Adiponectin regulates functions of gingival fibroblasts and periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Adiponectin is a cytokine constitutively produced by adipocytes and exhibits multiple biological functions by targeting various cell types. However, the effects of adiponectin on primary gingival fibroblasts and periodontal ligament cells are still unexplored. Therefore, we investigated the effects of adiponectin on gingival fibroblasts and periodontal ligament cells. MATERIAL AND METHODS: The expression of adiponectin receptors (AdipoR1 and AdipoR2) on human gingival fibroblasts (HGFs), mouse gingival fibroblasts (MGFs) and human periodontal ligament (HPDL) cells was examined using RT-PCR and western blotting. HGFs and MGFs were stimulated with interleukin (IL)-1ß in the presence or absence of adiponectin, and the expression of IL-6 and IL-8 at both mRNA and protein levels was measured by real-time PCR and ELISA, respectively. Furthermore, small interfering RNAs (siRNAs) in MGFs were used to knock down the expression of mouse AdipoR1 and AdipoR2. The effects of adiponectin on the expression of alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2) genes were evaluated by real-time PCR. Mineralized nodule formation of adiponectin-treated HPDL cells was revealed by Alizarin Red staining. RESULTS: AdipoR1 and AdipoR2 were expressed constitutively in HGFs, MGFs and HPDL cells. Adiponectin decreased the expression of IL-6 and IL-8 in IL-1ß-stimulated HGFs and MGFs. AdipoR1 siRNA in MGFs revealed that the effect of adiponectin on reduction of IL-6 expression was potentially mediated via AdipoR1. Adiponectin-treated HPDL cells promoted the expression of ALP and Runx2 mRNAs and up-regulated ALP activity. Furthermore, adiponectin enhanced mineralized nodule formation of HPDL cells. CONCLUSION: Our observations demonstrate that adiponectin exerts anti-inflammatory effects on HGFs and MGFs, and promotes the activities of osteoblastogenesis of HPDL cells. We conclude that adiponectin has potent beneficial functions to maintain the homeostasis of periodontal health, improve periodontal lesions, and contribute to wound healing and tissue regeneration.

Study of beamlets extracted from a multi-aperture and five-stage acceleration system.

A beam optics study using the ITER-relevant high intense negative ion beams, such as 1 MeV, 200 A/m2, has been performed experimentally and analytically using a multi-aperture and five-stage accelerator. Initially, multi-beamlets generated from this accelerator were deflected in various directions due to the magnetic field and space charge repulsion between beams and showed various divergences. These had limited the pulse length and the beam energy. Compensation methods of the beamlet deflections have worked effectively and contributed to achieving the ITER requirement, the divergence angle of <7 mrad, and the deflection angle of <1 mrad for 1 MeV beam. The beam pulse has been gradually extended from 1 to 100 s and is now going to a longer pulse based on these results. One of the remaining issues is to understand and suppress peripheral components of the beam, namely, the halo, and to reduce the local heat loads observed around the aperture edge. This halo component has been successfully distinguished from the beam core by using a newly developed beam emittance measurement system for high intense beams. By combining this measured beam emittance and the beam simulation, it was clarified for the first time that the halo components are generated in an area of 1 mm width from the aperture edge.

Activation of adenosine receptor on gingival fibroblasts.

CD73 (ecto-5'-nucleotidase) on human gingival fibroblasts plays a role in the regulation of intracellular cAMP levels through the generation of adenosine, which subsequently activates adenosine receptors. In this study, we examined the involvement of ecto-adenosine deaminase, which can be anchored to CD26 on human gingival fibroblasts, in metabolizing adenosine generated by CD73, and thus attenuating adenosine receptor activation. Ecto-adenosine deaminase expression on fibroblasts could be increased by pre-treatment with a lysate of Jurkat cells, a cell line rich in cytoplasmic adenosine deaminase. Interestingly, the cAMP response to adenosine generated from 5'-AMP via CD73 and the ability of 5'-AMP to induce hyaluronan synthase 1 mRNA were significantly decreased by the pre-treatment of fibroblasts with Jurkat cell lysate. This inhibitory effect was reversed by the specific adenosine deaminase inhibitor. These results suggest that ecto-adenosine deaminase metabolizes CD73-generated adenosine and regulates adenosine receptor activation.

Analgesic effect of fentiazac after tooth extraction or minor oral surgery.

