Human gingival fibroblasts produce nitric oxide in response to proinflammatory cytokines

BACKGROUND: Although nitric oxide (NO) synthesis is increased in periodontal disease (PD), little is known about the possible sources of production by gingival tissues. In fact, gingival tissues from patients with periodontitis demonstrate greater levels of inducible nitric oxide (iNOS) expression than healthy tissue. Macrophages are the source of the iNOS expression, with endothelial cells also contributing. In the present study, our hypothesis has been that human gingival fibroblasts (HGF) also have the ability to produce NO. We have established for the first time that HGF express increased levels of iNOS and modulate NO synthesis in response to proinflammatory cytokines that act synergistically. METHODS: NO production under basal conditions or following incubation with tumor necrosis factor (TNF-alpha), interleukin (IL)-1beta, and interferon (IFN)-gamma was assessed by measurement of stable NO metabolites, nitrite, and nitrate, in a microplate adaptation of the Griess assay. Total RNA was isolated from HGF for determination of iNOS mRNA levels. RESULTS: We have shown that NO production is elevated in HGF that are stimulated simultaneously by TNF-alpha, IL-1beta, and IFN-gamma. Northern blot analysis confirmed that the production of iNOS mRNA by HGF is upregulated in the presence of these cytokines. Addition of mercaptoethyl guanidine (MEG), a specific inhibitor of iNOS, profoundly reduced the production of NO in HGF. Non specific inhibitors of iNOS, L-NG-monomethyl arginine (L-NMMA), and L-arginine-methyl ester (L-NAME) had little or no effect on NO produced in HGF. CONCLUSIONS: These results suggest that elevated NO production could be important in the pathogenesis of PD, and also suggest the ability of an iNOS inhibitor to modulate the disease. Treatments with drugs to block the production of nitric oxide or block its effects might be therapeutically valuable.     

The gingival immune response to periodontal pathogens in juvenile periodontitis

A gingival explant culture system was utilized to evaluate the reactivity of local immunoglobulins produced by juvenile periodontitis tissue. Gingival explant culture supernatant fluids were screened, via a standardized dot-immunobinding assay, for antibodies reactive to: Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Campylobacter rectus, Eikenella corrodens, Peptostreptococcus micros, Peptostreptococcus anaerobius, Capnocytophaga ochracea, Eubacterium nodatum and Fusobacterium nucleatum and one nonoral microorganism, Porphyromonas asaccharolytica. Of the 75 juvenile periodontitis supernatant fluids tested, the organisms that reacted with the highest numbers of supernatant fluids were E. nodatum (72%) and A. actinomycetemcomitans (49%). More juvenile periodontitis than healthy tissue samples showed supernatant fluid reactivity to P. intermedia, C. ochracea, E. nodatum and P. micros. No significant difference was observed between the juvenile periodontitis group supernatant fluids reactivity and the supernatant fluids of the other periodontal disease groups tested. Cluster analysis revealed the association, as determined by supernatant fluid reactivity, of P. micros and C. ochracea in the juvenile periodontitis group. The data from this investigation are consistent with a hypothesis of multiple possible etiologies of periodontal destruction in juvenile periodontitis and other forms of periodontal diseases.     

牙龈成纤维细胞、牙周膜细胞对白细胞介素8的趋化反应

目的评价牙龈成纤维细胞、牙周膜细胞对白细胞介素8（interleukin-8,IL-8）的趋化反应。方法将体外培养第6代的牙龈成纤维细胞和牙周膜细胞（2.5×10     

The gingival immune response to Actinobacillus actinomycetemcomitans in juvenile periodontitis.

