Tasuku Kawano

Binding of Silurus asotus lectin to Gb3 on Raji cells causes disappearance of membrane-bound form of HSP70.

Heat shock proteins (HSPs) are divided into stress-inducible and constitutive types. Generally, HSP70 (stress inducible) and HSC70 (constitutive) are representative of their types, respectively. From the results of immunocytochemical analysis, both HSP70 and HSC70 were constitutively expressed in globotriaosylceramide (Gb3)-expressing Raji cells as well as Gb3-negative K562 cells. Furthermore, the membrane-bound form of HSP70 was present on the surfaces of two cell lines as patch and cap-like structures, and was recovered in the cholesterol rich microdomains (CRM) prepared from them. On the other hand, HSP70 was partially co-localized with Gb3 on the surface of Raji cells. This result suggested that HSP70 was not associated with all of Gb3 molecules but with Gb3 specifically located in the particular environment. The effect of Silurus asotus lectin (SAL), which is one of the rhamnose-binding lectins and specifically binds to Gb3, on the disappearance of membrane-bound HSP70 was dependent on whether Gb3 was present or not. These results suggested that the disappearance of membrane-bound HSP70 was caused by SAL binding to Gb3, that the reduction of membrane-bound HSP70 might result in the decrease in cell volume observed, and that the mechanism of SAL-induced HSP70 expression may differ from that of heat shock in Raji cells.

T Cell Subsets Related with a Sex Difference in IL-5 Production

Background: Before puberty, the prevalence and severity of asthma are higher in boys than in girls, but this pattern is reversed after puberty. The underlying mechanisms of these gender differences in asthma are not fully understood. Using murine models of allergic asthma, a sex difference in Th2 cytokine production has been suggested to contribute to the gender differences in asthma. Therefore, we determined which subsets of T cells are involved in the sex difference in Th2 cytokine production. Methods: Splenocytes from wild-type mice and CD4+ T cell-, CD8+ T cell-, and iNKT cell-deficient mice were stimulated with anti-CD3/CD28 antibodies for 3 days, and the concentrations of IL-4, IL-5, IL-13, and IFN-γ in the cultures were measured by ELISA. Results: IL-5, but not IL-4 and IL-13, concentrations in culture derived from female wild-type mice were significantly higher than those in male wild-type mice. The sex difference in IL-5 concentrations was not observed in the cultures of splenocytes from CD4+ and CD8+ T cell-deficient mice. The disappearance of the sex differences in CD4+ and CD8+ T cell-deficient mice was attributable to a decrease in IL-5 concentration in female mice and an increase in IL-5 concentration in male mice. In iNKT cell-deficient mice, the sex difference was still observed. There was no significant difference between the sexes in any type of mice with respect to IFN-γ production. Conclusions: There was a sex difference in IL-5 production by splenocytes stimulated by TCR activation. The difference might be attributable to sex differences in CD4+ and CD8+ T cell functions.

Higher Sensitivity of Male CD4+ T Cells to Suppressive Effects of CD8+ T Cells on IL-5 Production Compared to Female CD4+ T Cells

Background: Asthma prevalence and severity are higher in females than in males after puberty. The underlying mechanisms of this gender difference are not fully understood. More severe airway inflammation in female mice has been reported to be associated with higher levels of T helper type 2 (Th2) cytokines in asthma models. The aim of this study was to investigate sex differences in CD4+ and CD8+ T cell functions in Th2 cytokine production. Methods: Splenocytes from naive mice were stimulated with anti-CD3/CD28 antibodies and the proportions of CD4+ and CD8+ T cells were analyzed. CD4+ T cells were stimulated in the presence of CD8+ T cells. The concentrations of interleukin (IL)-5, IL-10 and interferon (IFN)-γ in the cultures were measured. Results: The concentration of IL-5, but not IFN-γ, was significantly higher in female splenocytes than in male splenocytes. There were no sex differences in the proportions of CD4+ and CD8+ T cells in the splenocytes. Although the IL-5 production levels in male and female CD4+ T cells were similar, IL-5 production in male CD4+ T cells, but not female CD4+ T cells, was suppressed by both male and female CD8+ T cells. While IL-5 and IL-10 were not detected in the cultures from both male and female CD8+ T cells, IFN-γ concentration in female CD8+ T cells was significantly higher than in male CD8+ T cells. Conclusions: The sex difference in the sensitivity of CD4+ T cells to CD8+ T cell suppression might contribute to the sex difference in IL-5 production by splenocytes.

