Effects of interleukin-4 on proteoglycan accumulation in human gingival fibroblasts

In inflammatory gingival diseases, cytokines have been demonstrated to play critical roles by coordinating the stimulation of immunological and connective tissue cells. The activities of these cells, degrading and remodeling extracellular matrices, constitute the major pathological and repair processes. Thus, elucidating cellular and molecular events occurring in inflamed connective tissues is crucial for the understanding and treatment of inflammation. In order to test a hypothesis that proinflammatory cytokines affect metabolism of major extracellular matrix molecules, we studied metabolism of proteoglycans (PGs) by human gingival fibroblasts (HGF) under the influence of interleukin-4 (IL-4) as a model of gingivitis. HGF in cell culture were metabolically radiolabeled using [3H]glucosamine and [35S]sulfate in the presence or absence of IL-4, and the labeled PGs were analyzed by chromatographic techniques. The incorporation of 35S into PGs increased with IL-4 both in media and cell layer. At 100 ng/ml of IL-4, the increment of 35S incorporation over control culture was 16-39% (p<0.001) in media and 12-35% (p=0.01) in cell layer. The 35S-labeled macromolecules were PGs containing heparan sulfate (HS) and chondroitin sulfate (CS) chains. From the molecular weight and glycosaminoglycan composition analyses, versican and perlecan-type and biglycan and decorin-type were very likely to be the major PG constituents both in media and cell layer. IL-4 stimulated synthesis of versican and perlecan-type more potently than biglycan and decorin-type. With IL-4 treatment, the ratio of CSPG/HSPG decreased in media and increased in cell layer. This ratio suggested that syndecan family HSPGs were also present in HGF. In conclusion, IL-4 stimulated accumulation of CS/HSPGs in human gingival fibroblasts.     

Contrasting responses of human gingival and periodontal ligament fibroblasts to bacterial cell-surface components through the CD14/Toll-like receptor system

We compared human periodontal ligament fibroblasts with human gingival fibroblasts isolated from the same donor to examine interleukin-8 (IL-8) responses of the cells to Salmonella lipopolysaccharide, a water-soluble peptidoglycan from Staphylococcus epidermidis and the synthetic muramyldipeptide, with special reference to the possible involvement of the CD14/Toll-like receptor (TLR) system of the cells in the responses. Human gingival fibroblasts expressed CD14 on their surfaces and strongly expressed CD14 mRNA, while human periodontal ligament fibroblasts showed considerably lower levels of expression in both respects. Both cells expressed mRNA of TLR-related molecules, i.e. TLR2, TLR4, MD-2 and MyD88, although human periodontal ligament fibroblasts expressed TLR2 more strongly than human gingival fibroblasts. Human gingival fibroblasts exhibited a stronger IL-8 response than human periodontal ligament fibroblasts to lipopolysaccharide, while human periodontal ligament fibroblasts exhibited a response comparable to, or slightly stronger than, that of human gingival fibroblasts to S. epidermidis peptidoglycan and muramyldipeptide. The IL-8 responses of both cells to lipopolysaccharide and S. epidermidis peptidoglycan were completely inhibited by antihuman CD14 monoclonal antibody (MAb). The responses of both cells to lipopolysaccaride were significantly inhibited by antihuman TLR4 MAb, while those to S. epidermidis peptidoglycan were inhibited by antihuman TLR2 MAb. In contrast, muramyldipeptide activated both types of cells in a TLR2- and TLR4-independent manner, although the activities of muramyldipeptide on human gingival fibroblasts, but not human periodontal ligament fibroblasts, were significantly inhibited by anti-CD14 MAb.     

Expression of fibrillins and tropoelastin by human gingival and periodontal ligament fibroblasts in vitro

The elastic system fibers consist of three different types, oxytalan, elaunin and elastic fibers, which differ in the relative content of microfibrils and elastin. In periodontal tissues, oxytalan fibers are known to be distributed in the periodontal ligament and gingiva, while elaunin and elastic fibers are present only in the gingiva. We examined the in vitro synthesis of microfibrils and elastin by human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (HPLF). The two kinds of HGF and HPLF were cultured in MEM containing 10% newborn calf serum for 30 days. Since fibrillin-1 and fibrillin-2 are the major components of microfibrils involved in elastogenesis, we investigated the synthesis of fibrillins and tropoelastin in the conditioned medium of HGF and HPLF. Western blot analysis revealed fibrillin-1 and fibrillin-2 to occur in the HGF and HPLF culture medium, HGF exhibiting a higher level of synthesis than HPLF. Tropoelastin, on the other hand, was detected only in the medium of HGF after day 24. In addition, analysis of RNA extracted from HGF and HPLF on day 30 showed that only HGF expressed mRNA encoding tropoelastin. Immunohistochemically, accumulation of tropoelastin in the perinuclear area was found only in HGF. These results show that HGF expressed microfibrils and elastin, while HPLF expressed only microfibrils for the experimental period, and suggest a biochemical basis for the different distribution of elastic system fibers of the gingiva and periodontal ligament in vivo.     

