title: Surface-associated material from the bacterium Actinobacillus actinomycetemcomitans contains a peptide which, in contrast to lipopolysaccharide, directly stimulates fibroblast interleukin-6 gene transcription.
abstract: The oral commensal Gram-negative bacterium Actinobacillus actinomycetemcomitans is believed to be the causative organism of localized juvenile periodontitis, a disease in which there is rapid loss of alveolar bone supporting the teeth. Previously, we have reported that gentle saline extraction of this bacterium removed a loosely adherent proteinaceous fraction from the cell surface of the bacterium, which we have termed surface-associated material. This material contained potent bone-resorbing activity. We now report that surface-associated material is also a potent stimulator of cytokines, and in particular, interleukin-6 (IL-6) synthesis, while the lipopolysaccharide from this bacterium is only a weak stimulator of IL-6 synthesis by fibroblasts and monocytes. In contrast to enteric lipopolysaccharide (LPS), which induces fibroblast IL-1, IL-6 and tumour necrosis factor (TNF) alpha synthesis, surface-associated material stimulated gingival fibroblasts to synthesize only IL-6, with no induction of IL-1 or TNF (the normal inducers of IL-6 synthesis). Reverse transcriptase PCR also failed to detect mRNA for IL-1 or TNF in surface-associated-material-stimulated fibroblasts, although both mRNAs were present in Escherichia coli LPS-stimulated cells. Neutralizing antibodies to IL-1 and/or TNF or the natural IL-1 receptor antagonist (IL-1ra) inhibited enteric LPS-induced IL-6 synthesis, but did not inhibit surface-associated-material-induced synthesis. In addition, dexamethasone, which completely suppressed LPS-induced IL-6 synthesis, only inhibited surface-associated-material-induced IL-6 synthesis by 50%. This suggests that the active constituent in the surface-associated material stimulates IL-6 gene transcription by a transcriptional control mechanism distinct to that of E. coli LPS. The IL-6 stimulating activity of the surface-associated material is inhibited by both heat and trypsin, suggesting that it is proteinaceous. The activity has been isolated using anion-exchange, reverse-phase and size-exclusion HPLC. The active moiety is a peptide of molecular mass 2kDa which may be the product of a bacterial short open reading frame.
authors.affiliation: Maxillofacial Surgery Research Unit, Eastman Dental Institute for Oral Health Care Sciences, University College London, UK.
authors.forename: K
authors.initials: K
authors.lastname: Reddi
authors.forename: S P
authors.initials: SP
authors.lastname: Nair
authors.forename: P A
authors.initials: PA
authors.lastname: White
authors.forename: S
authors.initials: S
authors.lastname: Hodges
authors.forename: P
authors.initials: P
authors.lastname: Tabona
authors.forename: S
authors.initials: S
authors.lastname: Meghji
authors.forename: S
authors.initials: S
authors.lastname: Poole
authors.forename: M
authors.initials: M
authors.lastname: Wilson
authors.forename: B
authors.initials: B
authors.lastname: Henderson
mesh_terms: D016976:Aggregatibacter actinomycetemcomitans / Q000254:growth & development / Q000276:immunology*; D000906:Antibodies / Q000494:pharmacology; D001862:Bone Resorption; D002462:Cell Membrane / Q000276:immunology; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis*; D003907:Dexamethasone / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000276:immunology; D005881:Gingiva / Q000166:cytology / Q000276:immunology*; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1 / Q000096:biosynthesis; D015850:Interleukin-6 / Q000096:biosynthesis*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000302:isolation & purification / Q000494:pharmacology*; D010455:Peptides / Q000302:isolation & purification / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D012795:Sialoglycoproteins / Q000096:biosynthesis; D014158:Transcription, Genetic / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
chemical_list: D000906:Antibodies; D016207:Cytokines; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1; D015850:Interleukin-6; D008070:Lipopolysaccharides; D010455:Peptides; D012333:RNA, Messenger; D012795:Sialoglycoproteins; D014409:Tumor Necrosis Factor-alpha; D003907:Dexamethasone