CD8+ T Cells Mediate Female-Dominant IL-4 Production and Airway Inflammation in Allergic Asthma.

The prevalence and severity of bronchial asthma are higher in females than in males after puberty. Although antigen-specific CD8+ T cells play an important role in the development of asthma through their suppressive effect on cytokine production, the contribution of CD8+ T cells to sex differences in asthmatic responses remains unclear. In the present study, we investigated the sex-specific effect of CD8+ T cells in the suppression of asthma using an ovalbumin mouse model of asthma. The number of inflammatory cells in bronchoalveolar lavage (BAL) fluid, lung type 2 T-helper cytokine levels, and interleukin-4 (IL-4) production by bronchial lymph node cells were significantly higher in female wild-type (WT) mice compared with male mice, whereas no such sex differences were observed between male and female cd8α-disrupted mice. The adaptive transfer of male, but not female, CD8+ T cells reduced the number of inflammatory cells in the recovered BAL fluid of male recipient mice, while no such sex difference in the suppressive activity of CD8+ T cells was observed in female recipient mice. Male CD8+ T cells produced higher levels of IFN-γ than female CD8+ T cells did, and this trend was associated with reduced IL-4 production by male, but not female, CD4+ T cells. Interestingly, IFN-γ receptor expression on CD4+ T cells was significantly lower in female mice than in male mice. These results suggest that female-dominant asthmatic responses are orchestrated by the reduced production of IFN-γ by CD8+ T cells and the lower expression of IFN-γ receptor on CD4+ T cells in females compared with males.

The involvement of central nervous system histamine receptors in psychological stress-induced exacerbation of allergic airway inflammation in mice

BACKGROUND Psychological stress is one of the major risk factors for asthma exacerbation. Although histamine in the brain acts as an excitatory and inhibitory neurotransmitter associated with psychological stress, the contribution of brain histamine to psychological stress-induced exacerbation of asthma remains unclear. The objective of this study was to investigate the role of histamine receptors in the CNS on stress induced asthma aggravation. METHODS We monitored the numbers of inflammatory cells and interleukin (IL)-13 levels in bronchoalveolar lavage fluid, airway responsiveness to inhaled methacholine, mucus secretion in airway epithelial cells, and antigen-specific IgE contents in sera in a murine model of stress-induced asthma treated with epinastine (an H1R antagonist), thioperamide (an H3/4R antagonist), or solvent. RESULTS All indicators of stress-induced asthma exacerbation were significantly reduced in stressed mice treated with epinastine compared with those treated with solvent, whereas treatment with thioperamide did not reduce the numbers of inflammatory cells in the stressed mice. CONCLUSIONS These results suggest that H1R, but not H3/4R, may be involved in stress-induced asthma exacerbations in the central nervous system.

Contribution of CD4+ T Cells and Dendritic Cells to Female-Dominant Antigen-Induced T Helper Type 2 Cytokine Production by Bronchial Lymph Node Cells

Background: After puberty, asthma severity is higher in women than in men. The underlying mechanisms of this gender difference are not fully understood. In murine models of allergic asthma, more severe airway inflammation in female mice is associated with higher levels of T helper type 2 (Th2) cytokines. The aim of this study was to investigate the contributions of CD4+ T cells and dendritic cells (DCs) to the differences in Th2 cytokine production between sexes. Methods: Bronchial lymph node (BLN) cells from ovalbumin (OVA)-sensitized male and female C57BL/6 mice were stimulated with OVA and anti-CD3/CD28 antibodies. The CD4+ T cells and DCs purified from BLN cells were cocultured with OVA in a sex-matched or mismatched fashion. The CD4+ T cells were also stimulated with anti-CD3/CD28 antibodies. The concentrations of interleukin (IL)-5, IL-4, IL-13 and interferon (IFN)-γ in the culture supernatants were measured. Results: The concentrations of IL-5, IL-4 and IL-13, but not IFN-γ, were significantly higher in female BLN cells stimulated with OVA than in male BLN cells. Sex differences were also observed in the CD4+ T cells stimulated with anti-CD3/CD28 antibodies, whereas only IL-4 was significantly different in the BLN cells stimulated with antibodies. IL-5 production by OVA-stimulated male and female CD4+ T cells, but not IL-4 or IL-13 production, was significantly increased in the coculture with female DCs when compared to the male DCs. Conclusions: The differences in Th2 cytokine production between sexes by the BLN cells may be attributable, at least in part, to the differing functions of CD4+ T cells and DCs between sexes.