The established and advanced lesions of juvenile periodontitis-localized form (JP) are predominated by B-lymphocytes and plasma cells. Local immune processes may participate in protective or immunopathologic roles in the pathogenesis of this disease. Actinobacillus actinomycetemcomitans (A.a.) is implicated as a primary etiologic agent in JP. An in vitro gingival explant culture system was utilized to study the specificity of immunoglobulins produced by diseased JP tissues. A dot-immunobinding assay demonstrated that 46% of the supernatant fluids (SF) from explant cultures of diseased tissues (n = 39) were positive for the presence of antibody to A.a. Y4, while 61% of autologous JP sera (n = 39) tested positive. For rapidly progressive (RP) and adult periodontitis (AP) SF, 50% and 40% were positive for A.a. Y4, respectively. Seventeen percent of SF from healthy tissue were positive for A.a. Y4. There was no significant difference between JP SF reactivities to A.a. Y4 when compared to reactivities of SF from AP and RP patients. Only 10% of JP SF were positive for Porphyromonas asaccharolytica, a non-oral control microorganism. The de novo biosynthesis of antibody in JP tissue, reactive with A.a. Y4, was demonstrated with Staph Protein A isolated 14C-labeled IgG (SPAG) and the use of a dot-immunobinding assay and autoradiography. The in vitro gingival tissue explant culture system described provides a useful model for the study of the synthesis and specificity of localized immunoglobulins produced by diseased tissues of JP patients.     

Nicotine-induced damage affects gingival fibroblasts in the gingival tissue of rats

Background: Very limited information is available from in vivo studies about whether smoking and/or nicotine affect gingival tissues in the absence of plaque. The purpose of this study is to evaluate the effect of the systemic administration of nicotine in the proliferation and counting of fibroblast‐like cells in the gingival tissue of rats. Methods: Thirty adult male Wistar rats were randomly assigned into two groups to receive subcutaneous injections of a saline solution (control group = group C) or nicotine solution (group N; 3 mg/kg) twice a day. The animals were euthanized 37, 44, or 51 days after the first subcutaneous injection. Specimens were routinely processed for serial histologic sections. Five fields of view in the connective tissue adjacent to the gingival epithelium and above the alveolar bone crest of the maxillary first molar were selected for the counting of fibroblast‐like cells. Data were statistically analyzed (P <0.05). Results: The intergroup analysis detected a lower number of fibroblast‐like cells in group N compared to group C on days 37 (2.65 ± 1.41 and 6.67 ± 3.25, respectively), 44 (2.70 ± 1.84 and 8.57 ± 2.37, respectively), and 51 (2.09 ± 1.41 and 7.49 ± 2.60, respectively) (P <0.05). The quantification of fibroblast‐like cells showed no significant difference (P >0.05) in the intragroup analysis of control and nicotine throughout experimental periods. In the intergroup analysis, group N had reduced proliferating cell nuclear antigen–positive fibroblasts compared to group C in all periods (P <0.05). Conclusion: The daily systemic administration of nicotine negatively affected, in vivo, the number and proliferation of fibroblast‐like cells in the gingival tissue of rats.     

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

Osteopontin adhesion receptors on gingival fibroblasts

Osteopontin (OPN) promotes attachment and spreading of cells in an RGD dependent fashion, suggesting that OPN interacts with integrins on cell surfaces. Here in, we show that LM-609, a monoclonal antibody to the α_vβ₃ integrin (a vitronectin receptor), inhibited OPN-mediated attachment of gingival fibroblasts. To characterize the cell surface receptors responsible for this interaction, we performed OPN-sepharose affinity chromatography using detergent extracts of ³⁵S-methionine or ^l25I-surface labeled gingival fibroblasts. Proteins bound to the OPN-matrix were eluted with EDTA and subjected to SDS-PAGE under reducing conditions. EDTA eluates from both ¹²⁵I-surface labeled and ³⁵Smethionine labeled extracts demonstrated prominent bands in the 90kDa and 50kDa regions, by both autoradiography and fluorography, respectively. These studies suggest that OPN is associated with other cell surface molecules in addition to α_vβ₃. Furthermore, these as yet to be characterized proteins, may prove to have a stronger affinity for OPN than α_vβ₃.     

Extracellular matrix in gingival fibroblasts]

Primary cultures of human gingival fibroblasts from patients of different age have been established. Histotype characterization has been confirmed by ultrastructural morphology and by the positivity of intermediate filament vimentin. Extracellular matrix expression has been analyzed by immunocitochemistry. Our data demonstrate that the extracellular matrix of human gingival fibroblasts is composed of type IV collagen, other than fibronectin and type I-III collagens.     

