Establishment of an allergic rhinitis model in mice for the evaluation of nasal symptoms

The purpose of our study was to establish a new model of allergic rhinitis in mice, eliciting symptoms such as sneezing, infiltration of eosinophils into the nasal mucosa, and antigen-specific IgE production. One of the major human T-cell epitopes in Cry j 1, an allergen of Japanese cedar pollen, is also a major murine T-cell epitope in B10.S mice. Thus we tried to establish an allergic rhinitis model in B10.S mice with Cry j 1 as the antigen. We sensitized B10.S mice subcutaneously with Cry j 1/alum three times at intervals of 1 week. Five weeks after the final sensitization, we challenged the mice by instilling Cry j 1 intranasally from the day after intranasal histamine pretreatment. Soon after, we counted the number of sneezes. We then evaluated the infiltration of eosinophils into the nasal tissues and also measured the serum levels of antigen-specific IgE antibody. In addition, we confirmed the effects of ketotifen fumarate and dexamethasone hydrochloride on these animals. In Cry j 1-sensitized B10.S mice, sneezes, eosinophil peroxidase (EPO) activity in nasal tissues, and Cry j 1-specific IgE clearly increased after intranasal histamine pretreatment and 5 days of continuous intranasal Cry j 1 challenge. Both ketotifen and dexamethasone inhibited the increase in sneezing, and dexamethasone also inhibited EPO activity and Cry j 1-specific IgE. Thus we succeeded in establishing a new model of allergic rhinitis in Cry j 1-sensitized B10.S mice, which exhibited sneezing, eosinophil infiltration into the nasal mucosa, and Cry j 1-specific IgE production.     

Pathogenesis of murine experimental allergic rhinitis: a study of local and systemic consequences of IL-5 deficiency

Recent studies have demonstrated an important role for IL-5-dependent bone marrow eosinophil progenitors in allergic inflammation. However, studies using anti-IL-5 mAbs in human asthmatics have failed to suppress lower airway hyperresponsiveness despite suppression of eosinophilia; therefore, it is critical to examine the role of IL-5 and bone marrow responses in the pathogenesis of allergic airway disease. To do this, we studied the effects of IL-5 deficiency (IL-5(-/-)) on bone marrow function as well as clinical and local events, using an established experimental murine model of allergic rhinitis. Age-matched IL-5(+/+) and IL-5(-/-) BALB/c mice were sensitized to OVA followed by 2 wk of daily OVA intranasal challenge. IL-5(-/-) OVA-sensitized mice had significantly higher nasal mucosal CD4(+) cells and basophilic cell counts as well as nasal symptoms and histamine hyperresponsiveness than the nonsensitized group; however, there was no eosinophilia in either nasal mucosa or bone marrow; significantly lower numbers of eosinophil/basophil CFU and maturing CFU eosinophils in the presence of recombinant mouse IL-5 in vitro; and significantly lower expression of IL-5Ralpha on bone marrow CD34(+)CD45(+) progenitor cells in IL-5(-/-) mice. These findings suggest that IL-5 is required for normal bone marrow eosinophilopoiesis, in response to specific Ag sensitization, during the development of experimental allergic rhinitis. However, the results also suggest that suppression of the IL-5-eosinophil pathway in this model of allergic rhinitis may not completely suppress clinical symptoms or nasal histamine hyperresponsiveness, because of the existence of other cytokine-progenitor pathways that may induce and maintain the presence of other inflammatory cell populations.     

