Neutrophil Elastase and Chronic Lung Disease.

Neutrophil elastase (NE) is a major inflammatory protease released by neutrophils and is present in the airways of patients with cystic fibrosis (CF), chronic obstructive pulmonary disease, non-CF bronchiectasis, and bronchopulmonary dysplasia. Although NE facilitates leukocyte transmigration to the site of infection and is required for clearance of Gram-negative bacteria, it also activates inflammation when released into the airway milieu in chronic inflammatory airway diseases. NE exposure induces airway remodeling with increased mucin expression and secretion and impaired ciliary motility. NE interrupts epithelial repair by promoting cellular apoptosis and senescence and it activates inflammation directly by increasing cytokine expression and release, and indirectly by triggering extracellular trap release and exosome release, which magnify protease activity and inflammation in the airway. NE inhibits innate immune function by digesting opsonins and opsonin receptors, degrading innate immune proteins such as lactoferrin, and inhibiting macrophage phagocytosis. Importantly, NE-directed therapies have not yet been effective in preventing the pathologic sequelae of NE exposure, but new therapies are being developed that offer both direct antiprotease activity and multifunctional anti-inflammatory properties.

New insights into the pathomechanism of cyclic neutropenia.

Cyclic neutropenia (CyN) is a hematologic disorder in which peripheral blood absolute neutrophil counts (ANCs) show cycles of approximately 21-day intervals. The majority of CyN patients harbor ELANE mutations, but the mechanism of ANC cycling is unclear. We performed analysis of bone marrow (BM) subpopulations in CyN patients at the peak and the nadir of the ANC cycle and detected high proportions of BM hematopoietic stem cells (HSCs) and hematopoietic stem and progenitor cells (HSPCs) at the nadir of the ANC cycle, as compared with the peak. BM HSPCs produced fewer granulocyte colony-forming unit colonies at the ANC peak. To investigate the mechanism of cycling, we found that mRNA expression levels of ELANE and unfolded protein response (UPR)-related genes (ATF6, BiP (HSPA5), CHOP (DDIT3), and PERK (EIF2AK3)) were elevated, but antiapoptotic genes (Bcl-2 (BCL2) and bcl-xL (BCL2L1)) were reduced in CD34+ cells tested at the ANC nadir. Moreover, HSPCs revealed increased levels of reactive oxygen species and gH2AX at the ANC nadir. We suggest that in CyN patients, some HSPCs escape the UPR-induced endoplasmic reticulum (ER) stress and proliferate in response to granulocyte colony-stimulating factor (G-CSF) to a certain threshold at which UPR again affects the majority of HSPCs. There is a cyclic balance between ER stress-induced apoptosis of HSPCs and compensatory G-CSF-stimulated HSPC proliferation followed by granulocytic differentiation.

Neutrophil elastase-mediated proteolysis of the tumor suppressor p200 CUX1 promotes cell proliferation and inhibits cell differentiation in APL.

AIMS: Neutrophil elastase (NE) is a critical proteolytic enzyme that is involved in cancer. We previously reported high NE expression in peripheral blood neutrophils from acute promyelocytic leukemia (APL) patients. The present study aimed to elucidate the specific role and mechanisms of NE in APL development. MATERIALS AND METHODS: NE expression was detected in APL bone marrow samples and analyzed in the BloodSpot database. CCK-8 assay and flow cytometry were used to assess cell proliferation and cell cycle distribution, respectively. The expression levels of proliferation and differentiation markers were measured by Western blotting and quantitative real-time PCR. The co-expression and interaction of NE and p200 cut-like homeobox 1 (CUX1) were evaluated by indirect immunofluorescence, co-immunoprecipitation, and in situ proximity ligation assay. KEY FINDINGS: NE was highly expressed in APL bone marrow and blood neutrophils. NE overexpression promoted the proliferation and inhibited the differentiation of NB4 cells, whereas NE downregulation achieved the opposite results in U937 cells. Mechanistically, NE interacted with and effectively hydrolyzed the tumor suppressor p200 CUX1. Rescue experiments revealed that p200 CUX1 upregulation reversed the functional influence of NE on APL cells. SIGNIFICANCE: NE-mediated proteolysis of the tumor suppressor p200 CUX1 promotes APL progression. NE/p200 CUX1 axis is a novel and promising therapeutic target for APL treatment.

