Showing papers by "Tomohiko Ogawa published in 2007"

Chemical structure and immunobiological activity of Porphyromonas gingivalis lipid A.

Abstract: In 1933, Boivin et al. extracted an endotoxin from Salmonella typhimurium for the first time, after which a variety of chemical and biological studies on endotoxins have been performed. In 1952, the structural and functional properties of endotoxic lipopolysaccharide (LPS), extracted by a hot phenol and water method devised by Westphal et al., were reported, which led to a number of studies of Gram-negative bacteria in regards to the host defense mechanism. Since 1960, the unique chemical structure and biological activity of Bacteroides species LPS have received a great deal of attention, and there is a long history of such studies. In addition, among oral bacterial strains that have received attention as causative periodontopathic bacteria, many have been classified as Bacteroides species. In particular, a number of researchers have investigated whether LPS of Porphyromonas gingivalis (formerly Bacteroides gingivalis), a black-pigmented oral anaerobic rod, is a virulent factor of the bacterium. The active center of the LPS of these Bacteroides species, the lipid A molecule, is known to be an active participant in endotoxic activation, though its other biological activities are weak, due to its unique chemical structure and action as an antagonist of LPS. On the other hand, many reports have noted that the LPS of those species activate cells in C3H/HeJ mice, which generally do not respond to LPS. We were the first to reveal the chemical structure of P. gingivalis lipid A and, together with other researchers, reported that P. gingivalis LPS and its lipid A have activities toward C3H/HeJ mice. Since that time, because of the popularity of Toll-like receptor (TLR) studies, a great deal of evidence has been reported indicating that P. gingivalis LPS and its lipid A are ligands that act on TLR2. In order to solve such problems as heterogeneity and contamination of the biologically active components of P. gingivalis lipid A, we produced a chemical synthesis counterpart of lipid A and test results indicated it to be a TLR4 agonist. Furthermore, in order to disprove the common belief that P. gingivalis LPS and its lipid A are TLR2 ligands, the TLR2-active component contained in a P. gingivalis LPS fraction was separated and purified, after which we showed its chemical structure to be a lipoprotein consisting of three fatty acid residues, thus answering a longstanding question regarding Bacteroides species LPS. In addition to the field of dentistry, many studies based on the misconception of "TLR2-active LPS/lipid A" still exist in the field of innate immunity. Based on the history of studies of ligands acting on TLR4, Bacteroides species LPS findings were reviewed and are presented here. In particular, we investigated P. gingivalis LPS and its lipid A.

Toll‐like receptor 4‐dependent recognition of structurally different forms of chemically synthesized lipid As of Porphyromonas gingivalis

Abstract: Summary Porphyromonas gingivalis is a Gram-negative anaerobic oral black-pigmented bacterium closely associated with chronic periodontitis. Lipopolysaccharide (LPS) derived from P. gingivalis is shown to be unusual because the LPS contains a greater number of lipid A species, such as tri-, tetra-, and/or penta-acylated lipid As. In this study, a lipid A possessing penta-fatty acyl chains derived from P. gingivalis strain 381 (compound PG-381–5FA) was synthesized, and examined for its immunobiological activities, compared with a tri-acylated lipid A (compound PG-381–3FA) synthesized previously. Compound PG-381-5FA, similar to compound PG-381-3FA, demonstrated weaker activity in a Limulus test as compared with Escherichia coli-type synthetic lipid A (compound 506). Compound PG-381-5FA, followed by compound PG-381-3FA, induced KC, interleukin-6, and tumour necrosis factor-α production in peritoneal macrophages from LPS-responsive C3H/HeN mice, but not in those from LPS-hyporesponsive C3H/HeJ mice. Furthermore, compound PG-381-5FA, as well as compound PG-381-3FA, activated nuclear factor-κB via Toll-like receptor (TLR)4/mD-2, but not TLR2, in a manner similar to compound 506, and worked as an antagonist for compound 506-induced cell activation. In the case of human peripheral blood mononuclear cells, compound PG-381-5FA showed much stronger IL-6-inducing activity than compound PG-381-3FA. The present results demonstrate that the chemical synthesis of a penta-acylated lipid A, mimicking the natural lipid A portion of LPS from P. gingivalis, is attributable to immune cell activation through TLR4, similar to that of compound 506.

Soluble CD14 Discriminates Slight Structural Differences between Lipid As That Lead to Distinct Host Cell Activation

Abstract: Soluble CD14 (sCD14) in serum is known to sensitize host cells to LPS. In the present study, the contributions of sCD14 and LPS-binding protein to a lipid A moiety from LPS preparations of periodontopathogenic Fusobacterium nucleatum sp. nucleatum were compared with that of Escherichia coli-type synthetic lipid A (compound 506). F. nucleatum lipid A was identified to be a hexa-acylated fatty acid composed of tetradecanoate (C(14)) and hexadecanoate (C(16)), similar to dodecanoate (C(12)) and C(14) in compound 506. The two lipid A specimens exhibited nearly the same reactivity in Limulus amoebocyte lysate assays, though F. nucleatum lipid A showed a weaker lethal toxicity. Both lipid A specimens showed nearly the same activities toward host cells in the absence of FBS, though compound 506 exhibited much stronger activity in the presence of FBS, sCD14, or sCD14 together with LPS-binding protein. Furthermore, native PAGE/Western immunoblot assays demonstrated that F. nucleatum lipid A had a weaker binding to sCD14 as compared with compound 506. These results suggest that sCD14 is able to discriminate the slight structural differences between these lipid As, which causes their distinct host cell activation activities.

Toll-like receptor 2-mediated dendritic cell activation by a Porphyromonas gingivalis synthetic lipopeptide.