A new nonsteroidal anti-inflammatory agent, fentiazac, was used for analgesia after tooth extractions and minor oral surgery in two Japanese dental hospitals. The drug was administered as a single oral dose of either 50 mg or 100 mg. The 50-mg dose provided rapid analgesic effect, but its effect lasted only two to three hours in a number of patients. At a dose of 100 mg, fentiazac proved effective for 85% of 53 patients, usually providing marked reduction of disappearance of pain within one hour or less. Among patients in whom pain reappeared, the mean time for recurrence was four hours, indicating a satisfactory duration of analgesic effect. One side effect--loss of appetite--was reported by one patient in the entire series of 71 subjects. It is concluded that fentiazac is a highly effective analgesic agent with a wide margin of safety for use after dental procedures that produce pain.

Neurotrophic effects on the electrical properties of cultured muscle produced by conditioned medium from spinal cord explants.

The passive electrical properties of cultured chick skeletal muscle are significantly altered when the muscle is co-cultured with spinal cord explants. A reduced transverse membrane resistance appears to be responsible for the smaller values of input resistance, space constant, and time constant observed in co-cultures relative to those observed in pure muscle cultures. In this report, we show that neuromuscular junctions are not required in order to observe this neurotrophic effect because medium from spinal cord explant cultures is capable of producing the same reduction in transverse membrane resistance as the co-culturing of spinal cord explants with muscle. Control medium from liver explant cultures has no effect on muscle passive electrical properties. These results indicate that a trophic substance which is capable of regulating the electrical properties of excitable cells is released into the culture medium by spinal cord explants.

Periodontal regeneration by FGF-2 (bFGF) in primate models.

We recently demonstrated that a topical application of basic fibroblast growth factor (FGF-2; bFGF) to alveolar bone defects in beagle dogs enhanced periodontal regeneration. The purpose of this study was further characterization of the biological effects of FGF-2 in non-human primates. Thirty-two inflamed furcation class II defects were surgically created in 4 male primates. The gelatinous carrier alone or the carrier containing 0.1 or 0.4% human recombinant FGF-2 was topically applied to the defects and compared with no treatment. Eight weeks after application, the periodontal regeneration in those defects was analyzed. In all FGF-2-treated sites, significant periodontal regeneration was dose-dependently observed in greater amounts than in the carrier-treated or non-treated sites. No instances of epithelial down-growth, ankylosis, or root resorption were observed in the FGF-2-treated sites. These results indicate that a topical application of FGF-2 can enhance considerable periodontal regeneration in surgically created furcation class II defects of non-human primates.

Induction of CD13 on T-lymphocytes by adhesive interaction with gingival fibroblasts.

Lymphocytes in peripheral blood do not express CD13 (aminopeptidase N), a membrane alanyl metallopeptidase. However, it has been demonstrated that locally infiltrated lymphocytes in chronic inflammatory sites can be CD13-positive, and possible involvement of stromal cell adherence in the induction of CD13 has been suggested. In this study, we examined whether T-lymphocyte/gingival-fibroblast interaction can activate T-lymphocytes to express CD13. CD13 expression was induced on PMA-activated T-lymphocytes only when they adhered directly to human gingival fibroblasts (HGF) at 2 hrs after the co-culture began, while an increase in the enzyme activity of CD13 was also confirmed in activated T-lymphocytes that had been co-cultured with HGF. Furthermore, CD13-positive T-lymphocytes were detected in inflamed gingival tissues in vivo. Analysis of these results indicates that direct interaction with HGF is essential for the induction of CD13 expression on T-lymphocytes that was also observed in periodontitis lesions.

CD44-hyaluronate interaction participates in the adherence of T-lymphocytes to gingival fibroblasts.

It has already been clarified that peripheral blood T-lymphocytes which had been activated with phorbol 12-myristate 13-acetate (PMA) acquired the ability to bind to human gingival fibroblasts (HGF) and that the adherence was mediated by VLA integrins. However, these studies also raised the possibility that molecules other than VLA integrins should be involved in the adherence between T-lymphocytes and HGF. In this study, the possible involvement of CD44, a hyaluronate receptor, in heterotypic cell-cell interactions was investigated. It was confirmed that PMA-activated T-lymphocytes strongly adhered to plate-coated hyaluronate and that the hyaluronate binding was clearly inhibited by the addition of OS/37, a newly established mAb specific for the hyaluronate-binding epitope on CD44. Interestingly, OS/37 also blocked the HGF binding of the activated T-lymphocytes when the adherence to HGF was assessed at 4 degrees C, at which temperature the adhesion of integrin molecules diminished, while that of CD44 functioned normally. Immunofluorescence staining revealed that hyaluronate was anchored along the cell surface of HGF. Furthermore, the binding of activated T-lymphocytes to HGF was significantly inhibited by the treatment of HGF with hyaluronidase. These results clearly demonstrated that CD44-hyaluronate interactions participated at least in part in the adhesiveness of T-lymphocytes to HGF.