Blue and Long-Wave Ultraviolet Light Induce in vitro Neutrophil Extracellular Trap (NET) Formation.

Neutrophil Extracellular Traps (NETs) are produced by neutrophilic granulocytes and consist of decondensed chromatin decorated with antimicrobial peptides. They defend the organism against intruders and are released upon various stimuli including pathogens, mediators of inflammation, or chemical triggers. NET formation is also involved in inflammatory, cardiovascular, malignant diseases, and autoimmune disorders like rheumatoid arthritis, psoriasis, or systemic lupus erythematosus (SLE). In many autoimmune diseases like SLE or dermatomyositis, light of the ultraviolet-visible (UV-VIS) spectrum is well-known to trigger and aggravate disease severity. However, the underlying connection between NET formation, light exposure, and disease exacerbation remains elusive. We studied the effect of UVA (375 nm), blue (470 nm) and green (565 nm) light on NETosis in human neutrophils ex vivo. Our results show a dose- and wavelength-dependent induction of NETosis. Light-induced NETosis depended on the generation of extracellular reactive oxygen species (ROS) induced by riboflavin excitation and its subsequent reaction with tryptophan. The light-induced NETosis required both neutrophil elastase (NE) as well as myeloperoxidase (MPO) activation and induced histone citrullination. These findings suggest that NET formation as a response to light could be the hitherto missing link between elevated susceptibility to NET formation in autoimmune patients and photosensitivity for example in SLE and dermatomyositis patients. This novel connection could provide a clue for a deeper understanding of light-sensitive diseases in general and for the development of new pharmacological strategies to avoid disease exacerbation upon light exposure.

The Role of Neutrophil Elastase Inhibitors in Lung Diseases.

In many respiratory diseases characterized by an intense inflammatory response, the balance between proteolytic enzymes (proteases, including elastases) and their inhibitors (proteinases inhibitors) is not neutral. Excess activity of neutrophil elastase (NE) and similar proteases has been reported to cause tissue damage and to alter the remodeling process in many clinical conditions such as pneumonia, respiratory distress, and acute lung injury (ALI). Several experimental NE inhibitors have been tested in preclinical and clinical studies of different conditions of inflammatory lung injury such as ALI and pneumonia, with contrasting results. This study reviews the literature regarding NE inhibitors in the field of respiratory diseases and reflects on possible future developments. In particular, we highlight potential gaps in the scientific evidence and discuss potential strategies for focusing investigation on antielastases in clinical practice through the selection of targeted populations and proper outcomes.

Neutrophil elastase enhances IL-12p40 production by lipopolysaccharide-stimulated macrophages via transactivation of the PAR-2/EGFR/TLR4 signaling pathway.

Proteinase-activated receptor 2 (PAR-2) and toll-like receptor 4 (TLR4) are involved in innate immune responses and signaling cross-talk between these receptor molecules has the potential to augment an ongoing inflammatory response. The aim of this study was to evaluate the possible cooperative influence of PAR-2 and TLR4 on IL-12p40 production by macrophages after stimulation with lipopolysaccharide (LPS). During culture, GM-CSF upregulated PAR-2 expression by macrophages in a time-dependent manner. Stimulation with LPS enhanced IL-12p40 production by macrophages in a concentration-dependent manner. While human neutrophil elastase (HNE) did not induce IL-12p40 production, pretreatment of macrophages with HNE synergistically increased the IL-12p40 protein level after LPS exposure. Silencing of TLR4 with small interfering RNA blunted the synergistic enhancement of IL-12p40 by HNE combined with LPS. Silencing of ß-arrestin 2, p22phox, or ERK1/2 also inhibited an increase of IL-12p40. Interestingly, transfection of macrophages with small interfering RNA duplexes for DUOX-2, EGFR, TLR4, or TRAF6 significantly blunted the increase of IL-12p40 in response to treatment with HNE plus LPS. U73122 and Rottlerin also inhibited the increased production of IL-12p40. In conclusion, HNE is involved in transactivation of TLR4 through activation of DUOX-2/EGFR and synergistically enhances IL-12p40 production by macrophages stimulated with LPS.

