Yukiyo Hirata

Forest bathing enhances human natural killer activity and expression of anti-cancer proteins.

In order to explore the effect of forest bathing on human immune function, we investigated natural killer (NK) activity; the number of NK cells, and perforin, granzymes and granulysin-expression in peripheral blood lymphocytes (PBL) during a visit to forest fields. Twelve healthy male subjects, age 37–55 years, were selected with informed consent from three large companies in Tokyo, Japan. The subjects experienced a three-day/two-night trip in three different forest fields. On the first day, subjects walked for two hours in the afternoon in a forest field; and on the second day, they walked for two hours in the morning and afternoon, respectively, in two different forest fields. Blood was sampled on the second and third days, and NK activity; proportions of NK, T cells, granulysin, perforin, and granzymes A/B-expressing cells in PBL were measured. Similar measurements were made before the trip on a normal working day as the control. Almost all of the subjects (11/12) showed higher NK activity after the trip (about 50% increased) compared with before. There are significant differences both before and after the trip and between days 1 and 2 in NK activity. The forest bathing trip also significantly increased the numbers of NK, perforin, granulysin, and granzymes A/B-expressing cells. Taken together, these findings indicate that a forest bathing trip can increase NK activity, and that this effect at least partially mediated by increasing the number of NK cells and by the induction of intracellular anti-cancer proteins.

EM703 improves bleomycin-induced pulmonary fibrosis in mice by the inhibition of TGF-β signaling in lung fibroblasts

BackgroundFourteen-membered ring macrolides have been effective in reducing chronic airway inflammation and also preventing lung injury and fibrosis in bleomycin-challenged mice via anti-inflammatory effects. EM703 is a new derivative of erythromycin (EM) without the bactericidal effects. We investigated the anti-inflammatory and antifibrotic effects of EM703 in an experimental model of bleomycin-induced lung injury and subsequent fibrosis in mice.MethodsSeven-week-old male ICR mice were used. All experiments used eight mice/group, unless otherwise noted in the figure legends. Bleomycin was administered intravenously to the mice on day 0. EM703 was orally administered daily to mice. All groups were examined for cell populations in the bronchoalveolar lavage (BAL) fluid and for induction of messenger RNA (mRNA) of Smad3 and Smad4 in the lung tissues by reverse transcriptase (RT)-polymerase chainreaction (PCR) on day 7. Fibroblastic foci were assessed histologically, and the hydroxyproline content was chemically determined in the lung tissues on day 28. We performed assay of proliferation and soluble collagen production, and examined the induction of mRNA of Smad3 and Smad4 by RT-PCR in murine lung fibroblast cell line MLg2908. We also examined Smad3, Smad4 and phosphorylated Smad2/3 (p-Smad2/3) protein assay by western blotting in MLg2908.ResultsBleomycin-induced lung fibrosis, and the infiltration of macrophages and neutrophils into the airspace were inhibited by EM703. The expression of Smad3 and Smad4 mRNA was clearly attenuated by bleomycin, but was recovered by EM703. EM703 also inhibited fibroblast proliferation and the collagen production in lung fibroblasts induced by Transforming growth factor-beta (TGF-β). The expression of Smad3 and Smad4 mRNA in murine lung fibroblasts disappeared due to TGF-β, but was recovered by EM703. EM703 inhibited the expression of p-Smad2/3 and Smad4 protein in murine lung fibroblasts induced by TGF-β.ConclusionThese findings suggest that EM703 improves bleomycin-induced pulmonary fibrosis in mice by actions of anti-inflammation and regulation of TGF-β signaling in lung fibroblasts.

Effect of phytoncide from trees on human natural killer cell function.

We previously reported that the forest environment enhanced human natural killer (NK) cell activity, the number of NK cells, and intracellular anti-cancer proteins in lymphocytes, and that the increased NK activity lasted for more than 7 days after trips to forests both in male and female subjects. To explore the factors in the forest environment that activated human NK cells, in the present study we investigate the effect of essential oils from trees on human immune function in twelve healthy male subjects, age 37–60 years, who stayed at an urban hotel for 3 nights from 7.00p.m. to 8.00a.m. Aromatic volatile substances (phytoncides) were produced by vaporizing Chamaecyparis obtusa (hinoki cypress) stem oil with a humidifier in the hotel room during the night stay. Blood samples were taken on the last day and urine samples were analysed every day during the stay. NK activity, the percentages of NK and T cells, and granulysin, perforin, granzyme A/B-expressing lymphocytes in blood, and the concentrations of adrenaline and noradrenaline in urine were measured. Similar control measurements were made before the stay on a normal working day. The concentrations of phytoncides in the hotel room air were measured. Phytoncide exposure significantly increased NK activity and the percentages of NK, perforin, granulysin, and granzyme A/B-expressing cells, and significantly decreased the percentage of T cells, and the concentrations of adrenaline and noradrenaline in urine. Phytoncides, such as α-pinene and β-pinene, were detected in the hotel room air. These findings indicate that phytoncide exposure and decreased stress hormone levels may partially contribute to increased NK activity.

