Role of cathepsin S in ozone-induced airway hyperresponsiveness and inflammation.

Ambient ozone has been linked to the worsening of symptoms of patients with obstructive diseases such as chronic obstructive pulmonary disease (COPD) and asthma. We investigated the role of cathepsin S on ozone-induced airway hyperresponsiveness (AHR) and inflammation, using the selective cathepsin S inhibitor, Compound A. Balb/c mice were exposed to ozone at a concentration of 3 ppm or air for 3 h, following administration by gavage of Compound A or vehicle. Bronchoalveolar lavage (BAL) was performed 3 h and 20-24 h following exposure, AHR was measured at 20-24 h only. Ozone exposure, compared to air exposure increased BAL cathepsin S levels, AHR and BAL inflammatory cells. Compound A (30 mg kg(-1) p.o.) dosing compared to vehicle dosing inhibited ozone-induced AHR (-logPC100 vehicle: -0.70+/-0.12, n=8 vs. cathepsin S inhibitor: -1.30+/-0.06, P<0.001, n=8) at 20-24 h and BAL neutrophilia at 3 h and 20-24 h (P<0.05, n=6). Ozone exposure increased levels of BAL cytokines IL-6, TNF-alpha and IFN-gamma. Compound A reduced IL-6 at 3 h and 20-24 h (P<0.05, n=5) and TNF-alpha, at 20-24 h (P<0.05, n=6). These data indicate an important role for cathepsin S in the regulation of ozone-induced AHR and neutrophil cell recruitment and suggest that cathepsin S may be a target in the treatment of oxidative stress-induced AHR and inflammation.

Dose-dependent inhibition of allergic inflammation and bronchial hyperresponsiveness by budesonide in ovalbumin-sensitised Brown-Norway rats.

Corticosteroids are known to inhibit bronchial hyperresponsiveness (BHR) and allergic inflammation but there is little information on its dose-dependence. We examined the effect of different doses of the glucocorticosteroid budesonide in an allergic model. Brown-Norway rats were sensitised to ovalbumin (OVA) and pretreated with an intra-gastric dose of budesonide (0.1, 1.0, or 10 mgkg(-1)). Exposure to OVA induced BHR, accumulation of eosinophils in the bronchoalveolar lavage (BAL) fluid and in the airways submucosa. Budesonide dose-dependently inhibited BAL fluid influx of lymphocytes, eosinophils and neutrophils, tissue eosinophils and lymphocytes and BHR. At 0.1 mgkg(-1), budesonide did not inhibit these parameters but at 1 mgkg(-1), BAL fluid eosinophils and T-cells, and submucosal T-cells were significantly reduced. At 10 mgkg(-1), budesonide suppressed BHR, BAL fluid inflammatory cells numbers and tissue eosinophilia. T-cell numbers were more related to BHR than eosinophil numbers. Budesonide inhibited both airway inflammation and BHR, but BAL fluid eosinophil cell counts may be dissociated from BHR.

Low-level subchronic exposure to wood smoke exacerbates inflammatory responses in allergic rats.

Epidemiological studies have implicated wood smoke as a risk factor for exacerbating asthma. However, comparisons of findings in animal models with those in humans are currently not possible, because detailed clinically relevant measurements of pulmonary function are not available in animal studies. Brown Norway rats were immunized with ovalbumin and exposed to either filtered air or wood smoke at 1 mg particulate matter/m(3) for 70 days and challenged with allergen during the last 4 days of exposure. Baseline values for dynamic lung compliance were lower while functional residual capacity was increased in rats exposed to wood smoke compared to rats exposed to filtered air. IFN-gamma levels were reduced and IL-4 levels increased in the bronchoalveolar lavage fluid and blood plasma, inflammatory lesions in the lungs were 21% greater, and airway mucous cells/mm basal lamina were non-significantly increased in rats exposed to wood smoke compared to controls. Collectively, these studies suggest that the pulmonary function was affected in rats by exposure to wood smoke and this decline was associated with only minor increases in inflammation of the lung. Therefore, this animal model may be useful to elucidate the mechanisms of the decline in pulmonary function caused by environmental pollutants when asthmatics are exposed to allergen.