Reduced secretion and altered proteolytic processing caused by missense mutations in progranulin.

Progranulin (GRN) is a secreted growth factor involved in various cellular functions, and loss-of-function mutations are a major cause of frontotemporal lobar degeneration (FTLD) with TDP-43 positive pathology. Most FTLD-related GRN mutations are nonsense mutations resulting in reduced GRN expression. Nonsynonymous GRN missense mutations have been described as risk factor for neurodegenerative brain diseases, but their pathogenic nature remains largely elusive. We identified a double missense mutation in GRN leading to amino acid changes p.D33E and p.G35R in an FTLD patient from Turkish origin. Biochemical and cell biological analysis of the double-mutation together with 2 so-far uncharacterized GRN missense mutations (p.C105R and p.V514M) revealed a reduced secretion efficiency of the GRN p.D33E/p.G35R and p.C105R proteins. Furthermore, loss of the conserved cysteine residue affects protein folding and altered proteolytic processing by neutrophil elastase and proteinase 3. Our data indicate that the described variants may cause a loss-of-function, albeit to a lesser extent than GRN null mutations, and hence could be considered as low-penetrant risk factors for neurodegenerative diseases.

The Role of Serine Proteases and Antiproteases in the Cystic Fibrosis Lung.

Cystic fibrosis (CF) lung disease is an inherited condition with an incidence rate of approximately 1 in 2500 new born babies. CF is characterized as chronic infection of the lung which leads to inflammation of the airway. Sputum from CF patients contains elevated levels of neutrophils and subsequently elevated levels of neutrophil serine proteases. In a healthy individual these proteases aid in the phagocytic process by degrading microbial peptides and are kept in homeostatic balance by cognate antiproteases. Due to the heavy neutrophil burden associated with CF the high concentration of neutrophil derived proteases overwhelms cognate antiproteases. The general effects of this protease/antiprotease imbalance are impaired mucus clearance, increased and self-perpetuating inflammation, and impaired immune responses and tissue. To restore this balance antiproteases have been suggested as potential therapeutics or therapeutic targets. As such a number of both endogenous and synthetic antiproteases have been trialed with mixed success as therapeutics for CF lung disease.

The serine protease inhibitor elafin maintains normal growth control by opposing the mitogenic effects of neutrophil elastase.

The serine protease inhibitor, elafin, is a critical component of the epithelial barrier against neutrophil elastase (NE). Elafin is downregulated in the majority of breast cancer cell lines compared with normal human mammary epithelial cells (HMECs). Here, we evaluated the role of elafin and NE on proliferation and tumorigenesis. Elafin is induced in growth factor-deprived HMECs as they enter a quiescent (G0) state, suggesting that elafin is a counterbalance against the mitogenic effects of NE in G0 HMECs. Stable knockdown of elafin compromises the ability of HMECs to maintain G0 arrest during long-term growth factor deprivation; this effect can be reversed by re-expression of wild-type elafin but not elafin-M25G lacking protease inhibitory function. These results suggest that NE, which is largely contributed by activated neutrophils in the tumor microenvironment, may be negatively regulating the ability of elafin to arrest cells in G0. In fact when purified NE was added to elafin-knocked down HMECs, these cells demonstrated greater sensitivity to the growth-promoting effects of purified NE. Activation of ERK signaling, downstream of toll-like receptor 4, was essential to the mitogenic effect of NE on HMECs. These findings were next translated to patient samples. Immunohistochemical analysis of normal breast tissue revealed robust elafin expression in the mammary epithelium; however, elafin expression was dramatically downregulated in a significant proportion of human breast tumor specimens. The loss of elafin expression during breast cancer progression may promote tumor growth as a consequence of increased NE activity. To address the role of NE in mammary tumorigenesis, we next examined whether deregulated NE activity enhances mammary tumor growth. NE knockout in the C3(1)TAg mouse model of mammary tumorigenesis suppressed proliferation and reduced the kinetics of tumor growth. Overall, the imbalance between NE and its inhibitors, such as elafin, presents an important therapeutic target in breast cancer.

