Role for Fimbriae and Lysine-Specific Cysteine Proteinase Gingipain K in Expression of Interleukin-8 and Monocyte Chemoattractant Protein inPorphyromonas gingivalis-Infected Endothelial Cells

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Abstract
Recent cross-sectional and prospective epidemiological studies have demonstrated an association between periodontal disease and atherosclerosis and human coronary heart disease. Previously, we have established that the periodontal pathogenPorphyromonas gingivalisis capable of invading aortic, heart, and human umbilical vein endothelial cells (HUVEC). Since atherosclerosis is a chronic inflammatory response initiated at the vascular wall, interactions ofP. gingivaliswith endothelial cells and the subsequent host cell response to infection may be important in the pathogenesis of atherosclerosis. In this study we examined the consequences ofP. gingivalisinfection of HUVEC on the expression of the chemokines interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1). HUVEC were found to constitutively produce low levels of IL-8 and MCP-1. The addition ofP. gingivalisfimbrillin-specific peptides, lipopolysaccharides (LPS), or heat-killed whole cell preparations to HUVEC stimulated modest IL-8 and MCP-1 responses. In contrast, coculture of HUVEC with liveP. gingivalisstrain A7436, 33277, or 381 abolished the IL-8 and MCP-1 responses. Inhibition of IL-8 and MCP-1 production was not dependent on bacterial adherence since similar results were obtained with the nonadherentP. gingivalis fimAmutant DPG3 or whenP. gingivaliswas preincubated with fimbrillin peptide antisera prior to the addition to HUVEC. Furthermore, treatment ofP. gingivalis-infected HUVEC with cytochalsin D, which preventedP. gingivalisinvasion, also abolished the constitutive IL-8 and MCP-1 responses. Treatment of HUVEC withE. coliLPS stimulated robust IL-8 and MCP-1 responses that were abolished when stimulated cells were cocultured with liveP. gingivalis. Analysis ofP. gingivalis-infected HUVEC cultures by an RNase protection assay revealed an increase in the IL-8 transcript relative to uninfected HUVEC. Pretreatment ofP. gingivaliswith protease inhibitors prior to the addition to HUVEC prevented the inhibition of IL-8 and MCP-1 production inP. gingivalis-infected HUVEC, indicating that the inhibition was proteolytically mediated. Coculture of HUVEC with aP. gingivalismutant deficient in lysine-specific cysteine proteinase (gingipain K [Kgp]) resulted in an increase in both IL-8 transcription and protein expression relative to that observed in HUVEC cocultured with theP. gingivaliswild-type strain. These results indicate thatP. gingivaliscan temporally modulate the chemokine response in endothelial cells through both fimbriae and gingipain-mediated mechanisms.

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