Yutaka Kusumoto

Lipopolysaccharide Interaction with Cell Surface Toll-like Receptor 4-MD-2: Higher Affinity than That with MD-2 or CD14

Toll-like receptors (TLRs) are innate recognition molecules for microbial products, but their direct interactions with corresponding ligands remain unclarified. LPS, a membrane constituent of gram-negative bacteria, is the best-studied TLR ligand and is recognized by TLR4 and MD-2, a molecule associated with the extracellular domain of TLR4. Although TLR4-MD-2 recognizes LPS, little is known about the physical interaction between LPS and TLR4-MD-2. Here, we demonstrate cell surface LPS–TLR4-MD-2 complexes. CD14 greatly enhances the formation of LPS–TLR4-MD-2 complexes, but is not coprecipitated with LPS–TLR4-MD-2 complexes, suggesting a role for CD14 in LPS loading onto TLR4-MD-2 but not in the interaction itself between LPS and TLR4-MD-2. A tentative dissociation constant (Kd) for LPS–TLR4-MD-2 complexes was ∼3 nM, which is ∼10–20 times lower than the reported Kd for LPS–MD-2 or LPS–CD14. The presence of detergent disrupts LPS interaction with CD14 but not with TLR4-MD-2. E5531, a lipid A antagonist developed for therapeutic intervention of endotoxin shock, blocks LPS interaction with TLR4-MD-2 at a concentration 100 times lower than that required for blocking LPS interaction with CD14. These results reveal direct LPS interaction with cell surface TLR4-MD-2 that is distinct from that with MD-2 or CD14.

Cathepsins are required for Toll-like receptor 9 responses

Toll-like receptors (TLR) recognize a variety of microbial products and activate defense responses. Pathogen sensing by TLR2/4 requires accessory molecules, whereas little is known about a molecule required for DNA recognition by TLR9. After endocytosis of microbes, microbial DNA is exposed and recognized by TLR9 in lysosomes. We here show that cathepsins, lysosomal cysteine proteases, are required for TLR9 responses. A cell line Ba/F3 was found to be defective in TLR9 responses despite enforced TLR9 expression. Functional cloning with Ba/F3 identified cathepsin B/L as a molecule required for TLR9 responses. The protease activity was essential for the complementing effect. TLR9 responses were also conferred by cathepsin S or F, but not by cathepsin H. TLR9-dependent B cell proliferation and CD86 upregulation were apparently downregulated by cathepsin B/L inhibitors. Cathepsin B inhibitor downregulated interaction of CpG-B with TLR9 in 293T cells. These results suggest roles for cathepsins in DNA recognition by TLR9.

The Radioprotective 105/MD-1 Complex Links TLR2 and TLR4/MD-2 in Antibody Response to Microbial Membranes

Low-affinity IgG3 Abs to microbial membranes are important for primary immune defense against microbes, but little is known about the importance of TLRs in their production. IgG3 levels were extremely low in mice lacking radioprotective 105 (RP105), a B cell surface molecule structurally related to TLRs. RP105−/− B cells proliferated poorly in response to not only the TLR4 ligand LPS but also TLR2 ligand lipoproteins, both of which mediate the immunostimulatory activity of microbial membranes. RP105−/− mice were severely impaired in hapten-specific Ab production against LPS or lipoproteins. CD138 (syndecan-1)-positive plasma cells were detected after lipid A injection in wild-type spleen but much less in RP105−/− spleen. RP105 ligation in vivo induced plasma cell differentiation. RP105 expression was ∼3-fold higher on marginal zone B cells than on follicular and B1 cells and was down-regulated on germinal center cells. These results demonstrate that a signal via RP105 is uniquely important for regulating TLR-dependent Ab production to microbial membranes.

Bacteroides gingivalis-specific serum IgG and IgA subclass antibodies in periodontal diseases

The level of serum IgM, IgG and IgA antibodies including IgG1. IgG2, IgG3, IgG4, IgA1 and IgA2 subclass‐specific antibodies to Bacteroides (Porphyromonas) gingivalis fimbriae and to lipopolysaccharide(LPS) were analysed in paiients with different forms of periodontal disease (PD) and control subjects by ELISA. Among PD subjects, sera obtained from adult periodonitis (AP). rapidly progressive periodontitis (RPP) and gingivitis contained high litres of fimbriae‐specific IgG antibodies (7500–15000 ELISA units) followed by IgA (90–700 units) and IgM (30–90 units). In contrast, sera from localized juvenile periodontitis (LJP) subjects exhibited much lower titres of fimbriae‐specific IgG (89 ± 11 units), IgA (31.5 units) and IgM (17 ± 3 units) antibodies. A similar response pattern was also seen in sera from normal subjects aged 35–41 years who practice normal oral hygiene, while sera of younger adults (aged 18–24) with superior hygiene did not have any antigen‐specific antibodies. Analysis of IgG subclass anti‐fimbriae responses revealed that the major response was IgG3 followed by IgG1, IgG2 and IgG4 in AP, RPP and gingivitis. Although lower, a similar pattern of IgG subclass titre was seen in LJP and normal subjects aged 35–41 years. When IgA subclass responses were measured in AP and RPP, higher titres of the fimbriae‐specific response were noted with IgAl when compared with IgA2. However, lower but approximately equal levels of fimbriae‐specific IgAl and IgA2 titres were seen in other PD groups. When anti‐B. gingivalis LPS‐specific responses were measured, the sera of AP patients contained high levels of IgG antibodies (2265 ± 224 units) followed by IgA (411 ± 90 units) and IgM (214 ± 56 units). Further. IgG anti‐LPS responses were mainly IgG2 followed by IgG4. IgG3 and IgGI. For IgA subclass responses, higher titres of anti‐LPS‐specific antibodies were noted in IgA2 subclass over IgAl. These results showed that higher anti‐B. gingivalis antibody responses occur in PD when compared with healthy individuals and protein and lipid‐carbohydrate antigens of B. gingivalis induce distinct patterns of antigen‐specific IgG and IgA subclass responses.

