Toshiaki Ara

Impaired colonization of the gonads by primordial germ cells in mice lacking a chemokine, stromal cell-derived factor-1 (SDF-1)

Primordial germ cells (PGCs) are the founders of sperm or oocytes. PGCs migrate through the tissues of the embryos and colonize the gonads during development. However, the cytokines essential for colonization of the gonads by PGCs in mammals remain unclear. Stromal cell-derived factor-1 (SDF-1, also called PBSF and CXCL12) is a member of chemokines, a family of structurally related chemoattractive cytokines. SDF-1 and its primary physiologic receptor CXCR4 have multiple essential functions in development including colonization of bone marrow by hematopoietic cells and neuron localization within cerebellum during embryogenesis as well as B lymphopoiesis and cardiovasculogenesis. Here, we have shown that PGCs have cell-surface expression of CXCR4 and that, in SDF-1−/− mice, PGCs undergo directed migration through tissues of embryos, but the numbers of PGCs in the gonads are significantly reduced. The proliferation of PGCs within the gonads seems normal in the mutant mice. These findings reveal the essential role for SDF-1 in murine PGC development likely by controlling colonization of the gonads by PGCs.

Long-Term Hematopoietic Stem Cells Require Stromal Cell-Derived Factor-1 for Colonizing Bone Marrow during Ontogeny

The physiological role of SDF-1 on hematopoietic stem cells (HSCs) remains elusive. We show that colonization of bone marrow by HSCs in addition to myeloid cells is severely impaired in SDF-1(-/-) embryos by a long-term repopulation assay. Colonization of spleen by HSCs was also affected, but to a lesser extent. Enforced expression of SDF-1 under the control of vascular-specific Tie-2 regulatory sequences could completely rescue the reduction of HSCs but not myeloid cells in SDF-1(-/-) bone marrow. SDF-1 was detected in the vicinity of the vascular endothelial cells in fetal bone marrow. SDF-1 plays a critical role in colonization of bone marrow by HSCs and myeloid cells during ontogeny, and the mechanisms by which SDF-1 functions are distinct between HSCs and myeloid cells.

A Role of CXC Chemokine Ligand 12/Stromal Cell-Derived Factor-1/Pre-B Cell Growth Stimulating Factor and Its Receptor CXCR4 in Fetal and Adult T Cell Development in Vivo

The functions of a chemokine CXC chemokine ligand (CXCL) 12/stromal cell-derived factor-1/pre-B cell growth stimulating factor and its physiologic receptor CXCR4 in T cell development are controversial. In this study, we have genetically further characterized their roles in fetal and adult T cell development using mutant and chimeric mice. In CXCL12−/− or CXCR4−/− embryos on a C57BL/6 background, accumulation of T cell progenitors in the outer mesenchymal layer of the thymus anlage during initial colonization of the fetal thymus was comparable with that seen in wild-type embryos. However, the expansion of CD3−CD4−CD8− triple-negative T cell precursors at the CD44−CD25+ and CD44−CD25− stages, and CD4+CD8+ double-positive thymocytes was affected during embryogenesis in these mutants. In radiation chimeras competitively repopulated with CXCR4−/− fetal liver cells, the reduction in donor-derived thymocytes compared with wild-type chimeras was much more severe than the reduction in donor-derived myeloid lineage cells in bone marrow. Triple negative CD44+CD25+ T cell precursors exhibited survival response to CXCL12 in the presence of stem cell factor as well as migratory response to CXCL12. Thus, it may be that CXCL12 and CXCR4 are involved in the expansion of T cell precursors in both fetal and adult thymus in vivo. Finally, enforced expression of bcl-2 did not rescue impaired T cell development in CXCR4−/− embryos or impaired reconstitution of CXCR4−/− thymocytes in competitively repopulated mice, suggesting that defects in T cell development caused by CXCR4 mutation are not caused by reduced expression of bcl-2.

Human gingival fibroblasts are critical in sustaining inflammation in periodontal disease.

