Actinobacillus actinomycetemcomitans lipopolysaccharide stimulates collagen phagocytosis by human gingival fibroblasts

Introduction:  Collagen phagocytosis by fibroblasts is involved in the intracellular pathway related to collagen breakdown in soft connective tissues. The possible role of lipopolysaccharide (LPS) in regulating this fibroblast function has not been elucidated so we investigated the effect of LPS from Actinobacillus actinomycetemcomitans , a periodontopathic bacterium, on collagen phagocytic activity in human gingival fibroblasts and associated regulatory mechanisms.
Methods:  LPS pretreatment stimulated binding of collagen-coated beads to cells and, subsequently, their internalization.
Results:  The LPS-activated collagen phagocytic process was enhanced in the presence of the soluble form of CD14 (sCD14) or LPS-binding protein (LBP), while the LPS/LBP treatment activated Akt and induced actin reorganization. Furthermore, these LPS/LBP-induced effects were partially suppressed by adding phosphatidyl-inositol-3 kinase (PI3K) inhibitors.
Conclusion:  These results suggest that A. actinomycetemcomitans LPS disturbs the homeostasis of collagen metabolism within gingival tissue by facilitating collagen phagocytosis by gingival fibroblasts, and serum sCD14 and LBP positively regulate the action of LPS. In addition, the PI3K/Akt signaling is thought to partially mediate the LPS/LBP-stimulated collagen phagocytic pathway, which may be dependent on actin cytoskeletal rearrangement.     

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。     

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.     

Immunochemical detection of CD14 on human gingival fibroblasts in vitro

The activation of monocytes and macrophages induced by lipopolysaccharide has been shown to contribute to the binding of lipopolysaccharide and lipopolysaccharide-binding protein complex to the cell surface CD14 molecule. To clarify the mechanism of the lipopolysaccharide-induced modulation of the function of gingival fibroblasts, we investigated the effect of anti-CD14 on interleukin 6 (IL-6) production on human gingival fibroblasts in vitro. Immunochemical staining revealed weak positivity for CD14 on fibroblasts from healthy gingiva, while strong positivity for CD14 was found on fibroblasts from inflamed gingiva. Western blot profiles of the fibroblasts and monocytes showed a CD14-positive reaction at 55 kDa. Fluorescein isothiocyanate-conjugated Escherichia coli lipopolysaccharide bound to fibroblasts more strongly in the presence of 10% fetal bovine serum than without serum. This binding, as well as IL-6 production, was blocked by anti-CD14 monoclonal antibody. The results showed that CD14 was present on human gingival fibroblasts, which suggests that lipopolysaccharide modulation of gingival fibroblast function depends on CD14.     

Cell cycle-specific growth inhibitory effect on human gingival fibroblasts of a toxin isolated from the culture medium of Actinobacillus actinomycetemcomitans

A toxin isolated from the growth medium of Actinobacillus actinomycetemcomitans by ammonium sulfate precipitation was shown to inhibit irreversibly the multiplication of human gingival fibroblasts. DNA histograms from flow cytometric measurements showed that the cells accumulated preferentially in the G2 phase of the cell cycle. Such cells exhibited sheetlike protrusions, and an increased frequency of micronuclei was evident in cells treated with low concentrations of the toxin. Toxin-treated cells were viable for several weeks, as shown by staining with trypan blue and fluorescein diacetate, and the general cell metabolism as measured by oxygen consumption was unimpaired.     

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.     

Capsular polysaccharide from Actinobacillus actinomycetemcomitans inhibits IL-6 and IL-8 production in human gingival fibroblast