内毒素对牙龈成纤维细胞mCD14表达的影响

目的:研究mCD14在牙龈成纤维细胞的膜表面分布及内毒素对mCD14在牙龈成纤维细胞膜表面表达的影响。方法:组织块法培养人牙龈成纤维细胞,利用免疫组化和Western blot方法研究mCD14在牙龈成纤维细胞的膜表面分布,同时观察内毒素对mCD14在牙龈成纤维细胞膜表面表达的影响。结果:免疫组织化学染色实验和蛋白印迹实验结果均表明mCD14在牙龈成纤维细胞的膜表面表达阳性,同时LPS可增强膜表面的mCD14的表达。结论:本实验表明正常人牙龈成纤维细胞可表达mCD14,LPS可以上调膜表面mCD14的表达,从而导致牙周损伤。     

Gamma-interferon enhances expression of CD14/MyD88 and subsequent responsiveness to lipopolysaccharide from Actinobacillus actinomycetemcomitans in human gingival fibroblasts

OBJECTIVES: CD14, toll-like receptor 4 (TLR4) and MyD88 have been shown to mediate responsiveness in host cells to lipopolysaccharide. We investigated here the regulatory effects of inflammatory cytokines on the expression of membrane CD14 (mCD14), TLR4 and MyD88, and on subsequent responsiveness to lipopolysaccharide from Actinobacillus actinomycetemcomitans in human gingival fibroblasts. MATERIALS AND METHODS: Following treatment with either interleukin-1beta, tumor necrosis factor-alpha (TNF-alpha) or gamma-interferon (IFN-gamma), expression of mCD14/TLR4 and MyD88 was determined by flow cytometry and western blotting, respectively. After pretreatment with IFN-gamma, cells were pre-incubated with either anti-CD14 antibody MY4 or anti-TLR4 antibody HTA125 and subsequently treated with A. actinomycetemcomitans lipopolysaccharide. Then, phosphorylation of mitogen-activated protein (MAP) kinases and IkappaBalpha was examined by western blotting, and production of interleukin-6 and interleukin-8 was measured by their respective enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: IFN-gamma stimulated expression of mCD14, whereas -1beta and TNF-alpha did not. Expression of MyD88 but not TLR4 was also enhanced by IFN-gamma. The lipopolysaccharide activated MAP kinases, such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38, and IkappaBalpha and stimulated production of interleukin-6 and interleukin-8. The lipopolysaccharide-stimulated interleukin-6 and interleukin-8 production was markedly inhibited by MY4 or HTA125. Pretreatment with IFN-gamma augmented the following activation of MAP kinases and IkappaBalpha and production of interleukin-6 and interleukin-8 in response to the lipopolysaccharide. CONCLUSIONS: These results suggest that the augmentation by IFN-gamma of the responsiveness to A. actinomycetemcomitans lipopolysaccharide, such as activation of MAP kinases and IkappaBalpha and terminal cytokine production in human gingival fibroblasts, may be partially mediated by up-regulation of CD14 and MyD88 expression.     

Contrasting responses of human gingival and periodontal ligament fibroblasts to bacterial cell‐surface components through the CD14/Toll‐like receptor system

