Eisuke Domae

ICAM-1–Dependent Homotypic Aggregates Regulate CD8 T Cell Effector Function and Differentiation during T Cell Activation

A hallmark of T cell activation in vitro and in vivo is the clustering of T cells with each other via interaction of the LFA-1 integrin with ICAM-1. The functional significance of these homotypic aggregates in regulating T cell function remains unknown. We used an APC-free in vitro activation system to demonstrate that stimulation of purified naive CD8 T cells results in enhanced expression of ICAM-1 on T cells that is sustained by the inflammatory cytokine IL-12 and associated with robust T cell aggregates. ICAM-1–deficient CD8 T cells proliferate normally but demonstrate a striking failure to aggregate. Interestingly, loss of ICAM-1 expression results in elevated levels of IFN-γ and granzyme B, as well as enhanced cytotoxicity. Similar results were obtained when anti–LFA-1 Ab was used to block the clustering of wild-type T cells. ICAM-1 ligation is not required for IFN-γ regulation, as clustering of ICAM-1–deficient CD8 T cells with wild-type T cells reduces IFN-γ expression. Analysis using a fluorescent reporter that monitors TCR signal strength indicates that T cell clustering limits T cell exposure to Ag during activation. Furthermore, T cell clustering promotes the upregulation of the CTLA-4 inhibitory receptor and the downregulation of eomesodermin, which controls effector molecule expression. Activation of ICAM-1–deficient CD8 T cells in vivo results in an enhanced percentage of KLRG-1+ T cells indicative of short-lived effectors. These results suggest that T cell clustering represents a mechanism that allows continued proliferation but regulates T cell effector function and differentiation.

Enamel Matrix Derivative Protein Stimulated Wound Healing via Phosphoinositide 3-Kinase

BACKGROUND Enamel matrix derivative (EMD) protein has been clinically used for periodontal regeneration, but the molecular mechanisms are not clear. Previous studies suggested that the activation of phosphoinositide 3-kinase (PI 3-kinase) plays a key role in facilitating cell migration. Given that the migration of osteoblasts is one of the key steps in the wound-healing processes, we hypothesized that EMD protein would stimulate osteoblast migration by activating PI 3-kinase. In this study, we tested this hypothesis using MG-63 cells as model systems to evaluate mechanisms of migration by stimulation with EMD protein. METHODS Confluent MG-63 cells were mechanically scratched using a sterilized 1-mm pipette tip that removed the cells within a circular area. The wells were incubated for 24 hours in various stimulation conditions (25, 50, or 100 microg/ml EMD protein) with or without the PI 3-kinase inhibitor wortmannin (1, 10, and 100 nM) or LY294002 (1, 10, and 100 microM). Migrated cells in the wound section were counted by randomly selecting three areas from one well. The activation of PI 3-kinase by EMD protein was evaluated by the phosphorylation of Akt using Western blot analysis. RESULTS Although EMD protein did not affect proliferation, it enhanced migration into wounds on MG-63 cells. We showed that EMD protein enhanced the phosphorylation of Akt in a dose-dependent manner. We demonstrated that the PI 3-kinase inhibitors wortmannin and LY294002 blocked migration into wounds and the phosphorylation of Akt enhanced by EMD protein in MG-63 cells. CONCLUSION These results demonstrated that the activation of PI 3-kinase plays a key role in the EMD protein-stimulated migration of osteoblasts.

RNA interference-mediated knockdown of Smad1 inhibits receptor activator of nuclear factor κB ligand expression induced by BMP-2 in primary osteoblasts

