Michael O'Mahony

Neutrophil Elastase Up-Regulates Cathepsin B and Matrix Metalloprotease-2 Expression

Neutrophil elastase (NE) activity is increased in many diseases. Other families of proteases, including cathepsins and matrix metalloproteases (MMPs), are also present at elevated levels in similar disease conditions. We postulated that NE could induce expression of cathepsins and MMPs in human macrophages. NE exposure resulted in macrophages, producing significantly greater amounts of cathepsin B and latent and active MMP-2. Cathepsin B and MMP-2 activities were decreased in Pseudomonas-infected NE knockout mice compared with wild-type littermates. We also demonstrate that NE can activate NF-κB in macrophages, and inhibition of NF-κB resulted in a reduction of NE-induced cathepsin B and MMP-2. Also, inhibition of TLR-4 or transfection of macrophages with dominant-negative IL-1R-associated kinase-1 resulted in a reduction of NE-induced cathepsin B and MMP-2. This study describes for the first time a novel hierarchy among proteases whereby a serine protease up-regulates expression of MMPs and cathepsins. This has important implications for therapeutic intervention in protease-mediated diseases.

Smoking is associated with shortened airway cilia.

Background Whereas cilia damage and reduced cilia beat frequency have been implicated as causative of reduced mucociliary clearance in smokers, theoretically mucociliary clearance could also be affected by cilia length. Based on models of mucociliary clearance predicting that cilia length must exceed the 6–7 µm airway surface fluid depth to generate force in the mucus layer, we hypothesized that cilia height may be decreased in airway epithelium of normal smokers compared to nonsmokers. Methodology/Principal Findings Cilia length in normal nonsmokers and smokers was evaluated in aldehyde-fixed, paraffin-embedded endobronchial biopsies, and air-dried and hydrated samples were brushed from human airway epithelium via fiberoptic bronchoscopy. In 28 endobronchial biopsies, healthy smoker cilia length was reduced by 15% compared to nonsmokers (p<0.05). In 39 air-dried samples of airway epithelial cells, smoker cilia length was reduced by 13% compared to nonsmokers (p<0.0001). Analysis of the length of individual, detached cilia in 27 samples showed that smoker cilia length was reduced by 9% compared to nonsmokers (p<0.05). Finally, in 16 fully hydrated, unfixed samples, smoker cilia length was reduced 7% compared to nonsmokers (p<0.05). Using genome-wide analysis of airway epithelial gene expression we identified 6 cilia-related genes whose expression levels were significantly reduced in healthy smokers compared to healthy nonsmokers. Conclusions/Significance Models predict that a reduction in cilia length would reduce mucociliary clearance, suggesting that smoking-associated shorter airway epithelial cilia play a significant role in the pathogenesis of smoking-induced lung disease.

Secretory leucocyte protease inhibitor inhibits interferon gamma-induced cathepsin s expression

We have demonstrated that bronchoalveolar lavage fluid from chronic obstructive pulmonary disease patients contains higher levels of interferon-γ compared with controls. Interferon-γ is a potent inducer of various cathepsins and matrix metalloproteases. Therefore, we postulated that interferon-γ could induce protease expression by macrophages in acute and chronic lung disease. Chronic obstructive pulmonary disease patients had greater levels of cathepsin S and matrix metalloprotease-12 in their bronchoalveolar lavage fluid. Macrophages incubated with chronic obstructive pulmonary disease bronchoalveolar lavage fluid exhibited increased expression of cathepsin S and matrix metalloprotease-12, which was inhibited by the addition of interferon-γ-neutralizing immunoglobulin. Human secretory leukocyte protease inhibitor is an 11.7-kDa cationic non-glycosylated antiprotease synthesized and secreted by cells at the site of inflammation. We have demonstrated that secretory leukocyte protease inhibitor can inhibit interferon-γ-induced cathepsin S production by macrophages. Pretreatment of macrophages with secretory leukocyte protease inhibitor inhibited interferon-γ-induced inhibitor κB β degradation and activation of nuclear factor κB. Secretory leukocyte protease inhibitor may prove to be therapeutically important as a potential inhibitor of protease expression in chronic obstructive pulmonary disease.

Anti-apoptotic effects of Z alpha-1 antitrypsin in human bronchial epithelial cells.

α1-antitrypsin (α1-AT) deficiency is a genetic disease which manifests as early-onset emphysema or liver disease. Although the majority of α1-AT is produced by the liver, it is also produced by bronchial epithelial cells, amongst others, in the lung. Herein, we investigate the effects of mutant Z α1-AT (ZAAT) expression on apoptosis in a human bronchial epithelial cell line (16HBE14o-) and delineate the mechanisms involved. Control, M variant α1-AT (MAAT)- or ZAAT-expressing cells were assessed for apoptosis, caspase-3 activity, cell viability, phosphorylation of Bad, nuclear factor (NF)-κB activation and induced expression of a selection of pro- and anti-apoptotic genes. Expression of ZAAT in 16HBE14o- cells, like MAAT, inhibited basal and agonist-induced apoptosis. ZAAT expression also inhibited caspase-3 activity by 57% compared with control cells (p = 0.05) and was a more potent inhibitor than MAAT. Whilst ZAAT had no effect on the activity of Bad, its expression activated NF-κB-dependent gene expression above control or MAAT-expressing cells. In 16HBE14o- cells but not HEK293 cells, ZAAT upregulated expression of cIAP-1, an upstream regulator of NF-κB. cIAP1 expression was increased in ZAAT versus MAAT bronchial biopsies. The data suggest a novel mechanism by which ZAAT may promote human bronchial epithelial cell survival.

Alpha-1 antitrypsin deficiency and computed tomography findings.

Objective: To investigate the severity of bronchiectasis and associated emphysema and the correlation with phenotype in patients with Alpha-1 antitrypsin deficiency. Methods: The scoring system of Ooi and his colleagues for bronchiectasis was modified to include the degree of dilatation of bronchi in affected segments and degree of emphysema. This was applied to 26 high-resolution computed tomography thorax scans of the study population. All criteria were scored on a scale of 0-3. Results: Nine patients (35%) were female and 17 (65%) were male. The median age was 56 years (range: 17-76 years). Twenty-one patients had a ZZ phenotype, 3 patients had an MZ phenotype, and 2 patients had an SZ phenotype. The median forced expiratory volume in 1 second/forced vital capacity ratio was 43% (range: 24%-87%). A total of 156 lobes were assessed, and 38 (24%) had evidence of bronchiectasis. The overall median total score in affected patients for the extent of bronchiectasis was 2, and all had a ZZ phenotype. Fourteen patients (54%) had a degree of dilatation score of 1 or more, all had a ZZ phenotype, and 4 (15%) had no evidence of emphysema. Bronchiectasis was seen most commonly affecting the upper lobes. Conclusion: The ZZ phenotype was associated with bronchiectasis most commonly affecting the upper lobes, with moderate emphysema throughout all lobes. Numbers of patients having the SZ and MZ phenotypes are too small to derive accurate conclusions, but none had evidence of bronchiectasis.