SURFACE-ASSOCIATED MATERIAL FROM THE BACTERIUM ACTINOBACILLUS-ACTINOMYCETEMCOMITANS CONTAINS A PEPTIDE WHICH, IN CONTRAST TO LIPOPOLYSACCHARIDE, DIRECTLY STIMULATES FIBROBLAST INTERLEUKIN-6 GENE-TRANSCRIPTION

The oral commensal Gram-negative bacterium Actinobacillus actinomycete mcomitans is believed to be the causative organism of localized juveni le periodontitis, a disease in which there is rapid loss of alveolar b one supporting the teeth. Previously, we have reported that gentle sal ine extraction of this bacterium removed a loosely adherent proteinace ous fraction from the cell surface of the bacterium, which we have ter med surface-associated material. This material contained potent bone-r esorbing activity. We now report that surface-associated material is a lso 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 fibro blast IL-1, IL-6 and tumour necrosis factor (TNF) alpha synthesis, sur face-associated material stimulated gingival fibroblasts to synthesize only IL-6, with no induction of IL-1 or TNF (the normal inducers of I L-6 synthesis). Reverse transcriptase PCR also failed to detect mRNA f or IL-1 or TNF in surface-associated-material-stimulated fibroblasts, although both mRNAs were present in Escherichia coli LPS-stimulated ce lls. Neutralizing antibodies to IL-1 and/or TNF or the natural IL-1 re ceptor antagonist (IL-1ra) inhibited enteric LPS-induced IL-6 synthesi s, 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 sur face-associated material stimulates IL-6 gene transcription by a trans criptional 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 act ivity has been isolated using anion-exchange, reverse-phase and size-e xclusion HPLC. The active moiety is a peptide of molecular mass 2 kDa which may be the product of a bacterial short open reading frame.