We compared human periodontal ligament fibroblasts with human gingival fibroblasts isolated from the same donor to examine interleukin‐8 (IL‐8) responses of the cells to Salmonella lipopolysaccharide, a water‐soluble peptidoglycan from Staphylococcus epidermidis and the synthetic muramyldipeptide, with special reference to the possible involvement of the CD14/Toll‐like receptor (TLR) system of the cells in the responses. Human gingival fibroblasts expressed CD14 on their surfaces and strongly expressed CD14 mRNA, while human periodontal ligament fibroblasts showed considerably lower levels of expression in both respects. Both cells expressed mRNA of TLR‐related molecules, i.e. TLR2, TLR4, MD‐2 and MyD88, although human periodontal ligament fibroblasts expressed TLR2 more strongly than human gingival fibroblasts. Human gingival fibroblasts exhibited a stronger IL‐8 response than human periodontal ligament fibroblasts to lipopolysaccharide, while human periodontal ligament fibroblasts exhibited a response comparable to, or slightly stronger than, that of human gingival fibroblasts to S. epidermidis peptidoglycan and muramyldipeptide. The IL‐8 responses of both cells to lipopolysaccharide and S. epidermidis peptidoglycan were completely inhibited by antihuman CD14 monoclonal antibody (MAb). The responses of both cells to lipopolysaccaride were significantly inhibited by antihuman TLR4 MAb, while those to S. epidermidis peptidoglycan were inhibited by antihuman TLR2 MAb. In contrast, muramyldipeptide activated both types of cells in a TLR2‐ and TLR4‐independent manner, although the activities of muramyldipeptide on human gingival fibroblasts, but not human periodontal ligament fibroblasts, were significantly inhibited by anti‐CD14 MAb.     

Expression of CD73/ecto-5'-nucleotidase on human gingival fibroblasts and contribution to the inhibition of interleukin-1alpha-induced granulocyte-macrophage colony stimulating factor production

BACKGROUND AND OBJECTIVES: CD73/5'-nucleotidase (5'-NT) is an ectoenzyme that participates in immune/inflammatory reactions. We examined the possible expression of CD73/5'-NT on human gingival fibroblasts (hGF), which are important to the immune/inflammatory system in periodontal tissue. METHODS AND RESULTS: We demonstrated that CD73/5'-NT was expressed on hGF by flow cytometry. We found that pre-treatment of hGF with 5'-AMP induced marked inhibition of granulocyte-macrophage colony-stimulating factor (GM-CSF) production from hGF upon stimulation with interleukin-1alpha (IL-1alpha) by enzyme-linked immunosorbent assay (ELISA). A specific inhibitor of 5'-NT, adenosine 5'-[alpha,beta-methylene] diphosphate blocked the inhibition of GM-CSF production, suggesting that adenosine converted from 5'-AMP acts on the inhibitory effects. The GM-CSF inhibition suggested that A3 receptor might be involved. The rank order of agonists was found to be (N6-benzyl-5'-N-ethylcarboxamidoadenosine) A3 receptor agonist > or = (2-chloroadenosine) non-selective agonist > (CGS-21680) A2A receptor agonist > adenosine > or = (N6-cyclohexyladenosine) A1 agonist. Further support for the main role of A3 receptor was the binding A3 antagonist [9-chloro-2-(2-furanyl)-5-([phenylacetyl]amino)[1,2,4]-triazolo[1,5-c]quinazdine] reversed the effect of adenosine, but no significant reverse was observed by A1 (1,3-dipropyl-8-cyclopentylxanthine), A2 [3,7-dimethyl-1-(2-propargyl)xanthine], A2A[8-(3-chlorostyryl)caffeine], and A2B (alloxazine) antagonists. The CD73/5'-NT expression was increased upon stimulation with gamma-interferon, but not other stimulants such as tumor necrosis factor-alpha, IL-4, lipopolysaccharide from Porphyromonas gingivalis and Escherichia coli, and fimbriae from P. gingivalis, and this increase was correlated with the enhanced GM-CSF inhibition by 5'-AMP but not adenosine. CONCLUSIONS: These findings suggested that CD73/5'-NT on hGF exerts an anti-inflammatory effects in periodontal disease by conversion from 5'-AMP to adenosine.     

Response of human gingival fibroblasts to prostaglandins

The effects of various prostaglandins (PGs) on the functions of human gingival fibroblasts (Gin-1 cells; ATCC CRL 1292) were examined by phase-contrast microscopy, cell-counting and radioautographic experiments. Tested PGs were PGA₁, PGA₂, PGB₁, PGB₂, PGD₂, PGE₁, PGE₂, PGF₁α, PGF₂α, PGI₂, 6-keto-PGF₁α, 9α-11α-methanoepoxy-PGF₂α, and thromboxane (TX) B₂. PGA₁, and PGD₂ at 30 μM caused morphological deformation of Gin-1 cells. All the PGs tested at 30 μM suppressed the proliferation of Gin-1 cells in the logarithmic growth phase. Furthermore, all the PGs tested at 10 μM suppressed DNA synthesis, collagen synthesis, and noncollagenous protein synthesis in confluent Gin-1 cells, while exerting no effect on GAG synthesis. The concentrations of PGs used are beyond those found in healthy gingiva. However, in periodontitis the local concentrations of some PGs within the gingiva are expected to be extremely elevated beyond the physiological level. These results suggest that PGs may play an important role as a negative regulator in metabolism and some pathologic gingival conditions by suppressing the functions of gingival fibroblasts.     

