Hideaki Shirasaki

Expression and localization of the cysteinyl leukotriene 1 receptor in human nasal mucosa

Background The cysteinyl leukotrienes (CysLT) are lipid mediators that have been implicated in the pathogenesis of allergic diseases. Pharmacological studies using CysLTs indicate that two classes of receptors, named CysLT1 and CysLT2 receptor, exist. The former is sensitive to the CysLT1 antagonist currently used to treat asthma and allergic rhinitis. Recently, the cDNA for human CysLT1 and CysLT2 receptor have been cloned, making it now possible to study the gene expression of CysLTs receptors.

Effects of glucocorticoids on infiltrating cells and epithelial cells of nasal polyps

Glucocorticoids are known to be effective in the treatment of nasal polyps (NPs). To examine the mechanisms of their effect, we evaluated 1) the ability of glucocorticoids to induce the apoptosis of eosinophils and T lymphocytes in NPs, and 2) the ability of dexamethasone to down-regulate epithelial cell functions that relate to eosinophilic inflammation. In vitro and in vivo, glucocorticoids increased the apoptosis of both eosinophils and T lymphocytes in NPs. Dexamethasone inhibited the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) from both NP epithelial cells that were unstimulated and NP epithelial cells that were stimulated with interleukin-4 or tumor necrosis factor a. These results suggest that the clinical efficacy of glucocorticoids on NPs may be due to 1) induction of apoptosis in both eosinophils and T lymphocytes that infiltrate NPs, and 2) down-regulation of epithelial GM-CSF production, which prolongs eosinophil survival.

Accumulation of CRTH2-positive leukocytes in human allergic nasal mucosa.

BACKGROUND Prostaglandin D2 (PGD2) has been thought to be a potent mediator involved in allergic rhinitis because PGD2 has been recovered from the nasal lavage fluid of patients with allergic rhinitis after allergen provocation and because PGD2 receptor antagonists relieved nasal allergic symptoms in an animal model of allergic rhinitis. The inflammatory effects of PGD2 are exerted through high-affinity interactions with 2 G protein-coupled receptors: D-prostanoid receptor 1 and chemoattractant-homologous receptor expressed on TH2 cells (CRTH2). CRTH2 may mediate the recruitment of leukocytes during a nasal allergic response. OBJECTIVE To evaluate the number of CRTH2-expressing cells in allergic and nonallergic human nasal mucosa by means of immunohistochemical analysis. METHODS Human turbinates were obtained after turbinectomy from 14 patients with nasal obstruction refractory to medical therapy. To identify cells expressing the CRTH2 protein, double immunostaining was performed using anti-CRTH2 antibody and monoclonal anti-leukocyte antibodies. RESULTS The immunohistochemical study revealed that anti-CRTH2 antibody labeled eosinophils, macrophages, mast cells, T lymphocytes, epithelial cells, and submucosal glands in the nasal mucosa. CRTH2 expressions of these leukocytes in allergic nasal mucosa are significantly up-regulated compared with those in nonallergic nasal mucosa. CONCLUSION These results suggest that CRTH2 may play an important role in the recruitment of leukocytes into allergic nasal mucosa.

Effects of a cysteinyl leukotriene antagonist, ONO-1078 (pranlukast), on total airway resistance after antigen challenge in sensitized guinea pigs.

Abstract.Objective and Design: To define the role of leukotriene (LT) in allergic rhinitis, we examined the effects of a cysteinyl (Cys) LT antagonist (ONO-1078, pranlukast).¶Material: Actively sensitized Dunkin-Hartley guinea pigs.¶Treatment: ONO-1078 (pranlukast), 3–100 mg/kg p.o. l h before antigen challenge.¶Methods: Nasal symptoms (sneezing, nasal scratches), changes of total airway resistance (TAR by plethysmography) and eosinophil infiltration into the nasal mucosa were determined following topical antigen (OA) challenge. Dunnets test (TAR and symptoms) and the Mann-Whitney U-test (eosinophils) were applied.¶Results: Control animals showed bi-phasic nasal responses, peaking 10 min and 240 min after the topical antigen challenge, respectively. While the early-phase response was characterized by nasal symptoms of sneezing and scratching accompanied by the increase in TAR, the late-phase was characterized by an increase in TAR accompanied by eosinophil infiltration into nasal mucosa. The nasal symptoms (sneezing and scratching) were not inhibited by pretreatment with ONO-1078 at doses up to 100 mg/kg (p.o., n = 15). Although early peak responses of TAR were not affected with even the highest dose (30 mg/kg, p.o., n = 6), late-phase TAR peak response (control: 174.8 ± 8.2%, n = 6) were significantly inhibited by 10 mg/kg (142.7 ± 15.8%; p < 0.05, n = 6) and 30 mg/kg (118.0 ± 6.6%; p < 0.01, n = 6) of ONO-1078 (p.o.). In addition, the eosinophil infiltration induced by the antigen was not inhibited by ONO-1078 (30 and 100 mg/kg, p.o., n = 6).¶Conclusions: Our results suggest that Cys LT may play an important role in the late-phase increase in TAR in the guinea pig model of allergic rhinitis.

Effect of glucocorticosteroids on tumour necrosis factor‐α‐induced intercellular adhesion molecule‐1 expression in cultured primary human nasal epithelial cells

Objective In order to confirm the direct effect of glucocorticosteroids on epithelial intercellular adhesion molecule‐1 (ICAM‐1) expression, we examined ICAM‐1 expression on primary cultured human nasal epithelial cells (HNECs) at both protein and mRNA levels.

Tumor necrosis factor increases MUC1 mRNA in cultured human nasal epithelial cells.

