Hideaki Nakae

CXCL12 and CXCR4 expression by human gingival fibroblasts in periodontal disease

CXCL12 is a CXC chemokine that is related to lymphocyte infiltration and angiogenesis in inflammatory sites such as arthritis. However, the expression and roles of CXCL12 in periodontal disease are uncertain. The aim of this study was to assess the expression of CXCL12 and its receptor, CXCR4, in periodontal tissue and to investigate the properties of CXCL12 and CXCR4 expression by human gingival fibroblasts (HGF). RT‐PCR analysis revealed that CXCL12 and CXCR4 mRNA were expressed in both normal gingival tissues and periodontal diseased tissues. Immunohistochemistry disclosed that CXCL12 was expressed and CXCR4 positive cells were found in both normal and periodontal diseased gingival tissues. Our in vitro experiments elucidated that HGF constitutively produced CXCL12, and the levels were enhanced by stimulation with tumour necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ), transforming growth factor‐β (TGF–β), regulated upon activation normal T cell expressed and secreted (RANTES) and macrophage inflammatory protein 3α (MIP‐3α). On the other hand, heat killed Porphyromonas gingivalis (P. gingivalis) and P. gingivalis LPS reduced the CXCL12 production by HGF. Flow cytometry analysis clarified that CXCR4 was highly expressed on HGF, and CXCR4 expression was abrogated by TNF‐α, IFN‐γ and P. gingivalis LPS. Moreover, CXCL12 induced vascular endothelial growth factor (VEGF) production by HGF. Our results demonstrated that CXCL12 might be related to CXCR4+ cells infiltration and angiogenesis both in normal periodontal tissues and periodontal diseased tissue. P. gingivalis, a known periodontal pathogen, inhibits the production of CXCL12 and the expression of CXCR4 by HGF. This fact means that P. gingivalis may inhibit CXCR4+ cells infiltration and neovascularization in periodontal tissue and escape from the immune response.

Increase of CCL20 expression by human gingival fibroblasts upon stimulation with cytokines and bacterial endotoxin.

We have demonstrated recently that CCL20 was expressed in periodontal diseased tissues and abundant CCR6 positive T cells infiltrated in periodontally diseased tissue. However, it is uncertain which cells can elicit CCL20 production. In the present study, we examined the properties of CCL20 production by human gingival fibroblasts (HGF) culture. Here, we report that interleukin‐1 beta (IL‐1β), tumour necrosis factor‐alpha (TNF‐α) and Escherichia coli lipopolysaccharide (LPS) can significantly induce the production of CCL20 by HGF. We found that TNF‐α and E. coli LPS enhanced the production of CCL20 by HGF treated with IL‐1β. In contrast, interferon‐gamma (IFN‐γ) dramatically diminished CCL20 production induced by IL‐1β. Moreover, we demonstrated that nuclear factor‐kappaB (NF‐κB), p38 mitogen‐activated protein kinase (MAPK) and extracellular signal‐regulated kinases (ERK) play an important role in mediating the production of CCL20 induced by IL‐1β and TNF‐α. On the other hand, we found that not only NF‐κB, p38 MAPK and ERK but also c‐Jun NH2‐terminal kinase (JNK) are involved in CCL20 production induced by E. coli LPS. Finally, we found that HGF express CCR6, CCL20 receptor, and CCL20 induced vascular endothelial growth factor (VEGF) by HGF. Taken together, these findings that HGF will be a source of CCL20 in periodontal tissue, and the CCL20 production will be controlled by proinflammatory cytokine and bacterial LPS in periodontally diseased tissue. Thus, CCL20 by HGF might be involved in inflammatory cells infiltration, and promote the progression of periodontal disease.