Abstract: A PG1828 gene-encoded triacylated lipoprotein was previously isolated from a Porphyromonas gingivalis lipopolysaccharide preparation as a Toll-like receptor (TLR) 2 agonist and its lipopeptide derivatives were synthesized based on the chemical structure. In the present study, granulocyte–macrophage colony stimulating factor-differentiated bone marrow-derived dendritic cells (BMDDCs) were stimulated separately with the P. gingivalis synthetic lipopeptide N-palmitoyl-S-[2-pentadecanoyloxy, 3-palmitoyloxy-(2R)-propyl]-l-Cys-Asn-Ser-Gln-Ala-Lys (PGTP2-RL) and its glyceryl stereoisomer (PGTP2-SL). Only PGTP2-RL activated BMDDCs from wild-type mice to secrete tumour necrosis factor-α, interleukin (IL)-6, IL-10 and IL-12p40, whilst PGTP2-RL-induced cytokine production was eliminated in TLR2 knockout (−/−) BMDDCs. BMDDCs from wild-type mice but not TLR2−/− mice responded to PGTP2-RL as well as Pam3CSK4 by increasing the expression of maturation markers, including CD80 (B7-1), CD86 (B7-2), CD40, CD275 (B7RP-1/inducible T-cell co-stimulatory ligand) and major histocompatibility complex class II. Taken together, these results indicate that the fatty acid residue at the glycerol position in the P. gingivalis lipopeptide plays a pivotal role in TLR2-mediated dendritic cell activation.

Chemical structure and immunobiological activity of lipid A from Serratia marcescens LPS

Abstract: The chemical structure and immunobiological activities of Serratia marcescens lipid A, an active centre of LPS, were investigated. LPS preparations of S. marcescens were extracted using a hot phenol/water method, after which purified lipid A specimens were prepared by weak acid hydrolysis, followed by normal phase and gel filtration chromatographic separation. The lipid A structure was determined by MS to be a diglucosamine backbone with diphosphates and five C14 normal chain acyl groups, including two acyloxyacyl groups at the 2 and 3 positions of the non-reducing side. S. marcescens lipid A and Escherichia coli-type synthetic lipid A (compound 506) exhibited definite reactivity in Limulus amoebocyte lysate assays. The lethal toxicity of S. marcescens lipid A was nearly comparable to that of compound 506, and both induced nuclear factor-κB activation in murine cells via Toll-like receptor (TLR)4/MD-2 but not TLR2, as well as various inflammatory cytokines in peritoneal macrophages of C3H/HeN mice but not C3H/HeJ mice. Furthermore, S. marcescens lipid A induced nearly the same amounts of tumour necrosis factor alpha, interleukin-6, and nitric oxide production by the murine alveolar macrophage cell line MH-S as compared with compound 506. These results indicate that S. marcescens possesses a penta-acylated lipid A, which is nearly identical to E. coli lipid A in regard to biological activities, while it also may be a crucial virulence factor of the bacterium.

Inflammatory stimuli regulate the binding of gingival fibroblasts to dendritic cells via integrin β2

Abstract: We investigated the mechanism and impact of various inflammatory stimuli on dendritic cell (DC)-gingival fibroblast (GF) adhesion. Human immature (im) DCs were generated from monocytes by culturing with interleukin (IL)-4 and granulocyte macrophage-colony stimulating factor (GM-CSF). GFs were outgrown from the human gingival specimen. DCs were co-cultured with GFs with/without pretreatment with various stimuli. Adhered cells were measured by fluorometer. Expression of adhesion molecules was analyzed by flow cytometry. Pretreatment of GFs with tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and Escherichia coli (Ec) lipopolysaccharides (LPS) significantly increased the adhesion to imDCs and enhanced intercellular adhesion molecule (ICAM)-1 expression. A signifi-cantly increased DC-GF adhesion was also observed when imDCs were pretreated with Ec LPS, Porphyromonas gingivalis (Pg) fimbriae, and peptideglycan but not with Pg LPS. Expression of LFA-1 and Mac-1 on DCs was not altered by the pretreatment with these stimuli. However, LFA-1 and Mac-1 blockade of imDC signifi cantly reduced the adhesion to TNF-α-stimulated GFs. These results showed that inflammatory stimuli increased the imDC-GF adhesion via lymphocyte function- associated antigen (LFA)-1/Mac-1-ICAM-1 ligation. Adhesion of DC to GFs may be important not only for the localization of DCs in the inflammatory periodontal lesion, but also for the modulation of immune responses.

Implication of immune interactions in bacterial virulence: is Porphyromonas gingivalis an “Invader” or “Stealth Element” in periodontal lesions?

Abstract: Porphyromonas gingivalis (Pg), a Gram-negative anaerobic blackpigmented rod bacterium, has been recognized as the most potent etiologic bacterium in human chronic periodontitis It possesses a variety of putative virulence factors providing both tissue destruction and host evasion including lipopolysaccharides (LPS), fimbriae, various proteinases, etc These factors actively participate in periodontal tissue destruction However, recent evidence suggests that Pg has also evolved mechanisms to inhibit or confuse host immune systems Thus, Pg is suggested to behave not only like an “active invader”, but also like a “stealth element” in periodontal lesions In the present study, repeated exposure of Pg components induced tolerance resulting in selective inhibition of cytokine production of both monocytes and gingival fibroblasts in a different fashion from that described for LPS in Escherichia coli It was also revealed that Pg LPS induced a unique dendritic cell subset with a CD14+CD16+ phenotype that exhibited weak maturation In animal studies, administration of live Pg or its LPS exerted a regulatory effect on systemic markers such as triglycerides or adiponectin Taken together, these findings suggest that Pg may be able to adapt to the local immune defense, contributing to the connection between systemic and periodontal disease