Hiroaki Takimoto

Immunomodulating Effects of Flavonoids on Acute and Chronic Inflammatory Responses Caused by Tumor Necrosis Factor α

Flavonoids have beneficial activities which modulate oxidative stress, allergy, tumor growth and viral infection, and which stimulate apoptosis of tumor cells. In addition to these activities, dietary flavonoids are able to regulate acute and chronic inflammatory responses. Here we describe new aspects of regulatory mechanisms by which flavonoids suppress production of tumor necrosis factor-alpha (TNF-alpha) by macrophages, microglial cells and mast cells stimulated with lipopolysaccharide (LPS) and others via toll-like receptors (TLRs), and TNF-alpha-mediated acute and chronic inflammatory responses. Treatment with flavonoids such as luteolin, apigenin, quercetin, genistein, (-)-epigallocatechin gallate, and anthocyanidin resulted in significant downregulation of LPS-elicited TNF-alpha and nitric oxide (NO) production and diminished lethal shock. In chronic diseases, pathogenesis of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis which is triggered by TNF-alpha, was improved by the oral administration of flavonoids after the onset of CIA. Here, we discuss that inhibitory effects of flavonoids on acute and chronic inflammation are due to regulation of signaling pathways, including the nuclear factor kappaB (NF-kappaB) activation and mitogen-activated protein (MAP) kinase family phosphorylation. FcetaRI expression by NF-kappaB activation was also reduced by flavonoids; while accumulation of lipid rafts, which is the critical step for signaling, was blocked by flavonoids. The intake of dietary flavonoids reduces acute and chronic inflammation due to blocking receptor accumulation and signaling cascades, and would assist individuals at high-risk from life-style related diseases.

Vγ1+ γδ T cells play protective roles at an early phase of murine cytomegalovirus infection through production of interferon-γ

Cytomegalovirus (CMV) causes severe opportunistic infection in immunocompromised hosts. The importance of conventional αβ T cells in protection against CMV infection has been well documented. However, the role of the second T‐cell population (which express the γδ T‐cell receptor) in CMV infection is not known. In the present study, we analysed the function and protective role of γδ T cells in a murine cytomegalovirus (MCMV) infection model. After intraperitoneal infection with MCMV, the number of γδ T cells increased in the liver and peritoneal cavity from day 3, and reached a peak on day 5. The γδ T cells showed an activated T‐cell phenotype and predominantly expressed Vγ1, which is known to be expressed by heat‐shock protein 65 (hsp 65)‐specific γδ T cells. Analysis of cytokine expression demonstrated that the MCMV‐induced γδ T cells expressed interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α) but not interleukin‐4 (IL‐4), implying their participation in the cell‐mediated immune response against MCMV. Depletion of γδ T cells by anti‐T‐cell receptor (TCR) γδ monoclonal antibody (mAb) treatment resulted in significant increase of virus titre and decrease of IFN‐γ in the liver on day 3 after MCMV infection, which further supports the importance of γδ T cells in early protection against infection. Finally, the MCMV‐induced γδ T cells produced IFN‐γin vitro in response to hsp 65. Our results suggest that γδ T cells participate in early protection against MCMV infection through recognition of hsp 65 and production of IFN‐γ.

Accelerated recovery from cyclophosphamide-induced leukopenia in mice administered a Japanese ethical herbal drug, Hochu-ekki-to.

The effect of a Japanese ethical herbal drug, Hochu-ekki-to (HOT), on recovery from leukopenia induced by cyclophosphamide (CY) was investigated. Daily oral administration of 1000 mg/kg HOT into CY-treated mice significantly prevented decrease of leukocyte numbers in the peripheral blood and accelerated recovery from leukopenia. Ginsenoside Rgl extracted from Ginseng radix, a major herb of HOT, was one of the active ingredients. HOT increased numbers of neutrophils and monocytes in the peripheral blood compared with CY-treated control. Moreover, HOT augmented the resistance against Pseudomonas aeruginosa infection. The number of colony-forming units in the spleen (CFU-S) also increased in HOT-treated mice. The frequencies of IL-3-, GM-CSF- and IFN-gamma-producing cells increased in the spleen, bone marrow, liver and IEL on HOT treatment, and HOT clearly augmented the expressions of IL-3, GM-CSF and IFN-gamma mRNA in the spleen, bone marrow, liver and IEL except IL-3 and IFN-gamma mRNA in the IEL. These results suggest that HOT enhances the production of hematopoietic lymphokines, stimulates the proliferation of hematopoietic progenitor cells and consequently accelerates recovery from leukopenia in CY-treated mice. Additionally, IFN-gamma which HOT-augmented the production may contribute the protective effect against the bacterial infection by activating of phagocyte cells.