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 by not 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.     

In vitro immunoexpression of extracellular matrix proteins in dental pulpal and gingival human fibroblasts

AIM: To compare the expression of extracellular matrix (ECM) components in human pulpal and gingival fibroblasts in vitro. METHODOLOGY: Cultured dental pulp fibroblasts and gingival mucosa fibroblasts were used. Tenascin (TN), fibronectin (FN), type I (col I) and III collagen (col III) and osteonectin (ONEC) were detected by immunofluorescence. Main morphological characteristics were also analysed by light microscopy (LM) and transmission electron microscopy. RESULTS: The results revealed different expression patterns of the proteins. TN and ONEC were only immunoexpressed by pulpal fibroblast cells, suggesting a role of these glycoproteins in formation of mineralized tissues. FN and col I were present in the cytoplasms of both cell types. No expression of col III was detected. Different morphological characteristics were visualized under LM, in which pulpal fibroblasts were spindle-shaped with a wide cytoplasm, while gingival fibroblast cells exhibited stellate/pyramidal configuration, with rounded nuclei. However, ultrastructurally, both cell lineages showed very well developed rough endoplasmatic reticulum and Golgi complex. CONCLUSIONS: Due to the immunodetection of TN and ONEC on pulpal fibroblasts, the present findings demonstrated that a pulpal fibroblast cell is similar to an osteoblastic cell rather than an undifferentiated mesenchymal cell, such as a gingival fibroblast cell. Functional differences between the two cell lines may then be suggested.     

淫羊藿苷、黄芩苷和大黄素抑制LPS对体外培养人牙龈成纤维细胞的作用

目的：探讨不同浓度淫羊藿苷、黄芩苷和大黄素对细菌内毒素脂多糖（lipopolysaccharide,LPS）刺激人牙龈成纤维细胞（human gingival fibroblasts,HGF）产生前列腺素E：（prostaglandin E2,PGE2）的影响。方法：以培养的人牙龈成纤维细胞为实验模型,用MTT试验检测淫羊藿苷、黄芩苷和大黄素抑制LPS对牙龈成纤维细胞毒性作用的效果,初步筛选其作用浓度：然后用酶联免疫法检测三种提取物对LPS刺激牙龈成纤维细胞产生重要的骨吸收因子PGE：的影响。结果：LPS对HGF有明显的细胞毒性,三种提取物均可显著提高细胞成活率。LPS作用6h后培养上清中PGE,含量上升,而同时加入淫羊藿苷、黄芩苷和大黄素各浓度组PGE：含量显著低于对照组qo〈o．01）。结论：淫羊藿苷、黄芩苷和大黄素均可抑制LPS对HGF的细胞毒性作用,并且可抑制LPS刺激HGF产生PGE2。     

Suppressive effect of the antimicrobial peptide LL-37 on expression of IL-6, IL-8 and CXCL10 induced by Porphyromonas gingivalis cells and extracts in human gingival fibroblasts

Porphyromonas gingivalis is a major periodontogenic bacterium and possesses immunostimulatory components, such as lipopolysaccharides (LPS) and fimbriae. The host antimicrobial peptide, LL-37, suppresses proinflammatory responses of immune cells but its effect on human gingival fibroblasts (HGFs) is not known. In this study, we assessed the effect of LL-37 on the proinflammatory responses of HGFs stimulated with P. gingivalis cells and their components. Live P. gingivalis cells did not induce proinflammatory responses of HGFs, and LL-37 did not alter these responses. However, LL-37 was able to suppress the killed P. gingivalis cell-induced secretion of interleukin (IL)-6 and IL-8. LL-37 also suppressed the expression of IL6, IL8, and CXCL10 genes that was induced by P. gingivalis components, including phenol-water extracts, lipid A, and fimbriae, and the induction of phosphorylation of p38 and extracellular signal-regulated kinase (ERK) by P. gingivalis lipopolysaccharide (LPS). CAMP was found to be expressed in oral epithelial cells but not in HGFs, despite stimulation with P. gingivalis components. Therefore, LL-37 can exert a suppressive effect on P. gingivalis-induced proinflammatory responses of HGFs in a paracrine manner, suggesting that excess inflammatory responses to P. gingivalis in the gingival tissue are suppressed by LL-37 in vivo.     