Objective--Mucins are high molecular weight glycoproteins which are normally expressed on the surface of a variety of epithelia. It is possible that shedding of such molecules from the epithelium could play a role in preventing bacterial colonization at the mucosal surface. Immunohistochemical and reverse transcriptase polymerase chain reaction (RT-PCR) analyses of human inferior turbinates have shown the existence of MUC1 mucin in nasal mucosa. However, the regulatory mechanisms of MUC1 mucin are poorly understood. In order to clarify the modulation of mucin gene expression, we developed a real-time semi-quantitative RT-PCR based on TaqMan fluorescence methodology to quantify MUC1 mRNA in primary cultured human nasal epithelial cells (HNECs).Material and Methods--HNECs were stimulated with recombinant human tumor necrosis factor (TNF)-α (20 pg/ml to 20 ng/ml) for specified time periods (0, 12, 24 and 48 h) and MUC1 mRNA was determined by means of semi-quantitative RT-PCR.Results--Significant increases in MUC1 gene expression in HNECs were initially detected at 12 h, peaking at 24 h after stimulation. TNF-mediated MUC1 mRNA expression at 24 h was significantly inhibited by co-incubation with human recombinant soluble TNF receptor.Conclusions--TNF-mediated MUC1 gene expression may contribute to the pathogenesis of human inflammatory upper airway disorders. Also, our mucin mRNA real-time PCR provides a quantitative method for investigating the regulation of mucin gene expression in both healthy and diseased samples.

Expression and Localization of Steroid Receptors in Human Nasal Mucosa

Objective—To investigate the expression of glucocorticoid receptor (GR), oestrogen receptor (ER), progesterone receptor (PR) and androgen receptor (AR) in nasal mucosa. Material and Methods—Human turbinates were obtained after turbinectomy from seven patients. The expression and localization of steroid receptors were examined using reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. Results—Using RT-PCR, GR and ERα mRNA were detected in all cases. In contrast, ERβ, PR and AR mRNA were found in five, four and six cases, respectively. Using immunohistochemistry, antibodies to GR showed the presence of GR within all cells of nasal mucosa, with the highest quantities of GR being localized in epithelial cells, submucosal glands and inflammatory leukocytes. Immunohistochemical analysis of sex steroid receptor revealed that anti-ERα antibody labelled mainly mast cells and anti-ERβ antibody labelled submucosal glands, and that no PR or AR expression was detected in any of the samples tested. Conclusions—The role of ER in mast cells and submucosal glands has not been well clarified. However, precise knowledge of the identity and distribution of sex steroid receptor should be of considerable interest in understanding the role of sex hormones in upper airway diseases such as allergic and non-allergic rhinitis.

Evaluation of the effects of antigen specific immunotherapy on chronic sinusitis in children with allergy.

The purpose of this study is to evaluate the clinical significance of allergy in children with chronic sinusitis. After allergic examinations, 52 sinusitis children were divided into allergic and non-allergic groups: 37 allergic children were treated with either the combination of antigen specific immunotherapy and medication with lysozyme chloride preparation (AI group, n = 20) or medication alone (AM group, n = 17); 15 non-allergic patients were also treated with lysozyme chloride preparation (NAM group). The treatment results including the radiographic improvements were significantly better in the AI group than in the AM or NAM group. The clinical effects of lysozyme chloride preparation tended to be better in the NAM group than in the AM group.

Arachidonate 5-Lipoxygenase Establishes Adaptive Humoral Immunity by Controlling Primary B Cells and Their Cognate T-Cell Help

In this study, we report the unique role of arachidonate 5-lipoxygenase (Alox5) in the regulation of specific humoral immune responses. We previously reported an L22 monoclonal antibody with which human primary resting B cells in the mantle zones of lymphoid follicles are well-defined. Proteomics analyses enabled identification of an L22 antigen as Alox5, which was highly expressed by naive and memory B cells surrounding germinal centers. Cellular growth of mantle cell lymphoma cells also seemed to depend on Alox5. Alox5(-/-) mice exhibited weak antibody responses specific to foreign antigens at the initial and recall phases. This was probably attributable to the low number of follicular and memory B cells and the functional loss of interleukin-21-mediated responses of follicular B cells. Moreover, Alox5(-/-) mice could not fully foster the development of follicular B helper T (Tfh) cells even after immunization with foreign antigens. Further experiments indicated that Alox5 affected mortality in experimentally induced enterocolitis in germ-prone circumstances, indicating that Alox5 would endow immunologic milieu. Our results illustrate the novel role of Alox5 in adaptive humoral immunity by managing primary B cells and Tfh cells in vivo.

Specific immune response of the adenoids to a respiratory antigen

The specific antibody response of the adenoids to a respiratory antigen was investigated, both in vitro and in vivo. The in vitro immunoglobulin (Ig) production of adenoidal and tonsillar lymphocytes in the same patient in 18 cases was measured by an enzyme-linked immunosorbent assay. Adenoidal lymphocytes produced more IgM than IgG or IgA under culture conditions without any mitogens, whereas IgG was the major Ig produced by tonsillar lymphocytes. The same results were obtained when cultured with pokeweed mitogen. Under culture conditions with Dermatophagoides farinae (mite) antigen, adenoidal lymphocytes produced only specific IgM class antibody, and at a level significantly greater than tonsillar lymphocytes. Through an in vivo study, we established experimental adenoidal tissue in the guinea pig by long-term exposure to an ovalbumin aerosol. Infiltration of cells producing the specific antibody against an ovalbumin was demonstrated in the epipharyngeal mucosa using immunofluorescent staining. Our results confirm that the adenoids play a role in specific immune responses to respiratory antigens.