Development of Dendritic Epidermal T Cells with a Skewed Diversity of γδTCRs in Vδ1-Deficient Mice

One of the most intriguing features of γδ T cells that reside in murine epithelia is the association of a specific Vγ/Vδ usage with each epithelial tissue. Dendritic epidermal T cells (DETCs) in the murine epidermis, are predominantly derived from the “first wave” Vγ5+ fetal thymocytes and overwhelmingly express the canonical Vγ5/Vδ1-TCRs lacking junctional diversity. Targeted disruption of the Vδ1 gene resulted in a markedly impaired development of Vγ5+ fetal thymocytes as precursors of DETCs; however, γδTCR+ DETCs with a typical dendritic morphology were observed in Vδ1−/− mice and their cell densities in the epidermis were slightly lower than those in Vδ1+/− epidermis. Moreover, the Vδ1-deficient DETCs were functionally competent in their ability to up-regulate cytokines and keratinocyte growth factor-expression in response to keratinocytes. Vγ5+ DETCs were predominant in the Vδ1−/− epidermis, though Vγ5− γδTCR+ DETCs were also detected. The Vγ5+ DETCs showed a typical dendritic shape, γδTCRhigh, and age-associated expansion in epidermis as observed in conventional DETCs of normal mice, whereas the Vγ5− γδTCR+ DETCs showed a less dendritic shape, γδTCRlow, and no expansion in the epidermis, consistent with their immaturity. These results suggest that optimal DETC development does not require a particular Vγ/Vδ-chain usage but requires expression of a limited diversity of γδTCRs, which allow DETC precursors to mature and expand within the epidermal microenvironment.

Suppression of IgE production in mice treated with a traditional Chinese medicine, Bu-zhong-yi-qi-tang (Japanese name: Hochu-ekki-to)

The ability of a traditional herbal medicine, Bu-zhong-yi-qi-tang (Japanese name: Hochu-ekki-to, HOT), to suppress IgE production was investigated. BALB/c mice were intraperitoneally immunized with aluminium hydroxide adsorbed with DNP-KLH (DNP-KLH + alum). When oral administration of HOT was begun just after immunization, the serum level of antigen-specific IgE was significantly decreased, although those of antigen-specific IgG1 and IgG2a were not influenced. In the culture of spleen cells obtained 14 days after immunization with DNP-KLH, antigen-specific IgE and IgG1 production by the cells of the HOT-treated mice was significantly suppressed compared to that in immunized mice. Furthermore, in the combination culture with CD4+ T cells and B cells separated from spleen cells, IgE production by the cells from immunized mice was inhibited by replacement of their corresponding cell population with either CD4+ T cells or B cells of HOT-treated mice. Additionally, production of interleukin 2 (IL-2) and IL-4 was significantly suppressed in HOT-treated mice but not that of IFN-gamma in comparison to the immunized mice. These results suggested that HOT decreased the IgE level in serum by inhibiting the development of IL-4-producing CD4+ T cells.

Involvement of Mannose Receptor in Glycopeptidolipid‐Mediated Inhibition of Phagosome‐Lysosome Fusion

We previously reported that glycopeptidolipid (GPL) isolated from Mycobacterium avium serovar 4 inhibited phagosome‐lysosome (P‐L) fusion when macrophages phagocytosed heat‐killed Staphylococcus aureus (SA). In the present study we analyzed the underlying inhibitory mechanism of GPL coated on SA. Elimination of oligosaccharide from GPL abrogated its inhibitory activity. GPL did not inhibit P‐L fusion of opsonized SA phagocytosed via complement receptors. The inhibitory activity of GPL was competitively reduced by the presence of α‐methyl‐D‐mannoside and anti‐mannose receptor antibody, suggesting that inhibition of P‐L fusion by GPL is mediated through mannose receptor. Recruitment of early endosome antigen 1 and Ca2+/calmodulin kinase II in human macrophage‐like THP‐1 cells were significantly suppressed by GPL, indicating that GPL inhibits steps for leading to the P‐L fusion.

