Kouya Yamaki

Prenatal exposure to bisphenol A up-regulates immune responses, including T helper 1 and T helper 2 responses, in mice

The effect of prenatal exposure to bisphenol A (BPA) on the immune system in mice was investigated. Virgin female mice were fed varying doses of BPA, on a daily basis, over a period of 18 days commencing on the day of pairing with stud males (day 0). On day 77, their male offspring of 8 weeks were immunized with hen egg lysozyme (HEL). Three weeks later, anti‐HEL immunoglobulin G (IgG) in sera, and proliferative responses of spleen cells to the antigen, were measured. Anti‐HEL IgG2a and interferon‐γ (IFN‐γ), secreted from splenic lymphocytes, were measured as indicators of T helper 1 (Th1) immune responses, while anti‐HEL IgG1 and interleukin‐4 (IL‐4) were measured as indicators of Th2 responses. The results showed that fetal exposure to BPA was followed by significant increases in anti‐HEL IgG as well as antigen‐specific cell proliferation. Both Th1 responses (including anti‐HEL IgG2a and IFN‐γ production) and Th2 responses (including anti‐HEL IgG1 and IL‐4 production) were augmented by prenatal exposure to BPA, although the augmentation of Th1 responses appeared to be greater than that of Th2 responses. Two‐colour flow cytometric analysis showed that mice exposed prenatally to BPA had 29% and 100% more splenic CD3+ CD4+ and CD3+ CD8+ cells, respectively, than control animals. Similar results were obtained from females whose mothers had consumed BPA during pregnancy. These results suggest that prenatal exposure to BPA may result in the up‐regulation of immune responses, especially Th1 responses, in adulthood.

Components of diesel exhaust particles differentially affect Th1/Th2 response in a murine model of allergic airway inflammation

Background Diesel exhaust particles (DEP) can enhance various respiratory diseases. However, it is unclear as to which components in DEP are associated with the enhancement. We investigated the effects of DEP components on antigen‐related airway inflammation, using residual carbonaceous nuclei of DEP after extraction (washed DEP), extracted organic chemicals (OC) in DEP (DEP–OC), and DEP–OC plus washed DEP (whole DEP) in the presence or absence of ovalbumin (OVA).

Enhancement of acute lung injury related to bacterial endotoxin by components of diesel exhaust particles

Background: Diesel exhaust particles (DEP) synergistically aggravate acute lung injury related to lipopolysaccharide (LPS) in mice, but the components in DEP responsible for this have not been identified. A study was undertaken to examine the effects of the organic chemicals (DEP-OC) and residual carbonaceous nuclei (washed DEP) derived from DEP on LPS related lung injury. Methods: ICR mice were divided into experimental groups and vehicle, LPS, washed DEP, DEP-OC, washed DEP+LPS, and DEP-OC+LPS were administered intratracheally. The cellular profile of the bronchoalveolar lavage (BAL) fluid, pulmonary oedema, lung histology, and expression of proinflammatory molecules and Toll-like receptors in the lung were evaluated. Results: Both DEP-OC and washed DEP enhanced the infiltration of neutrophils into BAL fluid in the presence of LPS. Washed DEP combined with LPS synergistically exacerbated pulmonary oedema and induced alveolar haemorrhage, which was concomitant with the enhanced lung expression of interleukin-1β, macrophage inflammatory protein-1α, macrophage chemoattractant protein-1, and keratinocyte chemoattractant, whereas DEP-OC combined with LPS did not. Gene expression of Toll-like receptors 2 and 4 was increased by combined treatment with washed DEP and LPS. The enhancement effects of washed DEP on LPS related changes were comparable to those of whole DEP. Conclusions: These results suggest that the residual carbonaceous nuclei of DEP rather than the extracted organic chemicals predominantly contribute to the aggravation of LPS related lung injury. This may be mediated through the expression of proinflammatory cytokines, chemokines, and Toll-like receptors.

