Activation of the epidermal growth factor receptor (EGFR) by a novel metalloprotease pathway

Neutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFa and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NF?B was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production.

Elafin, an Elastase-specific Inhibitor, Is Cleaved by Its Cognate Enzyme Neutrophil Elastase in Sputum from Individuals with Cystic Fibrosis

Elafin is a neutrophil serine protease inhibitor expressed in lung and displaying anti-inflammatory and anti-bacterial properties. Previous studies demonstrated that some innate host defense molecules of the cystic fibrosis (CF) and chronic obstructive pulmonary disease airways are impaired due to increased proteolytic degradation observed during lung inflammation. In light of these findings, we thus focused on the status of elafin in CF lung. We showed in the present study that elafin is cleaved in sputum from individuals with CF. Pseudomonas aeruginosa-positive CF sputum, which was found to contain lower elafin levels and higher neutrophil elastase (NE) activity compared with P. aeruginosa-negative samples, was particularly effective in cleaving recombinant elafin. NE plays a pivotal role in the process as only NE inhibitors are able to inhibit elafin degradation. Further in vitro studies demonstrated that incubation of recombinant elafin with excess of NE leads to the rapid cleavage of the inhibitor. Two cleavage sites were identified at the N-terminal extremity of elafin (Val-5—Lys-6 and Val-9—Ser-10). Interestingly, purified fragments of the inhibitor (Lys-6—Gln-57 and Ser-10—Gln-57) were shown to still be active for inhibiting NE. However, NE in excess was shown to strongly diminish the ability of elafin to bind lipopolysaccharide (LPS) and its capacity to be immobilized by transglutamination. In conclusion, this study provides evidence that elafin is cleaved by its cognate enzyme NE present at excessive concentration in CF sputum and that P. aeruginosa infection promotes this effect. Such cleavage may have repercussions on the innate immune function of elafin.

Delivery of rSLPI in a liposomal carrier for inhalation provides protection against cathepsin L degradation

Secretory leukocyte protease inhibitor (SLPI) is an endogenous serine protease inhibitor that protects the lungs from excessive tissue damage caused by leukocyte proteases released during inflammation. Recombinant SLPI (rSLPI) has shown potential as a treatment for inflammatory lung conditions. To date, its clinical application has been limited by rapid enzymatic cleavage by cathepsins and rapid clearance from the lungs after inhalation. In this study, rSLPI was encapsulated in 1,2-Dioleoyl-sn-Glycero-3-[Phospho-L-Serine] : Cholesterol (DOPS : Chol) liposomes for inhalation. Incubation of rSLPI with cathepsin L leads to complete loss of activity while encapsulation of rSLPI in DOPS : Chol liposomes retained 92.6 of its activity after challenge with cathepsin L. rSLPI-loaded liposomes were aerosolized efficiently using a standard nebulizer with a minimal loss of activity and stability. This formulation was biocompatible and encapsulation did not appear to diminish access to intracellular sites of action in in vitro cell culture studies. Liposome encapsulation of rSLPI therefore improves stability and potentially reduces the level and frequency of dosing required for therapeutic effect after inhalation.

It's Better to Stick to your own Kind: Teenagers' Views on Cross-community marriages in Northern Ireland

This article presents the attitudes of 80 teenagers-growing up in one of the most contested localities in Northern Ireland-to cross-community marriages, i.e. those between Catholics and Protestants. Research suggests that adults in interface areas continue to exhibit ethno-sectarian prejudices despite wider political developments such as the Good Friday Agreement. The teenagers perceived that their families would be largely unsupportive of cross-community unions but felt that their own views were much less prejudiced than those of their parents. However, while the majority of teenagers had no objections in principle to marrying outside their religious group, they outlined a number of practical difficulties which couples from cross-community unions would face. These included deciding where to live, in which religion, if any, to bring children up and where to send children to school. Most of the teenagers suggested that these potential problems would work against them marrying outside their own religious group. These practical dilemmas provide a more nuanced set of reasons for marrying within one's own community than dilemmas based on traditional prejudices and stereotypes and suggest that teenagers living in sectarian enclaves are more receptive to cross-community marriages than their parents.

