Makoto Hoshino

Bronchial subepithelial fibrosis and expression of matrix metalloproteinase-9 in asthmatic airway inflammation

BACKGROUND Bronchial asthma is characterized by airway structural changes, including mucosal inflammation and subepithelial collagen deposition. An imbalance between the matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) is thought to play a critical role in the synthesis or degradation of the extracellular matrix of the airway architecture. However, the relationship between subepithelial basement membrane thickness and these enzymes in asthma has not been determined. OBJECTIVE We compared the thickness of collagen and tenascin deposition and the expression of MMP-9 and TIMP-1 in bronchial biopsy specimens from subjects with asthma and control subjects. METHODS Bronchial biopsy specimens were obtained from 25 subjects with asthma and 10 healthy control subjects to estimate the extent of collagen and tenascin deposition in subepithelial reticular basement membrane by immunohistochemical staining. Using a computer-assisted image analysis system, we quantitated expression of both epithelial and submucosal MMP-9 and TIMP-1. The numbers of inflammatory cells were also determined. RESULTS Subjects with asthma exhibited greater thickness of collagen III (P <.01), collagen V (P <.01), and tenascin (P <.01) deposition in reticular basement membrane than did control subjects. The proportions of epithelium and submucosa immunoreactive to MMP-9 and TIMP-1 were significantly higher in the subjects with asthma than in the control subjects (each P <.001). Submucosal expression of MMP-9 was significantly higher than that of TIMP-1 in subjects with asthma (P <.01). Significant correlations were found between the number of myofibroblasts and thicknesses of collagen III (rs = 0. 70, P <.001), collagen V (rs = 0.67, P <.001), and tenascin (rs = 0. 58, P <.01) in subjects with asthma. On the other hand, the number of eosinophils was correlated with degree of mucosal expression of MMP-9 (rs = 0.43, P <.05) and TIMP-1 (rs = 0.69, P <.001). In subjects with asthma, a significant inverse correlation was found between subepithelial fibrosis and FEV1 (type III collagen, rs = -0. 89, P <.001; type V collagen, rs = -0.90, P <.001; tenascin, rs = -0. 88, P <.001), and airway responsiveness (type III collagen, rs = -0. 59, P <.01; type V collagen, rs = -0.47, P <.05; tenascin, rs = -0. 48, P <.05). CONCLUSION These findings suggest that collagen III, collagen V, and tenascin deposition in basement membrane in subjects with bronchial asthma are associated with increased expression of MMP-9, which may be produced by eosinophils, and that airway remodeling in subjects with asthma may be related to air-flow obstruction and airway hyperresponsiveness.

Inhaled corticosteroids decrease subepithelial collagen deposition by modulation of the balance between matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 expression in asthma.

BACKGROUND Bronchial asthma is characterized by airway wall remodeling. Matrix metalloproteinases (MMPs) are members of a family of proteolytic enzymes that degrade the extracellular matrix and that restrain the effects of their tissue inhibitors (TIMPs). Treatment with inhaled corticosteroids may prevent airway remodeling in asthma. However, the effects of corticosteroid treatment on MMPs and TIMPs in asthma are unknown. OBJECTIVE We examined the effects of inhaled beclomethasone dipropionate (BDP) on the expression of MMP-9 and TIMP-1 and subepithelial collagen deposition in bronchial biopsy specimens from 30 subjects with asthma. METHODS Inhaled BDP, 800 microg daily, or placebo was administered for 6 months in a double-blind, parallel-group study, and bronchial biopsies were performed before and after treatment. Biopsy specimens were examined for extent of collagen type III in the subepithelial basement membrane by means of immunohistochemistry, and expression of both epithelial and submucosal MMP-9 and TIMP-1 was quantitated. Numbers of inflammatory cells were also determined. RESULTS We observed significant decreases in collagen type III deposition (P <.01) and the expression of submucosal MMP-9 (P <.01) and a significant increase in the expression of submucosal TIMP-1 (P <.05) in the BDP group. Significant correlations were found between the subepithelial collagen type III deposition and epithelial (r (s ) = 0.37, P <.05) and submucosal expression of MMP-9 (r (s ) = 0.47, P <.01). Additionally, the number of many inflammatory cells and myofibroblasts in airway mucosa were significantly decreased in the BDP group. CONCLUSION Our findings suggest that corticosteroid treatment of asthma can reduce subepithelial collagen deposition by downregulation of MMP-9 expression and upregulation of TIMP-1 expression.

