Treponema denticola outer membrane enhances the phagocytosis of collagen-coated beads by gingival fibroblasts.
Human gingival fibroblasts (HGFs) degrade collagen fibrils in physiological processes by phagocytosis. Since Treponema denticola outer membrane (OM) extract perturbs actin filaments, important structures in phagocytosis, we determined whether the OM affects collagen phagocytosis in vitro by HGFs. Phagocytosis was measured by flow cytometric assessment of internalized collagen-coated fluorescent latex beads. Confluent HGFs pretreated with T. denticola ATCC 35405 OM exhibited an increase in the percentage of collagen phagocytic cells (phagocytosis index [PI]) and in the number of beads per phagocytosing cell (phagocytic capacity [PC]) compared with untreated controls. The enhancement was swift (within 15 min) and was still evident after 1 day. PI and PC of HGFs for bovine serum albumin (BSA)-coated beads were also increased, indicating a global increase in phagocytic processes. These results contrasted those for control OM from Veillonella atypica ATCC 17744, which decreased phagocytosis. The T. denticola OM-induced increase in bead uptake was eliminated by heating the OM and by depolymerization of actin filaments by cytochalasin D treatment of HGFs. Fluid-phase accumulation of lucifer yellow was enhanced in a saturable, concentration-dependent, transient manner by the T. denticola OM. Our findings were not due to HGF detachment or cytotoxicity in response to the T. denticola OM treatment since the HGFs exhibited minimal detachment from the substratum; they did not take up propidium iodide; and there was no change in their size, granularity, or content of sub-G1 DNA. We conclude that a heat-sensitive component(s) in T. denticola OM extract stimulates collagen phagocytosis and other endocytic processes such as nonspecific phagocytosis and pinocytosis by HGFs. (+info)
Lipoteichoic acid acts as an antagonist and an agonist of lipopolysaccharide on human gingival fibroblasts and monocytes in a CD14-dependent manner.
CD14 has been implicated as a receptor of lipoteichoic acid (LTA) and other bacterial components as well as lipopolysaccharide (LPS). Since the structures of LTAs from various gram-positive bacteria are heterogeneous, we analyzed the effects of LTAs on the secretion of interleukin-8 (IL-8) by high- and low-CD14-expressing (CD14(high) and CD14(low)) human gingival fibroblasts (HGF). While Bacillus subtilis LTA had an IL-8-inducing effect on CD14(high) HGF which was considerably weaker than that of LPS, Streptococcus sanguis and Streptococcus mutans LTAs had practically no effect on the cells. B. subtilis LTA had only a weak effect on CD14(low) HGF, as did LPS. S. sanguis and S. mutans LTAs at a 1,000-fold excess each completely inhibited the IL-8-inducing activities of both LPS and a synthetic lipid A on CD14(high) HGF. The effect of LPS was also inhibited by the presence of an LPS antagonist, synthetic lipid A precursor IVA (LA-14-PP), with a 100-fold higher potency than S. sanguis and S. mutans LTAs and by anti-CD14 monoclonal antibody (MAb). S. sanguis and S. mutans LTAs, LA-14-PP, and anti-CD14 MAb had no significant effect on phorbol myristate acetate-stimulated IL-8 secretion by HGF. These LTAs also inhibited the IL-8-inducing activity of B. subtilis LTA on CD14(high) HGF, as did LA-14-PP and anti-CD14 MAb. The antagonistic and agonistic functions of LTAs were also observed with human monocytes. Binding of fluorolabeled LPS to human monocytes was inhibited by S. sanguis LTA, although the inhibition was 100 times weaker than that of LPS itself, and anti-CD14 MAb inhibited fluorolabeled LPS and S. sanguis LTA binding. Binding of LTAs to CD14 was also observed with nondenaturing polyacrylamide gel electrophoresis. These results indicate that LTAs act as antagonists or agonists via a CD14-dependent mechanism, probably due to the heterogeneous structure of LTAs, and that an antagonistic LTA might be a useful agent for suppressing the periodontal disease caused by gram-negative bacteria. (+info)
Targeted disruption of fibronectin-integrin interactions in human gingival fibroblasts by the RI protease of Porphyromonas gingivalis W50.
Cell surface integrins mediate interactions between cells and their extracellular matrix and are frequently exploited by a range of bacterial pathogens to facilitate adherence and/or invasion. In this study we examined the effects of Porphyromonas gingivalis proteases on human gingival fibroblast (HGF) integrins and their fibronectin matrix. Culture supernatant from the virulent strain W50 caused considerably greater loss of the beta1 integrin subunit from HGF in vitro than did that of the beige-pigmented strain W50/BE1. Prior treatment of the W50 culture supernatant with the protease inhibitor Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) blocked its effects on cultured cells, indicating that this process is proteolytically mediated. Purified arginine-specific proteases from P. gingivalis W50 were able to mimic the effects of the whole-culture supernatant on loss of beta1 integrin expression. However purified RI, an alpha/beta heterodimer in which the catalytic chain is associated with an adhesin chain, was 12 times more active than RIA, the catalytic monomer, in causing loss of the alpha5beta1 integrin (fibronectin receptor) from HGF. No effect was observed on the alphaVbeta3 integrin (vitronectin receptor). The sites of action of RI and RIA were investigated in cells exposed to proteases pretreated with TLCK to inactivate the catalytic component. Use of both monoclonal antibody 1A1, which recognizes only the adhesin chain of RI, and a rabbit antibody against P. gingivalis whole cells indicated localization of RI on the fibroblasts in a clear, linear pattern typical of that seen with fibronectin and alpha5beta1 integrin. Exact colocalization of RI with fibronectin and its alpha5beta1 receptor was confirmed by double labeling and multiple-exposure photomicroscopy. In contrast, RIA bound to fibroblasts in a weak, patchy manner, showing only fine linear or granular staining. It is concluded that the adhesin component of RI targets the P. gingivalis arginine-protease to sites of fibronectin deposition on HGF, contributing to the rapid loss of both fibronectin and its main alpha5beta1 integrin receptor. Given the importance of integrin-ligand interactions in fibroblast function, their targeted disruption by RI may represent a novel mechanism of damage in periodontal disease. (+info)
The potential role of chemokines and inflammatory cytokines in periodontal disease progression.
