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Published ahead of print on January 14, 2005, doi:10.1165/rcmb.2004-0321OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 334-341, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0321OC

Induction of Human Airway Smooth Muscle Apoptosis by Neutrophils and Neutrophil Elastase

Ute Oltmanns, Maria B. Sukkar, Shaoping Xie, Matthias John and K. Fan Chung

Thoracic Medicine, National Heart and Lung Institute, Imperial College, London, United Kingdom; and Department of Pneumology, University Hospital Charité, Berlin, Germany

Correspondence and requests for reprints should be addressed to Professor K. Fan Chung, National Heart & Lung Institute, Imperial College, Dovehouse St., London SW3 6LY, UK. E-mail: f.chung{at}imperial.ac.uk


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Neutrophils are an important component of airway inflammation and may interact with human airway smooth muscle cells (HASMC). We investigated the effect of neutrophils and of neutrophil-derived proteases on HASMC survival. When co-incubated with neutrophils (0.1–1 x 106 cells/ml), attachment of human ASMC was reduced to 12.3 ± 4.3% compared with untreated controls after 72 h. HASMC showed nuclear condensation and fragmentation (41.6 ± 8.1% compared with baseline of 3.1 ± 0.4%), and the biochemical markers of apoptosis, annexin V binding (9.7 ± 0.7%; baseline 1.1 ± 0.3%) and cleaved caspase-3 expression, were observed. The proteolytic activity released by neutrophils was essential for the proapoptotic effect because inhibition of elastase activity by {alpha}1-antitrypsin and MeOSuc-Ala-Ala-Pro-Ala-CMK (MSACK) reduced HASMC apoptosis. Human neutrophil elastase (0.1–3 µg/ml) induced apoptosis of HASMC, as well as other neutrophil serine proteases, cathepsin G, and proteinase 3. Fibronectin degradation products were present in HASMC supernatants exposed to neutrophil-conditioned media and to neutrophil elastase. The local release of proteases from neutrophils present in airway smooth muscle cells may lead to HASMC apoptosis as a result of matrix degradation and loss of cell attachment. This may limit pathologic changes such as ASMC hyperplasia and extracellular matrix deposition seen in airway remodeling.

Key Words: apoptosis • airway smooth muscle • fibronectin • neutrophil elastase


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Persistent airway inflammation is a central feature of many respiratory disorders such as asthma and chronic bronchitis. Alterations of the airway structure, defined as airway remodeling, are also associated with these diseases and may contribute to airway wall thickening and airflow obstruction (1, 2). The events occurring in airway remodeling include modifications of extracellular matrix (ECM) deposition and composition, as well as changes in number and/or size of structural cells such as airway smooth muscle cells. In asthma, the remodeling process results in thickening of the airway wall with mucous gland hyperplasia, increased vessel area, and (most importantly) airway smooth muscle hyperplasia and hypertrophy (3). In chronic obstructive pulmonary disease (COPD), airway remodeling mainly affects small airways with epithelial metaplasia and submucosal fibrosis (1, 2). The cellular and molecular mechanisms accounting for these events remain unclear.

Interactions between inflammatory and mesenchymal cells are likely to be involved in the regulation of airway structure. Human airway smooth muscle cells (HASMC) are multifunctional cells that represent a major structural component of the airway wall, playing an important role in regulating airway caliber. In asthma, an increased muscle mass is probably the major cause for exaggerated airway narrowing (4). Airway smooth muscle may also contribute to tissue inflammation and remodeling by virtue of its synthetic capacity (5). Chemokines derived from HASMC, such as RANTES, interleukin (IL)-8, and monocyte chemotactic protein-1, amplify the recruitment and activation of inflammatory cells and prolong their survival in the airway microenvironment. Airway smooth muscle cells are also a rich source of growth factors (insulin growth factor [IGF], platelet-derived growth factor) and extracellular matrix components (laminin, collagen, fibronectin), and could modulate their own growth and the composition of their surrounding matrix through autocrine effects (5–7).