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.     

Activation of adenosine receptor on gingival fibroblasts

CD73 (ecto-5'-nucleotidase) on human gingival fibroblasts plays a role in the regulation of intracellular cAMP levels through the generation of adenosine, which subsequently activates adenosine receptors. In this study, we examined the involvement of ecto-adenosine deaminase, which can be anchored to CD26 on human gingival fibroblasts, in metabolizing adenosine generated by CD73, and thus attenuating adenosine receptor activation. Ecto-adenosine deaminase expression on fibroblasts could be increased by pre-treatment with a lysate of Jurkat cells, a cell line rich in cytoplasmic adenosine deaminase. Interestingly, the cAMP response to adenosine generated from 5'-AMP via CD73 and the ability of 5'-AMP to induce hyaluronan synthase 1 mRNA were significantly decreased by the pre-treatment of fibroblasts with Jurkat cell lysate. This inhibitory effect was reversed by the specific adenosine deaminase inhibitor. These results suggest that ecto-adenosine deaminase metabolizes CD73-generated adenosine and regulates adenosine receptor activation.     

Cytotoxic effects of gingival retraction cords on human gingival fibroblasts in vitro

The objective of this study was to determine the cytocompatibility of three different extracts of gingival retraction cords and to compare the cytotoxic effect of these materials on human gingival fibroblasts. Gingival retraction cords impregnated with aluminium sulphate (Gingi-Aid), dl-adrenaline HCl (Gingi-Pak) and non-drug-impregnated cord (Gingi-Plain) were eluted with culture medium for 10 min and 24 h. Cytotoxicity was judged using a tetrazolium bromide reduction assay. Our data demonstrated that gingival retraction cords applied alone almost completely inhibited cell viability (P < 0.05). In addition, the results also showed that the eluates from aluminium sulphate-impregnated cord, dl-adrenaline HCl-impregnated cord and non-drug-impregnated cord were cytotoxic to primary human gingival fibroblast cultures (P < 0.05). The cell viability of incubation of gingival fibroblasts containing 10-min eluates of aluminium sulphate, dl-adrenaline HCl and non-drug-impregnated cord was 61, 21 and 70%, respectively. The cell viability of incubation of gingival fibroblasts containing 24 h eluates of aluminium sulphate, dl-adrenaline HCl and non-drug-impregnated cord was 68, 58 and 72%, respectively. It was found that dl-adrenaline HCl-impregnated gingival retraction cord was the most toxic gingival retraction cord among the materials tested in all cultures (P < 0.05). The cytotoxicity decreased in an order of dl-adrenaline HCl-impregnated cord > aluminium sulphate-impregnated cord > non-drug-impregnated cord. The extent or degree of the cytotoxicity depended on the materials tested. Gingival retraction cords have significant potential for gingival toxicity. Careful management of gingiva retraction cords would lower the risk of potential gingival tissue damage during clinical application procedure and thus increase the success of prosthodontic procedures.     

Interleukin-6 production by human gingival fibroblasts

The ability of human gingival fibroblasts to synthesize interleukin-6 (IL-6) was studied using in vitro and immunohistochemical techniques. Culture supernatants of human gingival fibroblasts contained significant quantities of IL-6 activity which could be stimulated by fetal calf serum, recombinant interleukin-1 beta and lipopolysaccharide. The activity in the supernatants was specifically attributed to IL-6 since up to 97% of the activity could be inhibited by an anti-IL-6 antibody. Immunohistochemical studies on low-density human gingival fibroblast cultures indicated that the cells were associated with material reactive to the anti-IL-6 antibody. This localization was seen on the cell surface and in the cytoplasm of the cells. Immunoreactivity towards IL-6 was also noted in sections of human gingivae. Moderate staining was seen in the connective tissues and lower portions of the gingival epithelium, while intense staining was seen at foci of inflammation. The identification of IL-6 with human gingival tissues and cells implicates this lymphokine in the molecular events associated with the inflammatory periodontal diseases.