Lack of neutrophil elastase reduces inflammation, mucus hypersecretion, and emphysema, but not mucus obstruction, in mice with cystic fibrosis-like lung disease.

RATIONALE: Recent evidence from clinical studies suggests that neutrophil elastase (NE) released in neutrophilic airway inflammation is a key risk factor for the onset and progression of lung disease in young children with cystic fibrosis (CF). However, the role of NE in the complex in vivo pathogenesis of CF lung disease remains poorly understood. OBJECTIVES: To elucidate the role of NE in the development of key features of CF lung disease including airway inflammation, mucus hypersecretion, goblet cell metaplasia, bacterial infection, and structural lung damage in vivo. METHODS: We used the Scnn1b-Tg mouse as a model of CF lung disease and determined effects of genetic deletion of NE (NE(-/-)) on the pulmonary phenotype. Furthermore, we used novel Foerster resonance energy transfer (FRET)-based NE reporter assays to assess NE activity in bronchoalveolar lavage from Scnn1b-Tg mice and sputum from patients with CF. MEASUREMENTS AND MAIN RESULTS: Lack of NE significantly reduced airway neutrophilia, elevated mucin expression, goblet cell metaplasia, and distal airspace enlargement, but had no effect on airway mucus plugging, bacterial infection, or pulmonary mortality in Scnn1b-Tg mice. By using FRET reporters, we show that NE activity was elevated on the surface of airway neutrophils from Scnn1b-Tg mice and patients with CF. CONCLUSIONS: Our results suggest that NE plays an important role in the in vivo pathogenesis and may serve as a therapeutic target for inflammation, mucus hypersecretion, and structural lung damage and indicate that additional rehydration strategies may be required for effective treatment of airway mucus obstruction in CF.

Neutrophil elastase-mediated increase in airway temperature during inflammation.

BACKGROUND: How elevated temperature is generated during airway infections represents a hitherto unresolved physiological question. We hypothesized that innate immune defence mechanisms would increase luminal airway temperature during pulmonary infection. METHODS: We determined the temperature in the exhaled air of cystic fibrosis (CF) patients. To further test our hypothesis, a pouch inflammatory model using neutrophil elastase-deficient mice was employed. Next, the impact of temperature changes on the dominant CF pathogen Pseudomonas aeruginosa growth was tested by plating method and RNAseq. RESULTS: Here we show a temperature of ~38°C in neutrophil-dominated mucus plugs of chronically infected CF patients and implicate neutrophil elastase:α1-proteinase inhibitor complex formation as a relevant mechanism for the local temperature rise. Gene expression of the main pathogen in CF, P. aeruginosa, under anaerobic conditions at 38°C vs 30°C revealed increased virulence traits and characteristic cell wall changes. CONCLUSION: Neutrophil elastase mediates increase in airway temperature, which may contribute to P. aeruginosa selection during the course of chronic infection in CF.

The role of neutrophils in cystic fibrosis.