Social and Subcultural Capital among teenagers in Northern Ireland

This article makes a case for the inclusion of subcultural capital as an indictor of social capital networks in the lives of teenagers. It does so by critiquing approaches that assume that adult measures of social capital can be nonproblematically extended to account for stocks of social capital held by younger generations. To illustrate the fallacy of this approach, this article draws on data from the 2003 Northern Ireland Young Life and Times Survey (NIYLTS) and the indicators used to explore the relevance of social capital in the lives of teenagers. By ignoring concepts such as subcultural capital, surveys such as the NILYTS provide partial frameworks for understanding the complexities of young people's links to social capital networks and their inclusive and exclusive effects.

Decreased levels of secretory leucoprotease inhibitor in the Pseudomonas-infected cystic fibrosis lung are due to neutrophil elastase degradation

Secretory leucoprotease inhibitor (SLPI) is a neutrophil serine protease inhibitor constitutively expressed at many mucosal surfaces, including that of the lung. Originally identified as a serine protease inhibitor, it is now evident that SLPI also has antimicrobial and anti-inflammatory functions, and therefore plays an important role in host defense. Previous work has shown that some host defense proteins such as SLPI and elafin are susceptible to proteolytic degradation. Consequently, we investigated the status of SLPI in the cystic fibrosis (CF) lung. A major factor that contributes to the high mortality rate among CF patients is Pseudomonas aeruginosa infection. In this study, we report that P. aeruginosa-positive CF bronchoalveolar lavage fluid, which contains lower SLPI levels and higher neutrophil elastase (NE) activity compared with P. aeruginosa-negative samples, was particularly effective at cleaving recombinant human SLPI. Additionally, we found that only NE inhibitors were able to prevent SLPI cleavage, thereby implicating NE in this process. NE in excess was found to cleave recombinant SLPI at two novel sites in the NH(2)-terminal region and abrogate its ability to bind LPS and NF-kappaB consensus binding sites but not its ability to inhibit activity of the serine protease cathepsin G. In conclusion, this study provides evidence that SLPI is cleaved and inactivated by NE present in P. aeruginosa-positive CF lung secretions and that P. aeruginosa infection contributes to inactivation of the host defense screen in the CF lung.

Simvastatin Decreases Lipopolysaccharide-induced Pulmonary Inflammation in Healthy Volunteers

RATIONALE:
Simvastatin inhibits inflammatory responses in vitro and in murine models of lung inflammation in vivo. As simvastatin modulates a number of the underlying processes described in acute lung injury (ALI), it may be a potential therapeutic option.
OBJECTIVES:
To investigate in vivo if simvastatin modulates mechanisms important in the development of ALI in a model of acute lung inflammation induced by inhalation of lipopolysaccharide (LPS) in healthy human volunteers.
METHODS:
Thirty healthy subjects were enrolled in a double-blind, placebo-controlled study. Subjects were randomized to receive 40 mg or 80 mg of simvastatin or placebo (n = 10/group) for 4 days before inhalation of 50 microg LPS. Measurements were performed in bronchoalveolar lavage fluid (BALF) obtained at 6 hours and plasma obtained at 24 hours after LPS challenge. Nuclear translocation of nuclear factor-kappaB (NF-kappaB) was measured in monocyte-derived macrophages.
MEASUREMENTS AND MAIN RESULTS:
Pretreatment with simvastatin reduced LPS-induced BALF neutrophilia, myeloperoxidase, tumor necrosis factor-alpha, matrix metalloproteinases 7, 8, and 9, and C-reactive protein (CRP) as well as plasma CRP (all P < 0.05 vs. placebo). There was no significant difference between simvastatin 40 mg and 80 mg. BALF from subjects post-LPS inhalation induced a threefold up-regulation in nuclear NF-kappaB in monocyte-derived macrophages (P < 0.001); pretreatment with simvastatin reduced this by 35% (P < 0.001).
CONCLUSIONS:
Simvastatin has antiinflammatory effects in the pulmonary and systemic compartment in humans exposed to inhaled LPS.

Campaigns Against Incineration in Ireland - North and South: Framing Networking and Political Opportunities

An analysis of community-based campaigns opposing waste incineration and advocating 'zero waste' policies in Ireland, North and South.