OBJECTIVEThe aim of the study was to investigate whether Orai1 antibody intraperitoneal injection could improve the condition of allergic rhinitis (AR) in mice.

METHODSTwenty-four BALB/C mice (SPF grade) were classified into 4 groups (AR group, Control group, Experimental group 1 and experimental group 2) according to a random number table. A mouse model of AR was established (Control group was established by phosphate buffered solution), and experimental group 1 and Experimental group 2 were established through intraperitoneal injection of 100 μg and 150 μg Orai1 antibody respectively. The number of sneezing and rubbing and eosinophilia in mice were assessed after different doses of Orai1 antibody intraperitoneal injection were applied. Then Orai1 protein and its mRNA in nasal mucosa, histomine, eosionphil cation protein (ECP), interlukin (IL)-1β, IL-4, IL-5 and IL-6 and their mRNA in nasal lavage fluid (NLF) and nasal mucosa were evaluated using enzyme linked immunosorbent assay (ELISA) and real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). Furthermore, Orai1 protein and its mRNA in Th2 cells in peripheral blood, IL-4 and IL-5 in peripheral serum and their mRNAs in Th2 cells were also examined through ELISA and real-time RT-PCR. The data were analyzed by a statistical software of Graph Pad Prism 5.

RESULTSThere were significant differences in sneezing, nasal rubbing and local invading eosinophils in nasal mucosa after the treatment (t100 μg=7.88, t100 μg=9.92, t100 μg=4.30, respectively; t150 μg=16.43, t150 μg=16.31, t150 μg=9.35, respectively, all P-values<0.01). The Orai1 antibody intervention decreased contents of Orai1 in nasal mucosa, histomine, ECP, IL-1β, IL-4, IL-5 and IL-6. The contents of experimental group 1 were (0.186±0.015) μg/ml, (6.618±0.180) ng/ml, (2.555±0.031) ng/ml, (85.26±2.94) pg/ml, (55.12±1.21) pg/ml, (58.45±2.11) pg/ml and (77.12±2.13) pg/ml, respectively. The contents of experimental group 2 were (0.089±0.003) μg/ml, (4.501±0.310) ng/ml, (1.260±0.017) ng/ml, (48.49±2.12) pg/ml, (33.15±0.87) pg/ml, (38.24±0.95) pg/ml and (51.72±0.81) pg/ml, respectively. The differences were siginificant between group 1, group 2 and AR group(t value was 3.29, 10.44, 9.45, 17.53, 74.53, 87.06, 3.98; 8.54, 13.32, 23.00, 20.89, 80.73, 103.70, 13.34, all P<0.01). However, there were no significant differences in Orai1 protein and its mRNA in peripheral Th2 cells, IL-4 and IL-5 in peripheral serum and their mRNAs in Th2 cells (all P-values>0.05). In addition, the effect of 150 μg Orai1 antibody treatment was better than 100 μg one (all P-values<0.05).

CONCLUSIONOrai1 antibody intraperitoneal injection can improve the symptoms of AR mice, and alleviate the condition of allergic inflammation. Orai1 may become a novel aim in the AR study.

Animals ; Antibodies ; pharmacology ; Calcium Channels ; immunology ; metabolism ; Cytokines ; immunology ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Eosinophilia ; therapy ; Eosinophils ; immunology ; Immunotherapy ; Inflammation ; Injections, Intraperitoneal ; Mice ; Mice, Inbred BALB C ; Nasal Mucosa ; metabolism ; ORAI1 Protein ; RNA, Messenger ; Rhinitis, Allergic ; therapy ; Th2 Cells ; immunology