Cytokines differentially regulate ICAM-1 and VCAM-1 expression on human gingival fibroblasts

The expression of intercellular adhesion molecule‐1 (ICAM‐1) and vascular adhesion molecule‐1 (VCAM‐1) on human gingival fibroblasts (HGF) may be important for migration and retention of inflammatory cells in periodontally diseased tissue. This study aimed to assess which cytokines regulate ICAM‐1 and VCAM‐1 expression on HGF. Tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ enhanced both ICAM‐1 and VCAM‐1 expression on HGF. Interleukin (IL)‐1β mainly up‐regulated ICAM‐1 expression. On the other hand, IL‐4 and IL‐13 enhanced only VCAM‐1 expression on HGF. IL‐10 did not modulate both ICAM‐1 and VCAM‐1 expression. Transforming growth factor (TGF)‐β1 enhanced ICAM‐1 expression. However, TGF‐β1 inhibited the VCAM‐1 expression induced by TNF‐α or IL‐4. Both ICAM‐1 and VCAM‐1 expression by HGF was inhibited by nuclear factor‐kappaB (NF‐κB) activation inhibitor (MG‐132). Mitogen‐activated protein kinases (MAPK) inhibitors did not influence ICAM‐1 expression induced by TNF‐α. Interestingly, VCAM‐1 expression was enhanced by MEK inhibitor (PD98059) and c‐Jun NH2‐terminal kinase (JNK) inhibitor (SP600125). These results mean that the balance of cytokines in periodontally diseased tissue may be essential for control of ICAM‐1 and VCAM‐1 expression on HGF, and the balance of ICAM‐1 and VCAM‐1 expression might be important for regulation of leucocytes infiltration and retention in periodontally diseased tissue.

Proteoglycans of Bovine Cementum: Isolation and Characterization

The proteoglycans associated with the mineralized matrix of bovine cementum have been studied biochemically and their distribution within this tissue localized immunohistochemically. Both hyaluronate and proteoglycans were fractionated by DEAE-Sephacel ion-exchange chromatography. The proteoglycans eluted in three separate peaks of which two contained alkali labile protein associated with glycosaminoglycans, and one appeared as free glycosaminoglycan chains. Analysis of the glycosaminoglycans identified chondroitin sulfate as the predominant species, although minor quantities of dermatan sulfate and heparan sulfate were also identified. Agarose-acrylamide gel electrophoresis and Sepharose CL-6B molecular sieve chromatography of the proteoglycans indicated them to be smaller in size with respect to periodontal ligament and gingival proteoglycans, but similar to bone and dentine proteoglycans. Amino acid analyses indicated subtle differences between cementum and bone proteoglycans. Using a monoclonal antibody (9-A-2) which recognizes the unsaturated disaccharide of chondroitinase ACII-digested glycosaminoglycans, chondroitin sulfate was identified in the pericellular environment within the lacunae housing the cementoblasts as well as in the extracellular matrix of cementum.

Caries-related Bacteria and Cytokines Induce CXCL10 in Dental Pulp

Marked infiltration of inflammatory cells, such as activated T-cells, is observed in the progression of pulpitis; however, little is known about the mechanism of their recruitment into pulpal lesions. It has been recently demonstrated that CXC chemokine ligand 10 (CXCL10) chemoattracts CXC chemokine receptor 3 (CXCR3)-positive activated T-cells. We therefore examined whether CXCL10 is involved in the pathogenesis of pulpitis. CXCL10 mRNA expression levels in clinically inflamed dental pulp were higher than those in healthy dental pulp. Immunostaining results revealed that CXCL10 was detected in macrophages, endothelial cells, and fibroblasts in inflamed dental pulp, and that CXCR3 expression was observed mainly on T-cells. Moreover, cultured dental pulp fibroblasts produced CXCL10 after stimulation with live caries-related bacteria, peptidoglycans, and pro-inflammatory cytokines. In contrast, heat-killed bacteria did not induce CXCL10 secretion. These findings suggest that CXCL10-CXCR3 may play an important role in the pulpal immune response to caries-related bacterial invasion. Abbreviations: CXCL10, CXC chemokine ligand 10; CXCR3, CXC chemokine receptor 3; IFN, interferon; FBS, fetal bovine serum; LTA, lipoteichoic acid; PGN, peptidoglycan; IL, interleukin; TNF, tumor necrosis factor; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; CCL, C-C chemokine ligand; TLR, Toll-like receptor; NOD, nucleotide oligomerization domain; HDPF, human dental pulp fibroblasts.