Calpeptin通过抑制GATA3减轻变应性鼻炎大鼠炎症

摘要 目的：本研究旨在评估钙蛋白酶抑制剂calpeptin减轻变应性鼻炎大鼠炎症的作用并探讨其机制。方法：将20只雄性SD大鼠采用数字表法随机分为4组：正常组（Normal）、变应性鼻炎组（AR）、地塞米松干预AR组（DXMS+AR）、calpeptin干预AR组（Calpeptin+AR）。造模成功后,对大鼠AR症状进行行为学评分,对大鼠鼻黏膜组织切片以HE和PAS染色法观察鼻黏膜病理改变;对大鼠外周血以ELISA法检测总IgE、IL-4、IL-13水平;对大鼠鼻黏膜组织以免疫蛋白印迹法检测GATA3蛋白表达水平。单因素方差分析进行多组间比较,LSD- t检验进行组间两两比较。结果：与Normal组相比,AR组大鼠的鼻部过敏症状、鼻黏膜嗜酸粒细胞计数及外周血总IgE水平均升高,Calpeptin与地塞米松均能减轻气道炎症,减少嗜酸性粒细胞浸润,降低血清中OVA诱导的IgE的生成。探讨机制发现,酶联免疫吸附试验检测Th2细胞因子,与Normal组比较,AR组血清IL-4、IL-13水平均升高（P＜0.05）,而Calpeptin与地塞米松均能降低血清IL-4、IL-13水平（P＜0.05）。免疫蛋白印迹法检测大鼠鼻黏膜GATA3蛋白表达水平显示,与Normal组比较,AR组鼻黏膜 GATA3表达升高（P＜0.05）,而Calpeptin与地塞米松组鼻黏膜 GATA3表达均下降（P＜0.05）。结论：腹腔注射calpeptin能够减轻变应性鼻炎大鼠局部和全身过敏反应,其机制可能下调GATA3表达,影响Th2细胞的分化及细胞因子的分泌有关。     

Ethyl pyruvate attenuates murine allergic rhinitis partly by decreasing high mobility group box 1 release

High-mobility group box 1 (HMGB1) protein, a pro-inflammatory DNA-binding protein, meditates inflammatory responses through Toll-like receptor-4 signals and amplifies allergic inflammation by interacting with the receptor for advanced glycation end products. Previous studies have shown that HMGB1 is elevated in the nasal lavage fluids (NLF) of children suffering from allergic rhinitis (AR) and is associated with the severity of this disease. Furthermore, HMGB1 has been implicated in the pathogenesis of lower airway allergic diseases, such as asthma. Ethyl pyruvate (EP) has proven to be an effective anti-inflammatory agent for numerous airway diseases. Moreover, EP can inhibit the secretion of HMGB1. However, few studies have examined the effect of EP on AR. We hypothesized that HMGB1 plays an important role in the pathogenesis of AR and studied it using an AR mouse model. Forty BALB/c mice were divided into four groups: the control group, AR group, 50 mg/kg EP group, and 100 mg/kg EP group. The mice in the AR and EP administration groups received ovalbumin (OVA) sensitization and challenge, whereas those in the control group were given sterile saline instead of OVA. The mice in the EP administration group were given an intraperitoneal injection of EP 30 min before each OVA treatment. The number of nasal rubbings and sneezes of each mouse was counted after final treatment. Hematoxylin–eosin staining, AB-PAS staining, interleukin-4 and 13 in NLF, IgE, and the protein expression of HMGB1 were measured. Various features of the allergic inflammation after OVA exposure, including airway eosinophilia, Th-2 cytokine production, total IgE, and goblet cell hyperplasia were significantly inhibited by treatment with EP and the expression and release of HMGB1 were reduced after EP administration in a dose-dependent manner. These results indicate that HMGB1 is a potential therapeutic target of AR and that EP attenuates AR by decreasing HMGB1 expression.     