OBJECTIVE: To explore the changes of microRNAs in nasal mucosa after the specific immunotherapy (SIT) for allergic rhinitis (AR) in mice. METHOD: Female BALB/c mice, 6-8 weeks of age, were randomly divided into control group, model group and treatment group. AR model were established by intraperitoneal injection and intranasal challenge of ovalbumin and SIT was performed by inguinal subcutaneous injections. AR symptom scores were documented. The eosinophils (EOS) in the nasal mucosa were measured. Ovalbumin-specific IgE (OVA-sIgE) in the serum and expression of interferon-γ and interleukin-4 in the nasal lavage were measured by enzyme-linked immunosorbent assay meanwhile the ratio of interferon-γ and interleukin-4 was calculated. The microRNAs in the nasal mucosa were preliminary screened by microRNA gene microarray. Comparing with model group, the Fold changes of microRNA of the treatment group were ≥ 2.0 and the P < 0.05. MicroRNA target genes were predicted with GeneSpring 12.5 software. We took the intersection between genes in the signal pathway which associated with immune response,inflammation and target genes. The MEV-4-6-0 and Cytoscape_v2. 8. 2. software was applied to perform the cluster analysis and target gene regulatory networks maps. RESULT: The model of AR in mice and its SIT were successful. Comparing with the model group, the Fold changes of 15 microRNAs, of which 9 microRNAs were up-regulated and 6 microRNAs were down-regulated, were ≥ 2.0 in treatment group (P < 0.05). Cluste analysis showed clearly that microRNAs in the treatment group and model group respectively aggregated in two branches. The 15 microRNAs had 5302 target genes, of which, 451 genes were related more with SIT by the intersection. One microRNA can regulate many target genes, and one gene can also be affected by many microRNAs. Their synergistic effects may be involved in the mechanism of SIT. CONCLUSION The expressions of microRNAs are changed in nasal mucosa after SIT for AR in mice and we can speculate that microRNAs are involved in the process of SIT for AR. Bioinformatics methods can diminish the scope of target genes of microRNAs, which will help us studying the effect of changed microRNA on its relative target genes after SIT, and make us better understanding the mechanism of the disease and its SIT.
Administration, Intranasal ; Animals ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Eosinophils ; immunology ; Female ; Immunoglobulin E ; blood ; Immunotherapy ; Interferon-gamma ; immunology ; Interleukin-4 ; immunology ; Mice ; Mice, Inbred BALB C ; MicroRNAs ; metabolism ; Nasal Mucosa ; drug effects ; metabolism ; Ovalbumin ; Rhinitis, Allergic ; therapy

OBJECTIVE: To investigate the influence of budesonide on animal model of minimal persistent inflammation (MPI) of allergic rhinitis in rats and to investigate the changes of interleukin-12 (IL-12) in nasal mucosa. METHOD: Sixty Sprague-Dawley (SD) rats were randomly divided into four groups: group A (allergic rhinitis group), B (experimental group), C (MPI model group) and D (bland group) respectively, with fifteen animals in each group. Rats from group A,B and C were sensitized intraperitoneally by injection of suspension of ovalbumin (OVA) and aluminum hydroxide in 0.9% physiological saline. Then, repeated local booster sensitization with different concentration of OVA suspension (1% and 0.01%) or physiological saline into the nasal cavity of those rats were performed. For group D, physiological saline was used only. From 36th day, group B were given budesonide treatment for three weeks. A, C and D group were given normal saline nasal spray. Symptoms (sneezing) of rats after antigen challenge were observed and the infiltration of eosinophils (EOS) together with the expression of intercellular adhesion molecule 1 (ICAM-1) and IL-12 in the nasal epithelial cells were also examined. RESULT: When challenged with 1% OVA, the sneezing number of rats in group B was increased markedly than that in group D (P < 0.05). However, there was no difference between group B, A and C (P > 0.05). When challenged with 0.01% OVA and given budesonide, the symptom of sneezing almost disappeared in group B just like that in group D and there was no difference between the two groups (P > 0.05). Besides, there was still more EOS infiltrated in the nasal mucosa of rats in group C than that in group D (P < 0.05). There was no expression of ICAM-1 in nasal epithelium of rats in group D, nevertheless, ICAM-1 was found mildly expressed in group C. IL-12 expression was significantly increased compared with group A and group C, and was no significantly difference compared with bland group (P > 0.05). CONCLUSION Budesonide significantly inhibited the late reaction of animal model of minimal persistent inflammation (MPI) of allergic rhinitis in rats and increase the expression of IL-12 in MPI model.
Allergens ; Animals ; Budesonide ; pharmacology ; Disease Models, Animal ; Eosinophils ; immunology ; Inflammation ; drug therapy ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-12 ; metabolism ; Leukocyte Count ; Nasal Mucosa ; metabolism ; Ovalbumin ; Rats ; Rats, Sprague-Dawley ; Rhinitis, Allergic ; drug therapy

