OBJECTIVETo investigate the activation of Toll like receptor 4 (TLR4) on passively sensitized human airway smooth muscle cells (HASMCs) proliferation and the synthesis and secretion function.

METHODSThrough the cultivation of primary HASMCs, we studied TLR4 expression on cell surface, cell proliferation and transformation of parturient factor-beta1 (TGF-beta1) in asthma under the condition of synthesis and secretion level by passively sensitized HASMCs with asthma serum.

RESULTSCompared with the control group, in passive sensitized group and TNF-alpha group TLR4 expression were significantly increased (P < 0.01), significantly enhanced proliferation (P < 0.01), total protein concentration, IgE secretion and TGF-beta1 were significantly higher (P < 0.01); and all the above parameters were increased more significantly in TNF group compared with those in the target effect of passively group; and those parameters were significantly reduced in anti-TLR4 antibody group compared with those in the target effect both of passively sensitized group and TNF-alpha group.

CONCLUSIONTLR4 on passively sensitized HASMCs activated can induce the excessive proliferation of HASMCs and a large number of synthesis and secretion of TGF-beta1, resulting in changing airway micro-environment, which involved in airway remodeling in asthma.

Airway Remodeling ; Asthma ; metabolism ; pathology ; Bronchi ; cytology ; Cell Proliferation ; Cells, Cultured ; Humans ; Myocytes, Smooth Muscle ; cytology ; metabolism ; Toll-Like Receptor 4 ; immunology ; Transforming Growth Factor beta1 ; metabolism

BACKGROUNDAsthma is a complex disease involving genetic and environment interactions. Atopy is a strong risk factor for asthma. The airway epithelium not only forms a physical barrier but also provides immune defense against harmful materials. To explore the effects of airway epithelium on asthma, we hypothesized that environmental injuries could act on bronchial epithelial cells and damage the physical barrier, which might facilitate allergens to stimulate immunoreactions and play an important role in the pathogenesis of asthma.

METHODSThirty eight-week-old male Wistar rats were randomly divided into five groups with six rats in each group: control group, asthma group, ovalbumin (OVA) + OVA group, lipopolysaccharide (LPS) group and LPS + OVA group. In the control group, 0.9% saline was injected intraperitoneally on day 1. Fourteen days later, the rats were exposed to aerosolized 0.9% saline. In the asthma group, the rats were sensitized with an injection of 10 mg of OVA, followed by an aerosolized 2% OVA challenge 14 days later. The OVA + OVA group was sensitized by an inhalation 2% OVA, 20 minutes a day, from day 1 to day 7, and then OVA challenged in the same way as the asthma group. In the LPS group, LPS (200 µl, 1 µg/µl) was given by airway on day 1 and day 3, with a simultaneous aerosol inhalation of 2% OVA for 20 minutes a day from day 1 to day 7. Fourteen days later, the rats were challenged with saline as in the control group. While in the LPS + OVA group, LPS (200 µl, 1 µg/µl) was given by airway on day 1 and day 3, with a simultaneous aerosol inhalation of 2% OVA for 20 minutes a day from day 1 to day 7. Fourteen days later, the rats were challenged with OVA as in the asthma group. The expression of interleukin (IL)-4, interferon-gamma (IFN-γ) and thymic stromal lymphopoietin (TSLP) in the lungs was detected by reverse transcription polymerase chain reaction (RT-PCR) and the pulmonary pathological changes were also observed. The level of IL-4, IFN-γ and IgE in plasma was detected by enzyme-linked immunosorbent assay (ELISA). Bronchoalveolar lavage fluid (BALF) was collected to conduct differential cell counts. Flow cytometry analysis was also used to count Th1 and Th2 cells.

RESULTSThe pathological changes in the LPS + OVA group were similar to the asthma group, while in other groups, the pathological changes were not obvious. The ratio of lymphocytes in BALF, IL-4/IFN-γ in plasma and the expression of the TSLP and IL-4 in the asthma and LPS + OVA groups were higher than in the control group and the OVA + OVA group (P < 0.05). The level of IgE was higher in the asthma, LPS and LPS + OVA groups than in the control group and the OVA + OVA group (P < 0.05). By flow cytometry analysis, the Th1/Th2 ratio was lower in the LPS + OVA and asthma groups than in other groups (P < 0.05).

CONCLUSIONSThe experiment results show that the injury to the bronchial epithelial layer may be the initial event of allergic responses. This finding implies that a rational approach to therapeutics would be to increase the resistance of the airways to environmental injuries rather than concentrating on suppressing inflammation.