Transcriptional down-regulation of neurotrophin-3 in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) leads to progressive development of airflow limitation and is characterised by cough, mucus hypersecretion and inflammatory changes. These characteristic features of the disease may be modulated by neural mediators such as neurotrophins (NT). Here we examined the expression and transcriptional regulation of neurotrophins in bronchial biopsies of COPD patients and compared the data to control biopsies. Histology revealed characteristic changes in the COPD tissues, including remodelling of the epithelial lining. RT-PCR demonstrated the mRNA expression of neurotrophins in all biopsies. Immunohistochemistry confirmed the expression of different proteins. To assess changes in the transcriptional expression level, quantitative real-time PCR was carried out and revealed differential mRNA expression of neurotrophins, with marked down-regulation of NT-3 mRNA expression and constant levels of nerve growth factor (NGF), brain-derived nerve factor (BDNF), and NT-4/5 mRNA expression. The present data on neurotrophin-specific transcriptional down-regulation of NT-3 in human COPD indicate a pathophysiological role for neurotrophins in COPD.

Mitogen-activated protein kinase signalling pathways in IL-1 beta-dependent rat airway smooth muscle proliferation.

Asthma is associated with abnormal airway smooth muscle (ASM) growth that may contribute to airway narrowing and hyperresponsiveness. We investigated the role of mitogen-activated protein kinase (MAPK) pathway in IL-1beta induced ASM proliferation in the rat. Rat tracheal ASM cells were dissociated and maintained in culture. We examined the effect of selective MAPK inhibitors, SB239063 (a p38 MAPK inhibitor), U0126 (a mitogen-activated and extracellular regulated kinase kinase, MEK-1, inhibitor which inhibits downstream extracellular regulated kinase, ERK, activity), and SP600125 (a c-jun N-terminal kinase, JNK, inhibitor) on IL-1beta-induced proliferation. Proliferation of ASM cells was significantly increased following exposure to IL-1beta in a dose-dependent manner. p38, JNK and ERK MAPKs were activated by IL-1beta in a time-dependent manner, with peak activation time at 30, 60 min and at 6 h, respectively. This activation was inhibited by their respective inhibitors. SP600125 (20 microM) had no effect on IL-1beta-induced ERK and p38 phosphorylation. SB239063, U0126 and SP600125 dose-dependently inhibited IL-1beta-dependent proliferation at doses that inhibit the activities of p38, ERK and JNK MAPKs, respectively. No additive or synergistic effects were observed on proliferative responses with any combination of these compounds. In conclusion, the three major MAPK pathways, ERK as well as the p38 MAPK and JNK pathways, are independent regulators of IL-1beta-dependent proliferation of rat ASM.

Effect of an inhibitor of Jun N-terminal protein kinase, SP600125, in single allergen challenge in sensitized rats.

Jun N-terminal kinase (JNK) has been implicated in the pathogenesis of inflammatory diseases including asthma. We examined the effect of SP600125 (anthra [1,9-cd] pyrazol-6 (2H)-one), a novel inhibitor of JNK in a model of asthma. Brown-Norway rats were sensitized to ovalbumin and treated with SP600125 intraperitoneally (90 mg/kg in total). SP600125 inhibited allergen-induced, increased activity of phosphorylated c-jun but not of phosphorylated-MAPKAPK2, indicative of activation of p38 MAPK, in the lung. SP600125 inhibited macrophage (P < 0.04), lymphocyte (P < 0.05), eosinophil (P < 0.04) and neutrophil (P < 0.005) numbers in bronchoalveolar lavage. Eosinophil and T-cell accumulation in the airways, mRNA expression for interleukin-1beta, tumour necrosis factor-beta, interleukin-3, interleukin-4 and interleukin-5, serum levels of allergen-specific immunoglobulin E and bronchial hyperresponsiveness were not affected by SP600125. Selective inhibition of JNK reduced inflammatory cell egress into the airway lumen after single allergen exposure. The role of JNK mitogen-activated protein kinase activation may be limited in the pathogenesis of bronchial hyperresponsiveness after single allergen exposure.

Allergen-induced inflammation and airway epithelial and smooth muscle cell proliferation: role of Jun N-terminal kinase.