PURPOSE OF REVIEW: Neutrophils are known to dominate the pulmonary inflammatory process observed in cystic fibrosis (CF). An enduring paradox is how these large numbers of neutrophils fail to eradicate colonizing bacteria. Major advances in our understanding of neutrophil dysfunction in CF and its effect on the innate immune system are leading to advances in our understanding of the pathophysiology and leading directly to new therapies. RECENT FINDINGS: New mechanisms of neutrophil dysfunction have been described in CF including disabled cystic fibrosis transmembrane conductance regulator recruitment to phagosomes and novel mechanisms of protease-induced neutrophil dysfunction. Neutrophil elastase has been shown to be present in the airway very early in life in CF patients, and appears a biomarker of disease progression, predicting lung function decline and bronchiectasis. Elastase has also been shown to induce a pro-inflammatory state of senescence in bronchial epithelial cells in vitro and potentially in vivo. Inhibitors of neutrophil elastase are now entering clinical trials with promising results. New avenues of CF therapeutics are being explored including novel macrolides, CXCR2 antagonists and exogenous opsonins. SUMMARY: This article reviews the past 12 months of research that contributes to our understanding of the role of neutrophils and immune dysfunction in CF.

Promoting effect of neutrophils on lung tumorigenesis is mediated by CXCR2 and neutrophil elastase.

BACKGROUND: Tumor cells produce various cytokines and chemokines that attract leukocytes. Leukocytes can amplify parenchymal innate immune responses, and have been shown to contribute to tumor promotion. Neutrophils are among the first cells to arrive at sites of inflammation, and the increased number of tumor-associated neutrophils is linked to poorer outcome in patients with lung cancer. RESULTS: We have previously shown that COPD-like airway inflammation promotes lung cancer in a K-ras mutant mouse model of lung cancer (CC-LR). This was associated with severe lung neutrophilic influx due to the increased level of neutrophil chemoattractant, KC. To further study the role of neutrophils in lung tumorigenesis, we depleted neutrophils in CC-LR mice using an anti-neutrophil antibody. This resulted in a significant reduction in lung tumor number. We further selectively inhibited the main receptor for neutrophil chemo-attractant KC, CXCR2. Similarly, this resulted in suppression of neutrophil recruitment into the lung of CC-LR mice followed by significant tumor reduction. Neutrophil elastase (NE) is a potent elastolytic enzyme produced by neutrophils at the site of inflammation. We crossed the CC-LR mice with NE knock-out mice, and found that lack of NE significantly inhibits lung cancer development. These were associated with significant reduction in tumor cell proliferation and angiogenesis. CONCLUSION: We conclude that lung cancer promotion by inflammation is partly mediated by activation of the IL-8/CXCR2 pathway and subsequent recruitment of neutrophils and release of neutrophil elastase. This provides a baseline for future clinical trials using the IL-8/CXCR2 pathway or NE inhibitors in patients with lung cancer.

Neutrophil elastase induces MUC5AC secretion via protease-activated receptor 2.

Mucus hypersecretion is a major manifestation in patients with chronic inflammatory airway diseases, and mucin5AC (MUC5AC) protein is a major component of airway mucus. Previous studies have demonstrated that neutrophil elastase (NE) stimulates the secretion of MUC5AC from airway epithelial cells, however, the mechanism is poorly understood. NE is a known ligand for protein active receptors (PARs), which have been confirmed to participate in releasing MUC5AC in the airways. However, the role of PARs in NE-induced MUC5AC secretion remains unclear. We demonstrated that airway goblet-like Calu-3 cells express PAR1, PAR2, and PAR3 with a predominant level of PAR2. NE can increase PAR2 expression and MUC5AC release. In our study, we showed that NE binding to PAR2 can increase the cytosolic calcium concentration and subsequently activate PKC, leading to MUC5AC secretion. In order to investigate the mechanism of increased cytosolic calcium in Calu-3 cells, thapsigargin was used to exhaust the endoplasmic reticulum (ER) calcium pools, and 2-aminoethoxydiphenyl borate was used to inhibit the function of the store-operated calcium entry (SOCE) channels in the plasma membrane. We found that the NE-induced increase in intracellular calcium concentration is derived from release of the ER calcium pool and its subsequent calcium internal flux from the extracellular space via SOCE channels, which is dependent on sufficient levels of extracellular calcium.

Inhibition of neutrophil elastase attenuates airway hyperresponsiveness and inflammation in a mouse model of secondary allergen challenge: neutrophil elastase inhibition attenuates allergic airway responses.

