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) α 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 2 kDa which may be the product of a bacterial short open reading frame.