OBJECTIVE BMP-2 induces osteoblast differentiation and activates osteoclast formation. Here, we investigated the role of Smad1, a molecule that signals downstream of BMP-2, in mediating the effects of BMP-2 on osteoclast differentiation induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in osteoblasts. DESIGN The effects of 1,25(OH)2D3 and BMP-2 in osteoclasts were examined using polymerase chain reaction and Western blotting to measure changes in target gene and protein expression. Immunostaining was carried out to investigate the localization of the vitamin D receptor (VDR) in the nucleus in response to BMP-2. RESULTS Stimulation with both 1,25(OH)2D3 and BMP-2 resulted in significantly greater osteoclast formation and receptor activator of nuclear factor κB ligand (RANKL) mRNA expression compared to stimulation with 1,25(OH)2D3 alone. In addition, expression of the VDR protein was increased, enhancing the activity of 1,25(OH)2D3. Interestingly, knockdown of Smad1 resulted in reduced osteoclast formation, RANKL mRNA expression, and VDR protein expression compared with control cells. Costimulation with 1,25(OH)2D3 and BMP-2 enhanced VDR localization in the nucleus. CONCLUSIONS We found that BMP-2 induced Smad1 activation, thereby influencing the localization of VDR in the nucleus in the presence of 1,25(OH)2D3 and resulting in increased RANKL mRNA expression. These effects ultimately resulted in enhanced osteoclast differentiation.

Cytokine-mediated activation of human ex vivo-expanded Vγ9Vδ2 T cells

Vγ9Vδ2 T cells, the major subset of the human peripheral blood γδ T-cell, respond to microbial infection and stressed cells through the recognition of phosphoantigens. In contrast to the growing knowledge of antigen-mediated activation mechanisms, the antigen-independent and cytokine-mediated activation mechanisms of Vγ9Vδ2 T cells are poorly understood. Here, we show that interleukin (IL) -12 and IL-18 synergize to activate human ex vivo-expanded Vγ9Vδ2 T cells. Vγ9Vδ2 T cells treated with IL-12 and IL-18 enhanced effector functions, including the expression of IFN-γ and granzyme B, and cytotoxicity. These enhanced effector responses following IL-12 and IL-18 treatment were associated with homotypic aggregation, enhanced expression of ICAM-1 and decreased expression of the B- and T-lymphocyte attenuator (BTLA), a co-inhibitory receptor. IL-12 and IL-18 also induced the antigen-independent proliferation of Vγ9Vδ2 T cells. Increased expression of IκBζ, IL-12Rβ2 and IL-18Rα following IL-12 and IL-18 stimulation resulted in sustained activation of STAT4 and NF-κB. The enhanced production of IFN-γ and cytotoxic activity are critical for cancer immunotherapy using Vγ9Vδ2 T cells. Thus, the combined treatment of ex vivo-expanded Vγ9Vδ2 T cells with IL-12 and IL-18 may serve as a new strategy for the therapeutic activation of these cells.

Expression of Plectin-1 and Trichohyalin in Human Tongue Cancer Cells

In basal squamous cells, plectin-1 interacts with intermediate filaments, whereas trichohyalin, which is distributed primarily in the medulla and inner root sheath cells of human hair follicles, plays a role in strengthening cells during keratinization. Although both cytoskeletal proteins occur in trace amounts in human tongue epithelial cells, there are minimal data on their expression in human tongue primary cancer cells. We therefore investigated the expression of plectin-1 and trichohyalin in human tongue epithelial cell line (DOK) and tongue cancer cell line (BICR31) using western blotting and FITC-labeled immunocytochemistry techniques. DOK and BICR31 cells were cultivated to subconfluence in Dulbecco’s Modified Eagle’s Medium containing 0.4 μg/ml of hydrocortisone and 10% fetal bovine serum, and the levels of trichohyalin and plectin-1 were determined by western blot analysis and immunocytochemical staining. Trichohyalin expression was clearly observed, with no differences between DOK and BICR31 cells. Although DOK cells expressed trace levels of plectin-1, obvious plectin-1 bands were detected in western blot analyses of BICR31 cells. Immunocytochemical staining revealed that trichohyalin and plectin-1 localize in the cytoplasm. Trichohyalin was diffusely distributed in both cell lines, and colocalization of trichohyalin and cytokeratin 1/10 was observed in almost all BICR31 cells. There were no correlations between western blot and immunocytochemical data for trichohyalin. Conversely, correlations in immunochemical reactions for plectin-1 were observed. Most DOK cells showed no localization of plectin-1, but strong reactions were detected in the cytoplasm of BICR31 cells. These results indicate that trichohyalin is expressed by cancerous tongue epithelial cells during various stages of malignancy and that plectin-1 provides an index of malignancy.