BACKGROUND: Chronic asthma is often associated with neutrophilic infiltration in the airways. Neutrophils contain elastase, a potent secretagogue in the airways, nonetheless the role for neutrophil elastase as well as neutrophilic inflammation in allergen-induced airway responses is not well defined. In this study, we have investigated the impact of neutrophil elastase inhibition on the development of allergic airway inflammation and airway hyperresponsiveness (AHR) in previously sensitized and challenged mice. METHODS: BALB/c mice were sensitized and challenged (primary) with ovalbumin (OVA). Six weeks later, a single OVA aerosol (secondary challenge) was delivered and airway inflammation and airway responses were monitored 6 and 48 hrs later. An inhibitor of neutrophil elastase was administered prior to secondary challenge. RESULTS: Mice developed a two-phase airway inflammatory response after secondary allergen challenge, one neutrophilic at 6 hr and the other eosinophilic, at 48 hr. PAR-2 expression in the lung tissues was enhanced following secondary challenge, and that PAR-2 intracellular expression on peribronchial lymph node (PBLN) T cells was also increased following allergen challenge of sensitized mice. Inhibition of neutrophil elastase significantly attenuated AHR, goblet cell metaplasia, and inflammatory cell accumulation in the airways following secondary OVA challenge. Levels of IL-4, IL-5 and IL-13, and eotaxin in BAL fluid 6 hr after secondary allergen challenge were significantly suppressed by the treatment. At 48 hr, treatment with the neutrophil elastase inhibitor significantly reduced the levels of IL-13 and TGF-ß1 in the BAL fluid. In parallel, in vitro IL-13 production was significantly inhibited in spleen cells from sensitized mice. CONCLUSION: These data indicate that neutrophil elastase plays an important role in the development of allergic airway inflammation and hyperresponsiveness, and would suggest that the neutrophil elastase inhibitor reduced AHR to inhaled methacholine indicating the potential for its use as a modulator of the immune/inflammatory response in both the neutrophil- and eosinophil-dominant phases of the response to secondary allergen challenge.

Increased expression of senescence markers in cystic fibrosis airways.

Cystic Fibrosis (CF) is a chronic lung disease characterized by chronic neutrophilic airway inflammation and increased levels of neutrophil elastase (NE) in the airways. We have previously reported that NE treatment triggers cell cycle arrest. Cell cycle arrest can lead to senescence, a complete loss of replicative capacity. Importantly, senescent cells can be proinflammatory and would perpetuate CF chronic inflammation. By immunohistochemistry, we evaluated whether airway sections from CF and control subjects expressed markers of senescence, including p16(INK4a) (p16), a cyclin-dependent kinase inhibitor, phospho-Histone H2A.X (γH2A.X), and phospho-checkpoint 2 kinase (phospho-Chk2), which are also DNA damage response markers. Compared with airway epithelium from control subjects, CF airway epithelium had increased levels of expression of all three senescence markers. We hypothesized that the high load of NE in the CF airway triggers epithelial senescence by upregulating expression of p16, which inhibits cyclin-dependent kinase 4 (CDK4). Normal human bronchial epithelial (NHBE) cells, cultured in air-liquid interface were treated with NE (0, 200, and 500 nM) to induce visible injury. Total cell lysates were collected and evaluated by Western analysis for p16 protein expression and CDK4 kinase activity. NE significantly increased p16 expression and decreased CDK4 kinase activity in NHBE cells. These results support the concept that NE triggers expression of senescence markers in CF airway epithelial cells.

Role of neutrophil elastase in lung injury induced by burn-blast combined injury in rats.

