Yuichi Ohkawara

Transfer of granulocyte-macrophage colony-stimulating factor gene to rat lung induces eosinophilia, monocytosis, and fibrotic reactions.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine whose expression is increased in numerous respiratory diseases, particularly in asthma. However, the role of GM-CSF in the pathogenesis of these conditions in vivo remains unclear. Here, we report the functional activities of GM-CSF highly expressed in rat lung after intrapulmonary transfer of the gene coding for murine GM-CSF by using an adenoviral vector. This high, transient expression of GM-CSF led to the sustained but self-limiting accumulation of eosinophils and macrophages associated with tissue injury in the lung followed by varying degrees of irreversible fibrotic reactions observed in later stages. These results suggest that GM-CSF plays a previously unrealized role in the development of respiratory conditions characterized by eosinophilia, granuloma and/or fibrosis and provide the rationale for targeting this molecule in these diseases.

CD40 expression by human peripheral blood eosinophils.

In this study, we have investigated CD40 expression in human peripheral blood eosinophils and in human chronically inflamed nasal tissues, i.e., nasal polyps. We show by both reverse transcriptase-PCR and Northern blot analysis that eosinophils from allergic subjects express human CD40 mRNA. We also show that constitutive CD40 mRNA expression in eosinophils could be upregulated by exposure to IgA immune complexes and downregulated by IL-10 and the synthetic steroid budesonide. In addition, we demonstrate that eosinophils express CD40 protein by flow cytometry. Such expression is biologically functional as cross-linking CD40 with CD40 mAbs enhances eosinophil survival in a dose-dependent fashion; in addition, CD40 ligation stimulates eosinophils to release GM-CSF. CD40-mediated eosinophil survival was largely inhibited by an anti-GM-CSF neutralizing antibody suggesting GM-CSF involvement in the survival enhancing mechanism. CD40 mRNA was also detected in total RNA extracted from nasal polyp tissues but not in RNA isolated from normal nasal mucosa (inferior turbinate); by immunohistochemistry, we were able to detect immunoreactive CD40 protein in a variety of cell types in the polyp stroma, but primarily in eosinophils. These observations suggest previously unforeseen interactions between eosinophils and cells expressing the CD40 ligand and, thus, novel pathways by which eosinophils may contribute to the regulation of airway inflammation.

Gene transfer for cytokine functional studies in the lung: the multifunctional role of GM-CSF in pulmonary inflammation.

Using adenoviral‐mediated gene transfer techniques, the murine granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) transgene is efficiently targeted to and highly expressed by the respiratory epithelium of rat lung. This lung tissue‐directed expression of GM‐CSF induces accumulation of both eosinophils and macrophages at early stages and an irreversible fibrotic reaction at later stages. These tissue responses to GM‐CSF appear to be distinct from those induced by other proinflammatory cytokines, interleukin (IL) ‐5, IL‐6, macrophage inflammatory protein‐2 (MIP‐2), or RANTES overexpressed in the lung. These findings clearly demonstrate that GM‐CSF is more than a hematopoietic cytokine in the lung and may play a pivotal role in the multiple pathological processes underlying numerous respiratory illnesses, including asthma. In this overview, the differences in tissue responses induced by GM‐CSF and other individual cytokines are highlighted. In addition, the mechanisms by which GM‐CSF contributes to the development of eosinophilia, macrophage granuloma, and fibrosis are discussed in conjunction with the recent findings from us and others.

Disruption of antigen-induced inflammatory responses in CD40 ligand knockout mice.

The objective of this study was to investigate the contribution of the interaction between CD40 and its ligand (CD40L) to antigen-induced airways inflammatory responses. To this end, we used a model involving ovalbumin (OVA) sensitization followed by OVA aerosol challenge in CD40L knockout (KO) mice. OVA-specific IgE and IgG1 were detected in the serum of the sensitized control, but not in CD40L-KO mice. After antigen challenge, sensitized control mice developed airway inflammation that was primarily eosinophilic. This inflammatory response was dramatically reduced in CD40L-KO mice. In contrast, similar numbers of eosinophils were observed in both the bone marrow and the peripheral blood in the sensitized controls and mutant strains after antigen challenge. To investigate the mechanisms underlying these findings, we examined levels of the cytokines IL-5, IL-4, and TNFalpha in both bronchoalveolar lavage (BAL) and serum. Similar levels of IL-5 were detected in BAL and serum of control and CD40L-KO mice; however, negligible levels of IL-4 in BAL and serum and of TNFalpha in BAL were detected in CD40L-KO mice when compared with control mice. Furthermore, we demonstrated that endothelial cell expression of vascular cell adhesion molecule 1 in OVA-sensitized and -challenged CD40L-KO mice was, as detected by immunohistochemistry, markedly decreased compared with that observed in similarly treated control mice. In addition, we locally overexpressed IL-4 and TNFalpha by using an adenoviral (Ad)-mediated gene transfer approach. Intranasal administration of either Ad/TNFalpha or Ad/IL-4 into OVA-sensitized and -challenged CD40L-KO mice did not reconstitute airway eosinophilia. However, concurrent administration of Ad/TNFalpha and Ad/IL-4 upregulated endothelial expression of vascular cell adhesion molecule 1, and resulted in full reconstitution of the inflammatory response in the airways. Together, these findings demonstrate the importance of the CD40-CD40L costimulatory pathway in the full expression of the inflammatory response in the airways.