The by-products generated during sarin synthesis in the Tokyo sarin disaster induced inhibition of natural killer and cytotoxic T lymphocyte activity

More than 5000 passengers on Tokyo subway trains were injured by the nerve gas, sarin and its by-products. Analysis of phosphor-carrying metabolites of sarin and its by-products in urine samples from the victims suggested that they were exposed not only to sarin, but also by-products generated during sarin synthesis, i.e. diisopropyl methylphosphonate (DIMP) and diethyl methylphosphonate (DEMP). We suspected genetic after-effects due to sarin by-products, thus, we checked the frequency of sister chromatid exchange (SCE) and found that SCE was significantly higher in the victims than in a control group, and that DIMP and DEMP significantly induced human lymphocyte SCE in vitro. In the present study, to explore whether DIMP and DEMP, which induced a high frequency of SCE of lymphocytes, also affected the lymphocyte functions, we examined the effect of DIMP and DEMP on splenic natural killer (NK) and splenic cytotoxic T lymphocyte (CTL) activity in mice, and NK activity of human lymphocytes in vitro. We found that DIMP and DEMP significantly inhibited NK and CTL activity in a dose-dependent manner. The inhibition induced by DIMP was stronger than that by DEMP. The effect of DIMP and DEMP on the splenic NK activity of mice was stronger than on the splenic CTL activity, and the human lymphocytes is more sensitive to DIMP and DEMP than the splenocytes of mice.

EM, EM703 inhibit NF-kB activation induced by oxidative stress from diesel exhaust particle in human bronchial epithelial cells: Importance in IL-8 transcription

Diesel exhaust particle (DEP) is the major components of PM2.5, and much attention has focused on PM2.5 in relation to adverse health effects, and many pulmonary diseases. In the present study, we used a human bronchial epithelial cell (HBEC) line to investigate the anti-inflammatory effects of erythromycin (EM) and EM703 - a new derivative of erythromycin without antibacterial effects on the expressions of IL-8 caused by DEP exposure. DEP showed a dose-dependent stimulatory effect on IL-8 product in HBEC. Increases of IL-8 expression by DEP stimulation were significantly blocked by both EM and EM703 pretreatment. Furthermore, NF-κB and Nrf2 activation, the antioxidant enzymes such as HO-1, NQO-1 mRNA expression were increased by DEP exposure and these increases were blocked by both of EM and EM703 pretreatment. Our results suggest that, EM and EM703 may have an inhibitory effect on expression inflammatory cytokines in HBEC induced by DEP not only as an anti-inflammation but also an antioxidant drug. EM and EM703 might contribute to chemical prevention of the risk of pulmonary diseases induced by oxidative stress from environmental pollutant, such as DEP.

Immunotoxicity of N,N-diethylaniline in mice: effect on natural killer activity, cytotoxic T lymphocyte activity, lymphocyte proliferation response and cellular components of the spleen.

We previously found that N,N-diethylaniline increased the frequency of sister chromatid exchange (SCE) of human lymphocytes to about five times that of the control value, and was as toxic as cyclophosphamide used as a positive control for SCE. To explore whether N,N-diethylaniline affects the function of lymphocytes, we evaluated its immunotoxicity using CBA/N mice. The mice were divided into four groups and received 0, 100, 200, or 400 mg/kg body weight of N,N-diethylaniline by subcutaneous injection. The following items were investigated on days 3 and 7 after injection: body weight, weight of spleen, number of splenocytes, natural killer (NK) and cytotoxic T lymphocyte (CTL) activities, and concanavalin A (Con A)- and lipopolysaccharide (LPS)-stimulated lymphocyte proliferation using splenocytes. The following splenocyte phenotypes were also quantified by flow cytometry: (1) B cells; (2) total T cells; (3) CD4(+) and CD8(+) T cells; (4) NK; (5) macrophages and (6) nucleated erythrocytes. The splenic NK and CTL activities in exposed groups significantly decreased compared to the control in a dose-dependent manner and lymphocytes from the 200 and 400 mg/kg groups showed significantly higher spontaneous proliferation. The weight of the spleen and number of splenocytes were significantly higher in exposed groups than in the control. N,N-Diethylaniline also increased the percentages of macrophages, nucleated erythrocytes and B cells in the spleen. On the other hand, N,N-diethylaniline did not affect LPS-stimulated B cell and Con A-stimulated T cell proliferation, or the percentages of NK, total T, and CD4(+) and CD8(+) T cells in the spleen or the body weight of mice. The above findings indicated that N,N-diethylaniline selectively inhibited splenic NK and CTL activity and this inhibition was due to decreased NK and CTL functions, but not due to changes in the numbers of splenic NK and T cells.

