Masaya Takamoto

Exposure to Bisphenol A prenatally or in adulthood promotes T(H)2 cytokine production associated with reduction of CD4CD25 regulatory T cells.

Background Bisphenol A (BPA) is a widespread endocrine-disrupting chemical that can affect humans and animals. Objectives We investigated the effects of adult or prenatal exposure to BPA on T-helper (TH)1/TH2 immune responses and the mechanisms underlying these effects. Methods To evaluate the effects of exposure to BPA in adulthood, male Leishmania major–susceptible BALB/c and –resistant C57BL/6 mice were subcutaneously injected with 0.625, 1.25, 2.5, and 5 μmol BPA 1 week before being infected with L. major. To evaluate prenatal exposure, female mice were given BPA-containing drinking water at concentrations of 1, 10, and 100 nM for 2 weeks, then mated, and given BPA for another week. Male 10-week-old offspring were infected with L. major. Footpad swelling was assessed as a measure of the course of infection. Results Mice exposed to BPA prenatally or in adulthood showed a dose-dependent increase in footpad swelling after being infected with L. major. Exposure to BPA in adulthood significantly promoted antigen-stimulated production of interleukin (IL)-4, IL-10, and IL-13 but not interferon-γ (IFN-γ). However, mice prenatally exposed to BPA showed increased production of not only IL-4 but also IFN-γ. The percentages of CD4+CD25+ cells were decreased in mice exposed to BPA either prenatally or in adulthood. Effects of prenatal BPA exposure were far more pronounced than effects of exposure in adulthood. Conclusion BPA promotes the development of TH2 cells in adulthood and both TH1 and TH2 cells in prenatal stages by reducing the number of regulatory T cells.

A serine/threonine kinase, Cot/Tpl2, modulates bacterial DNA–induced IL-12 production and Th cell differentiation

A serine/threonine protein kinase, Cot/Tpl2, is indispensable for extracellular signal-regulated kinase (ERK) activation and production of TNF-alpha and PGE2 in LPS-stimulated macrophages. We show here that Cot/Tpl2 is also activated by other Toll-like receptor (TLR) ligands. Bacterial DNA rich in the dinucleotide CG (CpG-DNA), unlike LPS or synthetic lipopeptide, activated ERK in a Cot/Tpl2-independent manner. Peritoneal macrophages and bone marrow-derived DCs from Cot/Tpl2-/- mice produced significantly more IL-12 in response to CpG-DNA than those from WT mice. Enhanced IL-12 production in Cot/Tpl2-/- macrophages is, at least partly, regulated at the transcriptional level, and the elevated IL-12 mRNA level in Cot/Tpl2-/- macrophages is accompanied by decreased amounts of IL-12 repressors, such as c-musculoaponeurotic fibrosarcoma (c-Maf) and GATA sequence in the IL-12 promoter-binding protein (GA-12-binding protein; GAP-12) in the nucleus. Consistently, Cot/Tpl2-/- mice showed Th1-skewed antigen-specific immune responses upon OVA immunization and Leishmania major infection in vivo. These results indicate that Cot/Tpl2 is an important negative regulator of Th1-type adaptive immunity, that it achieves this regulation by inhibiting IL-12 production from accessory cells, and that it might be a potential target molecule in CpG-DNA-guided vaccination.

Marked eosinophilia in interleukin-5 transgenic mice fails to prevent Trichinella spiralis infection.

In order to study the role of eosinophils in the host defense against Trichinella spiralis infection, worm recovery after infection with T. spiralis was compared between interleukin-5 transgenic (IL-5 Tg) mice with a constant high level of peripheral eosinophils and nontransgenic C3H/HeN mice. No significant difference in the recovery of muscle larvae or adult worms in the small intestine, fecundity of female adult worms, or infectivity of newborn larvae was observed between nonimmunized C3H/HeN and IL-5 Tg mice or C3H/HeN and IL-5 Tg mice immunized with somatic antigen of T. spiralis. However, a significant difference was observed in the fecundity of female adult worms and recovery of muscle larvae between nonimmunized and immunized IL-5 Tg mice or C3H/HeN mice. These results demonstrate that having more eosinophils does not improve immunity against the various aspects of T. spiralis infection.

