Gingipains from Porphyromonas gingivalis increase the chemotactic and respiratory burst-priming properties of the 77-amino-acid interleukin-8 variant

Porphyromonas gingivalis, a gram-negative anaerobe which is implicated in the etiology of active periodontitis, secretes degradative enzymes (gingipains) and sheds proinflammatory mediators (e.g., lipopolysaccharides [LPS]). LPS triggers the secretion of interleukin-8 (IL-8) from immune (72-amino-acid [aa] variant [IL-8(72aa)]) and nonimmune (IL-8(77aa)) cells. IL-8(77aa) has low chemotactic and respiratory burst-inducing activity but is susceptible to cleavage by gingipains. This study shows that both R- and K-gingipain treatments of IL-8(77aa) significantly enhance burst activation by fMLP and chemotactic activity (P < 0.05) but decrease burst activation and chemotactic activity of IL-8(72aa) toward neutrophil-like HL60 cells and primary neutrophils (P < 0.05). Using tandem mass spectrometry, we have demonstrated that R-gingipain cleaves 5- and 11-aa peptides from the N-terminal portion of IL-8(77aa) and the resultant peptides are biologically active, while K-gingipain removes an 8-aa N-terminal peptide yielding a 69-aa isoform of IL-8 that shows enhanced biological activity. During periodontitis, secreted gingipains may differentially affect neutrophil chemotaxis and activation in response to IL-8 according to the cellular source of the chemokine.

Oxygen tension modulates the cytokine response of oral epithelium to periodontal bacteria

Background: There is an inverse relationship between pocket depth and pocket oxygen tension with deep pockets being associated with anaerobic bacteria. However, little is known about how the host tissues respond to bacteria under differing oxygen tensions within the periodontal pocket. Aim: To investigate the effect of different oxygen tensions upon nuclear factor-kappa B (NF-?B) activation and the inflammatory cytokine response of oral epithelial cells when exposed to nine species of oral bacteria. Materials and Methods: H400 oral epithelial cells were equilibrated at 2%, 10% or 21% oxygen. Cells were stimulated with heat-killed oral bacteria at multiplicity of infection 10:1, Escherichia coli lipopolysaccharide (15 µg/ml) or vehicle control. Interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-a) levels were measured by enzyme-linked immunosorbent assay and NF-?B activation was measured by reporter vector or by immunohistochemical analysis. Results: Tannerella forsythensis, Porphyromonas gingivalis and Prevotella intermedia elicited the greatest epithelial NF-?B activation and cytokine responses. An oxygen-tension-dependent trend in cytokine production was observed with the highest IL-8 and TNF-a production observed at 2% oxygen and lowest at 21% oxygen. Conclusions: These data demonstrate a greater pro-inflammatory host response and cell signalling response to bacteria present in more anaerobic conditions, and hypersensitivity of epithelial cells to pro-inflammatory stimuli at 2% oxygen, which may have implications for disease pathogenesis and/or therapy.

Non-enterobacterial endotoxins stimulate human coronary artery but not venous endothelial cell activation via toll-like receptor 2

To determine whether non-enterobacterial endotoxins, which are likely to constitute the majority of the circulating endotoxin pool, may stimulate coronary artery endothelial cell activation. Interleukin-8 secretion, monocyte adhesion, and E-selectin expression were measured in human umbilical vein endothelial cells (HUVECs) and coronary artery endothelial cells (HCAECs) challenged in vitro with highly purified endotoxins of common host colonisers Escherichia coli, Porphyromonas gingivalis, Pseudomonas aeruginosa, and Bacteroides fragilis. HCAECs but not HUVECs expressed Toll-like receptor (TLR)-2 and were responsive to non-enterobacterial endotoxins. Transfection of TLR-deficient HEK-293 cells with TLR2 or TLR4/MD2 revealed that while E. coli endotoxin utilised solely TLR4 to signal, the endotoxins, deglycosylated endotoxins (lipid-A), and whole heat-killed bacteria of the other species stimulated TLR2-but not TLR4-dependent cell-signalling. Blockade of TLR2 with neutralizing antibody prevented HCAEC activation by non-enterobacterial endotoxins. Comparison of each endotoxin with E. coli endotoxin in limulus amoebocyte lysate assay revealed that the non-enterobacterial endotoxins are greatly underestimated by this assay, which has been used in all previous studies to estimate plasma endotoxin concentrations. Circulating non-enterobacterial endotoxins may be an underestimated contributor to endothelial activation and atherosclerosis in individuals at risk of increased plasma endotoxin burden.