Compartmentalized transgene expression of granulocyte-macrophage colony- stimulating factor (GM-CSF) in mouse lung enhances allergic airways inflammation

To investigate the role of GM‐CSF in asthmatic airways inflammation, we have targeted GM‐CSF transgene to the airway cells in a mouse model of ovalbumin (OVA)‐induced allergic airways inflammation, a model in which there is marked induction of endogenous IL‐5 and IL‐4 but not GM‐CSF. Following intranasal delivery of a replication‐deficient adenoviral gene transfer vector (Ad), transgene expression was found localized primarily to the respiratory epithelial cells. Intranasal delivery of 0.03 × 109 plaque‐forming units (PFU) of AdGM‐CSF into naive BALB/c mice resulted in prolonged and compartmentalized release of GM‐CSF transgene protein with a peak concentration of ≈ 80 pg/ml detected in bronchoalveolar lavage fluid (BALF) at day 7, but little in serum. These levels of local GM‐CSF expression per se resulted in no eosinophilia and only a minimum of tissue inflammatory responses in the lung of naive mice, similar to those induced by the control vector. However, such GM‐CSF expression in the airways of OVA‐sensitized mice resulted in a much greater and sustained accumulation of various inflammatory cell types, most noticeably eosinophils, both in BALF and airway tissues for 15–21 days post‐OVA aerosol challenge, at which times airways inflammation had largely resolved in control mice. While the levels of IL‐5 and IL‐4 in BALF and the rate of eosinophil apoptosis were found similar between different treatments, there was an increased number of proliferative leucocytes in the lung receiving GM‐CSF gene transfer. Our results thus provide direct experimental evidence that GM‐CSF can significantly contribute to the development of allergic airways inflammation through potentiating and prolonging inflammatory infiltration induced by cytokines such as IL‐5 and IL‐4.

Potential role of interleukin-1 in allergen-induced late asthmatic reactions in guinea pigs: Suppressive effect of interleukin-1 receptor antagonist on late asthmatic reaction

Interleukin (IL)-1 is a pluripotential proinflammatory cytokine and is thought to be involved in the pathogenesis of bronchial asthma and late asthmatic reactions (LARs). To determine whether IL-1 plays a role in LAR, guinea pigs sensitized with Ascaris antigen were used. We evaluated IL-1 production by immunostaining with anti-IL-1 beta antibody and elucidated the action of IL-1 in LAR with recombinant IL-1 receptor antagonist. Immunostaining revealed that IL-1 beta-like immunoreactivity-positive cells increased in the airway walls and in bronchoalveolar lavage fluid after the antigen challenge. IL-1 receptor antagonist protein pretreatment reduced the generation of LAR in terms of pulmonary resistance. IL-1 receptor antagonist protein pretreatment did not change cellular components but reduced the percentage of hypodense eosinophils in bronchoalveolar lavage fluid. We also studied the direct effect of recombinant human IL-1 beta on pulmonary resistance and eosinophil activity measured as released eosinophil peroxidase activity. Recombinant human IL-1 beta did not change pulmonary resistance but primed eosinophils to release eosinophil peroxidase activity in response to platelet activating factor. Therefore these results suggest that IL-1 was produced in sensitized pulmonary tissue of guinea pigs by allergen exposure and played a role in the generation of LAR, at least partially by modulating the activation of eosinophils.

μ-opioid Receptor-Mediated Alterations of Allergen-Induced Immune Responses of Bronchial Lymph Node Cells in a Murine Model of Stress Asthma

BACKGROUND Psychological stress has a recognized association with asthma symptoms. Using a murine model of allergic asthma, we recently demonstrated the involvement of μ-opioid receptors (MORs) in the central nervous system in the stress-induced exacerbation of airway inflammation. However, the involvement of MORs on neurons and immunological alterations in the stress asthma model remain unclear. METHODS MOR-knockout (MORKO) mice that express MORs only on noradrenergic and adrenergic neurons (MORKO/Tg mice) were produced and characterized for stress responses. Sensitized mice inhaled antigen and were then subjected to restraint stress. After a second antigen inhalation, bronchoalveolar lavage cells were counted. Before the second inhalation, bronchial lymph node (BLN) cells and splenocytes from stressed and non-stressed mice were cultured with antigen, and cytokine levels and the proportions of T cell subsets were measured. RESULTS Stress-induced worsening of allergic airway inflammation was observed in wild-type and MORKO/Tg mice but not MORKO mice. In wild-type stressed mice, IFN-γ/IL-4 ratios in cell culture supernatants and the proportion of regulatory T cells in BLN cell populations were significantly lower than those in non-stressed mice. These differences in BLN cells were not observed between the stressed and non-stressed MORKO mice. Restraint stress had no effect on cytokine production or T cell subsets in splenocytes. CONCLUSIONS Restraint stress aggravated allergic airway inflammation in association with alterations in local immunity characterized by greater Th2-associated cytokine production and a reduced development of regulatory T cells, mediated by MORs.

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.

Gender differences in transcriptional regulation of IL‐5 expression by bronchial lymph node cells in a mouse model of asthma

Background and objective:  The severity of asthma after puberty is higher in women than in men. Increased numbers of eosinophils in the airways of female mice after antigen challenge was associated with increased levels of T helper (Th)2 cytokines at the site of inflammation, and in human and mouse studies, the profile of cytokines produced by immune cells from women showed greater Th2 predominance. The aim of this study was to investigate gender differences in the development of Th2 immune responses.

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.