Effect of the leukotriene receptor antagonist pranlukast on cellular infiltration in the bronchial mucosa of patients with asthma

BACKGROUND It has been reported that pranlukast reduces the antigen induced immediate and late phase asthmatic responses, airway hyperreactivity to acetylcholine, and pulmonary eosinophil accumulation in guinea pigs. A study was undertaken to test the hypothesis that pranlukast may reduce the number of inflammatory cells in the bronchial mucosa of patients with asthma. METHODS A double blind, placebo controlled study was performed in 17 mild to moderate asthmatic subjects to examine changes in inflammatory cell infiltration in response to pranlukast (225 mg orally twice per day for four weeks). Comparisons of the mean daily β2 agonist use, symptom score, FEV1 percentage predicted, and airway methacholine responsiveness were made before and after treatment. Using fibreoptic bronchoscopy, bronchial biopsy specimens were obtained before and after treatment with either pranlukast (n = 10) or placebo (n = 7). Immunohistology was performed using monoclonal antibodies for CD3, CD4, CD8, CD68, NP57, AA1, EG1, EG2, γGTP and CD19. RESULTS When the pranlukast and placebo treated groups were compared there were decreases in β2agonist use, symptom score, and airway methacholine responsiveness after pranlukast but no increase in FEV1 was seen. The clinical response in patients treated with pranlukast was accompanied by a reduction in CD3 (median difference –37, 95% confidence interval (CI) –69 to –1; p<0.05), CD4 (median difference –28, 95% CI –49 to –8; p<0.01), AA1 (median difference –15, 95% CI –26 to 0; p<0.05) and EG2 positive cells (95% CI –35 to 0; p<0.05), but not in EG1 positive eosinophils, γGTP positive cells, and CD19 positive plasma cells. CONCLUSIONS These results support the view that pranlukast may act by inhibition of bronchial inflammation in patients with asthma.

Inhaled corticosteroids decrease vascularity of the bronchial mucosa in patients with asthma.

Background Increased vascularity in airway mucosa is a distinctive feature of airway remodelling in asthma. While corticosteroids have proved most effective in modifying airway inflammation, the effect of inhaled corticosteroids on increased airway mucosal vascularity in asthmatics has been little studied.

Effect of AA-2414, a thromboxane A2 receptor antagonist, on airway inflammation in subjects with asthma

BACKGROUND Asthma is a chronic inflammatory disease of the airways. The chemokines are potent chemoattractants for eosinophils and other types of cells associated with allergic inflammation. AA-2414, a new thromboxane A2 receptor antagonist, reduces bronchial hyperresponsiveness in asthmatic subjects, but its mechanism of action is unclear. OBJECTIVE We tested the hypothesis that the beneficial effects of AA-2414 in asthma result from reduction in the number of inflammatory cells infiltrating the airway associated with inhibition of chemokine release. METHODS We studied bronchial biopsy specimens from 31 asthmatic subjects before and after oral treatment with AA-2414 (80 mg/day) or matched placebo for 4 months in a double-blind manner. Biopsy specimens were examined by immunohistochemistry. Each subject recorded symptom score and peak expiratory flow (PEF). Lung function and bronchial responsiveness to methacholine were measured before and after treatment. RESULTS After treatment, significant improvements in symptom score (P <.05), PEF (P <.01), diurnal variation of PEF (P <.01), and bronchial responsiveness (P <.01) were observed in the AA-2414 group compared with the placebo group. These improvements were accompanied by a significant decrease in the number of submucosal EG2(+) eosinophils (P <.05). There was also a reduction in the number of cells expressing RANTES (P <.05) and macrophage inflammatory protein (MIP)-1alpha (P <.05) in the epithelium and of cells expressing monocyte chemotactic protein-3 (P <.01), RANTES (P <.05), MIP-1alpha (P <.01), and eotaxin (P <.01) in the submucosa in the AA-2414 treatment group. A significant correlation was found between the number of EG2(+) eosinophils and numbers of monocyte chemotactic protein-3(+) (rs = 0.52, P <.005), MIP-1alpha+ (rs = 0.34, P <.05), and eotaxin+ cells (r s = 0.47, P <.01) in the submucosa. There was a significant negative correlation between the increase in bronchial responsiveness and the change in number of submucosal EG2(+) cells (rs = -0.65, P <.001). CONCLUSIONS These findings suggest that AA-2414 treatment of patients with asthma may inhibit activated eosinophil infiltration in part by modulating the expression of chemokines in bronchial tissues.