Activation of adenosine-receptor-enhanced iNOS mRNA expression by gingival epithelial cells.

A series of reports has revealed that adenosine has a plethora of biological actions toward a large variety of cells. In this study, we investigated the influence of adenosine receptor activation on iNOS mRNA expression in human gingival epithelial cells (HGEC) and SV-40-transformed HGEC. HGEC expressed adenosine receptor subtypes A1, A2a, and A2b, but not A3 mRNA. Ligation of adenosine receptors by a receptor agonist, 2-chloroadenosine (2CADO), enhanced iNOS mRNA expression by both HGEC and transformed HGEC. In addition, the adenosine receptor agonist enhanced the production of NO(2)(-)/NO(3)(-), NO-derived stable end-products. An enhanced expression of iNOS mRNA and NO(2)(-)/NO(3)(-) was also observed when SV40-transformed HGEC were stimulated with CPA or CGS21680, A1- or A2a-selective adenosine receptor agonists, respectively. These results provide new evidence for the possible involvement of adenosine in the regulation of inflammatory responses by HGEC in periodontal tissues.

Involvement of CD73 (ecto-5'-nucleotidase) in adenosine generation by human gingival fibroblasts.

Adenosine has various biological effects on human gingival fibroblasts (HGF) and epithelial cells closely associated with inflammation, such as cytokine production and cell adhesion. However, the mechanism of adenosine formation in periodontal tissues is not yet defined. In this study, we examined the involvement of CD73 (ecto-5'-nucleotidase) in adenosine generation by HGF. CD73 was detected on in vitro-maintained HGF by immunocytochemistry and flow cytometric analysis. Adenosine production was observed following the addition of 5'-AMP, the substrate of CD73-associated ecto-5'-nucleotidase. Moreover, the addition of 5'-AMP to cultured HGF resulted in the elevation of cyclic adenosine monophosphate (cAMP). The 5'-AMP-induced increase in intracellular cAMP level was inhibited markedly by xanthine amine congener, an adenosine receptor antagonist, and partially by alpha,beta-methylene adenosine 5'-diphosphate, an ecto-5'-nucleotidase inhibitor. These results suggest that CD73 on HGF is a critical enzyme responsible for the generation of adenosine, an immunomodulator that activates adenosine receptors.

Direct interaction between gingival fibroblasts and lymphoid cells induces inflammatory cytokine mRNA expression in gingival fibroblasts.

In inflamed periodontal lesions, dense infiltration of lymphocytes is usually observed in the extravascular periodontal connective tissue, adjacent to gingival fibroblasts. Our previous study revealed that activated lymphocytes can adhesively interact with gingival fibroblasts in vitro. In the present study, we investigated whether gingival fibroblasts are activated through direct interaction with lymphoid cells by monitoring the expression of inflammatory cytokine mRNA in human gingival fibroblasts (HGF). Co-culture with various human lymphoid cells in vitro resulted in a marked increase in the expression of IL-1alpha, IL-1beta, and IL-6 mRNA by the HGF. In addition, expression of the mRNA of the IL-1beta-converting enzyme (ICE), which is essential to produce the mature form of IL-1beta, was constitutively observed in the HGF, suggesting that mature IL-1beta is produced by these cells. When HGF were cultured with the culture supernatant of the lymphoid cells, the increase in the inflammatory cytokine mRNA expression was not observed. Similarly, when HGF and lymphoid cells were cultured in the same well but separated by a membrane which prevented direct contact between the cells, no increase in inflammatory cytokine mRNA expression was observed. These results strongly indicate that direct interaction between these heterotypic cell types transduces activation signals into HGF that induce an increase in inflammatory cytokine mRNA expression. Furthermore, IL-1beta mRNA expression in the HGF was synergistically increased when HGF directly interacted with lymphoid cells in the presence of exogeneous IL-1beta. The present study demonstrates that direct interaction between HGF and lymphoid cells stimulates HGF to increase inflammatory cytokine mRNA expression, and raises the possibility that heterotypic cell-cell interaction may facilitate local inflammatory reactions.

Separation of aromatic isomers on cyclophane-bonded stationary phases.

