Bone resorption and local interleukin-1alpha and interleukin-1beta synthesis induced by Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis lipopolysaccharide

Different types of periodontopathic bacterial lipopolysaccharide (LPS) exert various biological activities in vitro. However, whether or not these activities also occur in vivo remains unclear. Thus the present study investigates bone resorption, as well as local IL-1alpha and IL-1beta synthesis induced by Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis LPS in the periodontal tissue of mice. Both types of LPS were injected into mouse gingiva every 48 h and the animals were sacrificed 6 h after the 1st. 4th, 7th, 10th, 13th, 16th, 20th, or 24th injection. Bone resorption in the injected gingiva was histopathologically and histomorphometrically investigated and local concentrations of IL-1alpha and IL-1beta were detected using an enzyme-linked immunosorbent assay. The active resorption ratio was significantly higher in the group given the 10th injection of LPS from A. actinomycetemcomitans than in the group given P. gingivalis LPS. Furthermore, A. actinomycetemcomitans LPS stimulated significantly more synthesis of IL-1alpha than P. gingivalis LPS after the 4th and 10th injections. and of IL-1beta after the 4th, 7th, 10th, 13th, 16th and 20th injections. These results suggest that A. actinomycetemcomitans LPS is a more potent inducer of bone resorption and synthesis of IL-1alpha and IL-1beta in the short term than P. gingivalis LPS.     

Hemoglobin and LPS act in synergy to amplify the inflammatory response

Vascular disruption and bleeding during periodontitis likely increase the levels of hemoglobin in gingival crevicular fluid. The aim of this study was to investigate the effect of hemoglobin on the inflammatory responses of human macrophages stimulated with lipopolysaccharides (LPS) isolated from periodontopathogens. The production of interleukin-1 beta (IL-1beta), IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha) by macrophages following challenges with Porphyromonas gingivalis and Fusobacterium nucleatum LPS in the presence or absence of human hemoglobin was analyzed by ELISA. The effect of hemoglobin on LPS-binding to macrophages was evaluated with (3)H-LPS. Hemoglobin and LPS from periodontopathogens acted in synergy to stimulate the production of high levels of IL-1beta, IL-6, IL-8, and TNF-alpha by macrophages. Hemoglobin also enhanced LPS-binding to macrophages. This study suggests that hemoglobin contributes to increases in the levels of pro-inflammatory mediators in periodontal sites by acting in synergy with LPS from periodontopathogens, thus favoring the progression of periodontitis.     

Measurement of interleukin-1α and -1β in gingival crevicular fluid: Implications for the pathogenesis of periodontal disease

Samples of gingival crevicular fluid (GCF) were harvested from sites manifesting features characteristic of active disease including inflammation, periodontal attachment loss, and radiographic signs of alveolar bone destruction in untreated patients with advanced periodontitis. The presence and concentrations of interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) were measured using ELISAs specific for these cytokine molecules. IL-1 alpha and/or IL-1 beta were identified in the GCF of 15 of 15 patients having untreated periodontitis. Ninety percent (71 of 79) of the sites tested contained measureable amounts of IL-1, with IL-1 beta as the more frequently occurring form. IL-1 alpha levels ranged from 0.23 nM to 13.9 nM in the GCFs. IL-1 beta levels were between 0.04 nM and 5.28 nM. Marked reductions of total IL-1 levels were observed following effective treatment. Both forms of IL-1 messenger RNA (mRNA) were detected in 17 of 17 gingival tissue samples from 6 patients. These results demonstrate that IL-1 is produced and released locally in periodontal disease at concentrations sufficient to mediate tissue inflammation and bone resorption. IL-1 may serve as a marker of periodontal tissue destruction.     