A comparative study of the effects of ketotifen, disodium cromoglycate, and beclomethasone dipropionate on bronchial mucosa and asthma symptoms in patients with atopic asthma

Asthma is a chronic inflammatory disorder of the airways that is characterized by infiltration of many inflammatory cells into the bronchial mucosa. We compared the effects of ketotifen, disodium cromoglycate (DSCG), and beclomethasone dipropionate (BDP) on inflammatory cells in the bronchial mucosa and on the asthma symptoms of patients with atopic asthma. In this 12-week parallel study, 32 patients were randomly allocated to either the ketotifen group (2 mg day-1, n = 13), DSCG group (8 mg day-1, n = 9) or BDP (400 micrograms day-1, n = 10). Each subject recorded daily asthma symptoms and peak expiratory flow (PEF). Before and after treatment, pulmonary function and bronchial responsiveness to methacholine were evaluated, and fibreoptic bronchoscopy and biopsy were performed before and after treatment. Biopsy specimens were obtained by bronchoscopy. We performed immunohistochemistry using specific monoclonal antibodies for activated eosinophils (EG2), mast cells (AA1), and T cells (CD3, CD4, and CD8). Our clinical findings showed significant improvement in symptom score and bronchial responsiveness (P < 0.01) each) in all groups. Both the DSCG and the BDP groups had significantly better symptom scores than the ketotifen group (P < 0.05, both groups). PEF significantly increased in the DSCG group in comparison to the ketotifen (P < 0.01) and BDP (P < 0.05) groups, FEV1% increased significantly in the DSCG (P < 0.01) and BDP (P < 0.05) groups in comparison to the ketotifen group. Compared with their baseline values, treatment significantly decreased EG2+ activated eosinophils, and CD3+ and CD4+ T cells, in each group (P < 0.01). Both the DSCG (P < 0.05) and the BDP groups (P < 0.01) exhibited significant decreases in AA1+ mast cell count, but this was not observed in the ketotifen group. Comparing before- and after-treatment values, only the DSCG group exhibited a significant decrease in the number of CD8+ T cells (P < 0.01). Ketotifen, DSCG, and BDP all showed anti-inflammatory activity as determined by examination of the bronchial mucosa of asthmatic patients; and both the DSCG and BDP groups had better clinical responses than the ketotifen group.

TH2 cytokines and associated transcription factors as therapeutic targets in asthma.

The increasing levels of morbidity and mortality due to the rising prevalence of asthma and other allergic diseases have inspired investigations of several new molecular techniques to improve treatment. Recently, several preclinical studies have been published which utilize attributes or facets of DNA to address asthma therapy. These novel therapeutics include antisense oligonucleotides against TH2 cytokines and associated transcription factors. While no clinical experience has yet been reported for any of these areas of research in asthma, specific small molecule inhibitors of TH2 cell responses would be desirable for treatment of this chronic disease. Six transcription factors (c-Maf, NF-AT, NF-IL-6, AP-1, STAT-6 and GATA-3) have been implicated in the differentiation of TH2-type lymphocytes and therefore, in addition to TH2-type cytokines, represent therapeutic targets for asthma. This review will focus on new research involving suppression of Th2-type cytokines and associated transcription factors using antisense and decoy oligonucleotides. Recently, novel oligonucleotides have been devised to improve stability in vitro and in vivo stability against nucleases, and the efficacy of these approaches will also be presented.

Relationship between activated eosinophils of the bronchial mucosa and serum eosinophil cationic protein in atopic asthma.