Animals ; Bronchi ; pathology ; Cell Count ; Cytokines ; physiology ; Disease Models, Animal ; Epithelial Cells ; pathology ; Hypersensitivity ; etiology ; Immunoglobulin E ; blood ; Interferon-gamma ; blood ; Interleukin-4 ; blood ; Lipopolysaccharides ; toxicity ; Male ; Ovalbumin ; immunology ; Rats ; Rats, Wistar

OBJECTIVETo investigate the effect of prostaglandin D2 receptor antagonists on the airway inflammation in rats with asthma.

METHODSForty male SD rats were randomly divided into four groups: Group A (normal control), Group B (asthma group), Group C (CRTH2 antagonist BAYu3405 treatment group), Group D (DP1 antagonist BWA868C treatment group). Asthma was induced by ovalbumin (OVA) challenge. The rats in each group were sacrificed 24 h after the last challenge of OVA.DP1/CRTH2 receptors on eosinophils (EOS) were measured by radiological binding assay (RBA). The left lungs were used for histological examinations and bronchoalveolar lavage fluid (BALF) was collected from the right lungs. The total cell numbers, EOS absolute count and differential cell counts in BALF were performed. Serum concentrations of IL-4, 5 and IFN-gamma were measured by ELISA.

RESULTSRats in BAYu3405 treatment group showed profoundly decreased infiltrates of EOS and lymphocytes in the wall of bronchus when compared with those of asthma group and BWA868C treatment group. Serum concentrations of IFN-gamma in rats of BAYu3405 treatment group increased, but IL-4 and IL-5 decreased significantly when compared with those in rats of asthma group and BWA868C treatment group (P<0.01), and BALF EOS count was decreased significantly (P<0.01). Peripheral blood EOS count was higher than that in rats of normal control group, but was not significantly different from that in rats of asthma group and BWA868C treatment group. The combining capacity of CRTH2 and DP total combining capacity on EOS in asthma group, BAYu3405 treatment group and BWA868C treatment group were significantly higher than those in Group A (P<0.01). There was no significant difference in DP1 among all the groups (P>0.05).

CONCLUSIONCRTH2, but not DP1 antagonist can effectively ameliorate airway inflammation in rats with asthma.

Animals ; Asthma ; chemically induced ; drug therapy ; pathology ; Bronchi ; immunology ; pathology ; Carbazoles ; pharmacology ; therapeutic use ; Inflammation ; drug therapy ; Male ; Ovalbumin ; Prostaglandin D2 ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Immunologic ; antagonists & inhibitors ; Receptors, Prostaglandin ; antagonists & inhibitors ; Sulfonamides ; pharmacology ; therapeutic use

PURPOSE: Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that has been implicated in many aspects of the airway pathology in asthma. TNF-alpha blocking strategies are now being tried in asthma patients. This study investigated whether TNF-alpha blocking therapy inhibits airway inflammation and airway hyperresponsiveness (AHR) in a mouse model of asthma. We also evaluated the effect of TNF-alpha blocking therapy on cytokine production and adhesion molecule expression. MATERIALS AND METHODS: Ovalbumin (OVA) sensitized BALB/c female mice were exposed to intranasal OVA administration on days 31, 33, 35, and 37. Mice were treated intraperitoneally with soluble TNF-alpha receptor (sTNFR) during the OVA challenge. RESULTS: There were statistically significant decreases in the numbers of total cell and eosinophil in bronchoalveolar lavage fluid (BALF) in the sTNFR treated group compared with the OVA group. However, sTNFR-treatment did not significantly decrease AHR. Anti-inflammatory effect of sTNFR was accompanied with reduction of T helper 2 cytokine levels including interleukin (IL)-4, IL-5 and IL-13 in BALF and vascular cell adhesion molecule 1 expression in lung tissue. CONCLUSION: These results suggest that sTNFR treatment can suppress the airway inflammation via regulation of Th2 cytokine production and adhesion molecule expression in bronchial asthma.
Animals ; Anti-Asthmatic Agents/*therapeutic use ; Asthma/*drug therapy/*immunology ; Blotting, Western ; Bronchi/drug effects ; Bronchial Hyperreactivity ; Bronchoalveolar Lavage Fluid/immunology ; Enzyme-Linked Immunosorbent Assay ; Female ; Immunohistochemistry ; Inflammation/*drug therapy ; Interleukin-13/metabolism ; Interleukin-4/metabolism ; Interleukin-5/metabolism ; Mice ; Mice, Inbred BALB C ; Ovalbumin/pharmacology ; Tumor Necrosis Factor-alpha/*therapeutic use