In vivo induction of proinflammatory cytokines in mouse tissue by Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans

Periodontitis is a chronic inflammatory disease initiated by a multitude of bacteria. Persistent infection leads to generation of various inflammatory mediators, resulting in tissue destruction and osteoclastic resorption of the alveolar bone. This study describes a novel in vivo murine calvarial model to assess the effects of oral pathogens on the expression of three proinflammatory cytokines [interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha] which are involved in bone resorption. We chose Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans as prototype oral pathogens. We also tested the effects of Streptococcus gordonii, an oral commensal supragingival microorganism, considered a non-pathogen. Live bacteria were injected into subcutaneous tissue overlying the parietal bone of mice calvaria for 6 days. At the end of the experimental period, tissues overlying the calvaria were removed and analyzed for proinflammatory cytokine expression by Northern blotting. Cytokine mRNA was not detected in the tissue over the calvaria of control animals. In contrast, P. gingivalis and A. actinomycetemcomitans elicited mRNA expression of all three cytokines, TNFalpha being the highest (TNFalpha > > IL-1beta > IL-6). P. gingivalis was more potent than A. actinomycetemcomitans in inducing cytokine expression. In contrast, S. gordonii induced only low levels of mRNA for IL-1beta and TNFalpha but no IL-6 mRNA induction. These results suggest that oral microorganisms with access to host tissues elicit a battery of proinflammatory cytokines. There were clear differences in profiles and, interestingly, a commensal bacterium also stimulated bone resorptive cytokine expression in host tissues.     

Retinoic acid-inducible gene-I is induced in gingival fibroblasts by lipopolysaccharide or poly IC: possible roles in interleukin-1beta, -6 and -8 expression

Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH family of proteins, and little is known of its biological function in the oral region. We previously reported that interleukin 1beta (IL-1beta) induced RIG-I expression in gingival fibroblasts. In this study, we studied the mechanism of RIG-I expression induced by lipopolysaccharide (LPS) or double-stranded RNA (dsRNA) in gingival fibroblasts. We also addressed the role of RIG-I in the expression of IL-1beta, IL-6 and IL-8 in gingival fibroblasts stimulated with LPS or dsRNA. We stimulated cultured human gingival fibroblasts with LPS or dsRNA, and examined the expression of RIG-I mRNA and protein. The effect of cycloheximide, a protein synthesis inhibitor, on RIG-I induction by these stimuli was examined. The expression of IL-1beta, IL-6 and IL-8 in gingival fibroblasts transfected with RIG-I cDNA stimulated with LPS or dsRNA was examined. LPS or dsRNA induced the expression of mRNA and protein for RIG-I in concentration- and time-dependent manners. We also examined the localization of RIG-I, and found that it was expressed in cytoplasm. Cycloheximide did not suppress the LPS or dsRNA-induced RIG-I expression. Introduction of RIG-I cDNA into gingival fibroblasts resulted in enhanced expression of IL-1beta, IL-6 and IL-8; moreover, overexpression of RIG-I stimulated with LPS or dsRNA synergistically increased expression of IL-1beta, IL-6 and IL-8. RIG-I may have important roles in the innate immune response in the regulation of IL-1beta, IL-6 and IL-8 expression in gingival fibroblasts in response to LPS and dsRNA.     

Doxycycline reduces lipopolysaccharide-induced inflammatory mediator secretion in macrophage and ex vivo human whole blood models

Background: Tetracyclines have been extensively used as adjuncts in the treatment of some forms of periodontitis. The aim of this study was to evaluate the capacity of doxycycline to influence the secretion of inflammatory mediators in macrophage and ex vivo human whole blood models stimulated with periodontopathogen lipopolysaccharides (LPS). Methods: Monocyte-derived macrophages were treated with various concentrations of doxycycline prior to being stimulated with Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) LPS. The capacity of doxycycline to mediate the inflammatory response was also tested in an ex vivo whole blood model (whole blood isolated from periodontitis patients and healthy subjects) stimulated with Porphyromonas gingivalis LPS. The secretion of interleukin (IL)-1beta, -6, and -8 and tumor necrosis factor-alpha (TNF-alpha) in both models was assessed by enzyme-linked immunosorbent assays (ELISA). Changes in phosphorylation state of kinases induced by A. actinomycetemcomitans LPS and doxycycline in the macrophage model were characterized by a multiplex ELISA analysis. Results: The secretion of IL-1beta and -8 and TNF-alpha by macrophages decreased significantly (P <0.05) when they were pretreated with 2 muM doxycycline, whereas a concentration of 10 muM was required to significantly reduce IL-6 secretion. Pretreatment of macrophages with 10 muM doxycycline prior to A. actinomycetemcomitans LPS stimulation resulted in a marked decrease in the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 (-76%). In the whole blood model, doxycycline, more particularly at 10 muM, was also a potent inhibitor of the proinflammatory cytokine response. Conclusion: These two models provided clear evidence that some of the clinically proven benefits of doxycycline may be related to its ability to regulate inflammatory mediator release by host cells.     