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目的    探讨烟草提取液（ST）和尼古丁液对人牙龈成纤维细胞（human gingival fibroblast,HGF）的影响。方法    2009年6—12月于昆明医学院口腔实验室,用含不同浓度的ST和尼古丁液的培养液体外培养HGF 5 d,应用噻唑蓝（MTT）快速比色法,检测细胞生长增殖情况。结果    与对照组相比,ST质量浓度从5 g／L开始时,抑制HGF的增殖,差异有统计学意义（P < 0.05）,随着浓度的加大,抑制作用更明显,呈浓度依赖性。同样,高质量浓度的尼古丁液从0.01 g／L开始对细胞增殖有抑制作用,并呈浓度依赖性,差异有统计学意义（P < 0.05）。结论    高浓度的ST和尼古丁均能抑制HGF的增殖,低浓度时则对细胞的增殖无影响。烟草对口腔黏膜上皮细胞的增殖呈现抑制作用,并随着浓度的加大,抑制作用愈发明显。     

Adenosine regulates the production of interleukin-6 by human gingival fibroblasts via cyclic AMP/protein kinase A pathway

Adenosine has been reported to alter a variety functions of the cells that participate in inflammatory responses. However, the effect(s) of adenosine on human gingival fibroblasts (HGF), one of the immunomodulator cells in inflamed periodontal lesions, remains to be established. In this study, we examined the influence of adenosine on the production of interleukin (IL)‐6 by HGF. Ligation of adenosine receptors with adenosine or its related analogue, 2‐chloroadenosine (2‐CADO), increased IL‐6 production by HGF without any other stimuli. In addition, adenosine and 2‐CADO enhanced the cyclic AMP (cAMP) level in HGF as did prostaglandin E₁(PGE₁) and forskolin. Interestingly, these cAMP‐arising reagents and the permeable cAMP analogue, dibutyryl cAMP (dbtcAMP), also increased IL‐6 production by HGF. These results suggest that cAMP is involved in adenosine‐ induced IL‐6 production by HGF. Adenosine‐induced IL‐6 production was suppressed by protein kinase A (PKA) inhibitor, H89, indicating that cAMP/PKA pathway is involved in the induction. Moreover, the experiments using antagonists specific for adenosine receptor subtypes revealed that the adenosine‐induced IL‐6 production by HGF was, at least in part, mediated by the adenosine A2b receptor. These results provide new evidence for the possible effects of adenosine or its related analogue as an immunomodulator in inflammatory periodontal lesions.     

Expression and intracellular localization of progesterone receptors in cultured human gingival fibroblasts

OBJECTIVE: The aim of this immunocytochemical study was to characterize the expression and distribution of the progesterone receptor (PR) and estrogen receptor (ER) in gingival fibroblasts using culture cells derived from people at various ages. BACKGROUND: The reaction of female hormones is tissue or cell specific, and receptor availability in the cell is one of the major causes for the different reactions. Gingiva is a target tissue for female hormones; however, the characteristics of PR and ER in both the fibroblasts and the other component cells remain largely unknown. MATERIALS: Gingival tissue was obtained from six people at various ages and culture fibroblasts were established. At least three passages of each cell line were strained for PR and ER with monoclonal antibodies (Clone 1A6, Clone 1D5, respectively). RESULTS: PR positive cells were detected in all six cell lines through early passages to late ones, but ER were only observed in two of six samples with faint reactions. The staining intensity for PR was greater than for ER, but less than that shown in the MCF-7 breast cancer cells, positive control. In every positive control test, ER reactivity was equal to or higher than that of PR. During the interphase, significantly fewer positive fibroblasts occurred compared with negative fibroblasts, and positive nuclei were even fewer. Meanwhile, most of the mitotic cells were PR positive, showing intense localization around chromosomes and on microtubules. These findings suggest that gingival fibroblasts are fundamentally capable of expressing PR and transmit the signal to target genes. CONCLUSIONS: The present study may conclude that in either gender or at any age, gingival fibroblasts express PR rather low in level and do not necessarily localize PR in a nuclear dominant fashion, which is an essential feature for reproductive organ cells. The poor ER reactivity shown in the gingival fibroblasts was discussed in view of the receptor subtype.