Teruyuki Saho

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-la, IL-1β, and IL-6 mRNA by the HGF. In addition, expression of the mRNA of the IL-1β-converting enzyme (ICE), which is essential to produce the mature form of IL-1β, was constitutively observed in the HGF, suggesting that mature IL-lp 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-1β mRNA expression in the HGF was synergistically increased when HGF directly interacted with lymphoid cells in the presence of exogeneous IL-1β. 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.

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 NO2 -/NO3 -, NO-derived stable end-products. An enhanced expression of iNOS mRNA and NO2 -/NO3 - 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.

IL-15 up-regulates iNOS expression and NO production by gingival epithelial cells ☆

To investigate the biological activity of epithelial cells in view of host defense, we analyzed the mRNA expression of inducible NOS (iNOS) as well as NO production by human gingival epithelial cells (HGEC) stimulated with IL-15. RT-PCR analysis revealed that HGEC expressed IL-15 receptor alpha-chain mRNA. In addition, stimulation with IL-15 enhanced iNOS expression by HGEC through an increase of both mRNA and protein levels. Moreover, IL-15 up-regulated the production of NO(2)(-)/NO(3)(-), a NO-derived stable end product, from HGEC. The enhanced NO production by IL-15 was inhibited by AMT, an iNOS-specific inhibitor. These results suggest that IL-15 is a potent regulator of iNOS expression by HGEC and involved in innate immunity in the mucosal epithelium.

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°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.

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 α,β-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.

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.

Establishment of monoclonal antibody which blocks the adherence of activated-T lymphocytes to human gingival fibroblasts(HGF).

Molecular mechanism (s) involved in adhesive interactions between T lymphocytes and human gingival fibroblasts (HGF) were examined. Both human peripheral blood T lymphocytes and T cell leukemia line, Molt-4 demonstrated significant binding ability to HGF only when the cells were activated with phorbol 12-myristate 13-acetate (PMA) . In order to reveal the molecule (s) responsible for the adherence, a panel of monoclonal antibodies (mAbs) was prepared against PMA-activated Molt-4 and mAb, 4-145 was finally entablished on the basis of its ability to block the adherence of activated Molt -4 to HGF. Biochemical and functional analysis revealed that 4-145 recognizes an epitope on VLAβ1 chain. Furthermore, blocking experiments utilizing mAbs specific for VLAα chains demonstrated that VLA-4 plays an important role for mediating the adhesion of activated T lymphocytes to HGF.Results demonstrated in this study suggest that VLA integrins play critical roles not only in binding to activated endothelial cells in inflammatory sites but also in retention of activated T lymphocytes in inflamed periodontal lesions by mediating the adhesive interaction between T lymphocytes and HGF.

Rapid diagnosis of periodontitis based on enzymatic activity derived from periodontopathic bacteria. Evaluation of the efficacy of periodontal pocket curettage by SK-013.

本研究の目的は, 歯周病原菌が特異的に産生するペプチダーゼ活性を迅速に測定できる検査薬SK-013 kitを用いて, 歯周ポケット掻爬術の効果判定を行い, その有用性を評価することである。歯周炎患者64名のうち33名を歯周治療群 (T群), 31名を未治療群 (NT群) に分け, 1名につき1歯を被検歯として選択した。T群にはブラッシングによるプラークコントロール (初診時開始) および歯周ポケット掻爬術 (プラークコントロール後4週目) を行いその効果を臨床診査並びにSK-013kitによる酵素活性検査にて評価した (6, 8, 12週目) 。NT群については, 特に歯周治療は行わず初診時および12週目に臨床診査並びに酵素活性検査を行った。その結果, SK-013活性値は, プラークコントロールのみでは変化を示さなかったが, 歯周ポケット掻爬術後, GI, PD, AL, BOPと共に著しく減少した。NT群のSK-013活性は, いずれの検査時においても有意な変化は認められなかった。これらの結果よりSK-013kitによる酵素活性測定法は, 歯周治療効果をモニタリングするのに非常に高い有用性があることが示された。