Production of Rantes/CCL5 in human gingival fibroblasts challenged with tumor necrosis factor alpha

Chemokines are small-secreted proteins that stimulate the directional migration of leukocytes and thereby mediate the inflammatory process. The present study investigates the capacity of human gingival fibroblasts to produce the beta chemokine Rantes/CCL5. In situ hybridization, immunohistochemistry and ELISA were used to measure the induction of Rantes/CCL5 at the mRNA and protein levels, both in unstimulated gingival fibroblasts as well as in fibroblasts treated with the proinflammatory cytokines tumor necrosis factor (TNF)alpha or interleukin (IL)-1beta. TNFalpha in different concentrations (0.1-10 ng/ml) induced Rantes/CCL5 mRNA expression and protein production in 24-h cultures of human gingival fibroblasts. The expression of Rantes/CCL5-mRNA and protein production, induced by TNFalpha, was evident at 6 h and thereafter increased continuously during the study period (24 h). IL-1beta (3-300 pg/ml) also enhanced the production of Rantes/CCL5 in gingival fibroblasts. The amount of Rantes/CCL5 induced by IL-1beta (300 pg/ml), however, was less than that induced by TNFalpha (10 ng/ml). The study suggests that human gingival fibroblasts, by producing the chemokine Rantes/CCL5, participate in the regulation of the host response during the inflammatory process in the periodontal tissue.     

Interleukin-1: the principal osteolytic cytokine produced by keratocysts.

Fragments of keratocysts removed at operation were maintained in explant culture and the media were assayed for the biological activity of the potent osteolytic cytokines--interleukin (IL)-1, interleukin (IL)-6 and tumour necrosis factor (TNF). Media were also assayed for their ability to stimulate bone resorption. All six cysts examined released IL-1 and IL-6 bioactivity but TNF bioactivity was unmeasurable. Dialysed cyst media stimulated bone resorption and this could be completely inhibited by a monospecific antibody which neutralized IL-1 alpha and IL-1 beta. Immunohistochemical staining of cryostat sections of keratocysts revealed the presence of IL-1 alpha and IL-6 in cyst epithelial cells but not in other cell types. Sections did not react with antibodies to IL-1 beta or TNF. It is therefore proposed that IL-1 alpha is the major osteolytic cytokine produced by keratocysts and that IL-6 and IL-1 may contribute to keratocyst growth by promoting epithelial cell proliferation and bone resorption, respectively.     

The role of lipopolysaccharide in infectious bone resorption of periapical lesion

BACKGROUND: The role of lipopolysaccharide (LPS) in periapical lesion-induced bone resorption was investigated. Polymyxin B (PMB), a specific inhibitor of LPS, was evaluated to treat the apical lesion. METHODS: Lipopolysaccharide isolated from two common endodontic pathogens, Fusobacterium nucleatum and Porphyromonas endodontalis, stimulated mouse macrophage (J774) to release interleukin-1alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha) in a time-dependent manner. RESULTS: Combination of LPS further enhanced the stimulation. PMB inhibited these effects significantly. LPS also stimulated matrix metalloproteinase-1 (MMP-1) gene expression in J774, whereas anti-IL-1 alpha and anti-TNF-alpha antibodies, as well as PMB, diminished this effect. A disease model of periapical lesion was established in Wistar rat. Administration of PMB reduced the extent of lesion-associated bone resorption by 76% to approximately 80%, and simultaneously reduced the numbers of MMP-1-producing macrophages. CONCLUSIONS: It is suggested that LPS released from the infected root canal triggers the synthesis of IL-1 alpha and TNF-alpha from macrophages. These pro-inflammatory cytokines up-regulate the production of MMP-1 by macrophages to promote periapical bone resorption.     