Survival of HASMC strongly depends on the composition and integrity of their surrounding matrix (8). Detachment from this matrix results in the withdrawal of survival signals and may lead to apoptosis as shown in other anchorage-dependent cells, such as epithelial cells and fibroblasts (9, 10). Several proteinases, including leukocyte elastase, have the potential to cleave matrix molecules and cause detachment-induced apoptosis (11), a process that has been termed anoikis. Anoikis has been implicated in a wide range of tissue-homeostatic, developmental, and oncogenic processes. Resistance to anoikis is an important step in the development of metastatic cancer disease (12), and in the cardiovascular system anoikis may play an important role during tissue remodeling in heart failure, atherosclerosis, and aneurysms (13). However, the role of anoikis in airway remodeling is not known.

Neutrophils comprise an important cellular component of airway inflammation in COPD (14), and there is also evidence that neutrophils can be an important feature of bronchial inflammation in severe asthma (15, 16). A recent study reported increased numbers of neutrophils within the airway smooth muscle layer in COPD (17), indicating potential interactions between these cell types in airway inflammation. Products released by activated neutrophils, such as proteases, oxygen active radicals, and cytokines (such as IL-1, tumor necrosis factor-{alpha}, IL-6, and IL-8) may contribute to tissue inflammation and injury (18). Specifically neutrophil elastase, a serine protease, has been implicated in the pathophysiology of lung injury due to its capability to break down almost all ECM molecules (11, 19). Increased influx of neutrophils into the lungs of patients with COPD and severe asthma may be the result of neutrophil chemoattractants such as IL-8, and neutrophil-derived mediators may modify various functions of HASMC.

We hypothesized that neutrophils may affect airway smooth muscle viability by releasing matrix-degrading proteinases, such as neutrophil elastase, and causing detachment-induced apoptosis. Therefore, we studied the effect of neutrophils and of neutrophil-conditioned medium on the survival of HASMC.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Materials
Tissue culture reagents and drugs were obtained from Sigma (Poole, UK). Cell culture plastic ware was purchased from Falcon Labware (Becton Dickinson, Oxford, UK). Neutrophil elastase purified from human sputum was purchased from Elastin Products Co., Inc. (Owensville, MO). Cathepsin G and proteinase-3 were obtained from Athens Research and Technology, Inc. (Athens, GA). Annexin V apoptosis detection kit was from R&D Systems (Abingdon, UK). Mounting medium with DAPI was obtained from Vector Laboratories (Burlingame, CA). {alpha}1-Antitrypsin (Prolastin) was purchased from Bayer (Elkhart, IN) and MeOSuc-Ala-Ala-Pro-Ala-CMK (MSACK, Elastase Inhibitor II) from Calbiochem (La Jolla, CA). Antibodies to cleaved caspase-3 were purchased from New England BioLabs (Hertfordshire, UK), and to fibronectin and GAPDH from Biogenesis (Poole, UK). Protease inhibitor cocktail was obtained from Roche Diagnostic (Lewes, UK). All other chemical reagents were obtained from Sigma (Poole, UK).

Isolation and Culture of HASMC
Human airway smooth muscle was obtained from lobar or main bronchus from patients undergoing lung resection for carcinoma of the bronchus. The smooth muscle was dissected out under sterile conditions and placed in culture as previously described (20). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) supplemented with sodium pyruvate (1 mM), L-glutamine (2 mM), nonessential amino acids (1:100), penicillin (100 U/ml)/streptomycin (100 µg/ml), and amphotericin B (1.5 µg/ml) in a humidified atmosphere at 37°C in air/CO2 (95:5% vol/vol). At confluence, HASMC cultures exhibited a typical hill-and-valley appearance. Immunofluorescence techniques for calponin, smooth muscle {alpha}-actin, and myosin heavy chain revealed that more than 95% of the cells displayed the characteristics of smooth muscle cells in culture. HASMC at passages 3–7 from nine different donors were used in the studies.