We previously reported that a capsular polysaccharide (CP) from Actinobacillus actinomycetemcomitans Y4 induces bone resorption in a mouse organ culture system and osteoclast formation in mouse bone marrow cultures. However, the effects of A. actinomycetemcomitans Y4 CP on human gingival fibroblasts (HGF) are still unclear. The present study was undertaken to test the hypothesis that A. actinomycetemcomitans Y4 CP alters the production of inflammatory cytokines, such as interleukin-6 (IL-6) and IL-8 by HGF. When HGF were cultured with various concentrations of Y4 CP for 24 h, IL-6 and IL-8 production decreased in a concentration-dependent manner. Y4 CP (100 microg/ml) suppressed the release of IL-6 from 9.09 +/- 0.08 ng/ml to 0.34 +/- 0.21 ng/ml (P < 0.01) and IL-8 production decreased from 3.76 +/- 0.03 ng/ml to 0.09 +/- 0.01 ng/ml (P < 0.01). Y4 CP suppressed 70-80% of the release of IL-6 and IL-8 from HGF stimulated with Y4 lipopolysaccharide (LPS), too. Interestingly, anti-A. actinomycetemcomitans Y4 CP completely inhibited the effect of A. actinomycetemcomitans Y4 CP on IL-6 and IL-8 production from HGF. These results indicate that Y4 CP inhibits the release of IL-6 and IL-8 from HGF, suggesting that A. actinomycetemcomitans Y4 modulates the inflammatory response in periodontitis. Remarkably, this inhibitory effect was reversed by specific anti-A. actinomycetemcomitans Y4 CP suggesting an important relationship between the organism and the humoral host response.     

Effect of triclosan on interferon-gamma production and major histocompatibility complex class II expression in human gingival fibroblasts

BACKGROUND, AIMS: The effect of triclosan (2,4,4'-trichloro-2'-hydroxyl-diphenyl ether) on the production of interferon-gamma (IFN-gamma) and the expression of major histocompatibility complex (MHC) class II antigen was studied in human gingival fibroblasts isolated from 4 individuals. METHODS/RESULTS: All cell lines demonstrated high IFN-gamma production in 24-h cultures of human gingival fibroblasts stimulated by phytohemagglutinin (PHA) (5 microg/ml). Human gingival fibroblasts showed a high expression of MHC class II when stimulated with 500 and 1,000 pg/ml rIFN-gamma in 7-day cultures. Treatment of the cells with triclosan (0.5 microg/ml) reduced both IFN-gamma production and MHC class II expression in human gingival fibroblast cultures. Similar inhibitory effects on IFN-gamma production and MHC class II expression were observed when the anti-inflammatory agent dexamethazone (1 microM) was used. CONCLUSION: The present study further supports the view that the agent has an anti-inflammatory effect in addition to its antibacterial capacity.     

Synthesis of proinflammatory cytokines by human gingival fibroblasts in response to lipopolysaccharides and interleukin-1β

We have examined the ability of gingival fibroblasts (GF) to participate in inflammatory response and function as accessory immune cells. The accessory immune function of GF cells was evaluated by their ability to elaborate proinflammatory cytokines following stimulation with lipopolysaccharides and interleukin-1β (IL-β). Using three separate clonally derived and characterized human gingival fibroblast (GF) cell lines, we demonstrate that LPS from Actinobacillus actinomycetemcomitans (Aa) and Escherichia coli (Ec) induce mRNA and synthesis of proinflammatory cytokines, IL-1β, IL-6 and IL-8. IL-1β activation of GF cells showed that IL-1β not only induces the expression of IL-6, IL-8 and TNF-α, but also acts in an autocrine manner on GF cells and induces IL-1βexpression. Furthermore, the continuous presence of IL-1β in GF cell cultures did not down regulate the response of GF cells to IL-1β. Pretreatment of GF cells with IL-lβ resulted in the enhanced synthesis of TNF-α in response to additional IL-lβ. These findings indicate that GF cells, in addition to providing structural support, may also function as accessory immune cells and play an important role in the initial inflammatory reaction as well as in the amplification of immune response.     

DHA对人牙龈成纤维细胞生物学活性及炎症因子表达的作用

目的 探索二十二碳六烯酸(docosahexaenoic acid,DHA)对人牙龈成纤维细胞(human gingival fibroblasts,HGFs)生物学活性及炎症因子表达的作用.方法 分别采用活死细胞染色法、荧光染色法、流式细胞术观察DHA对细胞活性、细胞形态、细胞周期的影响;DHA预处理HGFs后,利用...     

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.