A cyclophane (CP66)-bonded silica gel stationary phase (CP66-SP) was prepared and the retention of water-insoluble hydrophobic compounds on it was investigated in comparison with that on the CP44-bonded stationary phase (CP44-SP) reported previously. Like CP44-SP, it retained aromatic compounds more strongly than the corresponding alicyclic compounds, as was expected by the cavity size of the cyclophane. The CP66-SP also showed isomer-selectivity for monosubstituted and disubstituted naphthalenes, but its selectivity was perfectly reversed to that of the CP44-SP. On the CP66-SP, isomers having methyl and ethyl groups at beta-position were eluted prior to those having groups at alpha-position, whereas on the CP44-SP beta-substituted naphthalenes were retained more strongly than alpha-substituted ones. Isomers of three- and four-ring aromatic compounds were also separated on these cyclophane-bonded stationary phases. The retention order on the CP66-SP was almost opposite to that on the CP44-SP; on the CP66-SP, the retention order was phenanthrene > anthracene, and chrysene > 1,2-benzanthracene > 2,3-benzanthracene, whereas on the CP44-SP, anthracene > phenanthrene, and 2,3-benzanthracene > chrysene > 1,2-benzanthracene. The retention mechanism of aromatic compounds is discussed on the basis of the structure of the cyclophane-involved complex.

Production of interleukin-8 and nitric oxide in human periapical lesions.

Bacterial infection of the pulp and root canal system leads to the recruitment of immunocompetent cells in the periapex and stimulates inflammatory cell responses to produce a variety of inflammatory mediators. Cytokines, reactive oxygen intermediates, and reactive nitrogen intermediates are frequently found at sites of acute inflammation. In this study, we measured the levels of interleukin (IL)-8 and nitric oxide (NO) in the periapical exudate (PE) from human periapical lesions and investigated the association of these mediators with the clinical symptoms of periapical periodontitis. PE samples were collected from root canals during routine endodontic treatments. The IL-8 concentration was measured by the enzyme-linked immunosorbent assay, and the NO level was measured as nitrite/nitrate concentration assayed by the Griess reaction. Detectable levels of IL-8 and nitrite/nitrate were found in 24 and 19 of 27 PE-samples, respectively. Although PE-IL-8 and nitrite/nitrate concentration showed a broad range, a significantly positive correlation was found between both mediators. Also, significantly higher IL-8 levels were found in PE from lesions that had painful symptoms at the sampling visit. However, there was no relationship between elevated NO levels and clinical symptoms. These results suggest that the up-regulation of IL-8 may have a critical role in the development of the symptoms of periapical disease.

Quantitative analysis of immunocompetent cells in human periapical lesions: correlations with clinical findings of the involved teeth.

Human periapical lesions develop as a result of a pathological immune response to continuous stimuli from infected root canals. This study identified the immunocompetent cells in such lesions immunohistochemically and quantified them to examine their interrelationships and correlations with clinical findings. The number of IgG-containing cells in CD4+ cell (Th/i)-rich lesions (> or = 55 CD4+ cells/2 x 10(4) microns 2) was significantly larger than in CD4+ cell-poor lesions (< 55 CD4+ cells/2 x 10(4) microns 2). This indicated that the CD4+ cells and the IgG-containing cells acted together against antigenic stimuli. The proportion of T cells in the mononuclear infiltrates varied with the size of the periapical lesions. The proportion of CD11+ cells (monocytes/macrophages) was significantly larger in lesions which showed a positive reaction to percussion or were tender on palpation than in the lesions without these symptoms. These observations suggest that T cells may play an important role in the development of periapical lesions and that CD11+ cells may be involved in the development of symptoms.

Antigen-presenting-cell function of interferon gamma-treated human gingival fibroblasts.