Isolation of Neutrophils and Preparation of Neutrophil-Conditioned Media
Peripheral venous blood was mixed with acid-citrate dextrose (1:6 vol/vol) and sedimented on dextran (6% in 0.9% NaCl) for 40 min. Neutrophils were separated by plasma-percoll density gradient centrifugation. Cells were washed twice with Hanks' balanced salt solution (HBSS) and resuspended in DMEM without serum.

Neutrophil-conditioned media were obtained by culturing neutrophils (0.1–1 x 106/ml) in serum-free DMEM for 24 h. Supernatants were collected and cells and debris were removed by centrifugation (1,700 rpm, 10 min). The neutrophil-conditioned media were immediately used for experiments.

HASMC Treatment
Before experiments, confluent cells were growth-arrested by FCS deprivation for 24 h in DMEM supplemented with sodium pyruvate (1 mM), L-glutamine (2 mM), nonessential amino acids (1:100), penicillin (100 U/ml)/streptomycin (100 µg/ml), amphotericin B (1.5 µg/ml), insulin (1 µM), transferrin (5 µg/ml), ascorbic acid (100 µM), and bovine serum albumin (0,1%). Cells were then incubated with fresh FCS-free medium containing 0.1–1.0 x 106 neutrophils/ml or human neutrophil elastase (HNE, 0.1–3.0 µg/ml) for the times indicated. Cells were further exposed to neutrophil-conditioned media (derived from 0.1–1.0 x 106 neutrophils/ml). In additional experiments, neutrophil-conditioned media (derived from 1 x 106 neutrophils/ml) and FCS-free media containing HNE (3 µg/ml) were incubated with {alpha}1-antitrypsin ({alpha}1-AT; 1–100 nM) and MeOSuc-Ala-Ala-Pro-Ala-CMK (MSACK, 1–100 µM) for 30 min at room temperature before adding to the cell cultures.

Cell Viability
HASMC viability was assessed by the mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan. Cells grown in 24-well plates were treated as indicated above, washed with PBS, and 400 µl MTT solution (1 mg/ml) was added to each well. After 1 h of incubation at 37°C, the MTT solution was removed and the converted dye was solubilized with DMSO. A sample (100 µl) from each well was then transferred in duplicate to a 96-well microplate and the OD was measured using a spectrophotometer set to 550 nm.

Detection of Neutrophil Apoptosis
Neutrophils were incubated at 1 x 106 cells/ml in DMEM without serum or conditioned medium derived from HASMC after 24 h of growth arrest. At the indicated time points, cytospin slides were prepared from the suspended neutrophils and stained with May-Gruenwald solution. Four hundred cells were counted and morphologically evaluated by light microscopy. Neutrophils with single-lobed condensed nuclei and intense staining were identified as apoptotic cells.

Morphologic Detection of HASMC Apoptosis
Nuclear morphology was assessed using 4,6-diamidino-2-phenylindole (DAPI)-staining. For these experiments, HASM cell smears were washed with cold PBS and fixed with 4% paraformaldehyde. Cells were permeabilized with 0.1% Triton X 100 in PBS, washed with PBS, and stained with 1.5 mg/ml DAPI solution. Cells were examined under a fluorescence microscope for morphologic features of apoptosis such as condensed or segmented chromatin. Rates of apoptosis were quantified by counting cells with apoptotic nuclei. A minimum of 400 cells was counted for each condition in every experiment and each experiment was performed in duplicate.