BACKGROUND AND OBJECTIVE A major factor in the pathogenesis of periodontal disease, which is one of the biofilm infectious diseases, is thought to be lipopolysaccharide (LPS), owing to its ability to cause inflammation and promote tissue destruction. Moreover, the elimination of pathogens and their component LPSs is essential for the successful treatment of periodontal disease. Lipopolysaccharide tolerance is a mechanism that prevents excessive and prolonged responses of monocytes and macrophages to LPS. Since persistence of inflammation is necessary for inflammatory cytokine production, cells other than monocytes and macrophages are thought to maintain the production of cytokines in the presence of LPS. In this study, we investigated whether human gingival fibroblasts (HGFs), the most abundant structural cell in periodontal tissue, might be able to maintain inflammatory cytokine production in the presence of LPS bynot displaying LPS tolerance. MATERIAL AND METHODS Human gingival fibroblasts were pretreated with LPS (from Porphyromonas gingivalis and Escherichia coli) and then treated with LPS, and the amounts of interleukin (IL)-6 and IL-8 in the cell culture supernatants were measured. The expression of negative regulators of LPS signalling (suppressor of cytokine signalling-1, interleukin-1 receptor-associated-kinase M and SH2 domain-containing inositol-5-phosphatase-1) was also examined in LPS-treated HGFs. RESULTS Human gingival fibroblasts did not display LPS tolerance but maintained production of IL-6 and IL-8 when pretreated with LPS, followed by secondary LPS treatment. Lipopolysaccharide-treated HGFs did not express negative regulators. CONCLUSION These results demonstrate that HGFs do not show LPS tolerance and suggest that this characteristic of HGFs sustains the inflammatory response in the presence of virulence factors.

Adrenomedullin/Cyclic AMP Pathway Induces Notch Activation and Differentiation of Arterial Endothelial Cells From Vascular Progenitors

Objective—The acquisition of arterial or venous identity is highlighted in vascular development. Previously, we have reported an embryonic stem (ES) cell differentiation system that exhibits early vascular development using vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2)-positive cells as common vascular progenitors. In this study, we constructively induced differentiation of arterial and venous endothelial cells (ECs) in vitro to elucidate molecular mechanisms of arterial-venous specification. Methods and Results—ECs were induced from VEGFR2+ progenitor cells with various conditions. VEGF was essential to induce ECs. Addition of 8bromo-cAMP or adrenomedullin (AM), an endogenous ligand-elevating cAMP, enhanced VEGF-induced EC differentiation. Whereas VEGF alone mainly induced venous ECs, 8bromo-cAMP (or AM) with VEGF supported substantial induction of arterial ECs. Stimulation of cAMP pathway induced Notch signal activation in ECs. The arterializing effect of VEGF and cAMP was abolished in recombination recognition sequence binding protein at the J&kgr; site deficient ES cells lacking Notch signal activation or in ES cells treated with &ggr;-secretase inhibitor. Nevertheless, forced Notch activation by the constitutively active Notch1 alone did not induce arterial ECs. Conclusions—Adrenomedullin/cAMP is a novel signaling pathway to activate Notch signaling in differentiating ECs. Coordinated signaling of VEGF, Notch, and cAMP is required to induce arterial ECs from vascular progenitors.

Membrane type 1-matrix metalloproteinase expression is regulated by E-cadherin through the suppression of mitogen-activated protein kinase cascade

To elucidate the role of E-cadherin in matrix metalloproteinases (MMPs) expression, we transfected to squamous carcinoma cells with E-cadherin cDNA. HN5 cells and mock-transfected HN5-neo cells expressed proMMP-2 and active MMP-2. E-cadherin-transfected HN5-EC cells produced comparable proMMP-2 but low active MMP-2; and membrane type 1-MMP (MT1-MMP) mRNA declined. Phosphorylated ERK, a marker of mitogen-activated protein (MAP) kinase cascade, also declined in HN5-EC cells. The addition of anti-E-cadherin antibody resulted in the disappearance of these alterations in HN5-EC cells. These results suggest that E-cadherin suppresses MAP kinase cascade and down-regulates MT1-MMP.

Aberrant TRPV1 Expression in Heat Hyperalgesia Associated with Trigeminal Neuropathic Pain

Trigeminal neuropathic pain is a facial pain syndrome associated with trigeminal nerve injury. However, the mechanism of trigeminal neuropathic pain is poorly understood. This study aimed to determine the role of transient receptor potential vanilloid 1 (TRPV1) in heat hyperalgesia in a trigeminal neuropathic pain model. We evaluated nociceptive responses to mechanical and heat stimuli using a partial infraorbital nerve ligation (pIONL) model. Withdrawal responses to mechanical and heat stimuli to vibrissal pads (VP) were assessed using von Frey filaments and a thermal stimulator equipped with a heat probe, respectively. Changes in withdrawal responses were measured after subcutaneous injection of the TRP channel antagonist capsazepine. In addition, the expression of TRPV1 in the trigeminal ganglia was examined. Mechanical allodynia and heat hyperalgesia were observed in VP by pIONL. Capsazepine suppressed heat hyperalgesia but not mechanical allodynia. The number of TRPV1-positive neurons in the trigeminal ganglia was significantly increased in the large-diameter-cell group. These results suggest that TRPV1 plays an important role in the heat hyperalgesia observed in the pIONL model.