We biopsied the bronchial mucosa of 20 atopic bronchial asthma patients and measured their serum eosinophil cationic protein (ECP) levels as a means of assessing whether serum ECP could be as a marker of airway eosinophil inflammation. We also investigated the relationship between serum ECP levels and airway hypersensitivity, pulmonary function, peak expiratory flow (PEF) and attack scores. EG2 monoclonal antibody was used to immunostain bronchial mucosal tissue, and positive cells were expressed as the number of positive cells per square millimeter of lamina propria mucosae. A significant correlation was found between bronchial mucosa EG2+ cell counts and serum ECP levels (r = 0.66, p < 0.01), but no clear relationships were detected between serum ECP levels and airway hypersensitivity, pulmonary function, PEF or attack scores. Based on the above findings, serum ECP levels reflect airway eosinophilic inflammation, but ECP levels must be considered in combination with other parameters in relation to the pathophysiology of asthma.

Effects of ketotifen on symptoms and on bronchial mucosa in patients with atopic asthma

Ketotifen is marketed throughout the world as an antiallergy drug, but whether it affects infiltration of inflammatory cells into airway mucosa is not known. We studied the effects of ketotifen on symptoms, pulmonary function, and airway inflammation in 25 patients with atopic asthma. Patients took ketotifen (1 mg twice daily) or a matching placebo for 8 weeks in a double‐blind, parallel‐group study. Data recorded on diary cards were used for 2 weeks before treatment began, and they were used for the last 2 weeks of treatment to study asthma symptoms, use of β2–agonists, and peak expiratory flow (PEF). Pulmonary function tests, bronchial responsiveness to methacholine, and fiberoptic bronchoscopy were performed before and after treatment. Biopsy specimens were obtained by bronchoscopy. Specimens were stained immunohistochemically with monoclonal antibodies against stored eosinophil cationic protein (EG1), the secreted form of eosinophil cationic protein (EG2), mast‐cell tryptase (AA1), neutrophil elastase (NP57), CD3, CD4, CD8, and CD25. The numbers of positively stained cells in the lamina propria were counted. Compared with the placebo, the ketotifen‐treated group exhibited significant improvement of asthma symptoms (P<0.05) and bronchial responsiveness (P<0.05). This was accompanied by a reduction of EG2+ eosinophils (P<0.05), CD3+ T cells (P<0.001), CD4+ T cells (P<0.01), and CD25+ activated T cells (P<0.01) in the bronchial mucosa. These results suggested that the beneficial effects of ketotifen in bronchial asthma may result from consequent inhibition of activated eosinophils and T‐cell recruitment into the airway. Moreover, ketotifen may relieve allergic inflammation in bronchial asthma.

The Effect of Azelastine on the Infiltration of Inflammatory Cells into the Bronchial Mucosa and Clinical Changes in Patients with Bronchial Asthma

BACKGROUND Azelastine is an oral antiallergic compound but there is no direct evidence of its anti-inflammatory actions in bronchial asthma. We examined the effect of azelastine on inflammatory cells infiltrating the bronchial mucosa and clinical symptoms in atopic asthma in a double-blind, parallel, randomized study. METHODS The study was completed by 13 subjects in the azelastine (4 mg/day) group and 11 subjects in the placebo group. Treatments were randomly administered for 3 months. Each subject recorded daily asthma symptoms and peak expiratory flow (PEF). Lung function and bronchial responsiveness to methacholine were measured before and after treatment. Fiberoptic bronchoscopy was performed and bronchial biopsies taken from segmental carinae before and after treatment with azelastine or placebo. Using each monoclonal antibody for eosinophils, mast cells, macrophages, and T lymphocytes we performed immunohistological staining on biopsy specimens. RESULTS Compared with the placebo-treated group, the azelastine-treated group exhibited significant improvement of asthma symptoms (p<0.01), PEF (p<0.01), PEF diurnal variation (p<0.001), excluding forced expiratory volume in 1 s or airway responsiveness. This was accompanied by a reduction of EG2(+) eosinophils (p<0.01), CD3(+) (p<0.001), CD4(+) (p<0.05), CD8(+) (p<0.05) T lymphocytes, and CD25(+)-activated T lymphocytes (p<0.001) in the bronchial mucosa. Significant correlations were found between clinical data and immunohistochemical parameters. CONCLUSION These results demonstrated that the beneficial effects ofazelastine in bronchial asthma may result from modulation of local bronchial inflammatory cell infiltration.