Flow Cytometric Analysis of Apoptosis
After exposure to test conditions, adherent HASMC were detached with trypsin and pooled with their respective supernatant to include cells that detached during the experiment. Annexin V binding was determined using an apoptosis detection kit according to the manufacturer's instruction (R&D Systems). To distinguish between apoptosis and necrosis, cells were double-stained with annexin V (green fluorescence) and propidium iodide (PI) (red fluorescence). Briefly, HASMC were washed in PBS once and suspended in binding buffer containing FITC-conjugated annexin V (0.25 µg/ml) and PI (5 µg/ml). The cell suspension was incubated in the dark for 15 min and then acquired (10,000 cells/sample) using a Becton-Dickinson FACScan flow cytometer. A total of 10,000 events was analyzed for each sample with Cell Quest software.

Elastase Activity from Neutrophils
The activity of neutrophil elastase was assayed using a colorimetric assay employing the chromogenic peptide substrate N-methoxy-succinyl-Ala-Ala-Pro-Val-p-nitroanilide (Sigma) for elastase. Briefly, supernatants from neutrophils (0.1–1.0 x 106/ml) cultured for 24 h were incubated with HEPES (0.1 M) buffer containing NaCl (0.5 M), 10% dimethylsulfoxide, and substrate (2 mM) at pH 7.5. The amount of p-nitroanilide liberated was measured spectrometrically at 405 nm. HNE at a concentration of 8 µg/ml was used as a positive control. To examine the effect of {alpha}1-AT (1–100 nM) and MSACK (1–100 µM) on elastase activity, supernatants from neutrophils were incubated with these inhibitors for 30 min before experiment.

Western Immunblot Analysis
Confluent HASMC were serum-deprived for 24 h and then exposed to neutrophil-conditioned media (derived from 1 x 106 neutrophils/ml), HNE (3 µg/ml), and cathepsin G or proteinase 3 (10 µg/ml each) in the presence and absence of {alpha}1-AT (100 nM) or MSACK (100 µM). After 24 h incubation time, cells were rinsed with ice-cold wash buffer (PBS containing 2 mM PMSF) and scraped off the culture dish. The harvested cells were pooled with their respective supernatant to include any detached apoptotic HASMC. HASMCs were pelleted by centrifugation (800 x g, 4°C, 5 min), washed twice in wash buffer, and lysed in radioimmunoprecipitation assay (RIPA) buffer (PBS containing 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1% Igepal, and 1 tablet protease inhibitor cocktail 10 ml-1 buffer). Samples were solubilized by sonication followed by centrifugation (10,000 x g, 4°C, 4 min). Protein concentrations were determined using a protein assay kit (BCA; Pierce, Rockford, IL). Lysates were boiled for 10 min and total protein extracts (40 µg/lane) were separated by SDS-polyacrylamide gel electrophoresis (SDS-Page) on a 4–12% acrylamide precast gel (Novex; Invitrogen, Paisley, UK). The separated proteins were transferred electrophoretically to a nitrocellulose membrane in transfer buffer (Novex) and the membrane was then blocked with 5% nonfat dry milk in TBS containing 0.1% Tween 20 (TBST) for at least 1 h at room temperature. Blots were then incubated overnight at 4°C with an anti-cleaved caspase-3 antibody in TBST containing 5% dried nonfat milk at a 1:1,000 dilution or an anti-fibronectin antibody in TBST containing 0.1% BSA. The next day, the membrane was washed five times with TBST and then incubated for 1 h with a 1:2,000 dilution of goat anti-rabbit horseradish peroxidase–conjugated secondary antibody in TBST containing 5% nonfat dry milk. The membrane was then washed as before and visualized by enhanced chemiluminescence (ECL; Amersham, Buckinghamshire, UK). Membranes were reprobed with a mouse anti-GAPDH monoclonal antibody (1:5,000; Biogenesis, Poole, UK) to show the amount of protein loaded. Signals were quantified by scanning densitometry using software from Ultra-Violet Products (UVP, Cambridge, UK). Densitometry data were normalized for GAPDH values.