Bisphenol A Promotes IL-4 Production by Th2 Cells

Background: It has been proposed that estrogen plays an important role in modulating the Th1/Th2 cytokine balance. From this viewpoint, chemicals with estrogenic responses were expected to possess similar immunoregulatory roles which have not been defined to date. To address this, we studied the effects of one of the estrogenic chemicals, bisphenol A (BPA), on the in vitro production of Th1 and Th2 cytokines. Methods: Mesenteric lymph node cells from Trichinella spiralis (Ts)-infected mice were incubated with serialfold dilutions of BPA under stimulation with Ts antigen. The Th2 cytokine production in the supernatant was determined by ELISA. The Th2 cytokine production by mesenteric lymph node cells from Ts-infected mice inoculated orally with BPA was compared with that of uninoculated mice infected with Ts. Results: The antigen-stimulated interleukin (IL)-4 production by Th2-dominant mesenteric lymph node cells from Ts-infected mice increased significantly by addition of 3 µM of BPA. The IL-5 production was not affected. The production of IL-4, but not that of IL-5, by splenocytes of Th2-skewed Leishmania major-infected BALB/c mice increased at concentrations of 3 and 10 µM of BPA. However, the interferon gamma production was not affected by BPA in Th1-skewed L. major-infected C57BL/6 mice. The production of IL-4 and IL-10, but not that of IL-13, markedly increased in Ts-infected mice inoculated orally with BPA. Conclusions: We demonstrated that the IL-4 production was increased both in vitro and in vivo by treatment with BPA. This suggests that BPA might cause allergic diseases by stimulating the IL-4 production by Th2 cells.

Eosinophilia, IgE production, and cytokine production by lung T cells in surface CD4-deficient mutant mice infected with Toxocara canis.

Mutant mice deficient in CD4+ T cells and their normal and heterozygous littermates were infected with Toxocara canis, and compared for eosinophilia, total and Toxocara‐specific immunoglobulin E (IgE) production, and in vitro cytokine production by lung cells. The numbers of eosinophils in the peripheral blood of normal and heterozygous mice peaked on days 10 and 21, although mutant mice showed eosinophilia with a peak on day 10. This indicates that the first peak on day 10 is CD4 independent and the second peak is CD4 dependent. Before infection, the levels of total IgE had no significant difference among the three groups of mice. Total and Toxocara‐specific IgE in all genotypes of mice increased after infection, and was the highest in normal mice and the lowest in mutant mice. In vitro production of interleukin (IL)‐5 and IL‐4 by total lung cells was the highest in normal mice and the lowest in mutant mice. CD4+ and CD4− CD8− T lymphocytes, but not CD8+ T lymphocytes produced IL‐5 and IL‐4 when incubated with anti‐CD3 monoclonal antibody (mAb) and lung‐adherent cells. These results indicated that IL‐5 and IL‐4 were produced mainly by CD4+ cells and partly by CD4− CD8− cells, but not by CD8+ cells. In addition, cytokine production by CD4+ cells was affected by the number of CD4 molecules on their surface.

Synergism of IL-3, IL-5, and GM-CSF on eosinophil differentiation and its application for an assay of murine IL-5 as an eosinophil differentiation factor

In order to elucidate the mechanisms of eosinophil differentiation, we examined the effects of combinations of interleukin 5 (IL-5) with IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) on in vitro differentiation into eosinophils from bone marrow cells of ICR mice. When the amount of added IL-5 was kept constant, IL-3 exhibited dose-dependent production of eosinophils both in the absence and presence of low doses of GM-CSF. In contrast, IL-5 plus GM-CSF showed highly enhanced production of eosinophils, and eosinophil production maximized at a concentration between 10 and 20 U/ml and decreased at higher concentrations. When IL-3 and GM-CSF were kept constant at concentrations of 20 U/ml and 10 U/ml, respectively, the number of eosinophils increased linearly in IL-5-dependent manner in a range from 0.3 to 30 U/ml. These results suggest that IL-3 and GM-CSF act synergistically with IL-5 in in vitro eosinophil differentiation. In addition, we propose a new method for quantifying eosinophil differentiation activity of IL-5.