Actinobacillus actinomycetemcomitans-induced expression of IL-1α and IL-1β in human gingival epithelial cells: role in IL-8 expression

Gingival epithelial cells (GEC) are the first cells of the host that encounter the periodontal pathogens. and therefore their role in the initiation of the inflammatory response is critical. We aimed to: 1) characterize the expression of interleukin (IL)- Ialpha and IL-Ibeta in human gingiva and cultured GEC: 2) demonstrate the ability of A. actinomycetemcomitans extracts to upregulate IL-1alpha, IL-1beta and IL-8 expression in GEC in vitro: and 3) characterize the role of IL-1alpha and IL-1beta in the induction of IL-8 expression in GEC in vitro. Ten gingival biopsies (5 inflamed and 5 controls) and cultured GEC were examined for IL-1alpha and IL-Ibeta using immunohistochemical techniques. GEC were also challenged with A. actinomycetemcomitans extracts or IL-1alpha, and secretion of IL-1 and IL-8 was determined by ELISA. In vivo, IL-lalpha and IL-1beta were localized in the gingival epithelium and the infiltrating leukocytes. In vitro, A. actinomycetemcomitans extracts induced a time-dependent expression of IL-1alpha, IL-1beta and IL-8 in GEC. IL-1 inhibitors did not affect A. actinomycetemcomitans-induced IL-8. although they inhibited IL-8 induced by IL-1alpha or IL-1beta. In conclusion, GEC are a major source of IL-1alpha and IL-1beta in the periodontium, which in turn induce additional inflammatory mediators such as IL-8. Therefore GEC can be a potential target for therapeutic intervention in the future.     

Cranberry components inhibit interleukin-6, interleukin-8, and prostaglandin E production by lipopolysaccharide-activated gingival fibroblasts

Periodontitis is a chronic inflammatory disease that affects the tooth supporting tissues. Gingival fibroblasts are the most abundant cells in periodontal tissues and participate actively in the host inflammatory response to periodontopathogens, which is known to mediate local tissue destruction in periodontitis. The aim of this study was to investigate the effect of a proanthocyanidin-enriched cranberry fraction, prepared from cranberry juice concentrate, on inflammatory mediator production by gingival fibroblasts stimulated by the lipopolysaccharide (LPS) of Aggregatibacter actinomycetemcomitans. Interleukin (IL)-6, IL-8, and prostaglandin E(2) (PGE(2)) production by fibroblasts treated with the cranberry fraction and stimulated by A. actinomycetemcomitans LPS was evaluated by enzyme-linked immunosorbent assay. Changes induced by A. actinomycetemcomitans LPS and the cranberry fraction in the expression and phosphorylation state of fibroblast intracellular signaling proteins were characterized by antibody microarrays. The LPS-induced IL-6, IL-8, and PGE(2) responses of gingival fibroblasts were inhibited by treatment with the cranberry fraction. This fraction was found to inhibit fibroblast intracellular signaling proteins, a phenomenon that may lead to a down-regulation of activating protein-1 activity. Cranberry components also reduced cyclooxygenase 2 expression. This study suggests that cranberry juice contains molecules with interesting properties for the development of new host-modulating therapeutic strategies in the adjunctive treatment of periodontitis.     