Inflammation is regulated by the expression of mediators that cause a number of pleiotropic events culminating in the recruitment of inflammatory cells and release of biologic mediators by leukocytes. If the inflammation is transient in nature, it can protect the host by activating defense mechanisms and initiating wound repair. However, if the inflammation is inappropriate, it can lead to considerable tissue damage. My colleagues and I have investigated the role of chemokines, particularly monocyte chemoattractant protein 1, in various pathological processes and the role of the proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF) in experimental periodontitis. I will discuss first the studies on chemokines and then the use of IL-1 and TNF blockers in inhibiting inflammation and bone loss in the periodontium. (+info)
Induction of prostaglandin release from macrophages by bacterial endotoxin.
This review summarizes the role of the monocytic responses to lipopolysaccharide as it relates to periodontal disease severity. Data are presented which illustrate that the levels of prostaglandin E2 (PGE2) secreted by systemic peripheral blood monocytes in culture, in the presence of bacterial endotoxins, are highly correlated with the levels observed in the gingival crevicular fluid. Furthermore, the different periodontal diagnostic categories have varying levels of monocytic and crevicular fluid PGE2, in juxtaposition with clinical disease severity. These data are consistent with the concept that there is close synchrony between the systemic responsiveness of peripheral blood monocytes with regard to prostanoid synthesis and the local levels of mediator present within the gingival crevice. (+info)
Production of beta-defensin antimicrobial peptides by the oral mucosa and salivary glands.
beta-Defensins are cationic peptides with broad-spectrum antimicrobial activity that are produced by epithelia at mucosal surfaces. Two human beta-defensins, HBD-1 and HBD-2, were discovered in 1995 and 1997, respectively. However, little is known about the expression of HBD-1 or HBD-2 in tissues of the oral cavity and whether these proteins are secreted. In this study, we characterized the expression of HBD-1 and HBD-2 mRNAs within the major salivary glands, tongue, gingiva, and buccal mucosa and detected beta-defensin peptides in salivary secretions. Defensin mRNA expression was quantitated by RNase protection assays. HBD-1 mRNA expression was detected in the gingiva, parotid gland, buccal mucosa, and tongue. Expression of HBD-2 mRNA was detected only in the gingival mucosa and was most abundant in tissues with associated inflammation. To test whether beta-defensin expression was inducible, gingival keratinocyte cell cultures were treated with interleukin-1beta (IL-1beta) or bacterial lipopolysaccharide (LPS) for 24 h. HBD-2 expression increased approximately 16-fold with IL-1beta treatment and approximately 5-fold in the presence of LPS. Western immunoblotting, liquid chromatography, and mass spectrometry were used to identify the HBD-1 and HBD-2 peptides in human saliva. Human beta-defensins are expressed in oral tissues, and the proteins are secreted in saliva; HBD-1 expression was constitutive, while HBD-2 expression was induced by IL-1beta and LPS. Human beta-defensins may play an important role in the innate defenses against oral microorganisms. (+info)
Transcriptional activation of mRNA of intercellular adhesion molecule 1 and induction of its cell surface expression in normal human gingival fibroblasts by Mycoplasma salivarium and Mycoplasma fermentans.
Lipoproteins in the cell membranes of both Mycoplasma salivarium and Mycoplasma fermentans were demonstrated to trigger the transcription of intercellular adhesion molecule-1 mRNA in normal fibroblasts isolated from human gingival tissue and to induce its cell surface expression by a mechanism distinct from that of Escherichia coli lipopolysaccharide. The lipid moiety of the lipoproteins was suggested to play a key role in the expression of the activity. (+info)
Overgrowth of oral mucosa and facial skin, a novel feature of aspartylglucosaminuria.
Aspartylglucosaminuria (AGU) is a lysosomal storage disorder caused by deficiency of aspartylglucosaminidase (AGA). The main symptom is progressive mental retardation. A spectrum of different mutations has been reported in this disease, one missense mutation (Cys163Ser) being responsible for the majority of Finnish cases. We were able to examine 66 Finnish AGU patients for changes in the oral mucosa and 44 of these for changes in facial skin. Biopsy specimens of 16 oral lesions, 12 of them associated with the teeth, plus two facial lesions were studied histologically. Immunohistochemical staining for AGA was performed on 15 oral specimens. Skin was seborrhoeic in adolescent and adult patients, with erythema of the facial skin already common in childhood. Of 44 patients, nine (20%) had facial angiofibromas, tumours primarily occurring in association with tuberous sclerosis. Oedemic buccal mucosa (leucoedema) and gingival overgrowths were more frequent in AGU patients than in controls (p<0.001). Of 16 oral mucosal lesions studied histologically, 15 represented fibroepithelial or epithelial hyperplasias and were reactive in nature. Cytoplasmic vacuolisation was evident in four. Immunohistochemically, expression of AGA in AGU patients' mucosal lesions did not differ from that seen in corresponding lesions of normal subjects. Thus, the high frequency of mucosal overgrowth in AGU patients does not appear to be directly associated with lysosomal storage or with alterations in the level of AGA expression. (+info)