Toll样受体2及Toll样受体4在大鼠实验性变应性鼻炎中的表达

目的研究Toll样受体2（Toll-like receptor2,TLR2）及Toll样受体4（TLR4）在实验性变应性鼻炎大鼠鼻黏膜中的表达及脂多糖（Lipopolysaccharide,LPS）对其表达的影响。方法SD大鼠48只随机分为正常对照组（A组）;变应性鼻炎组（B组）：经腹腔注射及鼻腔滴入卵清白蛋白（Ovalbumin,OVA）建立变应性鼻炎（Allergic rhinitis,AR）模型;变应性鼻炎＋LPS刺激组（C组）：大鼠激发成变应性鼻炎模型后再以LPS滴鼻。用逆转录聚合酶链反应（RT-PCR）方法检测鼻黏膜中TLR2 mRNA、TLR4 mRNA的表达。结果B、C组大鼠均成功激发为AR动物模型;各组鼻黏膜中均有TLR2 mRNA、TLR4 mRNA表达;各组间TLR2 mRNA的表达差异无统计学意义（P〉0．05）。B、C组TLR4 mRNA的表达较A组高（P〈0．01）;C组TLR4 mRNA表达较B组增高（P〈0．01）。结论AR大鼠有TLLR4的表达增高;LPS刺激后TLR4表达进一步增高,说明TLR4可能参与AR的发病。TLR2在AR大鼠中的表达未见增高;LPS刺激后。TLR2表达未见进一步增高,TLR2与变应性鼻炎的关系有待进一步研究。     

CRTH2 plays an essential role in the pathophysiology of Cry j 1-induced pollinosis in mice

PGD(2) is the major prostanoid produced during the acute phase of allergic reactions. Two PGD(2) receptors have been isolated, DP and CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells), but whether they participate in the pathophysiology of allergic diseases remains unclear. We investigated the role of CRTH2 in the initiation of allergic rhinitis in mice. First, we developed a novel murine model of pollinosis, a type of seasonal allergic rhinitis. Additionally, pathophysiological differences in the pollinosis were compared between wild-type and CRTH2 gene-deficient mice. An effect of treatment with ramatroban, a CRTH2/T-prostanoid receptor dual antagonist, was also determined. Repeated intranasal sensitization with Cry j 1, the major allergen of Cryptomeria japonica pollen, in the absence of adjuvants significantly exacerbated nasal hyperresponsive symptoms, Cry j 1-specific IgE and IgG1 production, nasal eosinophilia, and Cry j 1-induced in vitro production of IL-4 and IL-5 by submandibular lymph node cells. Additionally, CRTH2 mRNA in nasal mucosa was significantly elevated in Cry j 1-sensitized mice. Following repeated intranasal sensitization with Cry j 1, CRTH2 gene-deficient mice had significantly weaker Cry j 1-specific IgE/IgG1 production, nasal eosinophilia, and IL-4 production by submandibular lymph node cells than did wild-type mice. Similar results were found in mice treated with ramatroban. These results suggest that the PGD(2)-CRTH2 interaction is elevated following sensitization and plays a proinflammatory role in the pathophysiology of allergic rhinitis, especially pollinosis in mice.     

Mesenchymal stromal cells ameliorate acute allergic rhinitis in rats

Mesenchymal stromal cells (MSCs) have been extensively investigated as a potential antiinflammatory treatment in many inflammatory‐related diseases; however, it remains unclear whether MSCs could be used to treat acute allergic rhinitis. A rat model of allergic rhinitis was treated with MSCs. The effect of MSCs on the inflammation of allergic rhinitis was evaluated by sneezing, nose rubbing, the pathology of the nasal mucosa, and the expression of interleukin 4, tumour necrosis factor alpha, and immunoglobulin E in the serum of rats. Also, the population of MSCs isolated from umbilical cords of humans was evaluated to determine if they could inhibit the symptoms and inflammation of acute allergic rhinitis in a rat model. We observed that this population of cells inhibited sneezing, nose rubbing, and changes in the pathology of the nasal mucosa. Intriguingly, we observed that MSCs reduced the expression of interleukin 4, tumour necrosis factor alpha, and immunoglobulin E in the serum. Furthermore, MSCs reduced the expression of histamine and the recruitment of macrophages in the nasal mucosa of allergic rhinitis rats. We reasoned that the effect of MSCs on allergic rhinitis might be through its regulation of the secretion of related cytokines from macrophages during the process of acute allergic rhinitis. This work suggested that MSCs from the umbilical cords of humans could be used as a positive clinical therapy for the human disease.     