The bone resorbing activity released by gingival fibroblasts isolated from patients with periodontitis is independent of interleukin-1

Supernatants from gingival fibroblast cultures obtained from 14 patients with periodontal disease contained factor(s) capable of stimulating bone resorption in vitro, as assessed by the release of 45Ca from neonatal mouse calvariae. The possibility that the factor(s) was interleukin-1 alpha (IL-1 alpha), IL-1 beta or prostaglandin E2 (PGE2) was next investigated. The human fibroblast conditioned media (HFCM) stimulated PGE2 biosynthesis in bone. The stimulatory effect by HFCM on 45Ca release, however, was not affected by blocking prostaglandin biosynthesis with indomethacin. In contrast, 45Ca release induced by IL-1 alpha, IL-1 beta, thrombin and bradykinin was significantly reduced by indomethacin, whereas the effects of PTH and PTHrP were unaffected by indomethacin. The concentration of PGE2 in HFCM was too low to be solely responsible for the 45Ca release response. In addition, the amount of bone resorbing activity produced by the gingival fibroblasts was unaffected by cyclo-oxygenase inhibitors. Similar to IL-1 alpha and IL-1 beta, the stimulatory effect of HFCM was inhibited by gamma-interferon. HFCM did not stimulate cyclic AMP formation in the mouse calvarial bones. Antisera which specifically blocked human IL-1 alpha or IL-1 beta induced 45Ca release, and the specific IL-1 receptor antagonistic protein, did not inhibit the stimulatory effect of HFCM. These data show that gingival fibroblasts secrete bone resorbing factor(s) which is not due to IL-1 and which stimulates bone resorption by a prostaglandin- and cyclic AMP-independent mechanism.     

Enhanced prostaglandin biosynthesis in human gingival fibroblasts isolated from patients treated with phenytoin

Prostaglandin E2 (PGE2) formation was studied in human gingival fibroblasts derived from three epileptic patients before and after 9 months of phenytoin (PHT) therapy. Interleukin 1 (IL-1 alpha; 0.3-6.0 ng/ml), (IL-1 beta; 10-1000 pg/ml) and tumour necrosis factor (TNF alpha; 0.01-0.1 microgram/ml) dose-dependently stimulated the formation of PGE2 in 24 h cultures. In fibroblasts, derived after 9 months of PHT therapy, IL-1 alpha, IL-1 beta and TNF alpha induced a significantly higher formation of PGE2 compared to that in fibroblasts derived before PHT therapy. IL-1 beta induced a significantly higher release also of 3H-arachidonic acid (3H-AA) from prelabelled PHT fibroblasts compared to that in prelabelled gingival fibroblasts isolated before the drug therapy. Addition of exogenous AA caused a spontaneous increase of PGE2 formation in PHT fibroblasts compared to that in fibroblasts isolated before the PHT treatment. The results indicate that PHT medication results in an upregulation of prostanoid formation in gingival fibroblasts partly due to an increased phospholipase A2 activity and partly due to an increased cyclooxygenase activity.     

Keratinocyte growth factor (KGF)-1 and -2 protein and gene expression in human gingival fibroblasts

The onset and progression of periodontal disease is associated with significant changes in the epithelial component of the attachment complex. From the early to the advanced stages of periodontal disease increased epithelial cell proliferation, migration and invasion into the surrounding connective tissue takes place. Concomitantly there is a significant increase in proinflammatory cytokine expression in periodontal tissue and quantitative and qualitative changes in the subgingival microflora, including an increase in gram-negative microorganisms. One of the most significant virulence factors of these bacteria is lipopolysaccharide (LPS) connected to the outer membrane. Two important growth factors controlling epithelial behavior are Keratinocyte Growth Factor-1 (KGF-1) and -2 (KGF-2). Connective tissue cells express these growth factors, but only epithelial cells respond to them. We studied the effect of proinflammatory cytokines and LPS on gingival fibroblast expression of KGF-1 and KGF-2 in vitro. Gingival fibroblasts were found to express KGF-1 and -2 in culture but only KGF-1 protein and gene expression was stimulated by serum, in a concentration-dependent manner by proinflammatory cytokines IL-1alpha, IL-1beta, TNF-alpha and IL-6 and LPS isolated from Porphyromonas gingivalis and Escherichia coli. The local increase in proinflammatory cytokine expression and the accumulation of LPS in disease sites may therefore stimulate gingival fibroblast expression of KGF-1. We hypothesize that this local increase in KGF-1 expression may, via a paracrine mechanism, stimulate local epithelial cell proliferation, migration and invasion during the onset and progression of periodontitis.     