Effects of bisphenol A on antigen-specific antibody production, proliferative responses of lymphoid cells, and TH1 and TH2 immune responses in mice

We investigated the effect of bisphenol A (BPA), which binds estrogen receptors, on immune responses including production of antigen‐specific antibodies, proliferative responses of lymphoid cells, and Th1 and Th2 responses. For this investigation, mice were p.o. given varying doses including 3, 30, 300, and 3000 μg kg−1 of BPA immediately after immunization with hen egg lysozyme (HEL) (day 0) and then daily by day 20. On day 21, anti‐HEL IgG antibodies in sera and proliferative responses of spleen cells to the antigen were measured. Anti‐HEL IgG2a antibodies and IFN‐γ secreted from splenic lymphocytes were also measured as indicators of Th1 immune responses, while anti‐HEL IgG1 antibodies and IL‐4, as those of Th2 responses. The results showed that treatment with 3000 μg kg−1 of BPA was followed by a significant increase in anti‐HEL IgG as well as the antigen‐specific cell proliferation. Anti‐HEL IgG2a production and IFN‐γ secretion were significantly enhanced in mice treated with 300 and 30 μg kg−1 of BPA, respectively, while anti‐HEL IgG1 production and IL‐4 secretion were augmented in animals given 3000 and 300 μg kg−1 of the chemical, respectively. Augmentation of these immune responses was also observed in mice exposed to 0.3–30 μg kg−1 of estradiol, although Th1 responses appeared to be more sensitive to the sex hormone than Th2 responses. These results suggest that BPA may play a role in augmenting immune responses, especially Th1 responses.

Collagen antibody-induced arthritis in mice: development of a new arthritogenic 5-clone cocktail of monoclonal anti-type II collagen antibodies.

A cocktail of 4 monoclonal anti-type II collagen antibodies recognizing conserved epitopes located within the CB11 fragment (CII 124-402) of type II collagen is currently used as an arthritogenic antibody preparation for inducing collagen antibody induced arthritis (CAIA). In order to increase the arthritogenicity of this cocktail, we have developed 7 new monoclonal antibodies to anti-type II collagen from spleen cells of DBA/1J mice immunized with bovine type II collagen, and tested for their additional effect on the arthritogenicity over that of the current 4-clone cocktail. Three of the clones (CII-3, -5 and -6) bind to the LyC1 (CII 124-290) peptide of CB11 and 1 (CII-7) of the clones binds to CB9.7 (CII 898-1020), and highly cross-reacted with other species of type II collagen. This indicates that these clones recognize conserved epitopes within type II collagen, including mouse type II collagen. On the other hand, 2 other clones (CII-1 and -4) directed against CB9.7 and 1 clone (CII-2) against CB8 (CII 403-551) were less reactive with other species of type II collagen. The arthritogenicity of the current 4-clone cocktail was significantly increased by addition of a fifth clone, CII-3. No effects were observed with other clones. The arthritogenicity of this new 5-clone cocktail was 2-fold greater than the current 4-clone cocktail in all strains of mice tested: the CIA-responder strain DBA/1J, the CIA-resistant BALB/c (H-2(d)), the T-cell deficient C.B-17/l scid/scid and the CAIA-low responder C57BL/6 (H-2(b)) strain. These results clearly indicated the importance of epitope specificity of arthritogenicity of autoantibodies to type II collagen. Due to its enhanced arthritogenicity, this 5-clone cocktail is capable of inducing a more consistent and severe arthritis with lower doses compared to the current 4-clone cocktail, and will provide an effective new reagent for inducing arthritis in various strains of CAIA low responder mice.

Inhibition of the antigen-induced activation of RBL-2H3 cells by sinomenine

The present study was designed to investigate the effect of sinomenine (SIN), an alkaloid extracted from sinomenium acutum, on the antigen-induced activation of RBL-2H3. For this investigation, the RBL-2H3 cells were sensitized with dinitrophenyl (DNP)-specific IgE overnight in 1.0 ml of Eagles MEM (EMEM), and varying doses of SIN were added to the culture medium for 30 min and challenged with dinitrophenyl-human serum albumin (DNP-HSA) to induce mast cell degranulation before supernatants were collected. The effects of SIN on antigen-induced release of beta-hexosaminidase were measured by enzymatic assay, calcium influx by FACS, cytokines by ELISA, and signaling events by immunoblotting. The results showed that treatment with SIN was followed by a decrease in FcepsilonRI-mediated mast cell release of beta-hexosaminidase, production of IL-4 and TNF-alpha, phosphorylation of Gab2 (Scaffolding adapter Grb2-associated binder 2), Akt and p38 mitogen-activated protein kinase (MAPK). In addition, SIN had no effect on the phosphorylation of LAT and no significant difference on calcium mobilization was observed between control and SIN treated group. These results suggested that SIN might suppress the antigen-induced activation of RBL-2H3 cells via a Ca2+ independent pathway.