OBJECTIVE: To investigate the expression and role of Interleukin-33 (IL-33) and ST2 in the nasal polyps of human Eosinophilic and non-Eosinophilic chronic rhinosinusitis with nasal polyps (ECRS and non-ECRS). METHOD: IL-33 and ST2 protein expression in nasal polyps of ECRS and non-ECRS as well as in seemingly normal mucosa of the inferior turbinate tissue was investigated by immunohistochemical staining and messenger RNA (mRNA) expression of IL-33 and ST2 was assessed by realtime polymerase chain reaction (PCR) in 27 subjects with ECRS, 33 subjects with non-ECRS, and 11 control subjects. RESULT: (1) The ST2 was found both in nasal polyps of ECRS and non-ECRS,especially in ECRS, yet hardly found in the normal mucosa of the inferior turbinate tissue; (2) The expression of ST2 mRNA in nasal polyps of ECRS was higher than that in non-ECRS and normal inferior turbinate tissue, and the difference was both prominent in statistics (P<0.01); (3) The expression patterns of IL-33 at both mRNA and protein levels were not significantly different among the three groups (P>0.05). CONCLUSION The IL-33 and its receptor ST2 were both expressed in human nasal polyps including ECRS and non-ECRS, meanwhile the expression patterns of ST2 at both mRNA and protein levels were significantly higher in nasal polyps of ECRS. The current study suggests that IL-33 and its receptor ST2 may play important roles in the pathogenesis of chronic rhinosinusitis with nasal polyps, especially in ECRS through the increased expression of ST2 in Eosinophils as a hypothesis.
Chronic Disease ; Eosinophils ; immunology ; Humans ; Interleukin-1 Receptor-Like 1 Protein ; Interleukin-33 ; metabolism ; Nasal Mucosa ; metabolism ; Nasal Polyps ; immunology ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Receptors, Cell Surface ; metabolism ; Rhinitis ; immunology ; Sinusitis ; immunology ; Turbinates ; metabolism

OBJECTIVE: To explore the role of the innate immune factors TLR2 and TLR4 in the pathogenesis of chronic rhinosinusitis (CRS) by detecting their expression in different clinical types of CRS and the normal control group. METHOD: Immunohistochemistry was used to detect the expression of TLR2 and TLR4 respectively in 21 cases (chronic rhinosinusitis with nasal polyps, CRSwNP) group, 15 cases (chronic rhinosinusitis without nasal polyos, CRSsNP) group, 11 cases recurrent CRSwNP group and 13 cases control group. Positive cells were counted under the microscope artificially, Mann-Whitney U analysis was applied for the ranked data, and one-way anova analysis was adopted to analyze the experimental group and control group. RESULT: (1) TLR2 and TLR4 expression had the same characteristics. Expression mainly concentrated in parts of the whole layer of epithelial basement membrane, cytoplasm of glandular cells, very few inflammatory cells such as monocytes and plasma cells in the cytoplasm, sometimes unknown cell nuclei positive expression. (2) The glandular cells were stained manual counting and color grading. TLR2 and TLR4 packet application Wilcoxon rank test Mann-Whitney U test analysis was not statistically significant (P > 0.05), measurement data within the group variance statistical difference between the groups (P < 0.05). CONCLUSION The Nasal mucosa can produce the innate immune factors TLR2 and TLR4. The different expression of TLR2 and TLR4 in the various clinical types of CRS suggests that they play the certain role in the pathogenesis of CRS.
Chronic Disease ; Epithelial Cells ; immunology ; metabolism ; Female ; Humans ; Immunohistochemistry ; Male ; Nasal Mucosa ; immunology ; metabolism ; Nasal Polyps ; immunology ; metabolism ; Rhinitis ; immunology ; metabolism ; Sinusitis ; immunology ; metabolism ; Toll-Like Receptor 2 ; metabolism ; Toll-Like Receptor 4 ; metabolism

