Toll-like receptors (TLRs) 2 and 4 are signal transducers for lipopolysaccharide, the major proinflammatory constituent in the outer membrane of Gram-negative bacteria. We observed that membrane lipoproteins/lipopeptides from Borrelia burgdorferi, Treponema pallidum, and Mycoplasma fermentans activated cells heterologously expressing TLR2 but not those expressing TLR1 or TLR4. These TLR2-expressing cells were also stimulated by living motile B. burgdorferi, suggesting that TLR2 recognition of lipoproteins is relevant to natural Borrelia infection. Importantly, a TLR2 antibody inhibited bacterial lipoprotein/lipopeptide-induced tumor necrosis factor release from human peripheral blood mononuclear cells, and TLR2-null Chinese hamster macrophages were insensitive to lipoprotein/lipopeptide challenge. The data suggest a role for the native protein in cellular activation by these ligands. In addition, TLR2-dependent responses were seen using whole Mycobacterium avium and Staphylococcus aureus, demonstrating that this receptor can function as a signal transducer for a wide spectrum of bacterial products. We conclude that diverse pathogens activate cells through TLR2 and propose that this molecule is a central pattern recognition receptor in host immune responses to microbial invasion.

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The lipopeptide FSL-1 [S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe, Pam 2 CG DPKHPKSF] synthesized on the basis of the N-terminal structure of a Mycoplasma salivarium lipoprotein capable of activating normal human gingival fibroblasts to induce the cell surface expression of ICAM-1 revealed an activity to induce production of monocyte chemoattractant protein 1, interleukin-6 (IL-6), and IL-8. FSL-1 also activated macrophages to produce tumor necrosis factor alpha as the Mycoplasma fermentansderived lipopeptide MALP-2 (Pam 2 CGNNDESNISFKEK), a potent macrophage-activating lipopeptide, did. The level of the activity of FSL-1 was higher than that of MALP-2. This result suggests that the difference in the amino acid sequence of the peptide portion affects the activity because the framework structure other than the amino acid sequence of the former is the same as that of the latter. To determine minimal structural requirements for the activity of FSL-1, the diacylglyceryl Cys and the peptide portions were examined for this activity. Both portions did not reveal the activity. A single amino acid substitution from Phe to Arg and a fatty acid substitution from palmitic acid to stearic acid drastically reduced the activity. Similar results were obtained in measuring the NF-B reporter activity of FSL-1 to human embryonic kidney 293 cells transfected with Toll-like receptor 2 and 6, together with a NF-B-dependent luciferase reporter plasmid. These results suggest that both the diacylglyceryl and the peptide portions of FSL-1 are indispensable for the expression of biological activities and for the recognition by Toll-like receptors 2 and 6 and that the recognition of FSL-1 by Toll-like receptors 2 and 6 appears to be hydrophobic.Various bacterial cell wall components such as lipopolysaccharides (LPS), lipoteichoic acid, peptidoglycans, and lipoproteins (LP) have been shown to activate macrophages, fibroblasts, or lymphocytes to induce production of cytokines (16). Escherichia coli LP were first characterized and sequenced by Braun (9), and they have been demonstrated to be biologically active (5)(6)(7)(8)20). The part of LP responsible for biological activity is the N-terminal lipopeptide moiety, the structure of which is S-(2,3-bispalmitoyloxypropyl)-N-palmitoyl-Cys-SerSer-Asp-Ala-(Pam 3 CSNNA-) (7).Mycoplasmas, wall-less microorganisms, also possess LP capable of activating macrophages or fibroblasts (11,27,28,31,32). Mühlradt et al. (27,28) recently identified a 2-kDa lipopeptide, MALP-2, from Mycoplasma fermentans that is capable of activating monocytes/macrophages, and these authors determined the structure to be S-(2,3-bispalmitoyloxypropyl) Cys-Gly-Asn-Asn-Asp-Glu-Ser-Asn-Ile-Ser-Phe-Lys-Glu-Lys (Pam 2 CGNNDESNISFKEK). We have also found that Mycoplasma salivarium LP activate normal human gingival fibroblasts (HGF) to induce production of inflammatory cytokines and surface expression of ICAM-1 and have purified a 44-kDa LP (LP44) responsible for the activity (32). The structure of the N-ter...

Toll-like receptors (TLRs) 2 and 4 have recently been identified as possible signal transducers for various bacterial ligands. To investigate the roles of TLRs in the recognition of periodontopathic bacteria by the innate immune system, a Chinese hamster ovary (CHO) nuclear factor-B (NF-B)-dependent reporter cell line, 7.7, which is defective in both TLR2-and TLR4-dependent signaling pathways was transfected with human CD14 and TLRs. When the transfectants were exposed to freeze-dried periodontopathic bacteria, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Capnocytophaga ochracea, and Fusobacterium nucleatum, and a non-oral bacterium, Escherichia coli, all species of the bacteria induced NF-B-dependent CD25 expression in 7.7/huTLR2 cells. Although freeze-dried A. actinomycetemcomitans, F. nucleatum, and E. coli also induced CD25 expression in 7.7/huTLR4 cells, freeze-dried P. gingivalis did not. Similarly, lipopolysaccharides (LPS) extracted from A. actinomycetemcomitans, F. nucleatum, and E. coli induced CD25 expression in 7.7/huTLR4 cells, but LPS from P. gingivalis and C. ochracea did not. Furthermore, LPS from P. gingivalis and C. ochracea attenuated CD25 expression in 7.7/huTLR4 cells induced by repurified LPS from E. coli. LPS from P. gingivalis and C. ochracea also inhibited the secretion of interleukin-6 (IL-6) from U373 cells, the secretion of IL-1␤ from human peripheral blood mononuclear cells, and ICAM-1 expression in human gingival fibroblasts induced by repurified LPS from E. coli. These findings indicated that LPS from P. gingivalis and C. ochracea worked as antagonists for human TLR4. The antagonistic activity of LPS from these periodontopathic bacteria may be associated with the etiology of periodontal diseases.