Macrolide antibiotics like azithromycin increase lipopolysaccharide-induced IL-8 production by human gingival fibroblasts

ObjectiveMacrolide antibiotics are reported to modulate the production of cytokines in various type of cells. We examined the effect of macrolide antibiotics on inflammatory cytokines (IL-6 and IL-8) and chemical mediator (PGE2) and also matrix metalloproteinases (MMPs) productions by human gingival fibroblasts (HGFs) treated with lipopolysaccharide (LPS).MethodsThe effect of macrolide antibiotics [erythromycin (EM), azithromycin (AZM) and josamycin (JOM)] on HGFs proliferation were examined by MTT assay. HGFs were treated with LPS from Porphyromonas gingivalis (PgLPS) and macrolide antibiotics, and IL-6, IL-8 and PGE2 levels were evaluated by ELISA. MMPs were detected by gelatin zymography.ResultsAZM slightly but significantly decreased HGFs proliferation, while EM and JOM did not affected. AZM increased PgLPS-induced IL-8 production dose-dependently, while AZM did not alter IL-6 and PGE2 productions. EM and JOM did not altered PgLPS-induced IL-6, IL-8 and PGE2 productions. All macrolide antibiotics did not alter MMPs production. These results indicate that macrolide antibiotics have no direct anti-inflammatory effect. However, the use of the inhibitors of cell signaling pathway failed to reveal the mechanism that AZM enhanced PgLPS-induced IL-8 production.ConclusionThese results suggest macrolide antibiotics have an indirect anti-inflammatory effect as a result of their antimicrobial properties. Because AZM increased LPS-induced IL-8 production by HGFs, the possibility is considered that neutrophils may be migrated to periodontal tissue and phagocytize the periodontopathic bacteria more efficiently.

The effects of cigarette exposure on rat salivary proteins and salivary glands

OBJECTIVE Passive smoking is the involuntary inhalation of cigarette smoke (CS) and has an adverse impact on oral health. We examined the effect of CS exposure on saliva and salivary glands (SGs). METHODS Cigarette smoke-exposed rats were intermittently housed in an animal chamber with whole-body exposure to CS until killed. Whole saliva was collected before CS exposure (0 day), and 15 and 30 days after the start of CS exposure. Saliva secretion was stimulated by administration of isoproterenol and pilocarpine after anesthesia. SGs were collected on 31 days. RESULTS The increase in body weight of the CS-exposed rats was less than that of the control rats. Salivary flow rates did not differ at 0, 15 or 30 days after the start of CS exposure. However, the amylase and peroxidase activities and total protein content in the saliva were significantly lower in 15-day CS-exposed rats than in 15-day control rats. Histological examination of the SGs of CS-exposed rats showed vacuolar degeneration, vasodilation and hyperemia. CONCLUSION These results suggest that CS exposure has adverse impacts on salivary composition and SGs, which could aggravate the oral environment.

Protein kinase A enhances lipopolysaccharide-induced IL-6, IL-8, and PGE2 production by human gingival fibroblasts

ObjectivePeriodontal disease is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss in severe clinical cases. Interleukin (IL)-6, IL-8, and the chemical mediator prostaglandin E2 (PGE2) are known to play important roles in inflammatory responses and tissue degradation.Recently, we reported that the protein kinase A (PKA) inhibitor H-89 suppresses lipopolysaccharide (LPS)-induced IL-8 production by human gingival fibroblasts (HGFs). In the present study, the relevance of the PKA activity and two PKA-activating drugs, aminophylline and adrenaline, to LPS-induced inflammatory cytokines (IL-6 and IL-8) and PGE2 by HGFs were examined.MethodsHGFs were treated with LPS from Porphyromonas gingivalis and H-89, the cAMP analog dibutyryl cyclic AMP (dbcAMP), aminophylline, or adrenaline. After 24 h, IL-6, IL-8, and PGE2 levels were evaluated by ELISA.ResultsH-89 did not affect LPS-induced IL-6 production, but suppressed IL-8 and PGE2 production. In contrast, dbcAMP significantly increased LPS-induced IL-6, IL-8, and PGE2 production. Up to 10 μg/ml of aminophylline did not affect LPS-induced IL-6, IL-8, or PGE2 production, but they were significantly increased at 100 μg/ml. Similarly, 0.01 μg/ml of adrenaline did not affect LPS-induced IL-6, IL-8, or PGE2 production, but they were significantly increased at concentrations of 0.1 and 1 μg/ml. In the absence of LPS, H-89, dbcAMP, aminophylline, and adrenaline had no relevance to IL-6, IL-8, or PGE2 production.ConclusionThese results suggest that the PKA pathway, and also PKA-activating drugs, enhance LPS-induced IL-6, IL-8, and PGE2 production by HGFs. However, aminophylline may not have an effect on the production of these molecules at concentrations used in clinical settings (8 to 20 μg/ml in serum). These results suggest that aminophylline does not affect inflammatory responses in periodontal disease.