Proinflammatory effects of tumour necrosis factor-like weak inducer of apoptosis (TWEAK) on human gingival fibroblasts.

Tumour necrosis factor (TNF)‐like weak inducer of apoptosis (TWEAK), a member of the TNF family, is a multi‐functional cytokine that regulates cellular proliferation, angiogenesis, inflammation and apoptosis. In this study, we investigated TWEAK expression in periodontally diseased tissues and the effect of TWEAK on human gingival fibroblasts (HGF). Reverse transcription–polymerase chain reaction (RT–PCR) analysis and immunohistochemistry revealed that TWEAK and the TWEAK receptor, fibroblast growth factor‐inducible 14 (Fn14), mRNA and protein were expressed in periodontally diseased tissues. HGF expressed Fn14 and produced interleukin (IL)‐8 and vascular endothelial growth factor (VEGF) production upon TWEAK stimulation in a dose‐dependent manner. The IL‐8 and VEGF production induced by TWEAK was augmented synergistically by simultaneous stimulation with transforming growth factor (TGF)‐β1 or IL‐1β. IL‐1β and TGF‐β1 enhanced Fn14 expression in a dose‐dependent manner. Moreover, TWEAK induced intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) expression on HGF in a dose‐dependent manner. The ICAM‐1 expression induced by TWEAK was augmented by TGF‐β1. On the other hand, the TWEAK‐induced VCAM‐1 expression was inhibited by TGF‐β1. Phosphatidylinositol 3‐kinase (PI3K) and nuclear factor‐kappaB (NF‐κB) inhibitor inhibit both ICAM‐1 and VCAM‐1 expression induced by TWEAK. However, mitogen‐activated protein kinase (MEK) and c‐Jun NH2‐terminal kinase (JNK) inhibitor enhanced only VCAM‐1 expression on HGF. These results suggest that TWEAK may be involved in the pathophysiology of periodontal disease. Moreover, in combination with IL‐1β or TGF‐β1, TWEAK may be related to the exacerbation of periodontal disease to induce proinflammatory cytokines and adherent molecules by HGF.

Catechins Inhibit CCL20 Production in IL-17A-Stimulated Human Gingival Fibroblasts

CC chemokine ligand 20 (CCL20) plays a pivotal role in the recruitment of Th17 cells and thus in the development of periodontal disease. Epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), the major catechins in green tea, have multiple beneficial effects, but the effects of catechins on CCL20 production in human gingival fibroblasts (HGFs) are not known. In this study, we investigated the mechanisms by which EGCG and ECG inhibit interleukin (IL)-17A-induced CCL20 production in human gingival fibroblasts. IL-17A increased CCL20 production in HGFs in a concentration-dependent manner. EGCG and ECG prevented IL-17A-mediated CCL20 production in HGFs. Inhibitors of p38 mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK) decreased IL-17A-induced CCL20 production. EGCG and ECG prevented IL-17A-induced phosphorylation of p38 MAPK and ERK in HGFs. In addition, EGCG and ECG attenuated IL-17 receptor expression on HGFs. These data provide a novel mechanism through which the green tea flavonoids catechins could be used to provide direct benefits in periodontal disease.

Catechins inhibit CXCL10 production from oncostatin M-stimulated human gingival fibroblasts.