Immunobiological activities of synthetic peptide segments of fimbrial protein from Porphyromonas gingivalis

Several oligopeptide segments of fimbrial subunit protein (fimbrilin) of Porphyromonas gingivalis strain 381 were synthesized and tested for immunobiological activities. Peptides F3(31-50; amino acid residue numbers 31 to 50, based on the amino acid sequence of the fimbrilin proposed by Dickinson et al., Infect. Immun., 170, 1658, 1988), F12(212-231) and F17(312-331) were found to be immunodominant epitopes of this fimbrial protein as revealed by ELISA. Furthermore, peptides F5(71-90) and F17(312-331) were demonstrated to agglutinate rabbit erythrocytes, and were mitogenic for BALB/c spleen cells but not thymocytes. These peptides enhanced the number of fimbria-specific antibody-secreting cells in BALB/c spleen cell cultures, and induced cytokines such as tumor necrosis factor-alpha and interleukin-6 production in human monocyte/macrophage cultures. The data demonstrate that these defined peptide segments are responsible for the immunostimulating portions within the fimbrial protein molecule.

Direct Interaction between Gingival Fibroblasts and Lymphoid Cells Induces Inflammatory Cytokine mRNA Expression in Gingival Fibroblasts

In inflamed periodontal lesions, dense infiltration of lymphocytes is usually observed in the extravascular periodontal connective tissue, adjacent to gingival fibroblasts. Our previous study revealed that activated lymphocytes can adhesively interact with gingival fibroblasts in vitro. In the present study, we investigated whether gingival fibroblasts are activated through direct interaction with lymphoid cells by monitoring the expression of inflammatory cytokine mRNA in human gingival fibroblasts (HGF). Co-culture with various human lymphoid cells in vitro resulted in a marked increase in the expression of IL-la, IL-1β, and IL-6 mRNA by the HGF. In addition, expression of the mRNA of the IL-1β-converting enzyme (ICE), which is essential to produce the mature form of IL-1β, was constitutively observed in the HGF, suggesting that mature IL-lp is produced by these cells. When HGF were cultured with the culture supernatant of the lymphoid cells, the increase in the inflammatory cytokine mRNA expression was not observed. Similarly, when HGF and lymphoid cells were cultured in the same well but separated by a membrane which prevented direct contact between the cells, no increase in inflammatory cytokine mRNA expression was observed. These results strongly indicate that direct interaction between these heterotypic cell types transduces activation signals into HGF that induce an increase in inflammatory cytokine mRNA expression. Furthermore, IL-1β mRNA expression in the HGF was synergistically increased when HGF directly interacted with lymphoid cells in the presence of exogeneous IL-1β. The present study demonstrates that direct interaction between HGF and lymphoid cells stimulates HGF to increase inflammatory cytokine mRNA expression, and raises the possibility that heterotypic cell-cell interaction may facilitate local inflammatory reactions.

Agonistic Antibody to TLR4/MD-2 Protects Mice from Acute Lethal Hepatitis Induced by TNF-α

LPS is recognized by a heterodimer consisting of TLR4 and its coreceptor MD-2. LPS signal causes excessive inflammation and tissue damage. In this study, we show that a mAb to TLR4/MD-2 protected mice from acute lethal hepatitis caused by LPS/d-galactosamine. The protective effect of the mAb was not due to inhibition of LPS response, because serum TNF-α, which was induced by LPS and caused lethal hepatitis, was 10 times up-regulated by the mAb pretreatment. Moreover, this mAb induced antiapoptotic genes in liver in a TLR4/MD-2-dependent manner. These results demonstrated that an agonistic mAb to TLR4/MD-2 protected mice from LPS/d-galactosamine-induced acute lethal hepatitis by delivering a protective signal activating NF-κB through TLR4/MD-2.