Gender differences in inflammatory proteins and pathways in seasonal allergic rhinitis

In model organisms, thousands of genes differ in expression between females and males. It is not known if differences on a similar scale are found in humans nor how this relates to disease. However, in allergic disease gender differences in the levels of both inflammatory cells and proteins have been shown. In this study, we found lower nasal fluid allergen-specific IgE in women than men with seasonal allergic rhinitis (SAR). This led to genome-wide analyses of gene expression in allergen-challenged CD4⁺ cells from patients with SAR before and after treatment with cortisone. Before treatment, 975 genes differed in expression between women and men: 337 were higher in women. After treatment only 428 genes and one pathway differed in expression. The genes that differed in expression between women and men were over-represented in 10 pathways. Five of the pathways regulated chemotaxis. All five were less active in women. One of the pathways was induced by the eosinophilic chemokine CCL4. Analysis of nasal fluid CCL4 protein confirmed lower levels in women with seasonal allergic rhinitis, before and during the pollen season. By contrast, nasal fluid CCL3 levels did not differ between the genders. In summary, this study shows gender differences in specific inflammatory pathways and proteins in patients with seasonal allergic rhinitis. Further studies are warranted to examine if such differences have diagnostic and therapeutic implications in allergic diseases.     

Macrophage migration inhibitory factor (MIF) contributes to the development of allergic rhinitis

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine whose expression has been found to be critical to the generation of antigen-specific immune responses. Recent studies suggested that MIF played a role in the initiation and maintenance of allergic diseases. To elucidate MIF's role in the pathogenesis of allergic rhinitis (AR), we sensitized MIF-deficient gene knockout (KO) mice and wild-type (WT) mice intraperitoneally with ovalbumin (OVA) and compared their clinical symptoms and allergic responses after intranasal challenge. Antigen-induced nasal symptoms were significantly reduced in MIF KO mice compared to WT mice. Histological examination of nasal mucosa showed that the number of infiltrating eosinophils in MIF KO mice was significantly lower than that in WT mice (P < 0.05). The concentration of TNF-alpha in nasal mucosa was also significantly lower in MIF KO mice than in WT mice (P < 0.05). We have demonstrated that the absence of MIF affects several aspects of experimental AR. One mechanism by which these effects might be mediated is by down regulating TNF-alpha. The block of allergic inflammation in MIF KO mice suggests that MIF may play a role in the allergic response.     

Inhalation of toluene diisocyanate vapor induces allergic rhinitis in mice

Diisocyanates are the leading cause of occupational asthma, and epidemiological evidence suggests that occupational rhinitis is a comorbid and preceding condition in patients who develop asthma. The goal of the present studies was to develop and characterize a murine model of toluene diisocyanate (TDI)-induced rhinitis. Female C57BL/6 mice were exposed to workplace-relevant concentrations of TDI vapor via inhalation for 4 h/day for 12 days with or without a 2-wk rest period and TDI challenge. Mice exposed 12 consecutive weekdays to 50 parts per billion TDI vapor showed elevated total serum IgE and increased TDI-specific IgG titers. Breathing rates were decreased corresponding with increased inspiratory time. TDI exposure elevated IL-4, IL-5, IL-13, and IFN-gamma mRNA expression in the nasal mucosa, suggesting a mixed Th1/Th2 immune response. Expressions of mRNA for proinflammatory cytokines and adhesion molecules were also up-regulated. These cytokine changes corresponded with a marked influx of inflammatory cells into the nasal mucosa, eosinophils being the predominant cell type. Removal from exposure for 2 wk resulted in reduced Ab production, cytokine mRNA expression, and cellular inflammation. Subsequent challenge with 50 parts per billion TDI vapor resulted in robust up-regulation of Ab production, cytokine gene expression, as well as eosinophilic inflammation in the nasal mucosa. There were no associated changes in the lung. The present model shows that TDI inhalation induces immune-mediated allergic rhinitis, displaying the major features observed in human disease. Future studies will use this model to define disease mechanisms and examine the temporal/dose relationship between TDI-induced rhinitis and asthma.     