Statistics
Data are presented as mean ± SEM. Data were compared using one-way ANOVA followed by Bonferroni's t test post hoc to determine statistical differences. A p value < 0.05 was considered significant. SigmaStat software (Jandel Scientific, Germany) was used for statistical analysis.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Effect of PMN on HASMC Viability
In the presence of neutrophils, HASMC morphology changed rapidly from a flattened monolayer to a stellate cell shape with progressive loss of cell–cell contact and detachment from the underlying matrix over a time period of 72 h (Figures 1A and 1B). The number of remaining viable adherent cells was assessed by MTT assay (Figures 1C and 1D). The number of adherent viable HASMC decreased with increasing numbers of neutrophils in the culture dish. After addition of 1 x 106 neutrophils/ml to the cell cultures, HASMC numbers decreased to a minimum of 12.3 ± 4.3% compared with untreated controls over a time period of 72 h. Stimulation of neutrophils with FMLP (1 µg/ml) before addition to HASMC did not enhance this process (data not shown).



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  		Figure 1. Neutrophils and neutrophil-conditioned media reduce HASMC attachment and viability. A and B show light micrographs of HASMC incubated for 24 h in the absence (A) and presence (B) of neutrophil-conditioned media (derived from 1 x 106 cells/ml). Cells were visualized using a Zeiss Axiovert 25 CA microscope (x100 magnification; Herts, UK). (C) Time course (0–72 h) of HASMC detachment induced by neutrophils and neutrophil-conditioned media (1 x 106 cells/ml). Viable adherent HASMC were quantified by using the MTT test. The same experiment was performed incubating HASMC with various concentrations of neutrophils or neutrophil-conditioned media for 24 h (D). Results are expressed as percentage of untreated control cells (mean ± SEM, n = 4). **P < 0.01 compared with untreated HASMC. (E) Time course of neutrophil apoptosis (1 x 106/ml) in the presence of DMEM or HASMC-conditioned media. Apoptosis was determined by assessing nuclear morphology. Data (n = 4) are presented as the percentage of all cells counted (mean ± SEM). ***P < 0.001, *P < 0.05 compared with neutrophils suspended in DMEM.

 
To examine whether cell–cell contact was involved in neutrophil-induced HASMC detachment, conditioned media from 0.1–1 x 106 neutrophils/ml cultured for 24 h without stimulation was added to HASMC cultures. In these experiments, HASMC numbers were reduced to the same extent as in the presence of neutrophils. Therefore, cell–cell contact was not essential to the induction of cell detachment, and neutrophil-conditioned media was used for the subsequent experiments.

To assess whether the presence of HASMC modifies neutrophil survival, the number of apoptotic neutrophils suspended in HASMC-conditioned medium was determined and compared with neutrophils incubated in DMEM (Figure 1E). The number of apoptotic nuclei increased from 4.9 ± 1.2% immediately after isolation to 70.1 ± 6.9% after 24 h when neutrophils were suspended in DMEM. In comparison, neutrophils incubated in conditioned medium from HASMC showed a lower rate of apoptosis at 8 h (20.5 ± 3.2%, P < 0.05) and 24 h (22.8 ± 3.7%, P < 0.001).

Neutrophils Induce Apoptosis in HASMC
The movement of phospatidylserine to the extracellular membrane surface is a characteristic event of cells undergoing apoptosis. To investigate whether the reduction in viability and detachment of HASMC in the presence of neutrophil-conditioned media involves apoptosis, binding of annexin V to exposed phosphatidylserines on HASMC membranes was measured by flow cytometry. Because annexin V does not discriminate between apoptotic and necrotic cells, we used propidium iodide to identify necrotic cells. The percentage of annexin V–positive and PI-negative HASMC was significantly increased after addition of neutrophil-conditioned media derived from 0.3 x 106 PMN/ml compared with untreated HASMC. Maximum annexin V binding was detected in the presence of conditioned media derived from 1.0 x 106 neutrophils/ml after 72 h (Figures 2A–2E).