OBJECTIVE: Neutrophil elastase (NE) takes part in the pathogenesis of acute lung injury. However, its role in lung injury of burn-blast combined injury is unclear. Our objective was to assess the role of NE, and effect of sivelestat, a specific NE inhibitor, in lung injury induced by burn-blast combined injury in rats. METHODS: One hundred and sixty male Sprague-Dawley rats were randomly subjected to burn-blast combined injury (BB) group, burn-blast combined injury plus sivelestat treatment (S) group or control (C) group. Blood gas, protein concentration and NE activity in bronchoalveolar lavage fluid (BALF), pulmonary myeloperoxidase (MPO) activity, serum concentrations of TNF-α and IL-8, etc. were investigated from 0 h to 7 d post-injury. RESULTS: In BB group, PaO2 decreased, while NE activity in BALF, total protein concentration in BALF, pulmonary MPO activity and W/D ratio, serum concentrations of TNF-α and IL-8 increased with neutrophil infiltration, progressive bleeding and pulmonary oedema. Compared with BB group, sivelestat treatment decreased the NE activity and ameliorated the above indexes. CONCLUSION: Sivelestat, exerts a protective effect in lung injury after burn-blast combined injury through inhibiting NE activity to decrease pulmonary vascular permeability, neutrophil sequestration, and production of TNF-α and IL-8.

Neutrophil elastase degrades cystic fibrosis transmembrane conductance regulator via calpains and disables channel function in vitro and in vivo.

RATIONALE: Cystic fibrosis transmembrane conductance regulator (CFTR) protein is a chloride channel regulating fluid homeostasis at epithelial surfaces. Its loss of function induces hypohydration, mucus accumulation, and bacterial infections in CF and potentially other lung chronic diseases. OBJECTIVES: To test whether neutrophil elastase (NE) and neutrophil-mediated inflammation negatively impact CFTR structure and function, in vitro and in vivo. METHODS: Using an adenovirus-CFTR overexpression approach, we showed that NE degrades wild-type (WT)- and ΔF508-CFTR in vitro and WT-CFTR in mice through a new pathway involving the activation of intracellular calpains. MEASUREMENTS AND MAIN RESULTS: CFTR degradation triggered a loss of function, as measured in vitro by channel patch-clamp and in vivo by nasal potential recording in mice. Importantly, this mechanism was also shown to be operative in a Pseudomonas aeruginosa lung infection murine model, and was NE-dependent, because CFTR integrity was significantly protected in NE(-/-) mice compared with WT mice. CONCLUSIONS: These data provide a new mechanism and show for the first time a link between NE-calpains activation and CFTR loss of function in bacterial lung infections relevant to CF and to other chronic inflammatory lung conditions.

Novel mechanism of aortic aneurysm development in mice associated with smoking and leukocytes.

OBJECTIVE: The purpose of this study was to evaluate potential mechanisms promoting abdominal aortic aneurysm development with tobacco smoke (TS) exposure. METHODS AND RESULTS: Experiments used the elastase perfusion model of abdominal aortic aneurysms with smoke-free controls. The effect of TS exposure was evaluated in C57/Bl6 mice, after broad-spectrum matrix metalloproteinase inhibition with doxycycline and in mice deficient in matrix metalloproteinase-9, matrix metalloproteinase-12, Cathepsin-S, and Neutrophil Elastase. Preparations of washed marrow, spleen, and peripheral blood leukocytes were transferred to smoke-free mice from 6-week TS-exposed mice or smoke-free mice. All mice were euthanized 14 days after elastase perfusion, and the percentage of change in aortic diameter (%Δ aortic diameter) was calculated. Electron microscopy of aortic tissue from animals exposed to TS without elastase exposure did not demonstrate any ultrastructural changes. Neither doxycycline nor any specific elastase deficiency was effective at preventing an increase in %Δ aortic diameter in TS-exposed animals. Smoke exposure for 6 weeks increased the %Δ aortic diameter after a smoke-free interval of up to 6 weeks before elastase perfusion. Leukocyte preparations from TS-exposed mice localized to abdominal aortic aneurysms and increased the %Δ aortic diameter in smoke-free mice. CONCLUSIONS: The effect of TS on the development of abdominal aortic aneurysms is not dependent on the activity of elastolytic enzymes and persists for long periods despite cessation of TS. Alterations in leukocyte response to aortic injury appear to mediate this effect.