CXC chemokine ligand 10 (CXCL10) plays a pivotal role in the recruitment of Th1 cells and, thus, in the development of periodontal disease. Epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), the major catechins derived from green tea, have multiple beneficial effects, but the effects of catechins on CXCL10 production from human gingival fibroblasts (HGFs) is not known. In this study, we investigated the mechanisms by which EGCG and ECG inhibit oncostatin M (OSM)-induced CXCL10 production in HGFs. HGFs constitutively expressed glycoprotein 130 and OSM receptor beta (OSMR beta), which are OSM receptors. OSM increased CXCL10 production in a concentration-dependent manner. EGCG and ECG prevented OSM-mediated CXCL10 production by HGFs. Inhibitors of p38 mitogen-activated protein kinase, c-Jun N-terminal kinase (JNK), phosphatidylinositol-3-OH kinase and signal transducer and activator of transcription (STAT)3 decreased OSM-induced CXCL10 production. EGCG significantly prevented OSM-induced phosphorylation of JNK, Akt (Ser473) and STAT3 (Tyr705 and Ser727). ECG prevented phosphorylation of JNK and Akt (Ser473). In addition, EGCG and ECG attenuated OSMR beta expression on HGFs. These data provide a novel mechanism through which the green tea flavonoids, catechins, can provide direct benefits in periodontal disease.

Expression of fractalkine (CX3CL1) and its receptor, CX3CR1, in periodontal diseased tissue

The regulatory role of chemokines and chemokine receptors on specific leucocyte recruitment into periodontal diseased tissue is poorly characterized. We observed that leucocytes infiltrating inflamed gingival tissue expressed marked levels of CX3CR1. In periodontal diseased tissue, the expression of fractalkine and CX3CR1 mRNA was detected by reverse transcription‐polymerase chain reaction (RT‐PCR) and further, fractalkine was distributed mainly on endothelial cells, as shown by immunohistochemistry. Moreover, we can detect CX3CR1‐expressing cells infiltrated in periodontal diseased tissue by immunohistochemical staining. Furthermore, fractalkine production by human umbilical vein endothelial cells (HUVEC) was up‐regulated by pathogen‐associated molecular patterns (PAMPs), including Porphyromonas gingivalis lipopolysaccharide (LPS). Thus, these findings suggested that CX3CR1 and the corresponding chemokine, fractalkine may have an important regulatory role on specific leucocyte migration into inflamed periodontal tissue.

Proinflammatory effects of muramyldipeptide on human gingival fibroblasts

BACKGROUND AND OBJECTIVE Because human gingival fibroblasts (HGFs) are the predominant cells in periodontal tissues, we hypothesized that HGFs are contributed to receptors for components of bacteria. In this study, we focused on expression and function of nucleotide binding oligomerization domain 2 (NOD2) in HGFs, which is a mammalian cytosolic pathogen recognition molecule. MATERIAL AND METHODS Expression of NOD2 in HGFs was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry. Production of interleukin (IL)-6, IL-8, cc chemokine ligand2, cxc chemokine ligand10 (CXCL10) and CXCL11 from HGFs was examined by enzyme-linked immunosorbent assay (ELISA). We used RT-PCR and immunohistochemistry to detect the NOD2 expression in human gingival tissues. RESULTS We found clear NOD2 expression in HGFs. Upon stimulation with NOD2 agonist, muramyldipeptide (MDP), production of proinflammatory cytokines was enhanced. Moreover, MDP-induced production of proinflammatory cytokines was inhibited in a different manner by mitogen-activated protein kinase inhibitors and phosphatidylinositol 3-kinase inhibitor. Furthermore, MDP enhanced CXCL10 and CXCL11 productions by tumor necrosis factor-alpha (TNF-alpha)- or interferon-gamma (IFN-gamma)-stimulated HGFs, although MDP alone did not induce these chemokines. TNF-alpha and IFN-gamma increased NOD2 expression in HGFs. In addition, we detected NOD2 expression in mononuclear cells and HGFs in periodontally diseased tissues. CONCLUSION These findings indicate that MDP which induces production of cytokines and chemokines from HGFs is related to the pathogenesis of periodontal disease.