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  		Figure 2. Annexin V binding in HASMC exposed to neutrophil-conditioned media was evaluated by flow cytometry. (A) Representative histogram blot showing the intensity of annexin V binding in HASMC exposed to neutrophil-conditioned media (1 x 106 neutrophils/ml) compared with untreated HASMC. The graph in B shows the dose response in annexin V staining of HASMC exposed to neutrophil-conditioned media (0.1–1 x 106 neutrophils/ml, n = 6). HASMC were double-stained with annexin V and PI to distinguish between apoptotic and necrotic cells. (C) Percentage of annexin V–positive and PI-negative HASMC exposed to neutrophil-conditioned media (1 x 106 neutrophils/ml) over a time period of 72 h (n = 3). Data are shown as mean ± SEM. ***P < 0.001, **P < 0.01 compared with untreated HASMC. D and E show representative flow cytometry dot-plots of FITC-Annexin V/PI dual color flow cytometry for untreated HASMC (D) and HASMC exposed to neutrophil-conditioned media (1 x 106 neutrophils/ml; E) for 72 h. Cells in the lower quadrants demonstrate intact cytoplasmic membrane integrity (PI-negative) and represent viable (bottom left quadrant, annexin V–negative) or apoptotic cells (bottom right quadrant, annexin V–positive). Cells in the top left (annexin V–negative/PI-positive) and top right quadrant (annexin V–positive/PI-positive) show necrotic and late apoptotic cells, respectively.

 
To confirm the above observation, nuclear morphology of HASMC was examined by fluorescence microscopy for signs of apoptosis. Apoptotic nuclei are condensed or fragmented with higher apparent intensity because of the concentrated chromatin. Untreated HASMC showed uniform DAPI staining (Figure 3A). As cells were treated with increasing concentrations of neutrophil-conditioned media, the number of condensed or fragmented nuclei increased (Figure 3B). The highest percentage of HASMC nuclei with apoptotic markers (41.6 ± 8.1%) was reached after 72 h in conditioned media derived from 1.0 x 106 neutrophils/ml (Figures 3C and 3D).



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  		Figure 3. Morphologic detection of apoptosis in HASMC by fluorescence microscopy (magnification: x400). Representative micrographs showing DAPI staining on HASMC under control (A) and treated (B) (neutrophil-conditioned media derived from 1 x 106 cells/ml, 72 h incubation time) conditions. Arrows are indicating HASMC with condensed nuclei after treatment with neutrophil-conditioned media. C shows the dose–response and D the time course of HASMC apoptosis in the presence of neutrophil-conditioned media. Cells from five different donors were used for experiments. Data are presented as the percentage of all cells counted (mean ± SEM). ***P < 0.001, **P < 0.01 compared with untreated HASMC.

 
Effect of Elastase Inhibitors on Neutrophil-Induced HASMC Apoptosis
To further identify factors responsible for neutrophil-induced HASMC apoptosis, elastase activity in the neutrophil-conditioned media was measured and blocked with the elastase inhibitors {alpha}1-AT and MSACK before addition to HASMC cultures. Elastase activity in conditioned media derived from 1.0 x 106 neutrophils/ml reached 42.2 ± 6.2% of the positive control (Figure 4A). Incubation of neutrophil-conditioned media with these inhibitors reduced elastase activity in a dose-dependent manner. {alpha}1-AT (100 nM) completely blocked elastase activity in neutrophil-conditioned media, whereas MSACK (100 µM) reduced elastase activity in the same samples by 77.4% (Figure 4B).