A number of case reports on occupational asthma caused by herbal medicines have been issued, for example, on Sanyak, Chunkung, Banha, and Brazilian ginseng. Recently, cases of occupational asthma induced by Sanyak and Korean ginseng have been reported, but the pathogenic mechanisms involved are unknown. This study was carried out to evaluate the immunologic mechanism underlying Korean ginseng-induced occupational asthma. A patient engaged in Korean ginseng wholesale was referred for recurrent dyspnea, wheezing, and nasal symptoms, which were aggravated at work. Allergen bronchial provocation testing to Korean ginseng extract showed a typical immediate response, and skin prick testing to Korean ginseng extract also showed a strong positive response. Moreover, serum-specific IgE levels to Korean ginseng extract were significantly higher than in controls. Enzymelinked immunosorbent assay (ELISA) inhibition tests showed a dose-dependent inhibition by Korean ginseng, but not by Dermatophagoides farinae, wheat flour, or Chinese balloon flower. Sodium dodecylsulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) and immunoblotting revealed four specific Immunoglobulin E (IgE) binding components at 26, 30, 47, and 60 kDa, which were not bound by control sera. These results strongly suggest that occupation asthma induced by Korean ginseng is induced via an IgE-mediated mechanism.
Animals ; Asthma/diagnosis/*etiology/*immunology ; Bronchi/metabolism ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay/methods ; Flour ; Flowers ; Humans ; Hypersensitivity/*diagnosis ; Immunoglobulin E/analysis/*chemistry ; Korea ; Occupational Diseases/diagnosis/*etiology/*immunology ; Panax/*adverse effects ; Pyroglyphidae/metabolism ; *Skin Tests

Asthma was induced by the sensitization and challenge with ovalbumin (OVA) in mice. B-cell activating factor (BAFF) plays a role in mature B cell generation and maintenance. Here, we investigated whether, BAFF expression was changed in OVA-induced mice and whether the control of BAFF expression level alleviates the symptom of bronchial asthma. BAFF expression was detected in alveolar-associated cells surrounding bronchi of OVA-induced mouse lung tissues. BAFF protein was also increased in OVA-induced mouse serum. The increased BAFF transcripts was detected in OVA-induced mouse splenocytes. OVA-induced asthma was associated with the increased number of eosinophils in bronchoalveolar lavage fluid (BALF). When TACI:Fc scavenging soluble BAFF was injected to OVA-induced mice, a significant inhibition was detected in the thickness of airway smooth muscle and glycol-containing cellular elements in airway that were visualized by hematoxylin/eosin Y and periodic acid-Schiff staining, respectively. In addition, when mice were treated with TACI:Fc protein, BAFF protein level was decreased in alveolar-associated cells surrounding bronchi of OVA-induced mouse lung tissues compared to control mice. When compared to OVA-induced control, TACI:Fc treatment reduced the percentage of non-lymphoid cells and no changes were detected in lymphoid cell population. Hypodiploid cell formation in BALF was decreased by OVA-challenge but it was recovered by TACI:Fc treatment. Collectively, data suggest that asthmatic symptom could be alleviated by scavenging BAFF and then BAFF could be a novel target for the develpoment of anti-asthmatic agents.
Animals ; Apoptosis ; Asthma/chemically induced/*drug therapy/immunology ; B-Cell Activating Factor/*biosynthesis ; Bronchi/metabolism/pathology ; Bronchoalveolar Lavage Fluid/cytology ; Eosinophils/pathology ; Female ; Humans ; Immunoglobulin Fc Fragments/*genetics ; Immunoglobulin G/*genetics ; Lymphocytes/pathology ; Mice ; Mice, Inbred BALB C ; *Ovalbumin ; Pulmonary Alveoli/metabolism ; Recombinant Fusion Proteins/genetics/*therapeutic use ; Spleen/metabolism ; Transmembrane Activator and CAML Interactor Protein/*genetics