Chronic cellular inflammation and airway wall remodelling with subepithelial fibrosis and airway smooth muscle (ASM) cell hyperplasia are features of chronic asthma. Jun N-terminal kinase (JNK) may be implicated in these processes by regulating the transcriptional activity of activator protein (AP)-1. We examined the effects of an inhibitor of JNK, SP600125 (anthra [1,9-cd] pyrazole-6 (2 H)-one), in a model of chronic allergic inflammation in the rat. Rats sensitised to ovalbumin (OA) were exposed to OA-aerosol every third day on six occasions and were treated with SP600125 (30 mg kg-1 b.i.d; 360 mg in total) for 12 days, starting after the second through to the sixth OA exposure. We measured eosinophilic and T-cell inflammation in the airways, proliferation of ASM cells and epithelial cells by incorporation of bromodeoxyuridine (BrdU), and bronchial responsiveness to acetylcholine. SP600125 significantly reduced the number of eosinophils (P<0.05) and lymphocytes (P<0.05) in bronchoalveolar lavage fluid, suppressed eosinophilic (P<0.05) and CD2+ T-cell (P<0.05) infiltration within the bronchial submucosa, and the increased DNA incorporation in ASM (P<0.05) and epithelial cell incorporation (P<0.05). SP600125 did not alter bronchial hyper-responsiveness observed after chronic allergen exposure. Pathways regulated by JNK positively regulate ASM cell proliferation and allergic cellular inflammation following chronic allergen exposure.

Effects of cyclosporin A and a rapamycin derivative (SAR943) on chronic allergic inflammation in sensitized rats.

Immunomodulators such as cyclosporin A (CsA) and SAR943 (32-deoxorapamycin) inhibit single allergen-induced allergic inflammation such as eosinophilic and lymphocytic infiltration and mRNA expression for interleukin (IL)-4 and IL-5. We examined the effects of CsA and SAR943, administered orally, on asthmatic responses in a rat model of chronic allergic inflammation. Sensitized Brown-Norway (BN) rats were exposed to ovalbumin (OVA) aerosol every third day on six occasions. CsA (5 mg/kg/day), SAR943 (2.5 mg/kg/day) or vehicle (Neoral) was administered orally, once a day, from days 10 to 21 (a total of 12 doses). We measured eosinophilic and T-cell inflammation in the airways, proliferation of airway cells by incorporation of bromodeoxyuridine (BrdU) and bronchial responsiveness to acetylcholine. CsA had no effects, while SAR943 inhibited airway smooth muscle (ASM, P < 0.05) and epithelial cell (P < 0.01) BrdU incorporation, and the number of CD4+ T cells (P < 0.05), without effects on BHR. ASM thickness was not significantly increased following chronic allergen exposure. Therefore, CsA and SAR943 have no effect on chronic eosinophilic inflammation, while SAR943, but not CsA, had a small effect on the proliferation of ASM and epithelium.

Distribution of respiratory mucin proteins in human nasal mucosa.

OBJECTIVES/HYPOTHESIS: The upper respiratory tract is involved in many acute and chronic respiratory tract diseases that present with the symptom of mucus hypersecretion. Mucin genes that encode for the backbone of glycoproteins contribute to the viscoelastic property of airway mucus. We examined the cellular expression and distribution of two major respiratory mucus-forming glycoproteins, MUC5AC and MUC5B, in normal human nasal tissues. METHODS: Immunohistochemical analysis using polyclonal antibodies against the mucins MUC5AC and MUC5B was performed in normal human nasal tissues. RESULTS: An abundant staining of submucosal mucus gland and epithelial goblet cells for MUC5B was found. Immunohistochemical analysis of MUC5AC showed staining of surface epithelium goblet cells, whereas there was no staining of glandular cells. Comparison of the expression to lower airways revealed a similar pattern of expression of both mucins. CONCLUSIONS: The data in the present study demonstrated the localization of the two major respiratory mucin proteins in human nasal mucosa with a similar distribution of expression of MUC5AC and MUC5B in normal upper and lower airways. Mucin protein expression parallels that of mucin messenger RNA expression.

Repeated allergen inhalation induces phenotypic modulation of smooth muscle in bronchioles of sensitized rats.

