Rintaro Yamanishi

Occurrence of IgE antibody-recognizing N-linked glycan moiety of a soybean allergen, Gly m Bd 28K.

Background: It has been reported that N-linked glycan moieties of glycoproteins function as IgE-reactive determinants. Gly m Bd 28K, a soybean allergen, was a glycoprotein with glycan moieties, which are supposed to be the Man3GlcNAc2 backbone with the β1→2 xylose and α1→3 fucose branches. The purpose of the present study was to examine the IgE-binding ability of the glycan moiety of Gly m Bd 28K in the binding reaction with patients’ sera. Methods: A peptide containing the glycan moiety was prepared from Gly m Bd 28K by digestion with lysyl endopeptidase. The binding site of the glycan moiety was determined by amino acid sequence analyses. The glycan moiety of the allergen was characterized using anti-horseradish peroxidase antibody (anti-HRP) recognizing the N-linked glycan moieties of glycoproteins. The binding of patients’ IgE antibodies with their glycan moiety was examined by an immunostaining technique using the glycopeptide and its deglycosylated peptide derived from Gly m Bd 28K. Results: The binding site of the glycan moiety in Gly m Bd 28K was shown to be its Asn20 residue. Gly m Bd 28K did react with anti-HRP and the sera of soybean-sensitive patients, but the binding of IgE antibodies was inhibited by the preincubation with anti-HRP. Moreover, the glycopeptide also reacted with the sera of soybean-sensitive patients, but its deglycosylated peptide did not react with any IgE antibodies of patients’ sera. Conclusions: The specific IgE antibodies recognizing the N-linked glycan moieties of Gly m Bd 28K and other glycoproteins with homologous glycan moieties occur in the sera of soybean-sensitive patients. It was indicated that the N-linked glycan moieties such as that of Gly m Bd 28K may be one of the common IgE-reactive determinants distributed in various plant food proteins.

Effect of a conjugated quercetin metabolite, quercetin 3-glucuronide, on lipid hydroperoxide-dependent formation of reactive oxygen species in differentiated PC-12 cells

To assess the efficacy of conjugated quercetin metabolites as attenuators for oxidative stress in the central nervous system, we measured the 13-hydroperoxyoctadecadienoic acid (13-HPODE)-dependent formation of reactive oxygen species (ROS) in pheochromocytoma PC-12 cells in the presence of quercetin 3-O-β-glucuronide (Q3GA) and related compounds. A 2′,7′-dichlorofluorescin (DCFH) assay showed that Q3GA significantly suppressed the formation of ROS, when it was coincubated with 13-HPODE (coincubation system). However, it was less effective than quercetin aglycon in the concentration range from 0.5 to 10 μM. In an experiment in which the cells were incubated with the test compounds for 24 h before being exposed to 13-HPODE, Q3GA was also effective in suppressing the formation of ROS in spite that little Q3GA was taken up into the cells. These results suggest that antioxidative metabolites of quercetin are capable of protecting nerve cells from attack of lipid hydroperoxides.

Effect of Quercetin and Its Conjugated Metabolite on the Hydrogen Peroxide-induced Intracellular Production of Reactive Oxygen Species in Mouse Fibroblasts

To clarify the antioxidative role of quercetin metabolites in cellular oxidative stress, we measured the inhibitory effects of the quercetin aglycon and quercetin 3-O-β-D-glucuronide (Q3GA), which is one of the quercetin metabolites in the blood after an intake of quercetin-rich food, on the production of hydrogen peroxide (H2O2)-induced intracellular reactive oxygen species in mouse fibroblast 3T3 cultured cells. When the cells were exposed to H2O2 in the presence of quercetin or Q3GA, Q3GA was found to be less effective than quercetin. In the case of a pretreatment with quercetin or Q3GA before the exposure, Q3GA, but not the quercetin aglycon, exerted an inhibitory effect, although its cellular uptake was unlikely. The quercetin aglycon appeared to fail in its antioxidative effect due to metabolic conversion into isorhamnetin conjugates, with substantial oxidative degradation resulting from the pretreatment. It is, therefore, suggested that quercetin metabolites take part in the protection of intracellular oxidative stress induced by the extraneous attack of H2O2.

β-Carotene and β-cryptoxanthin but not lutein evoke redox and immune changes in RAW264 murine macrophages

The mechanism of immunological benefits induced by carotenoids has not been fully elucidated. Here, we investigated some of the immunity-related properties of beta-carotene and two other carotenoids, beta-cryptoxanthin, and lutein, on the murine macrophages cell line RAW264. beta-Carotene added to the culture medium accumulated in the cells in a time- and dose-dependent manner. The accumulation was positively correlated with cellular lipid peroxidation, demonstrating the pro-oxidative activity of beta-carotene, and also with the synthesis of glutathione, an intracellular antioxidant. Conversely, accumulation of beta-carotene was negatively correlated with the transcription of immune-active molecules, such as IL-1beta, IL-6, and IL-12 p40, in cells stimulated by LPS and INF-gamma. The transcription of the pro-inflammatory cytokines IL-1beta and IL-6 was more sensitive to the accumulation of beta-carotene than was IL-12 p40. The accumulation of beta-cryptoxanthin in cells resulted in effects similar to those of beta-carotene. However, lutein accumulated minimally and did not significantly affect the cells. These results demonstrate that beta-carotene, and beta-cryptoxanthin as well, can accumulate in RAW264 cells and induce changes in intracellular redox status, which in turn regulate the immune function of macrophages.