Several studies have suggested the involvement of an autoimmune mechanism in aspirin (ASA)-intolerant asthma. To test this hypothesis, we measured the levels of circulating autoantibodies, such as IgG and IgA to tissue transglutaminase (TGase), IgG to cytokeratins (CKs) 8, 18, and 19, Clq-binding immune complex (CIC), and antinuclear antibody (ANA), in the sera of 79 patients with ASA-intolerant asthma (Group I) and those of two control groups, consisting of 61 patients with ASA-tolerant asthma (Group II) and 88 healthy control subjects (Group III) by means of ELISA. Significantly higher prevalences of IgG antibodies to CK18 (13.9%) and CK19 (17.7%) were noted in Group I, as compared with Group III (p<0.05 for all) not with Group II. Regarding the prevalences of other autoantibodies, the levels of ANA (1.3%), IgG to TGase (3.8%), and CIC (24.7%) in Group I were not significantly different from those in Groups II and III. Significant correlations were found between positivities for the anti-CK18 and anti-CK19 autoantibodies and the PC20 methacholine values in the analysis of asthma Groups I and II vs. normal controls, (p=0.001 and p=0.003, respectively). Further studies are needed to explore the potential involvement of an autoantibody-mediated mechanism in the clinical manifestation of bronchial asthma.
Middle Aged ; Male ; Keratins/chemistry ; Inflammation ; Humans ; Female ; *Drug Resistance ; Child ; Case-Control Studies ; Bronchi/*pathology ; Autoantibodies/*chemistry ; Asthma/*drug therapy/*immunology ; Aspirin/*pharmacology ; Anti-Inflammatory Agents, Non-Steroidal/pharmacology ; Aged

BACKGROUNDImiquimod is an imidazoquinoline, which class of compounds are known to have antiviral and antitumoural properties. In recent studies, it was shown that imiquimod modulates the T helper cell type Th1/Th2 response by inducing the production of Th1 cytokines like IFN-gamma, and by inhibiting the Th2 cytokines like interleukin (IL)-4. Several investigators have shown that T-bet and GATA-3 are master Th1 and Th2 regulatory transcription factors. This study investigated whether imiquimod treatment inhibited airway inflammation by modulating transcription factors T-bet and GATA-3.

METHODSThirty-six male SD rats were randomly divided into a control group, an asthmatic group, and an imiquimod group, which was exposed to an aerosol of 0.15% imiquimod. Twenty-four hours after the last ovalbumin (OVA) challenge, airway responsiveness was measured and changes in airway histology were observed. The concentrations of IL-4, IL-5 and IFN-gamma in bronchoalveolar lavage fluid (BALF) and serum were measured by enzyme linked immunosorbent assay (ELISA). The mRNA expressions of IL-4, IL-5, IFN-gamma, T-bet and GATA-3 in lung and in CD4(+) T cells were determined by reverse transcription polymerase chain reaction (RT-PCR). The protein expressions of T-bet and GATA-3 were measured by Western blot.

RESULTSIt was demonstrated that imiquimod 1) attenuated OVA induced airway inflammation; 2) diminished the degree of airway hyperresponsiveness (AHR); 3) decreased the Th2 type cytokines and increased Th1 type cytokines mRNA and protein levels; 4) modulated the Th1/Th2 reaction by inhibiting GATA-3 production and increasing T-bet production.

CONCLUSIONImiquimod treatment inhibits OVA induced airway inflammation by modulating key master switches GATA-3 and T-bet that result in committing T helper cells to a Th1 phenotype.

Administration, Inhalation ; Aminoquinolines ; administration & dosage ; therapeutic use ; Animals ; Asthma ; drug therapy ; metabolism ; Bronchi ; pathology ; Bronchial Hyperreactivity ; drug therapy ; metabolism ; Cytokines ; biosynthesis ; Eosinophils ; physiology ; GATA3 Transcription Factor ; genetics ; Gene Expression Regulation ; drug effects ; Lung ; pathology ; Male ; Ovalbumin ; immunology ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; T-Box Domain Proteins ; Transcription Factors ; genetics

OBJECTIVETo study the expression of signal transducer and activator of transcription 6 and its mRNA in rat asthma model and the modulatory effect of dexamethasone (DXM).

METHODSThirty male SD rats were randomly divided into three groups: the control group, asthma group and DXM group. The rats in each group were sacrificed 24 h after the last challenge. In the experiment, the rat model of asthma was established by ovalbumin (OVA) challenge method. The lung tissue was taken from the left lung, and bronchoalveolar lavage fluid (BALF) was collected from the right lung. The total cell numbers, eosinophils (EOS) count and differentiated cell counts in BALF were performed on different count fluids. The concentrations of IL-4 in serum and BALF were measured by using sandwich ELISA. The protein expressions of STAT6 were detected with immunohistochemical techniques. The mRNA expressions of STAT6 were detected with in situ hybridization.