OBJECTIVE: In this study, we investigated the anti-inflammation effects of Xuebijing in OVA-induced murine allergic rhinitis model. Furthermore, we determined whether heme oxygenase (HO)-1 is required for the protective activity of Xuebijing. METHOD: Airways of OVA-sensitized mice exposed to OVA challenge developed eosinophilia, mucus hypersecretion and increased cytokine levels. Levels of interleukin IL-4, IL-5, IL-13, and tumor necrosis factor (TNF)-alpha in nasal lavage fluid were measured using enzyme-linked immunosorbent assays (ELISAs). Lung tissue and nasal mucosa sections were stained with Mayer's hematoxylin and eosin for assessment of cell infiltration and mucus production, Immunohistochemistry, Real-time PCR and Western Blot analyses for HO-1 protein expression. RESULT: Orally administered Xuebijing significantly inhibited the number of OVA-induced inflammatory cells and IgE production, along with reduced T-helper (Th) 2 cytokine levels, such as IL-4, IL-5 and IL-13, improved the level of IFN-gamma, in nasal lavage fluid. In addition, Xuebijing induced a marked decrease in OVA-induced inflammatory cell infiltration and mucus production in nasal and lung tissues. These effects were correlated with HO-1 mRNA and protein induction. CONCLUSION Our results indicate that Xuebijing protects against OVA-induced airway inflammation, at least in part, via HO-1 upregulation.
Animals ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Eosinophilia ; metabolism ; Heme Oxygenase-1 ; metabolism ; Immunoglobulin E ; immunology ; Inflammation ; drug therapy ; metabolism ; Interleukin-13 ; metabolism ; Interleukin-4 ; metabolism ; Interleukin-5 ; metabolism ; Membrane Proteins ; metabolism ; Mice ; Nasal Mucosa ; metabolism ; Rhinitis, Allergic ; Rhinitis, Allergic, Perennial ; chemically induced ; drug therapy ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVE: The aim of this study was to explore the changes of the extracellular matrix in nasal mucosa by a guinea pig model of prolonged allergic-induced rhinitis. METHOD: Thirty-two male Hartley guinea pigs were randomly divided into four groups: allergen challenged groups (Group 2 w, Group 6 w and Group 12 w) and a control group. Ovalbumin-sensitized guinea pigs were repeatedly challenged with allergen twice a week from 2 weeks to 12 weeks. Matched control groups were challenged with physiological saline. Nasal mucosa were obtained from the animals killed. Hematoxylin-Eosin, Masson's trichrome, and immunohistochemical staining against transforming growth factor-beta1 (TGF-beta1), Collagen III and Collagen I were performed to nasal mucosa. RESULT: (1) Pathological examination showed obvious infiltration of eosinophils and the enlarged thickness of epithelial layer of nasal mucosa in the experiment groups. (2) The area ratios of blue stained in the extracellular matrix of nasal mucosa were increased. The area ratios of blue stained were statistically different in Group 6 w and Group 12 w compared with the control group. (3) The increasing absorbance of TGF-beta1 were statistically different in the experiment groups with the control group. The absorbance of Collagen III and Collagen I showed a rising trend along prolonged allergen challenged in the experiment groups. CONCLUSION Prolonged allergen challenge and the inflammation of nasal mucosa, can lead to the increasing of the inflammation relevant factors and the deposit of collagen in the extracellular matrix of nasal mucosa.
Allergens ; immunology ; Animals ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Eosinophils ; immunology ; Extracellular Matrix ; immunology ; metabolism ; pathology ; Guinea Pigs ; Inflammation ; Male ; Nasal Mucosa ; immunology ; metabolism ; pathology ; Rhinitis, Allergic, Perennial ; immunology ; metabolism ; pathology ; Transforming Growth Factor beta1 ; metabolism

Orai1 is the key subunit of the Ca2+-release-activated Ca2+ channel. Our previous report has demonstrated that Orai1 expression in the airway was upregulated in the ovalbumin (OVA)-induced allergic rhinitis (AR) mouse models. To observe whether inhibition of Orai1 expression in the airway could suppress symptoms in a murine model of AR and to assess the impacts of this inhibition on the responses of local and systemic immunocytes, we administered recombinant lentivirus vectors that encoded shRNA against ORAI1 (lenti-ORAI1) into the nostrils of OVA-sensitized mice before the challenges, and analyzed its effect on allergic responses, as compared with the unsensitized mice and untreated AR mice. Administration of lenti-ORAI1 into the nasal cavity successfully infected cells in the epithelial layer of the nasal mucosa, and significantly decreased the frequencies of sneezing and nasal rubbing of the mice. Protein levels of leukotriene C4, OVA-specific IgE, and IL-4 in the nasal lavage fluid and serum and eosinophil cation protein in the serum were also significantly reduced by lenti-ORAI1, as were the mRNA levels of these factors in the nasal mucosa and spleen. These data suggested that administration of lenti-ORAI1 into the nasal cavity effectively decreased Orai1 expression in the nasal mucosa, alleviated AR symptoms, and partially inhibited the hyperresponsiveness of the local and systemic immune cells including T cells, B cells, mast cells and eosinophils that are involved in the pathogenesis of AR.
Animals ; Calcium Channels/analysis/*genetics/immunology ; *Down-Regulation ; Eosinophil Cationic Protein/blood/genetics ; Glutathione Transferase/blood/genetics/immunology ; Immunoglobulin E/blood/genetics/immunology ; Interleukin-4/blood/genetics/immunology ; Lentivirus/genetics ; Mice ; Mice, Inbred BALB C ; Nasal Mucosa/immunology/metabolism ; Ovalbumin/immunology ; RNA, Messenger/genetics ; RNA, Small Interfering/*administration & dosage/genetics ; Rhinitis, Allergic, Perennial/*genetics/immunology ; Spleen/immunology/metabolism ; *Transfection