Polymorphonuclear leukocytes (PMN) are the first cells that migrate into periodontal tissues and gingival crevices in response to invading pathogens. It was recently demonstrated that PMN have the ability to synthesize and release cytokines following appropriate stimulation, while it is not clear whether these capacities are directly related to periodontal destructive processes. We therefore investigated the amounts of the cytokines interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), IL-8 and IL-1 receptor antagonist (IL-1ra) secreted by PMN from healthy donors following stimulation with lipopolysaccharide (LPS) from 4 periodontopathic bacteria, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Capnocytophaga ochracea and Fusobacterium nucleatum, and the non-oral bacterium Escherichia coli. A actinomycetemcomitans, F. nucleatum and E. coli LPS stimulated the release of significantly greater amounts of IL-1 beta, TNF-alpha and IL-8 than the control unstimulated PMN (p < 0.01). The levels of IL-1 beta, TNF-alpha and IL-8 released from cells stimulated with P. gingivalis or C. ochracea LPS were significantly lower than those of cells stimulated with A. actinomycetemcomitans or E. coli LPS (p < 0.05). On the other hand, substantially greater amounts of IL-1ra were released from PMN stimulated with each LPS and from control unstimulated PMN during the first 6 h, and then significantly greater amounts of IL-1ra were secreted by PMN stimulated with A. actinomycetemcomitans and E.coli LPS during the following 12 h (p < 0.01). The inhibitory effects of IL-1ra on the biological activity of IL-1 in the supernatants of PMN were examined by the thymocyte comitogen proliferation assay. The supernatants of PMN stimulated with each LPS showed less biological IL-1 activity as compared with the same doses of recombinant human IL-1 beta detected by enzyme-linked immunosorbent assay. Furthermore, no activity was detected in the supernatants of PMN stimulated with P. gingivalis or C. ochracea LPS. These findings demonstrated that LPS from periodontopathic bacteria were capable of stimulating PMN to release not only pro-inflammatory cytokines but also their inhibitors such as IL-1ra. Different secretion levels of these cytokines and their biological activities induced by the various LPS might be important in the onset and progression of periodontal diseases.

The present study investigated the expression of Toll‐like receptor (TLR) 2, TLR4, cluster of differentiation (CD) 14 and CD1a in human periodontitis gingiva using immunohistochemical methods. The specimens were classified according to the degree of inflammation into three groups (mild, moderate and severe). We established three zones in which to evaluate the ratios of TLR2‐, TLR4‐, CD14‐ and CD1a‐positive cells to total cells in the connective tissues of each section. TLR2 and TLR4 were expressed in human periodontal tissues, and the ratio of TLR2‐positive cells was highest overall in zone 1 (connective tissue subjacent to pocket epithelium) of the severe group and that of TLR4‐positive cells was higher in the severe group than in the other groups. These results suggest that TLR2 and TLR4 participate in the innate immune response to stimulation by bacterial products in periodontal tissues. The ratio of CD14‐positive cells was lowest overall in zone 1 of the severe group and that of CD1a was higher in the severe group than in the other groups. These results suggest that CD14 may be down‐regulated during the development of inflammation and/or dendritic cells might infiltrate chronically inflamed gingival tissue.

Chronic periodontitis is a silent infectious disease prevalent worldwide and affects lifestyle-related diseases. Therefore, efficient screening of patients is essential for general health. This study was performed to evaluate prospectively the diagnostic utility of a blood IgG antibody titer test against periodontal pathogens. Oral examination was performed, and IgG titers against periodontal pathogens were measured by ELISA in 1,387 individuals. The cut-off value of the IgG titer was determined in receiver operating characteristic curve analysis, and changes in periodontal clinical parameters and IgG titers by periodontal treatment were evaluated. The relationships between IgG titers and severity of periodontitis were analyzed. The best cut-off value of IgG titer against Porphyromonas gingivalis for screening periodontitis was 1.682. Both clinical parameters and IgG titers decreased significantly under periodontal treatment. IgG titers of periodontitis patients were significantly higher than those of healthy controls, especially in those with sites of probing pocket depth over 4 mm. Multiplied cut-off values were useful to select patients with severe periodontitis. A blood IgG antibody titer test for Porphyromonas gingivalis is useful to screen hitherto chronic periodontitis patients.

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