Repeated ovalbumin (OA) or saline exposure of sensitized Brown Norway rats was examined on agonist reactivity, airway smooth muscle (ASM) content, and contractile protein expression in small bronchioles at 24 h, 7 days, and 35 days after challenge. OA increased ASM content (P < 0.05 vs. saline) at 24 h, which resolved by 7 days. Maximum developed tension (T(max)) to carbachol, KCl, and 4-beta-phorbol 12,13-dibutyrate was increased (P < 0.05) by OA in bronchioles at 24 h but was abrogated after correction for ASM. Differences in T(max) were not present at 7 days. In contrast, at 35 days, T(max) was increased (P < 0.05) after correction for ASM. Smooth muscle (sm)-alpha-actin, sm-myosin heavy chain (MHC) isoform 1, calponin, smoothelin-A, and sm-myosin light chain kinase expression were reduced (P < 0.05) by OA at 24 h in bronchioles but not in trachealis. Consistent with contraction findings, no difference in expression of these proteins was detected at 7 days. At 35 days, however, with the exception of sm-alpha-actin, their abundance was again reduced (P < 0.05) by OA. Nonmuscle MHC and beta-actin were unchanged throughout by OA. These findings indicate persistent changes in contractile protein content, consistent with ASM phenotypic modulation in vivo, which occur in response to repeated OA inhalation. Thus, OA exposure induces structural changes in bronchiole ASM content and in agonist responsiveness ex vivo that resemble remodeling in asthma.

Role of nitric oxide in chronic allergen-induced airway cell proliferation and inflammation.

Chronic cellular inflammation and airway wall remodeling with subepithelial fibrosis and airway smooth muscle thickening are features of chronic asthma. We determined the role of nitric oxide in the pathogenesis of allergen-induced airway cell proliferation and inflammation by studying the effects of a relatively selective prodrug inhibitor of nitric-oxide synthase type 2 (NOS2), L-N6-(1-iminoethyl)-lysine-5-tetrazole amide (SC-51). Brown-Norway rats were sensitized to ovalbumin and were exposed to ovalbumin aerosol every 3rd day on six occasions and were treated orally with either vehicle or SC-51 (10 mg. kg(-1); 12 doses). We measured inflammatory cell accumulation in the airways and proliferation of cells by incorporation of bromodeoxyuridine. There was an increase in the total number of airway smooth muscle cells expressing bromodeoxyuridine from 1.3% of airway smooth muscle cells in saline exposed to 5.4% after allergen-exposure (P < 0.001) and airway epithelial cells from 3.3 cells/mm basement membrane to 9.6 after allergen-exposure (P < 0.001). SC-51 had no effect on airway smooth muscle or epithelial cell proliferation. SC-51 attenuated the allergen-induced increase in major basic protein (MBP+) eosinophil (P < 0.05) and CD4+ T-cell (P < 0.05) accumulation. We conclude that nitric oxide derived during allergic inflammation is involved in the expression of eosinophilic inflammation and not in epithelial or airway smooth muscle cell DNA synthesis induced by chronic allergen exposure.

Role of nitric oxide in allergic inflammation and bronchial hyperresponsiveness.

The role of nitric oxide (NO) in allergic inflammation and bronchial hyperresponsiveness is unclear. We studied a selective prodrug nitric oxide synthase (NOS)-2 inhibitor, L-N(6)-(1-iminoethyl)lysine 5-tetrazole amide (SC-51). In ovalbumin-sensitized and challenged rats, exhaled NO levels increased by 3 h following challenge (3.73 +/- 0.74 ppb; P < 0.05), peaking at 9 h (11.0 +/- 2.75; P < 0.01) compared to saline controls (1.87 +/- 0.26; P < 0.05 and 2.81 +/- 0.18; P < 0.01). Immunoreactive lung NOS2 expression was increased in ovalbumin-challenged rats compared with ovalbumin-sensitized, saline-challenged rats at 8 h post-challenge. SC-51 (10 mg/kg; p.o.) inhibited allergen-induced increase in exhaled NO levels to 1.3 +/- 0.17 ppb. SC-51 inhibited bronchial hyperresponsiveness in ovalbumin-sensitized and challenged rats (P < 0.05). In sensitized non-exposed rats, SC-51 increased bronchial responsiveness (P < 0.05). SC-51 reduced the allergen-induced increase in bronchoalveolar lavage neutrophils, but caused a nonsignificant reduction in bronchial mucosal eosinophil numbers. NO generated through NOS2 contributes to allergen-induced bronchial hyperresponsiveness but not to bronchial eosinophilia, indicating that these are independently expressed.