Effect of sinomenine on collagen-induced arthritis in mice

The present study was designed to investigate the effect of sinomenine (SIN), an alkaloid extracted from sinomenium acutum on collagen-induced arthritis (CIA) in mice. For this investigation, mice were s.c. immunized with type II collagen (CII) emulsified with complete Freunds adjuvant (day 0). Varying doses of SIN were orally administered daily commencing on day 0 daily over a period of 55 days. The severity of arthritis was evaluated according to clinical score, the effect of SIN on immune responses were determined by measurement of proliferative responses of spleen cells, antibody levels in serum and cytokine assays. Anti-CII IgG2a and IFN-γ were measured as indicators of Th1 immune responses and anti-CII IgG1, IgE and IL-5 as those of Th2 responses. IL-10 and TGF-β were measured as indicators of T cell regulator responses. The results showed that treatment with SIN was followed by decreases in the incidence and severity of CIA, anti-CII IgG and the antigen-specific splenocyte proliferation. Production of all isotypes of antibodies including anti-CII IgG2a, IgG1 and IgE as well as secretion of cytokines such as IFN-γ and IL-5 were suppressed by SIN. In addition, SIN enhanced the secretion of TGF-β while it had no obvious effect on production of IL-10. These results suggest that the anti-arthritic effect of SIN may be related to the suppression of both Th1 and Th2 immune responses. TGF-β may at least in part contribute to the suppression of Th1 as well as Th2 immune responses.

Preventive and therapeutic effects of rapamycin, a mammalian target of rapamycin inhibitor, on food allergy in mice

Because few curative treatments are available for food allergy, we investigated the therapeutic potential of rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, on mouse food allergy.

Comparison of inhibitory activities of zinc oxide ultrafine and fine particulates on IgE-induced mast cell activation

The effects of ultrafine and fine particles of zinc oxide (ZnO) on IgE-dependent mast cell activation were investigated. The rat mast cell line RBL2H3 sensitized with monoclonal anti-ovalbumin (OVA) IgE was challenged with OVA in the presence or absence of ZnO particles and zinc sulfate (ZnSO4). Degranulation of RBL2H3 was examined by the release of β-hexosaminidase. To understand the mechanisms responsible for regulating mast cell functions, the effects of ZnO particles on the levels of intracellular Zn2+, Ca2+, phosphorylated-Akt, and global tyrosine phosphorylation were also measured. IgE-induced release of β-hexosaminidase was obviously attenuated by ultrafine ZnO particles and ZnSO4, whereas it was very weakly inhibited by fine ZnO particles. The intracellular Zn2+ concentration was higher in the cells incubated with ultrafine ZnO particles than in those with fine ZnO particles. Consistent with inhibitory effect on release of β-hexosaminidase, ultrafine ZnO particles and ZnSO4, but not fine ZnO particle, strongly attenuated the IgE-mediated increase of phosphorylated-Akt and tyrosine phosphorylations of 100 and 70 kDa proteins in RBL2H3 cells. These findings indicate that ultrafine ZnO particles, with a small diameter and a large total surface area/mass, could release Zn2+ easily and increase intracellular Zn2+ concentration efficiently, thus decreasing FcεRI-mediated mast cell degranulation through inhibitions of PI3K and protein tyrosine kinase activation. Exposure to ZnO particles might affect immune responses, especially in allergic diseases.

Effects of nanoparticles on lung physiology in the presence or absence of antigen.

Ambient particulate matter (PM) exacerbates allergic airway diseases. Our previous study showed that diesel exhaust particles, the main constituents in urban PM, enhance airway hyperresponsivness in mice. In addition, health effects of PM with a diameter of less than 100 nm, called nanoparticles, have been reported, and we have also demonstrated that carbon nanoparticles exacerbate antigen-related airway inflammation. The present study investigates the effects of pulmonary exposure to two sizes of carbon nanoparticles on lung physiology and lung expression of Muc5ac in the presence or absence of antigen in mice. Nanoparticles alone or ovalbumin (OVA) alone moderately enhanced cholinergic airway reactivity, as assessed by total respiratory system resistance (R) and Newtonian resistance (Rn). In the nanoparticle + OVA groups, all the parameters for lung responsiveness, such as R, compliance, elastance, Rn, tissue damping, and tissue elastance, were worse than those in the vehicle group, the corresponding nanoparticle groups or the OVA group. The lung mRNA level for Muc5ac was significantly higher in the OVA group than in the vehicle group, and further increased in the nanoparticle + OVA groups than in the OVA or the nanoparticle groups. These data suggest that carbon nanoparticles can enhance lung hyperresponsiveness, especially in the presence of antigen. The effects may be mediated, at least partly, through the enhanced lung expression of Muc5ac.