Inhibition of Immunoglobulin E Production in Allergic Model Mice by Supplementation with Vitamin E and β-Carotene

A diet containing different amounts of vitamin E (α-tocopherol; 0.5 mg, 5 mg, 10 mg or 50 mg per 100 g diet) was supplemented to BALB/c mice for 6 weeks. These mice were subcutaneously immunized twice with ovalbumin (OVA). A passive cutaneous anaphylaxis (PCA) analysis demonstrated that the mice fed on the diet containing 5 mg of vitamin E produced the highest level of the OVA-specific immunoglobulin E (IgE) antibody. A lower level of serum IgE was found in the mice supplemented with 0.5 mg, 10 mg and 50 mg of vitamin E. A sandwich ELISA analysis showed that the pattern of the total IgE antibody level among these four groups was the same as that of the allergen-specific IgE. In a separate experiment, 5 mg of vitamin E and/or 50 mg of β-carotene was supplemented to the basal diet containing vitamin E as α-tocopherol acetate (5 mg) in order to evaluate the effect of their combination on OVA-specific and total IgE production in the mice. The supplementation with β-carotene alone had no effect on OVA-specific or total IgE production. In contrast, supplementation with vitamin E plus β-carotene effectively suppressed both the antigen-specific and total IgE antibodies. The serum vitamin E and β-carotene levels were increased by supplementation with the respective compounds. These results strongly suggest that the combination of dietary vitamin E and β-carotene suppressed IgE production and would therefore help to prevent the type-I allergic reaction.

β-Carotene Modulates the Immunological Function of RAW264, a Murine Macrophage Cell Line, by Enhancing the Level of Intracellular Glutathione

The activities of β-carotene on redox status and the immune functions of RAW264 cells, a murine macrophage cell line, were investigated. Supplementation with β-carotene for RAW264 cells resulted in apparently inconsistent redox indices: lipid peroxidation was enhanced but intracellular oxidation was moderately attenuated. Attenuated intracellular oxidation was endorsed by an increase in glutathione accompanied by up-regulated transcription of a subunit of γ-glutamylcysteine synthetase, the rate-limiting enzyme for glutathione synthesis. α-Tocopherol, which can quench lipid peroxidation by free radical, neither inhibited that by β-carotene nor influenced the intracellular redox status. Lipopolysaccharide-stimulated transcriptions of IL-1β and IL-12 p40 in RAW264 were inhibited by β-carotene but not by α-tocopherol. These results indicate that β-carotene, which can modulate the intracellular redox status of macrophages by enhancing the level of intracellular glutathione, is related to the immune functions of macrophages.

Adjuvant activity of alum in inducing antigen specific IgE antibodies in BALB/c mice: a reevaluation.

The IgE production was compared in the presence and absence of aluminum hydroxide gel (alum). Without alum, the IgE production was induced within a suitable range of the antigen dosage; however, alum enhanced it. Alum did not affect the minimum requirement for the antigen dosage, indicating that alum may not take part in the efficiency of antigen presentation.

Feeding with Both β-Carotene and Supplemental α-Tocopherol Enhances Type 1 Helper T Cell Activity among Splenocytes Isolated from DO11.10 Mice

β-Carotene and/or supplemental α-tocopherol were fed to DO11.10 mice to investigate their effect on the immune function of naive splenocytes. A high secretion of interleukin-12 and interferon-γ in response to the ex vivo primary antigen presentation occurred only when both were fed. This is consistent with the suppressed immunoglobulin E production under the similar condition described in our previous report.

Ingested β-Carotene Enhances Glutathione Level and up-Regulates the Activity of Cysteine Cathepsin in Murine Splenocytes

To elucidate health benefits of β-carotene, especially on immunity, we measured redox-related indices in spleen cells from BALB/c mice supplemented with various amounts of β-carotene. In mice supplemented with β-carotene in their diet, glutathione, an intracellular anti-oxidation agent, increased in their splenocytes. This change was highly correlated with the accumulation of β-carotene, but not with that of retinol. The increase in glutathione was accompanied by an increase in mRNA for γ-glutamylcysteine synthetase, a rate-limiting enzyme for glutathione synthesis. The higher the glutathione content was in the spleen cells, the higher the activity of cysteine cathepsin became in crude antigen-presenting cells contained in the spleen. These data suggest that accumulated β-carotene in splenocytes, without being metabolized, caused an increase in the intracellular glutathione level, thereby anti-oxidatively supporting the activity of redox-sensitive lysosomal protease, which is involved in antigen-presentation.

Ingested quercetin but not rutin increases accumulation of hepatic β‐carotene in BALB/c mice

Beta-carotene is a carotenoid with a range of reported health benefits besides vitamin A activity. If the enzymatic conversion of beta-carotene to retinal is suppressed in the digestive tract, residual beta-carotene that reaches the tissues increases. We evaluated the function of quercetin and rutin (quercetin-3-rutinoside) to increase the accumulation of beta-carotene in vitro and in vivo in BALB/c mice. When the conversion of beta-carotene by a preparation of the murine small intestine was measured in vitro, the addition of quercetin or rutin considerably inhibited the conversion. When the levels of hepatic beta-carotene and retinoids were measured among three groups of mice fed a diet supplemented with beta-carotene plus quercetin or rutin or beta-carotene alone (four to six mice per group), quercetin increased the level of beta-carotene and decreased the level of retinol, whereas rutin did not. These results demonstrate that quercetin can suppress the conversion of beta-carotene which develops in the cytosol of small intestinal epithelial cells, and that rutin whose rutinose-moiety prevents being absorbed in the small intestine cannot suppress the conversion in vivo. This study offers a novel insight into the interaction between flavonoids and carotenoids with respect to the health benefits from the latter.