lipid-a and Periodontal-Diseases

Porphyromonas gingivalis produces different LPS isoforms with significant structural variations of their lipid A and O-antigen moieties that can affect its pro-inflammatory and bone-resorbing potential. We show here, for the first time, that P. gingivalis LPS isolated from W83 strain is highly sialylated and possesses significantly reduced inflammatory potential compared with less sialylated ATCC 33277 strain LPS. Nevertheless, the reduction in the endotoxin activity is not mediated by the presence of sialic acid LPS moieties as the sialic acid-free LPS produced by the mutant W83 strain exhibits a similar inflammatory potential to the wild type strain. Furthermore, our findings suggest that the interaction between the sialic acid LPS moieties and the inhibitory CD33 receptor is prevented by endogenously expressed sialic acid on the surface of THP-1 cells that cannot be out-competed by sialic acid containing P. gingivalis LPS. The present study also highlights the importance of endogenous sialic acid as a 'self-associated molecular pattern' and CD33 receptors in modulation of innate immune response as human gingival fibroblasts, which do not express CD33 receptors, and desialylated THP-1 cells have both been found to have much higher spontaneous IL-8 production than naïve THP-1 cells.

Topics: Bacteroidaceae Infections; Cell Line; Fibroblasts; Gingiva; Host-Pathogen Interactions; Humans; Immunity, Innate; Interleukin-8; Lipid A; Lipopolysaccharides; Monocytes; Mutation; N-Acetylneuraminic Acid; O Antigens; Periodontal Diseases; Porphyromonas gingivalis; Protein Processing, Post-Translational; Sialic Acid Binding Ig-like Lectin 3

The purpose of this study was to examine the effects of lipid A-associated proteins from Porphyromonas gingivalis, a major cause of inflammatory periodontal disease, on the production of nitric oxide and expression of inducible nitric oxide synthase in the murine macrophage cell line, RAW264.7. We also attempted to throw light on the signaling mechanisms involved in P. gingivalis lipid A-associated protein-induced nitric oxide production.. The lipid A-associated proteins from P. gingivalis 381 were prepared by standard hot phenol-water extraction of endotoxin isolated by the butanol method. Nitric oxide production was assayed by measuring the accumulation of nitrite in culture supernatants. Western blot analysis of inducible nitric oxide synthase and analysis of reverse transcription-polymerase chain reaction products were carried out.. We found that P. gingivalis lipid A-associated proteins can induce inducible nitric oxide synthase expression and stimulate the release of nitric oxide without additional stimuli, and we demonstrated that multiple signaling pathways, such as nuclear factor-kappaB, microtubule polymerization, protein tyrosine kinase, protein kinase C, and mitogen-activated protein kinase cascades, are involved in P. gingivalis lipid A-associated protein-stimulated nitric oxide production. The production of nitric oxide required l-arginine.. The present study clearly shows that P. gingivalis lipid A-associated proteins fully induced inducible nitric oxide synthase expression and nitric oxide production in RAW264.7 cells in the absence of other stimuli. The ability of P. gingivalis lipid A-associated proteins to promote the production of nitric oxide may be important in the pathogenesis of inflammatory periodontal disease.

Topics: Animals; Bacterial Proteins; Cell Line; Endotoxins; Enzyme Induction; Lipid A; Mice; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Periodontal Diseases; Porphyromonas gingivalis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Lipopolysaccharide (LPS) derived from the periodontal pathogen Porphyromonas gingivalis has been reported to differ structurally and functionally from enterobacterial LPS. These studies demonstrate that in contrast to protein-free enterobacterial LPS, a similarly purified preparation of P. gingivalis LPS exhibited potent Toll-like receptor 2 (TLR2), rather than TLR4, agonist activity to elicit gene expression and cytokine secretion in murine macrophages and transfectants. More importantly, TLR2 stimulation by this P. gingivalis LPS preparation resulted in differential expression of a panel of genes that are normally induced in murine macrophages by Escherichia coli LPS. These data suggest that (i) P. gingivalis LPS does not signal through TLR4 and (ii) signaling through TLR2 and through TLR4 differs quantitatively and qualitatively. Our data support the hypothesis that the shared signaling pathways elicited by TLR2 and by TLR4 agonists must diverge in order to account for the distinct patterns of inflammatory gene expression.

Topics: Animals; Drosophila Proteins; Escherichia coli; Gene Expression; Interleukin-12; Lipid A; Lipopolysaccharides; Macrophages, Peritoneal; Membrane Glycoproteins; Mice; Mice, Inbred C3H; Periodontal Diseases; Porphyromonas gingivalis; Receptors, Cell Surface; Signal Transduction; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptors; Tumor Necrosis Factor-alpha

Apoptosis (programmed cell death) is a mechanism by which superfluous or damaged cells undergo changes that lead to selective removal from organ systems by phagocytic cells. Certain bacterial products delay apoptosis in neutrophils (PMNs). In this study, PMNs were incubated for up to 8 h with varying concentrations of lipopolysaccharide (LPS), lipid A or capsular polysaccharide isolated from 3 strains of Porphyromonas gingivalis (Pg) (strains HG-184, A7A1-28 and 381). Assay runs included controls containing cells and medium but no bacterial products. Fluorescence microscopy was used to evaluate apoptotic changes. PMNs exhibited a time-dependent increase in the number of apoptotic cells. When cells were cultured in the presence of LPS from any of the 3 Pg strains, apoptosis was delayed in a dose-dependent fashion (p < 0.05). The effects of these LPS preparations were similar to each other and to Escherichia coli 0111:B4 LPS. Lipid A from the 3 Pg strains also delayed apoptosis (p < 0.05), but was less potent than LPS or synthetic lipid A. Capsular polysaccharide had no significant effect on apoptosis (p > 0.05). Thus, LPS and lipid A from P. gingivalis appear to modulate the functional lifespan of PMNs. This could potentiate the inflammatory and destructive components of periodontal diseases.