CXCR1+CD4+ T cells in human allergic disease

Chemokine receptors play an important role in the migration of leukocytes to sites of allergic inflammation in humans. In this study, we have identified increased expression of the chemokine receptor CXCR1 on CD4+ T lymphocytes derived from patients with atopic disease compared with normal donors. Enhanced expression of CXCR1 by atopic donors was identified on freshly isolated peripheral blood cells and on expanded cell populations derived from nasal mucosal biopsies and from the periphery. Identification of CXCR1 expression on CD4 cells in the nasal mucosa was confirmed by double immunofluorescence. In addition, expression of CXCR1 was dramatically decreased in patients undergoing successful treatment of allergic rhinitis by specific immunotherapy. CXCR1 provided a functional receptor capable of regulating T cells in the context of allergic disease, since expression of CXC chemokine ligand 8 was up-regulated at the site of allergic inflammation and freshly isolated CXCR1+CD4+ cells from atopic donors showed an enhanced functional response to this ligand. CXCR1 expression on CD4+ T cells was increased in vitro in response to the pro-Th2 cytokine IL-4. Phenotypic analysis reveals that IFN-gamma expression was lower in the CXCR1+CD4+ cells. The identification of CXCR1 as a marker of allergic rhinitis reveals a possible target for therapeutic intervention in atopic disease.     

Expression of Toll-like Receptor 9 in nose, peripheral blood and bone marrow during symptomatic allergic rhinitis

Background

Allergic rhinitis is an inflammatory disease of the upper airway mucosa that also affects leukocytes in bone marrow and peripheral blood. Toll-like receptor 9 (TLR9) is a receptor for unmethylated CpG dinucleotides found in bacterial and viral DNA. The present study was designed to examine the expression of TLR9 in the nasal mucosa and in leukocytes derived from different cellular compartments during symptomatic allergic rhinitis.

Methods

The study was based on 32 patients with seasonal allergic rhinitis and 18 healthy subjects, serving as controls. Nasal biopsies were obtained before and after allergen challenge. Bone marrow, peripheral blood and nasal lavage fluid were sampled outside and during pollen season. The expression of TLR9 in tissues and cells was analyzed using immunohistochemistry and flow cytometry, respectively.

Results

TLR9 was found in several cell types in the nasal mucosa and in different leukocyte subpopulations derived from bone marrow, peripheral blood and nasal lavage fluid. The leukocyte expression was generally higher in bone marrow than in peripheral blood, and not affected by symptomatic allergic rhinitis.

Conclusion

The widespread expression of TLR9 in the nasal mucosa along with its rich representation in leukocytes in different compartments, demonstrate the possibility for cells involved in allergic airway inflammation to directly interact with bacterial and viral DNA.     

CD23-dependent transcytosis of IgE and immune complex across the polarized human respiratory epithelial cells

IgE-mediated allergic inflammation occurs when allergens cross-link IgE on the surface of immune cells, thereby triggering the release of inflammatory mediators as well as enhancing Ag presentations. IgE is frequently present in airway secretions, and its level can be enhanced in human patients with allergic rhinitis and bronchial asthma. However, it remains completely unknown how IgE appears in the airway secretions. In this study, we show that CD23 (FcεRII) is constitutively expressed in established or primary human airway epithelial cells, and its expression is significantly upregulated when airway epithelial cells were subjected to IL-4 stimulation. In a transcytosis assay, human IgE or IgE-derived immune complex (IC) was transported across a polarized Calu-3 monolayer. Exposure of the Calu-3 monolayer to IL-4 stimulation also enhanced the transcytosis of either human IgE or the IC. A CD23-specific Ab or soluble CD23 significantly reduced the efficiency of IgE or IC transcytosis, suggesting a specific receptor-mediated transport by CD23. Transcytosis of both IgE and the IC was further verified in primary human airway epithelial cell monolayers. Furthermore, the transcytosed Ag-IgE complexes were competent in inducing degranulation of the cultured human mast cells. Because airway epithelial cells are the first cell layer to come into contact with inhaled allergens, our study implies CD23-mediated IgE transcytosis in human airway epithelial cells may play a critical role in initiating and contributing to the perpetuation of airway allergic inflammation.