Increased Susceptibility to Allergic Asthma with the Impairment of Respiratory Tolerance Caused by Psychological Stress

Background: Bronchial asthma is characterized by type 2 T helper (Th2) cell inflammation, essentially due to a breakdown of immune tolerance to harmless environmental allergens. Etiologically, experiences of psychological stress can be associated with a heightened prevalence of asthma. However, the mechanisms underlying stress-related asthma development are unclear. In this study, we examined whether psychological stress increases susceptibility to allergic asthma by downregulating immune tolerance. Methods: Female BALB/c mice were sensitized with ovalbumin/alum, followed by ovalbumin inhalation. Ovalbumin inhalation induced immune tolerance before sensitization occurred. Some mice were exposed to restraint stress during tolerance induction or sensitization. Asthma development was evaluated by airway responsiveness, inflammation, cytokine expression, and IgE synthesis. Sensitization was evaluated by measuring proliferation and cytokine production by splenocytes. The effects of stress exposure on the numbers and functions of dendritic cells and regulatory T (Treg) cells in bronchial lymph nodes and spleens were evaluated. To investigate the role of endogenous glucocorticoid in inhibiting immune tolerance after stress exposure, we examined the effects of (i) a glucocorticoid-receptor antagonist administered prior to stress exposure, and (ii) exogenous glucocorticoid (instead of stress exposure). Results: Asthmatic responses and Th2-biased sensitization, which were suppressed in tolerized mice, re-emerged in tolerized mice stressed during tolerance induction in association with decreased tolerogenic dendritic and Treg cell numbers. The effects of stress exposure on tolerized mice were abolished by administering a glucocorticoid-receptor antagonist and reproduced by administering exogenous glucocorticoid without stress. Conclusions: Our findings suggested that psychological stress can potentially increase allergic asthma susceptibility by inhibiting immune tolerance.

ETosis-derived DNA trap production in middle ear effusion is a common feature of eosinophilic otitis media

Eosinophilic otitis media (EOM) is a recently recognized intractable middle ear disease in Japan and has been focused on in Western country.1 EOM is characterized by the strong infiltration of eosinophils in highly viscous ear effusion (eosinophilic mucin) and in the middle ear mucosa.2,3 Unlike conventional chronic otitis media, EOM is generally resistant to all pharmacological therapies except for systemic steroid treatment. Surgical techniques are often warranted to improve the drainage pathway so as to remove obstructive eosinophilic mucin. Patients with EOM have a high risk of conductive hearing loss in the early stage, and half of all patients develop an increased bone-conduction threshold, leading to deafness in 6% of cases.2,4 The high levels of toxic eosinophil granule proteins observed in the eosinophilic mucin are suggested to be pathogenic and to cause epithelial damage.4 Eosinophil degranulation is mediated by several mechanisms, namely exocytosis, piecemeal degranulation, and cytolysis. Cytolysis is a well recognized pathology in diverse eosinophil-associated diseases, including EOM,4 and was suggested to represent a full activation of eosinophils,5 although its precise mechanism is not understood. A recent finding showed that extracellular trap cell death (ETosis), a novel form of cell death, mediates eosinophil cytolytic degranulation.6 Eosinophil ETosis (EETosis) is characterized by its striking final morphology: the release of intact granules and web-like chromatin structures (DNA traps/extracellular traps: ETs) through the breakdown of nuclear and plasma membranes. It does not feature apoptotic signatures such as phosphatidylserine exposure on the cell surface and DNA fragmentation. We have previously reported the presence of eosinophil ETs (EETs) in the middle ear secretions obtained from a few EOM patients.7 To clarify whether the presence of cytolysis and ETs is common in EOM, we conducted a case series study. All patients met the diagnostic criteria of Iino et al.2 and were seen by experienced ENT doctors at Yamagata City Hospital Saiseikan, Yamagata University Faculty of Medicine, and Tohoku Medical and Pharmaceutical University between February 2010 and August 2017. Informed consent was obtained under protocols approved by the Institutional Review Board. Clinical features of the patients with EOM are summarized in Table 1. The patients were 4 men and 4 women who ranged in age from 25 to 72 years. Audiometry was performed by audiologists using a pure-tone audiometer (AA-76, Rion, Tokyo, Japan). All patients had comorbid chronic