Role of Macrophages in Acute Murine Cytomegalovirus Infection

It has been recognized that macrophages play an important role in controlling virus infection in experimental animal models. To evaluate the role of macrophages in acute murine cytomegalovirus infection, macrophages in the spleen and the liver were eliminated by an intravenous injection of liposomes containing a cytolytic agent, dichloromethylene diphosphonate. The depletion of macrophages led to a significant increase of virus titer in the spleen and lungs in both susceptible BALB/c and resistant C57BL/6 mice during the first three days after intravenous infection. In the spleen, the increase of virus titer in macrophage‐depleted BALB/c mice was much greater than that in NK cell‐depleted mice. These results suggest that macrophages contribute to protection mainly by the mechanisms which are independent of NK cells during the first three days after infection. The increase of virus titer in macrophage‐depleted C57BL/6 mice was as great as that in NK cell‐depleted mice because of the high contribution of NK cells to protection in C57BL/6 mice. In the liver in both strains of mice, the effects of macrophage depletion on virus titer were not as much as those in the spleen and lungs. Furthermore, the local depletion of peritoneal macrophages resulted in a great increase of virus titer in the spleen at three days after intraperitoneal infection. We conclude that macrophages greatly contribute to decreasing the virus load in some organs possibly through either or both intrinsic and extrinsic mechanisms in the early phase of primary infection with murine cytomegalovirus.

Biological properties of lipopolysaccharides from Bordetella species.

Biological activities of lipopolysaccharides (LPS) extracted from Bordetella pertussis, B. parapertussis and B. bronchiseptica were compared with those of Escherichia coli LPS. The LPS preparations from B. pertussis showed biological activities comparable to those of E. coli LPS in terms of lethal toxicity in galactosamine-sensitized mice, pyrogenicity in rabbits, mitogenicity in C3H/He spleen cell cultures, macrophage activation, and induction of tumour necrosis factor. All the activities of LPS preparations from B. parapertussis, except mitogenicity, were lower than those of E. coli LPS. LPS from B. parapertussis gave the greatest mitogenic action of all those tested. Biological activities stronger than or comparable to those of E. coli LPS were observed for LPS from B. bronchiseptica.

Activation of murine peritoneal macrophages by intraperitoneal administration of a traditional chinese herbal medicine, xiao-chai-hu-tang (Japanese name: Shosaiko-To)

Macrophage activation by a traditional Chinese herbal medicine, xiao-chai-hu-tang (Japanese name: shosaiko-to), was investigated. Intraperitoneal (i.p.) administration of shosaiko-to into (BALB/c x DBA/2)F1 mice resulted in marked activation of macrophages with respect to phagocytic and lysosomal enzyme activities (acid phosphatase and N-acetyl-beta-D-glucosaminidase) compared with the control. The maximal responses were induced by an i.p. injection of 3 mg shosaiko-to 4 days previously. Enhanced activities induced by shosaiko-to were also seen in C3H/HeJ mice, which is a non-responder strain to bacterial lipopolysaccharide (LPS). Significant macrophage accumulation in the peritoneal cavity and increased lysosomal enzyme activities were observed in mice injected with shosaiko-to. Shosaiko-to exhibited significant cytostasis-inducing activity. In addition, the administration of shosaiko-to led to a moderate expression of Ia antigen on the surface of peritoneal macrophages. These results suggest that shosaiko-to is a potent macrophage activator.

Suppressive Effects of the Flavonoids Quercetin and Luteolin on the Accumulation of Lipid Rafts after Signal Transduction via Receptors

Quercetin (QUER) and luteolin (LUTE) are dietary flavonoids capable of regulating the production of cytokines, such as tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). However, their mechanisms of action are not fully understood. In lipopolysaccharide-triggered (LPS)-triggered signaling via Toll-like receptor 4 (TLR4), QUER and LUTE suppresses not only the degradation of the inhibitor of κB (IκB), with resultant activation of nuclear factor-κB (NF-κB), but also the phosphorylation of p38 and Akt in bone marrow-derived macrophages that have been stimulated with LPS. We report here that, in TNF-α-induced signaling, QUER and LUTE significantly suppressed the production of IL-6 and activation of NF-κB. Accumulation of lipid rafts, the initial step in the signaling pathway, was significantly inhibited when macrophages were treated with QUER or with LUTE prior to exposure to LPS. Similarly, the accumulation of lipid rafts was inhibited by the flavonoids when B cells were activated via the membrane IgM and when T cells were activated via CD3. In contrast, QUER and LUTE did not inhibit the activation of phorbol myristate acetate-induced NF-κB in macrophages. Our observations suggest that QUER and LUTE interact with receptors on the cell surface and suppress the accumulation of lipid rafts that occurs downstream of the activation of the receptors.