Corrigendum to “Effects of gallotannin on osteoclastogenesis and the p38 MAP kinase pathway” [Orthod. Waves 75 (2016) 105–113]

Yukiko Hosoyama D.D.S. , Eisuke Domae D.D.S., Ph.D. *, Seiji Goda D.D.S., Ph.D. , Naoyuki Matsumoto D.D.S., Ph.D. d Graduate School of Dentistry, Department of Orthodontics, Osaka Dental University, 8-1 Kuzuhahanazonocho, Hirakata, Osaka 573-1121, Japan Department of Biochemistry, Osaka Dental University, 8-1 Kuzuhahanazonocho, Hirakata, Osaka 573-1121, Japan Department of Oral Science, Graduate School of Dentistry, Kanagawa Dental University, Yokosuka, Kanagawa 2388580, Japan Department of Orthodontics, Osaka Dental University, 8-1 Kuzuhahanazonocho, Hirakata, Osaka 573-1121, Japan

Effects of gallotannin on osteoclastogenesis and the p38 MAP kinase pathway

Abstract Purpose Osteoclasts are multinucleated giant cells that specialize in bone resorption and work together with bone-forming osteoblasts to maintain bone homeostasis. However, excessive osteoclast activation accounts for bone diseases, such as osteoporosis and periodontitis. In previous studies, natural small-molecule compounds have been shown to regulate osteoclastogenesis and osteoclast functions. Here we demonstrate that gallotannin, a hydrolyzable plant tannin, suppresses osteoclast differentiation. Methods We first used an ex vivo bone marrow culture system containing both osteoclast precursors and surrounding cells, thereby resembling physiological conditions, to evaluate the suppressive effect of gallotannin. We also used a RANKL-induced osteoclastogenesis assay containing only osteoclast precursors to confirm the suppressive effect of gallotannin in the absence of effects from other cells. Results The suppressive effect of gallotannin was associated with the reduced RANKL-mediated induction of NFATc1, a critical transcription factor involved in osteoclast differentiation. We further confirmed that gallotannin reduced the p38 MAPK pathway activation, which is mediated by M-CSF and RANKL. This pathway suppression might underlie the suppression of NFATc1 production and subsequent reduction in osteoclast differentiation. Conclusion Our data indicate that the natural small-molecule compound gallotannin might be useful as a novel anti-bone resorptive agent.

Enamel matrix derivative protein enhances production of matrixmetalloproteinase-2 by osteoblasts

BackgroundMatrix metalloproteinases (MMPs) degrade the extracellular matrix (ECM) and regulate remodeling and regeneration of bone. Enamel matrix derivative (EMD) protein has been used clinically for periodontal regeneration, although its molecular mechanisms are not clear. We evaluated the role of matrix metalloproteinases (MMPs) in regulating EMD-dependent degradation of gelatin on oeoblast-like cell line MG63.MethodsMG-63 cells (osteoblast cell line) were incubated with 100 μg/ml EMD protein in the presence or absence of MMP-2 tissue inhibitor for 20 h followed by incubation on DQ-gelatin-coated plates for 4 h. MG-63 cells (1 × 106) were preincubated with SB203580 for 30 min at 37°C and were then placed in 100 μg/ml EMD protein for 24 h. Conditioned media were collected and detected by Western blot analysis.ResultsEMD protein enhanced cell-mediated degradation of gelatin, which was inhibited by the MMP inhibitor TIMP-2. Furthermore, MMP-2 was produced by MG63 cells in response to EMD protein in a P38 MAPK-dependent manner. In addition, blocking of p38 MAPK activation by SB203580 significantly inhibited generation of the active form of MMP-2.ConclusionP38 MAPK pathway promotes expression MMP-2 in EMD activated osteoblasts, which in turn stimulates periodontal regeneration by degrading matrix proteins in periodontal connective tissue.