The present study was carried out to examine the antigen-presenting cell (APC) functions of human gingival fibroblasts (HGF) elicited with IFN gamma. Stimulation of HGF with IFN gamma clearly induced HLA-DR expression and enhanced expression of intercellular adhesion molecule-1 (ICAM-1) on HGF. Despite the phenotypical resemblance of IFN gamma-treated HGF to so-called APC, HLA-DR positive HGF were unable to induce proliferation of allo-reactive peripheral blood T cells (PBT) isolated from different donors. The failure of IFN gamma-treated HGF to stimulate unprimed allo-reactive PBT was not due to the lack of production of IL-1 or the immunosuppressive effect of PGE2 from HGF. On the other hand, the fact that detectable expression of CD80, ligand for CD28, was not found on IFN gamma-treated HGF may at least in part explain the ineffective function of HGF as APC. Interestingly, IFN gamma-treated HGF induced proliferation of primed allo-reactive CD4+ T cells in a HLA-DR dependent manner, suggesting that IFN gamma-treated HGF may have the ability to stimulate pre-activated T cells. We then confirmed that high levels of IFN gamma mRNA were detectable in inflamed gingival tissue. Although it cannot be concluded from this study that HGF are incapable of effectively presenting antigenic peptides to autologous T cells bearing appropriate T cell receptors, present results suggest that HGF may be affected by locally-secreted IFN gamma and that the IFN gamma-stimulated HGF may play a role in regulating immune responsiveness in inflammatory periodontal lesions.

Interferon-gamma-dependent immunosuppressive effects of human gingival fibroblasts.

Immunoregulatory functions of human gingival fibroblasts (HGF) were examined. As in fibroblasts isolated from other tissues, HGF were activated with interferon-gamma (IFN-gamma) to express HLA-DR molecules. Despite the fact that the IFN-gamma-treated HGF showed phenotypical resemblance to so-called antigen-presenting cells (APC), the IFN-gamma-treated HGF were ineffective stimulators of alloreactive peripheral T cells. Conversely, IFN-gamma-treated HGF dramatically inhibited the proliferative responses of allogeneic APC (allo-APC) or phytohaemagglutinin (PHA)-stimulated T cells. Immunosuppressive effects of culture supernatant (CS) of IFN-gamma-treated HGF were low and were completely abrogated by the addition of indomethacin. Moreover, the production of prostaglandin E2 (PGE2) by HGF was not affected by IFN-gamma. These results suggest that IFN-gamma-dependent immunosuppressive effects of HGF were not due to PGE2 produced by HGF. In order to investigate further the mechanism(s) of IFN-gamma-dependent immunosuppressive effects of HGF, activated T cells and IFN-gamma-treated HGF were separately cultured in the same well by collagen films which were assembled in cylindrical cells and disturbed physical interactions between T cells and HGF. The proliferative responses of T cells which directly contacted with IFN-gamma-treated HGF were inhibited more significantly than those of T cells which did not contact with IFN-gamma-treated HGF. This suggests that IFN-gamma-dependent immunosuppressive effects of HGF were mediated by direct interactions between T cells and activated HGF. The present results suggest that IFN-gamma-stimulated HGF would modulate the immune responses of locally infiltrated T cells in periodontal lesions.

Very late antigen integrins are involved in the adhesive interaction of lymphoid cells to human gingival fibroblasts.

To date, it is still unclear how the trafficking and retention of activated lymphocytes in periodontal lesions are regulated. In this study, we investigated the molecular basis for the adhesive interactions between lymphocytes and human gingival fibroblasts (HGF). Peripheral blood T lymphocytes (PBT) exhibited binding ability, but only when the calls were activated with phorbol 12-myristate 13-acetate (PMA). Among several human cell lines tested, PMA-stimulated Molt-4, a human T-cell leukaemia line, also displayed significant binding ability to HGF. In order to clarify the molecule(s) involved in this cell-cell interaction, a panel of monoclonal antibodies (mAb) was prepared to PMA-activated Molt-4 and one clone, 4-145, was selected on the basis of its ability to block the binding of PMA-activated Molt-4 to HGF. Moreover, 4-145 inhibited the binding of not only activated Molt-4 but also activated PBT and other cell types to HGF. Biochemical and flow cytometric analyses revealed that 4-145 probably recognizes the beta 1 chain of very late antigen (VLA) integrins. Blocking experiments using mAb specific for the alpha-chain of VLA integrins demonstrated the involvement of alpha 4 (VLA-4) and, to a lesser extent, alpha 5 (VLA-5) chains in the adhesive interactions between T cells and HGF. Despite the significant involvement of VLA integrins in the adhesive interaction between PBT and HGF, the binding of PBT to human dermal fibroblasts (HDF) was not abrogated by 4-145, suggesting that HGF and HDF differ in their requirement of VLA integrins for adhesion to activated PBT. Furthermore, the fact that vascular cell adhesion molecule-1 (VCAM-1), one of the ligands of VLA-4, was not detected on HGF by flow cytometry and anti-fibronectin (FN) Ab did not block the adhesive interaction to HGF suggests that not-yet-identified ligand(s) for VLA-4 might be present on HGF.