-antigens is intrinsically maintained at a constant rate. Because the levels of self-antigens as well as the phenotype of these DCs are thought to be important in the strength of immune responses, the results may imply that the constant rate of trafficking of self-antigens is required for maintaining homeostatic conditions, such as self-tolerance.

Ascorbic acid promotes osteoclastogenesis from embryonic stem cells.

Ascorbic acid (AA) is known to regulate cell differentiation; however, the effects of AA on osteoclastogenesis, especially on its early stages, remain unclear. To examine the effects of AA throughout the process of osteoclast development, we established a culture system in which tartrate-resistant acid phosphate (TRAP)-positive osteoclasts were induced from embryonic stem cells without stromal cell lines. In this culture system, the number of TRAP-positive cells was strongly increased by the addition of AA during the development of osteoclast precursors, and reducing agents, 2-mercaptoethanol, monothioglycerol, and dithiothreitol, failed to substitute for AA. The effect of AA was stronger when it was added during the initial 4 days during the development of mesodermal cells than when it was added during the last 4 days. On day 4 of the culture period, AA increased the total cell recovery and frequency of osteoclast precursors. Magnetic cell sorting using anti-Flk-1 antibody enriched osteoclast precursors on day 4, and the proportion of Flk-1-positive cells but not that of platelet-derived growth factor receptor alpha-positive cells was increased by the addition of AA. These results suggest that AA might promote osteoclastogenesis of ES cells through increasing Flk-1-positive cells, which then give rise to osteoclast precursors.

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 kappaB 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.

Lipopolysaccharide-induced osteoclastogenesis in Src homology 2-domain phosphatase-1-deficient viable motheaten mice.

Osteoclasts are hemopoietic cells that participate in bone resorption and remodeling. Receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) are critical for development of osteoclasts. The Toll-like receptor (TLR) family shares some of the downstream signaling with RANK. The TLR4 ligand, lipopolysaccharide (LPS), is reported to accelerate bone lysis; however, signaling via TLRs has never been reported to induce osteoclastogenesis without RANKL. In this study we showed that significant numbers of mature osteoclasts were generated from protein tyrosine phosphatase Src homology 2-domain phosphatase-1-defective Hcph(me-v)/Hcph(me-v) (me(v)/me(v)) bone marrow cells in the presence of M-CSF and LPS without addition of RANKL in culture. This M-CSF plus LPS-induced osteoclastogenesis was not inhibited by an anti-TNFalpha antagonistic antibody or by osteoprotegerin, a decoy receptor for RANKL. The replacement of RANKL by TLR ligands only occurred with LPS. Other ligands, a peptidoglycan for TLR2 or an unmethylated CpG oligonucleotide for TLR9, did not support osteoclast generation. The osteoclast precursors as well as RANKL-responsive osteoclast precursors were present in the Kit-positive cell-enriched fraction of bone marrow cells. Although me(v)/me(v) bone marrow cells required a comparable concentration of RANKL or TNFalpha as wild-type cells for the initiation of osteoclastogenesis, the numbers of multinucleated osteoclasts in me(v)/me(v) bone marrow cultures were significantly increased by the equivalent dose of RANKL or TNFalpha in the presence of M-CSF. These results indicate that a defect of Src homology 2-domain phosphatase-1 function not only accelerates physiological osteoclast development by RANKL/RANK, but also acquires a novel pathway for osteoclastogenesis by LPS.

Distinct antigen trafficking from skin in the steady and active states.

In antigen trafficking from the skin, it has been postulated that Langerhans cells/dendritic cells are activated after capturing exogenous antigens, up-regulate the expression of the chemokine receptor, CCR7, and migrate into lymphoid organs in response to the signaling of a chemokine, CCL21, which is expressed in lymphatic vessels and T cell zone stromal cells. Here we demonstrate that there is a distinct pathway of antigen trafficking from skin in the steady state that is independent of CCL21-CCR7 signaling. Employing melanin granules as an endogenous traceable antigen, we developed a system for visualizing antigen trafficking using mice with melanocytosis in the skin. We found the abrogation of antigen trafficking into regional lymph nodes (LN) in CCL21-Ser-deficient paucity of lymph node T cells (plt) mice in the active state induced by lipopolysaccharide injection, corresponding with previous reports, but normal accumulation of antigen in regional LN under steady-state conditions. These findings suggest that self-antigen is trafficking constitutively using pathway(s) other than that of the active state and the constitutive trafficking might regulate self-reactivity of the immune system.

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 Csfm(op) 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.

Regulation of osteoclast development by Notch signaling directed to osteoclast precursors and through stromal cells.

Osteoclasts are derived from hematopoietic precursor cells belonging to the monocyte/macrophage lineage. Osteoclast development has been reported to be regulated by several molecules such as macrophage colony-stimulating factor (M-CSF), receptor activator of nuclear factor (NF)-kappaB ligand (RANKL), and a decoy receptor of RANKL, osteoprotegerin (OPG). Recently, it was demonstrated that the Notch signaling pathway regulates myeloid differentiation and antagonizes cell fate determination, however, the effect of Notch signaling on the osteoclast lineage has not been reported. In this study, we examined the effect of signaling via Notch receptors on the differentiation into osteoclasts by using cells from the bone marrow, spleen, and peritoneal cavity, and a cloned macrophagelike cell line. Osteoclastogenesis was inhibited by an immobilized Notch ligand, Delta-1. The dish-adherent bone marrow cells precultured with M-CSF expressed both Mac-1 and M-CSF receptors, c-Fms; osteoclastogenesis of these cells was efficiently inhibited. The immobilized Delta-1 also down-regulated the surface c-Fms expression, while the c-Fms gene expression was not changed. Genes for Notch receptors and Notch ligands are expressed in not only hematopoietic cells but also stromal cells that support osteoclast development. Constitutively active Notch1-transfected stromal cells showed increased expression of RANKL and OPG genes, and strong inhibition of M-CSF gene expression, resulting in reduction of their ability to support osteoclast development. Taken together, these findings indicate that Notch signaling affects both osteoclast precursors and stromal cells and thereby negatively regulates osteoclastogenesis.