Aggregatibacter actinomycetemcomitans Omp29 Is Associated with Bacterial Entry to Gingival Epithelial Cells by F-Actin Rearrangement

The onset and progressive pathogenesis of periodontal disease is thought to be initiated by the entry of Aggregatibacter actinomycetemcomitans (Aa) into periodontal tissue, especially gingival epithelium. Nonetheless, the mechanism underlying such bacterial entry remains to be clarified. Therefore, this study aimed to investigate the possible role of Aa outer membrane protein 29 kD (Omp29), a homologue of E. coli OmpA, in promoting bacterial entry into gingival epithelial cells. To accomplish this, Omp29 expression vector was incorporated in an OmpA-deficient mutant of E. coli. Omp29+/OmpA− E. coli demonstrated 22-fold higher entry into human gingival epithelial line cells (OBA9) than Omp29−/OmpA− E. coli. While the entry of Aa and Omp29+/OmpA− E. coli into OBA9 cells were inhibited by anti-Omp29 antibody, their adherence to OBA9 cells was not inhibited. Stimulation of OBA9 cells with purified Omp29 increased the phosphorylation of focal adhesion kinase (FAK), a pivotal cell-signaling molecule that can up-regulate actin rearrangement. Furthermore, Omp29 increased the formation of F-actin in OBA9 cells. The internalization of Omp29-coated beads and the entry of Aa into OBA9 were partially inhibited by treatment with PI3-kinase inhibitor (Wortmannin) and Rho GTPases inhibitor (EDIN), both known to convey FAK-signaling to actin-rearrangement. These results suggest that Omp29 is associated with the entry of Aa into gingival epithelial cells by up-regulating F-actin rearrangement via the FAK signaling pathway.

Generation of specific antibody-secreting cells in salivary glands of BALB/c mice following parenteral or oral immunization with Porphyromonas gingivalis fimbriae

The humoral immune response and induction of antigen-specific antibody-secreting cells in mucosal lymphoid tissues were examined in mice immunized subcutaneously or orally with fimbrial protein purified from Porphyromonas gingivalis strain 381. A group of BALB/c mice was immunized subcutaneously with P. gingivalis fimbriae and semisynthetic adjuvant GM-53 in Freunds incomplete adjuvant on days 0 and 28. Another group of mice was immunized perorally with liposome containing fimbriae and GM-53 on days 0, 1, 27 and 28. In the mice immunized subcutaneously, salivary anti-fimbrial IgM antibodies were detected transiently on day 5, followed by the appearance of specific IgG and IgA antibodies on day 14. Higher concentrations of salivary IgG and IgA anti-fimbrial antibodies were found after the second immunization. Fimbria-specific IgM and IgG spot-forming cells (SFC) were detected in cervical lymph nodes of the immunized mice by the ELISPOT method. Fimbria-specific IgA SFC but not IgM and IgG appeared in the parotid and submandibular glands of subcutaneously immunized mice. On the other hand, mice immunized by gastric intubation generated almost exclusively salivary anti-fimbrial IgA antibodies. In agreement with this finding, increased numbers of antigen-specific IgA but not IgM and IgG SFC were seen in parotid and submandibular glands, but not in cervical lymph nodes of orally immunized mice. It can be concluded that systemic or oral immunization with fimbrial antigen induces distinct immune responses in specific lymphoid tissues or in the salivary glands in respect of their temporal sequences and the numbers of plasma cells secreting antigen-specific and non-specific immunoglobulins.

Regulatory effects of Spirulina complex polysaccharides on growth of murine RSV‐M glioma cells through Toll‐like receptor 4

This study is the first to report that Spirulina complex polysaccharides (CPS) suppress glioma growth by down‐regulating angiogenesis via a Toll‐like receptor 4 signal. Murine RSV‐M glioma cells were implanted s.c. into C3H/HeN mice and TLR4 mutant C3H/HeJ mice. Treatment with either Spirulina CPS or Escherichia coli (E. coli) lipopolysaccharides (LPS) strongly suppressed RSV‐M glioma cell growth in C3H/HeN, but not C3H/HeJ, mice. Glioma cells stimulated production of interleukin (IL)‐17 in both C3H/HeN and C3H/HeJ tumor‐bearing mice. Treatment with E. coli LPS induced much greater IL‐17 production in tumor‐bearing C3H/HeN mice than in tumor‐bearing C3H/HeJ mice. In C3H/HeN mice, treatment with Spirulina CPS suppressed growth of re‐transplanted glioma; however, treatment with E. coli LPS did not, suggesting that Spirulina CPS enhance the immune response. Administration of anti‐cluster of differentiation (CD)8, anti‐CD4, anti‐CD8 antibodies, and anti‐asialo GM1 antibodies enhanced tumor growth, suggesting that T cells and natural killer cells or macrophages are involved in suppression of tumor growth by Spirulina CPS. Although anti‐interferon‐γ antibodies had no effect on glioma cell growth, anti‐IL‐17 antibodies administered four days after tumor transplantation suppressed growth similarly to treatment with Spirulina CPS. Less angiogenesis was observed in gliomas from Spirulina CPS‐treated mice than in those from saline‐ or E. coli LPS‐treated mice. These findings suggest that, in C3H/HeN mice, Spirulina CPS antagonize glioma cell growth by down‐regulating angiogenesis, and that this down‐regulation is mediated in part by regulating IL‐17 production.