Porphyromonas gingivalis is one of the most important periodontal pathogens and has been to known to invade various types of cells, including endothelial cells. The present study investigated the mechanisms involved in the internalization of P. gingivalis in human umbilical vein endothelial cells (HUVEC). P. gingivalis internalization was reduced by clathrin and lipid raft inhibitors, as well as a siRNA knockdown of caveolin-1, a principal molecule of lipid raft-related caveolae. The internalization was also reduced by perturbation of actin rearrangement, while microtubule polymerization was not required. Furthermore, we found that Src kinases are critical for the internalization of P. gingivalis into HUVEC, while neither Rho family GTPases nor phosphatidylinositol 3-kinase are required. Taken together, this study indicated that P. gingivalis internalization into endothelial cells involves clathrin and lipid rafts and requires actin rearrangement associated with Src kinase activation

Aggregatibacter actinomycetemcomitans is the most important etiologic agent of aggressive periodontitis and can interact with endothelial cells. Monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) are chemokines, playing important roles in periodontal pathogenesis. In our current study, the effects of A. actinomycetemcomitans on the production of MCP-1 and IL-8 by human umbilical vein endothelial cells (HUVEC) were investigated. A. actinomycetemcomitans strongly induced the gene expression and protein release of both MCP-1 and IL-8 in a dose- and time-dependent manner. Dead A. actinomycetemcomitans cells were as effective as live bacteria in this induction. Treatment of HUVEC with cytochalasin D, an inhibitor of endocytosis, did not affect the mRNA up-regulation of MCP-1 and IL-8 by A. actinomycetemcomitans. However, genistein, an inhibitor of protein tyrosine kinases, substantially inhibited the MCP-1 and IL-8 production by A. actinomycetemcomitans, whereas pharmacological inhibition of each of three members of mitogen-activated protein (MAP) kinase family had little effect. Furthermore, gel shift assays showed that A. actinomycetemcomitans induces a biphasic activation (early at 1-2 h and late at 8-16 h) of nuclear factor-κB (NF-κB) and an early brief activation (0.5-2 h) of activator protein-1 (AP-1). Activation of canonical NF-κB pathway (IκB kinase activation and IκB-α degradation) was also demonstrated in these experiments. Although lipopolysaccharide from A. actinomycetemcomitans also induced NF-κB activation, this activation profile over time differed from that of live A. actinomycetemcomitans. These results suggest that the expression of MCP-1 and IL-8 is potently increased by A. actinomycetemcomitans in endothelial cells, and that the viability of A. actinomycetemcomitans and bacterial internalization are not required for this effect, whereas the activation of protein tyrosine kinase(s), NF-κB, and AP-1 appears to play important roles. The secretion of high levels of MCP-1 and IL-8 resulting from interactions of A. actinomycetemcomitans with endothelial cells may thus contribute to the pathogenesis of aggressive periodontitis.

Enterococcus faecalis, a gram-positive bacterium, has been implicated in endodontic infections, particularly in chronic apical periodontitis. Proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), are involved in the pathogenesis of these apical lesions. E. faecalis has been reported to stimulate macrophages to produce TNF-α. The present study investigated the mechanisms involved in TNF-α production by a murine macrophage cell line, RAW 264.7 in response to exposure to E. faecalis. Both live and heat-killed E. faecalis induced high levels of gene expression and protein release of TNF-α. Treatment of RAW 264.7 cells with cytochalasin D, an inhibitor of endocytosis, prevented the mRNA up-regulation of TNF-α by E. faecalis. In addition, antioxidant treatment reduced TNF-α production to baseline levels. Inhibition of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase also significantly attenuated E. faecalis-induced TNF-α expression by RAW 264.7 cells. Furthermore, activation of NF-κB and AP-1 in RAW 264.7 cells was also stimulated by E. faecalis. These results suggest that the phagocytic uptake of bacteria is necessary for the induction of TNF-α in E. faecalis-stimulated macrophages, and that the underlying intracellular signaling pathways involve reactive oxygen species, ERK, p38 MAP kinase, NF-κB, and AP-1.

Porphyromonas gingivalis is a major etiologic agent of chronic periodontitis and cytokines produced by macrophages play important roles in the pathogenesis of periodontal diseases. In this study we investigated the cytokine response of phorbol myristate acetatedifferentiated THP-1 cells exposed to P. gingivalis. Compared with the prominent cell wall components of P. gingivalis (lipopolysaccharide and the major fimbrial protein FimA), live P. gingivalis stimulated much higher levels of cytokine production. In addition, whereas low multiplicity of infection challenges (MOI=10) of P. gingivalis 381 stimulated high levels of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β, high dose challenges with this bacterium (MOI = 100) resulted in a substantially diminished production of MCP-1 and IL-6. Moreover, high MOI P. gingivalis challenges achieved only low levels of induction of MCP-1 and IL-6 mRNA. The decreased production of MCP-1 and IL-6 appeared to be mediated by P. gingivalis proteases, because high MOI challenges with congenic protease mutant strains of this microorganism (MT10 and MT10W) did not result in a diminished production of MCP-1 and IL-6. Similar to its protease mutant strains, leupeptin (a protease inhibitor)- treated P. gingivalis at high doses induced high levels of MCP-1 production. To examine the mechanisms underlying the diminished production of MCP-1 by P. gingivalis proteases, the activation of mitogen-activated protein (MAP) kinases and NF-xB was compared between the 381 and MT10W strains. Whilst high doses of both 381 and MT10W similarly activated the three members of the MAP kinase family, the DNA binding activity of NF-xB, as revealed by gel shift assays, was greatly increased only by MT10W. Taken together, our data indicate that P. gingivalis stimulates the production of high levels of TNF-α, IL-1β, IL-6, and MCP-1 but that high dose challenges with this bacterium result in a diminished production of MCP-1 and IL-6 via the protease-mediated suppression of NF-B activation in THP-1 macrophagic cells.

Periodontopathogens including Porphyromonas gingivalis interact with host periodontal cells and the excessive subsequent host responses contribute a major part to the development of periodontal diseases. Cyclooxygenase(COX)-2-synthesized has detrimental activities in terms of periodontal pathogenesis. The present study investigated induction of COX-2 expression by P. gingivalis in human monocytic THP-1 cells. Live P. gingivalis increased expression of COX-2, but not that of COX-1, which was demonstrated at both mRNA and protein levels. Elevated levels of were released from P. gingivalis-infected THP-1 cells. Pharma-cological inhibition of p38 mitogen-activated protein kinase(MAPK) and extracellular signal-regulated kinase(ERK) substantially attenuated P. gingivalis-induced COX-2 mRNA expression. Indeed, activation of p38 MAPK and ERK was observed in P. gingivalis-infected THP-1 cells. Also, P. gingivalis induced activation of nuclear factor-κB (NF-κB)which is an important transcription factor for COX-2. These results suggest that COX-2 expression is up regulated in P. gingivalis-infected monocytic cells, at least in part, via p38 MAPK, ERK, and NF-κB.