Effect of oral exposure to fenitrothion and 3-methyl-4-nitrophenol on splenic cell populations and histopathological alterations in spleen in Wistar rats

Fenitrothion (FNT) is used throughout the world as an insecticide in agriculture. To investigate the effect of FNT on the splenocytes and the underlying mechanism, FNT and its main metabolite, 3-methyl-4-nitrophenol (MNP), were administered orally to Wistar rats in daily doses of 0, 5 and 10 mg/kg, 4-5 days/week for 9 weeks. Splenocytes were harvested from control and exposed rats, and the following cell phenotypes were quantified by flow cytometry: (1) B cells (PE-CD45RA), (2) T cells (FITC-CD3), (3) T cell subsets (PE-CD4 and PerCPCD8), (4) natural killer (NK) cells (FITC-CD161a), (5) macrophages (FITC-CD11b), and (6) granulocyte (PE-granulocyte). Body weight, weight of the spleen, and histopathological alterations of spleens were also examined. The percentage of splenic CD8+ T cells and the ratio of CD8/CD4 in the group receiving 10 mg/kg FNT, and the percentages of splenic CD3+ and CD8+ T cells in the group receiving 10 mg/kg MNP were significantly decreased compared with those in the controls. FNT exposure also significantly decreased the weight of the spleen and body weight. In addition, apoptotic lymphocytes in spleen were observed in FNT-exposed rats under transmission electron microscope. However, FNT and MNP exposures did not affect splenic NK cells, B cells, macrophages, and granulocytes. The above findings indicate that FNT and MNP may selectively affect splenic T cells in rats.

Insomnia as a sequela of sarin toxicity several years after exposure in Tokyo subway trains.

More than 5,000 passengers on Tokyo subway trains were injured with toxic chemicals including the nerve gas “sarin” on March 20, 1995. The purpose of this study was to identify the effect of sarin exposure on insomnia in a cross-sectional study. A self-administered questionnaire concerning sleep-related items was distributed to victims of sarin exposure in October and November, 2003. Questionnaires were completed by 161 of the 163 participants (98.8%), who were selected from 1,500 subjects. Among them, the authors selected 75 women 30 to 69 years of age. Control participants were collected from inhabitants living in Maebachi City, Gunma Prefecture, Japan. For the younger exposed group (under 50 yr. of age), percentages of poor sleep, difficulty falling asleep, intermittent awakening, early morning awakening, a feeling of light overnight sleep, and insomnia were significantly higher than those for the control group. In contrast, the older exposed group (ages 50 to 69 years) had significantly higher prevalence of poor sleep, a feeling of light overnight sleep, and early morning awakening for the exposed group when compared with the control group. The high prevalence of insomnia and insomnia-related factors for victims especially under 50 years of age suggests a need for research on sleep quality after sarin exposure. Although posttraumatic stress disorder is assumed to be a psychological effect of exposure to a toxic substance, a cause-and-effect relationship has not been established.

Effects of subchronic inhalation exposure to ethyl tertiary butyl ether on splenocytes in mice.

Ethyl tertiary-butyl ether (ETBE) is a motor fuel oxygenate used in reformulated gasoline. The current use of ETBE in gasoline or petrol is modest but increasing. To investigate the effects of ETBE on splenocytes, mice were exposed to 0 (control), 500 ppm, 1750 ppm, or 5000 ppm of ETBE by inhalation for 6 h/day for 5 days/wk over a 6- or 13-week period. Splenocytes were harvested from the control and exposed mice, and the following cell phenotypes were quantified by flow cytometry: (1) B cells (PerCP-Cy5.5-CD45R/B220), (2) T cells (PerCP-Cy5-CD3e), (3) T cell subsets (FITC-CD4 and PE-CD8a), (4) natural killer (NK) cells (PE-NK1.1), and (5) macrophages (FITC-CD11b). Body weight and the weight of the spleen were also examined. ETBE-exposure did not affect the weight of the spleen or body weight, while it transiently increased the number of RBC and the Hb concentration. The numbers of splenic CD3+, CD4+, and CD8+ T cells, the percentage of CD4+ T cells and the CD4+/CD8+ T cell ratio in the ETBE-exposed groups were significantly decreased in a dose-dependent manner. However, ETBE exposure did not affect the numbers of splenic NK cells, B cells, or macrophages or the total number of splenocytes. The above findings indicate that ETBE selectively affects the number of splenic T cells in mice.

Recombinant human progranzyme 3 expressed in Escherichia coli for analysis of its activation mechanism

Gr3 is reported to play an important role in defense against viral infection. Although it is known that Gr3 is synthesized as a proenzyme and activated in the cytotoxic granules of NK cells and CTL, the activation mechanism is not clearly understood. In an attempt to analyze the activation mechanism of human Gr3, a recombinant pro‐Gr3 was expressed in the periplasm of E. coli and purified to homogeneity. On SDS‐PAGE the recombinant pro‐Gr3 showed a slightly higher molecular weight than the enzymatically active Gr3, because the former possesses a small propeptide at its N‐terminal. The recombinant pro‐Gr3 was enzymatically inactive. It could be activated by treatment with cathepsin C, which concomitantly decreased the molecular weight to that of active Gr3. The proteolytic reaction of cathepsin C did not continue after one dipeptide had been removed, indicating that the recombinant pro‐Gr3 had the native conformation without any refolding process. The recombinant pro‐Gr3 would be a valuable tool for analyzing the activation mechanism and exploring other activating enzymes besides cathepsin C.