Mechanisms of eosinophilia in Toxocara canis infected mice: In vitro production of interleukin 5 by lung cells of both normal and congenitally athymic nude mice

Mechanisms of eosinophilia were compared between in vitro bone marrow cell cultures of congenitally athymic (nu/nu) mice and their heterozygous littermates (nu/+). Cultures of 5 × 104 bone marrow cells using interleukin 3 (IL‐3), IL‐5 and granulocyte‐macrophage colony‐stimulating factor showed that nu/nu and nu/+ mice mimicked each other in eosinophil production both before and after infection with Toxocara canis. Eosinophil differentiating activity (EDA) was detected in media conditioned by spleen cells and lungs of T. canis infected nu/+ mice, although nu/nu mice showed EDA only in lung‐conditioned medium. EDA, detected both in infected nu/nu and nu/+ mice, was inhibited by an anti‐IL‐5 monoclonal antibody. These results indicate that IL‐5 may be produced by lung cells of both nu/nu and nu/+ mice as well as by spleen cells of nu/+ mice infected with T. canis, which is the reason why nu/nu mice infected with T. canis exhibit blood eosinophilia.

STAT6 signalling is important in CD8+ T‐cell activation and defence against Toxoplasma gondii infection in the brain

Signal transducer and activator of transcription (STAT) 6 is a molecule involved in interleukin (IL)‐4 and ‐13 signalling. We investigated the role of STAT6 signalling in Toxoplasma gondii‐infected mice using STAT6‐deficient (STAT6−/−) and wild‐type (WT) mice. A significantly larger number of cysts were recovered from the brain in STAT6−/− than in WT mice on days 28 and 56 post‐infection. CD8+ T cells in cerebrospinal fluid and spleen stimulated with T. gondii antigen produced higher levels of interferon (IFN)‐γ in WT than in STAT6−/− mice. CD8+ T‐cell function, estimated by expression of CD25 and cytotoxic activity, was lower in STAT6−/− than in WT mice. Transfer of CD8+ but not CD4+ T cells, purified from infected WT mice, into STAT6−/− mice successfully prevented formation of cysts in the brain. However, transfer of naïve CD8+ T cells from WT into STAT6−/− mice did not show either activation of CD8+ T cells or a decrease in the number of cysts in the brain. Transfer of splenic adherent cells from WT into STAT6−/− mice induced activation of CD8+ T cells and decreased the number of cysts in the brain. Expression of CD86 on splenic dendritic cells and IL‐12 p40 production were weaker in STAT6−/− than in WT mice after T. gondii infection. These results indicate that STAT6 signalling is important in CD8+ T‐cell activation, possibly through regulation of antigen‐presenting cells, which could suppress T. gondii infection in the brain.

IFN-γ deficiency worsen Pneumocystis pneumonia with Th17 development in nude mice

Pneumocystis pneumonia (PCP) occurs frequently in patients with immunodeficiency syndromes, especially AIDS. In order to investigate the role of IFN-gamma on PCP, nude mice deficient in IFN-gamma (GKO nude) and their wild-type ones (WT nude) were infected with murine Pneumocystis. Nine weeks later they were sacrificed, and cytokines in BALF and lung histopathology were compared between them. Cyst burden was greater in GKO than in WT nude mice. Histopathology in the lung was severer and granulomatous lesions were observed more frequently in GKO nude mice. Levels of IL-17 were higher in BALF of GKO than in that of WT nude mice. Greater number of CD4(+) T cells from lungs of infected GKO nude mice produced IL-17 than those from WT ones. These results suggest that deficiency in IFN-gamma induces the differentiation of Th17 and that IL-17 is responsible for inflammatory response in PCP.

Inhibition of interleukin 5 production with no influence on interleukin 4 production by an anti-allergic drug, tranilast, in Toxocara canis-infected mice.

Tranilast is well-known as a useful drug for allergic diseases. This drug is believed to exhibit its therapeutic effects by inhibiting the release of chemical mediators from mast cells and basophils. Effects of tranilast on T helper type 2 (Th2) cytokine production were investigated in mice infected with Toxocara canis (Tc). Tranilast reduced interleukin (IL)-5 production in a dose-dependent manner but not IL-4 production at all in lung and spleen cells from Tc-infected mice cultured under stimulation with excretory-secretory antigen. Obvious IL-5 mRNA expression was observed at week 1 in the lung alone, and IL-4 mRNA expression was detected at similar levels at weeks 1-6 of infection in both lung and spleen. IL-5 but not IL-4 mRNA expression in the lung was significantly inhibited by daily administration of 100 mg/kg of tranilast for 1 week. This treatment also reduced the serum IL-5 level. Thus, tranilast inhibited IL-5 but not IL-4 production either in vitro or in vivo. The results imply that IL-5 and IL-4 production by Th2 cells may be controlled through different mechanisms.