BACKGROUNDAllergic rhinitis (AR) is a Th2 dominant cytokine response. Chloride channel-3 (ClC-3) plays an important role in nasal mucosal edema and inflammatory pathologic changes in AR. Antiallergic herbal agents (AHA) are antiallergic herbal products. In the previous study, we have demonstrated that AHA clearly inhibited allergic medium and relieved allergic reaction of AR. The aim of this study was to evaluate the function of ClC-3 and discuss the possible therapeutic effects of AHA on immune microenvironment in AR.

METHODSAHA were produced and used to treat AR. An animal model of an AR rabbit was established by ovalbumin (OVA). The rhinitis rabbits were randomly divided into three groups: AHA treated group (AHATG), model group (MG) and healthy control group (HCG). The expressions of ClC-3 protein were examined by immunohistochemical method. The mucosal epithelial cells of all the rabbit groups were primarily cultured with tissue culture method in vitro with or without rhIL-4 or rhIL-2. Furthermore, the expressions of ClC-3 mRNA were detected by real-time PCR. The levels of monocyte chemotactic factor-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1) protein in culture supernatants were measured by ELISA.

RESULTSThe expressions of ClC-3 mRNA increased more in mucosal epithelial cells of MG than those in AHATG and HCG (P < 0.01). The levels of ClC-3 mRNA, MCP-1 and VCAM-1 protein in culture supernatants of MG were significantly higher than those in the other two groups (P < 0.01). Those were significantly increased in MG untreated 12 hours later than those in other two groups (P < 0.01). The expressions of ClC-3 mRNA, MCP-1 and VCAM-1 protein in culture supernatants of MG and HCG treated with rhIL-4 were significantly higher than those in the AHATG treated with rhIL-4 (P < 0.01). The levels of ClC-3 mRNA, MCP-1 and VCAM-1 protein in culture supernatants of all groups treated with rhIL-2 showed no significant changes (P > 0.05).

CONCLUSIONSAHA can inhibit the secretions of ClC-3, MCP-1 and VCAM-1 in mucosal epithelia and improve inflammatory reaction of AR. ClC-3 plays an important role in the secretion of cytokines and mucosal inflammatory response in AR. RhIL-4 can enhance the secretion of ClC-3, MCP-1 and VCAM-1 in mucosal epithelial cells, especially during the AR process. These enhanced effects of rhIL-4 were significantly suppressed by AHA. The secretions of ClC-3, MCP-1 and VCAM-1 can not be induced obviously by rhIL-2 in mucosal epithelial cells in AR.

Animals ; Anti-Allergic Agents ; pharmacology ; Chemokine CCL2 ; metabolism ; Chloride Channels ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Immunohistochemistry ; Male ; Mucous Membrane ; drug effects ; immunology ; metabolism ; Nasal Mucosa ; drug effects ; immunology ; metabolism ; Polymerase Chain Reaction ; Rabbits ; Random Allocation ; Rhinitis ; chemically induced ; immunology ; metabolism ; Vascular Cell Adhesion Molecule-1 ; metabolism

OBJECTIVE: To observe expression and distribution of histamine H4 receptor in nasal mucosa in normal people and allergic rhinitis patients,and understand role of histamine H4 receptor in allergic rhinitis. METHOD: Select normal people and allergic rhinitis patients each 10, take the nasal mucosa, detect expression and distribution of histamine H4 receptor at proteins and transcription level respectively by immunohistochemical method and RT-PCR, and compared. RESULT: Histamine H4 receptor at proteins and transcription level were found in normal nasal mucosa (25 509 +/- 6 441, 0.42 +/- 0.08), increased significantly in nasal mucosa of allergic rhinitis patients (49 676 +/- 8 541, 0.69 +/- 0.11, P < 0.05), which in structural cells and immune cells. CONCLUSION Histamine H4 receptors exist in normal nasal mucosa, its express significantly enhance, flew histamine H4 receptor may be mediated histamine in pathogenesis of allergic rhinitis ,who is one of the ligands of histamine.
Case-Control Studies ; Humans ; Nasal Mucosa ; immunology ; metabolism ; pathology ; Receptors, G-Protein-Coupled ; metabolism ; Receptors, Histamine ; metabolism ; Receptors, Histamine H4 ; Rhinitis, Allergic, Perennial ; immunology ; metabolism ; pathology