RESULTS(1) The total cell counts in BALF, the absolute counts of EOS, and the ratios of eosinophils to the total cell numbers (EOS%) of asthma group were all significantly higher than those of the control group (P < 0.01). The total cell counts in BALF, the absolute counts of EOS, and EOS% of DXM group were all significantly lower than those of asthma group (P < 0.01). (2) The concentrations of IL-4 in BALF and serum of asthma group [(25.7 +/- 7.4) ng/L, (34.2 +/- 10.5) ng/L] were significantly higher than those of control group [(8.6 +/- 3.0) ng/L, (12.1 +/- 2.9) ng/L] (P < 0.01). The concentrations of IL-4 in BALF and serum of DXM group were significantly lower than those of asthma group. (3) Immunohistochemistry showed that the protein content of STAT6 around the bronchus of asthma group (0.171 +/- 0.025) was significantly higher than that of the control group (0.082 +/- 0.022) (P < 0.01), while that of DXM group (0.114 +/- 0.013) was significantly lower than that of asthma group. The epithelial cells were the cells. In situ hybridization showed that the mRNA expression of STAT6 around the bronchus of asthma group (0.180 +/- 0.013) was significantly higher than that of the control group (0.091 +/- 0.012) (P < 0.01), while that of DXM group (0.114 +/- 0.010) was significantly lower than that of asthma group. (4) There was a significant correlation between the concentration of IL-4 in BALF, the content of STAT6 and STAT6 mRNA, respectively, in the epithelial cells of bronchus. There was a significant correlation between the absolute numbers of EOS and EOS% in BALF, the content of STAT6 and STAT6 mRNA, respectively, in the epithelial cells of bronchus.

CONCLUSIONSSTAT6 protein and STAT6 mRNA were found strongly expressed in rat asthma model and the epithelial cells were the chief expressing cells. Dexamethasone had an inhibitory effect on airway inflammatory cells infiltration. It significantly depressed STAT6 and mRNA expression. Which may be a key process in modulatory mechanism of asthma.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Asthma ; drug therapy ; genetics ; metabolism ; Bronchi ; cytology ; Bronchoalveolar Lavage Fluid ; cytology ; Cell Count ; Dexamethasone ; pharmacology ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Eosinophils ; drug effects ; Epithelial Cells ; drug effects ; metabolism ; Glucocorticoids ; pharmacology ; Immunohistochemistry ; In Situ Hybridization ; Interleukin-4 ; metabolism ; Male ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; STAT6 Transcription Factor ; genetics ; immunology ; metabolism

This investigation was designed to confirm IL-8 production from human bronchial epithelial cells with toluene diisocyanate (TDI) exposure and to examine the effects of pro-inflammatory cytokine and dexamethasone. We cultured Beas-2B, a bronchial epithelial cell line with TDI-HSA conjugate and compared with those without conjugate. IL-8 in the supernatant was measured by ELISA. To evaluate the effect of proinflammatory cytokines, peripheral blood mononuclear cells (PBMC) were collected from TDI- and non-TDI asthma patients, and were added to the epithelial cell culture. Dexamethasone or antibodies to TNF-alpha and IL-1beta were pre-incubated with PBMC supernatant. There was a significant production of IL-8 from bronchial epithelial cells with addition of TDI-HSA conjugate in a dose-dependent manner, which was significantly augmented with addition of PBMC supernatant. Higher production of IL-8 was noted with addition of PBMC supernatant from TDI-asthma patients than in those from non-TDI asthma patients. IL-1beta and IL-1beta/TFNalpha antibodies were able to suppress the IL-8 productions. Pre-treatment of dexamethasone induced dose-dependent inhibition of the IL-8 production. These results suggest that the IL-8 production from bronchial epithelial cells contribute to neutrophil recruitment occurring in TDIinduced airway inflammation. IL-1beta released from PBMC of TDI-induced asthma patients may be one of the pro-inflammatory cytokines to enhance IL-8 production.
Asthma/immunology ; Bronchi/*cytology/metabolism ; Cell Line ; Dexamethasone/pharmacology ; Epithelial Cells/cytology/*drug effects/*metabolism ; Glucocorticoids/pharmacology ; Human ; Interleukin-8/*metabolism ; Leukocytes, Mononuclear/cytology/metabolism ; Support, Non-U.S. Gov't ; Toluene 2,4-Diisocyanate/*toxicity