Topics: Apoptosis; Bacterial Capsules; Cell Survival; Dose-Response Relationship, Drug; Escherichia coli; Humans; Lipid A; Lipopolysaccharides; Microscopy, Fluorescence; Neutrophils; Periodontal Diseases; Phagocytes; Polysaccharides, Bacterial; Porphyromonas gingivalis; Time Factors

There is evidence that the lipid A-associated proteins (LAPs) of enteric bacteria can induce the synthesis of interleukin 1 (IL-1) and therefore may be important virulence factors. Porphyromonas gingivalis is now recognized as a major pathogen in the chronic inflammatory periodontal diseases and it has previously been reported that a crude LAP fraction from this organism could induce IL-1 and interleukin 6 (IL-6) synthesis. In the present study the chemical and biological properties of the LAPs of this bacterium have been further characterized. Analysis by SDS-PAGE has shown that the LAPs comprise nine proteins of molecular masses 81, 68, 48, 47, 28, 25, 20, 17 and 16 kDa. These LAPs, at concentrations as low as 100 ng ml(-1), were shown to stimulate human gingival fibroblasts, human peripheral blood mononuclear cells and whole human blood to produce the pro-inflammatory cytokine IL-6. The cytokine-inducing activity of the LAPs was reduced after heat-inactivation and trypsinization, suggesting that the activity was not due to contaminating LPS. To establish which proteins in this mixture were the active cytokine inducers, the LAPs were separated by electrophoresis on polyacrylamide gels. The majority of the activity was associated with the 17 kDa LAP. N-terminal sequence analysis demonstrated that this protein was homologous to an internal region of a conserved adhesin domain contained within a family of P. gingivalis extracellular proteins including the RI protease, Lys-gingipain, porphypain and haemagglutinin A. In addition to a role in adherence, the adhesin domain(s) of these proteins may also have cytokine-inducing properties. Furthermore, it has also been shown that the previously observed degradation of cytokines by P. gingivalis may be attributable to the catalytic domain of the RI protease. Thus, different domains within the same molecule appear to have opposing actions on pro-inflammatory cytokine levels and the balance between these two activities may influence the cytokine status of the periodontium in patients with the common chronic inflammatory conditions known as the periodontal diseases.

Topics: Amino Acid Sequence; Bacterial Proteins; Bacteroidaceae Infections; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Endotoxins; Fibroblasts; Gingiva; Humans; Interleukin-1; Interleukin-6; Lipid A; Lipopolysaccharides; Molecular Sequence Data; Periodontal Diseases; Porphyromonas gingivalis

When neutrophils are incubated with bacterial lipopolysaccharide (LPS), they become primed for enhanced release of superoxide anion (O2-) in response to stimulation by FMLP. We investigated the human neutrophil-priming activity of LPS from the periodontal pathogens, Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi) and Actinobacillus actinomycetemcomitans (Aa) in comparison with that of LPS from Escherichia coli (E. coli). The optimum conditions for LPS to prime neutrophils were assessed for every LPS and found to be as follows: Neutrophils were incubated with LPS in the presence of 10% heat-inactivated plasma and 1 mM EDTA at 37 degrees C for 30 min and then stimulated with 1 microM FMLP at 37 degrees C for 7 min. Under these conditions, half-maximum priming was observed at 6.2 ng/ml Pg-LPS, 45 ng/ml Pi-LPS, 1.5 ng/ml Aa-LPS and 1.5 ng/ml E. coli-LPS. The priming activity of each LPS was neutralized by polymyxin B. Anti-CD14 monoclonal antibody inhibited priming by all LPS. The priming by Aa-LPS and E. coli-LPS was inhibited by LA-14-PP, a synthetic lipid A precursor IVA, but that by Pg-LPS and Pi-LPS was not. Priming by tumor necrosis factor alpha was not affected by polymyxin B, anti-CD14 antibody or LA-14-PP. Gelation of Limulus amebocyte lysate occurred at 10 pg/ml Pg-LPS, 30 pg/ml Pi-LPS, 3 pg/ml Aa-LPS and 3 pg/ml E. coli-LPS. Thus LPS from different periodontal pathogens primed neutrophils with different efficacy.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aggregatibacter actinomycetemcomitans; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Escherichia coli; Glycolipids; Humans; Limulus Test; Lipid A; Lipopolysaccharide Receptors; Lipopolysaccharides; N-Formylmethionine Leucyl-Phenylalanine; Neutrophil Activation; Periodontal Diseases; Polymyxin B; Porphyromonas gingivalis; Prevotella intermedia; Respiratory Burst

In this comparative study, samples of root substance from healthy and periodontally diseased human teeth were analyzed for fatty acids by means of a gas-chromatographic technique. The sample preparation procedure allowed specimens from individual teeth to be analyzed. The content of the fatty acids C16:0, C16:1, C18:0, and C18:1 in the superficial layer of the periodontally diseased teeth was significantly higher than that of healthy teeth. In the inner layer, there was no such difference. Four different 3-hydroxy fatty acids (3-OH C14:0, 3-OH C15:0, 3-OH C16:0, and 3-OH C17:0) were identified by combined gas-chromatography/mass-spectrometry in the tooth substance of periodontally diseased teeth. This indicates the presence of lipid A, which is the toxic component of bacterial lipopolysaccharides.

Topics: Chromatography, Gas; Dental Cementum; Dentin; Fatty Acids; Humans; Lipid A; Mass Spectrometry; Periodontal Diseases; Tooth Root