Autocrine regulation of osteoclast formation and bone resorption by IL-1 alpha and TNF alpha.

Bone resorption is regulated by the cytokines within marrow cells that mediate osteoclast formation and activation. IL-1 and TNF induce bone resorption by stimulating the production of osteoclast-like multinucleated cells and by increasing the bone-resorbing activity of formed osteoclasts. This study was designed to detect IL-1 and TNF in osteoclasts in vitro and to determine whether these cytokines up-regulate osteoclast differentiation and bone resorption. The production of IL-1 alpha, -beta, and TNF alpha, beta in osteoclasts was examined immunohistochemically and by in situ hybridization. In the co-culture of C57BL/6N mouse bone marrow and MC3T3-G2/PA6 cells, a colony of osteoclasts formed, and IL-1 alpha and TNF alpha were detected. However, IL-1 beta and TNF beta were not detected. To investigate the role of IL-1 alpha and TNF alpha from osteoclasts, we enumerated TRAP-positive cells and measured the resorption pit areas in the presence of antibodies against IL-1 alpha and TNF alpha. The addition of antibodies against IL-1 alpha and TNF alpha to the co-culture system decreased the number of TRAP-positive colonies at seven days after incubation (anti-IL-1 alpha, 25.0 +/- 2.3%; anti-TNF alpha, 41.7 +/- 3.7%; anti-IL-1 alpha + anti-TNF alpha, 40.5 +/- 1.3%; and control, 100%), and the ratio of mononuclear to multinuclear cells had changed (anti-IL-1 alpha, 90:10; anti-TNF alpha, 75:25; anti-IL-1 alpha+ anti-TNF alpha, 88:12; and control, 60:40). The total pit areas per dentin slice also decreased with the addition of antibodies (anti-IL-1 alpha, 28,828; anti-TNF alpha, 49,249; anti-IL-1 alpha + anti-TNF alpha, 30,685; and control, 303,139 mm2). These results suggest that local production of IL-1 alpha and TNF alpha by osteoclasts is an important mechanism for regulating the osteoclast differentiation and bone resorptive process.     

Study of cytokine production in inflamed human gingival tissues in periodontitis. Interleukin-1 (IL-1 alpha, beta) and tumor necrosis factor (TNF alpha)

It seems to be generally agreed that periodontal disease is a local manifestation of a systemic immune response. Interleukin-1 (IL-1), which has multiple biologic activities, is detected in the gingival sulcus fluid of periodontitis sites. Recent investigations have revealed that IL-1 and tumor necrosis factor (TNF) are analogous to osteoclast activating factor and promote bone resorption. These findings have suggested the possibility that IL-1 and TNF may play a significant role in the initiation and development of periodontal disease. However, it remains to be determined whether these cytokines influence periodontal tissue breakdown in periodontitis. To elucidate the mechanisms of tissue breakdown in periodontitis, we examined cytokine production by human periodontitis gingival tissue. Twelve periodontitis patients were included in this study. Control subjects with healthy periodontium consisted of nine individuals. Gingival samples were biopsied from inflamed or healthy gingival tissues. Biopsy specimens were dissected into fragments 3 mm in diameter and plated onto 24 well culture plates with RPMI 1640 medium. IL-1 activity was measured by a growth inhibition assay using melanoma cell line A 375. An enzyme-linked immunosorbent assay (ELIZA) was used for measuring levels of human IL-1 alpha, IL-1 beta. TNF alpha activity was measured by a growth inhibition assay using cell line LM2D6. IL-1 activity was detected in significantly (p less than 0.001) higher levels in culture supernatants from gingival tissues in periodontitis (48.0 +/- 23.3 units/ml) than in control tissues (2.3 +/- 0.6 units/ml), however, levels of IL-1 activity were not associated with periodontal pocket depth or extent of alveolar bone resorption in periodontitis.(ABSTRACT TRUNCATED AT 250 WORDS)