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  		Figure 4. Elastase activity in neutrophil-conditioned media derived from 0.1–1 x 106 cells/ml (A). Data are expressed as percentage (mean ± SEM) of positive control (HNE 8 µg/ml). (B) Inhibition of elastase activity in neutrophil-conditioned media (derived from 1 x 106 cells/ml) by {alpha}1-AT (1–100 nM) and MSACK (1–100 µM). Data are expressed as percentage of elastase activity in neutrophil-conditioned media derived from 1 x 106 cells/ml (mean ± SEM). C and D show the effect of pre-incubation of neutrophil-conditioned media (1 x 106 cells/ml) with the elastase inhibitors {alpha}1-AT (100 nM) and MSACK (100 µM) on annexin V binding and nuclear morphology in HASMC. ***P < 0.001, **P < 0.01 compared with HASMC treated with neutrophil-conditioned media. Data are derived from three independent experiments. (E) Supernatants of HASMC treated with neutrophil-conditioned media (1 x 106 cells/ml) or HNE (3 µg/ml) for 24 h in the presence or absence of elastase inhibitors were analyzed for fibronectin fragmentation by Western blot. A representative example of four identical experiments is shown. Arrows indicate the presence of small fibronectin fragments ranging from 220–29 kD.

 
Blocking elastase activity in neutrophil-conditioned media with {alpha}1-AT (100 nM) markedly reduced the apoptotic response of HASMC to neutrophil-conditioned media. Annexin V binding on HASMC induced by neutrophil-conditioned media decreased from 12.1 ± 1.3% to 2.7 ± 0.6% (P < 0.001) in the presence of {alpha}1-AT (Figure 4C). The number of apoptotic nuclei in the presence of neutrophil-conditioned media also declined from 51.8 ± 3.9% to 20.9 ± 3.6% (P < 0.01) by pretreatment with {alpha}1-AT (Figure 5D). MSACK was much less effective than {alpha}1-AT in reducing the apoptotic response of HASMC to neutrophil-conditioned media. The number of annexin V–positive cells was reduced to only 8.3 ± 1.4% and the amount of apoptotic nuclei to 40.9 ± 5.6%.



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  		Figure 5. HNE induces apoptosis in HASMC. A and B show the dose-dependent effect of HNE on annexin V binding and nuclear morphology in HASMC. Cells from three to four different donors were used for experiments. Data are presented as the percentage of all cells counted (mean ± SEM). ***P < 0.001, **P < 0.01, *P < 0.05 compared with untreated HASMC. {ddagger}{ddagger}P < 0.001, {ddagger}P < 0.05 compared with cells exposed to HNE 3 µg/ml. C–E show representative flow cytometric histograms of annexin V binding in HASMC treated with HNE with and without elastase inhibitors.

 
To test the hypothesis that elastase-induced HASMC apoptosis involved degradation of ECM proteins, we assessed fibronectin fragmentation by Western blot (Figure 4E). In conditioned media of untreated HASMC, only limited fibronectin degradation products were detected. In contrast, both, neutrophil-conditioned media– and HNE-induced fibronectin proteolysis, with the production of small fragments ranging from 220–30 kD. The presence of {alpha}1-AT prevented fibronectin degradation by neutrophil-conditioned media or HNE. MSACK reduced but did not prevent fibronectin degradation.

HNE Induces HASMC Apoptosis
To further confirm that neutrophil-derived elastases induce HASMC apoptosis, cells were incubated with increasing concentrations of HNE, the main elastase released by neutrophils. Apoptosis was assessed by flow cytometry and fluorescence microscopy. HNE (0.1–3 µg/ml) induced a dose-dependent increase in annexin V binding by HASMC with a maximum (13.0 ± 2.5%) reached at a concentration of 3 µg/ml. The number of apoptotic nuclei increased to 75.5 ± 5.5% in the presence of 3 µg HNE/ml. The percentage of HASMC that stained positive for annexin V/negative for PI and the number of HASMC with apoptotic nuclei declined significantly after pretreatment with {alpha}1-AT and MSACK (Figures 5A–5E).

Neutrophil Elastase–Induced Apoptosis of HASMC Involves Cleavage of Caspase-3
Caspase-3 is a member of a family of cysteine proteases that play a key role in pathways leading to apoptosis. Activation of caspase-3 requires cleavage into its activated subunits. Figure 6 shows that, using a specific antibody for the large fragment (17/19 kD) of caspase-3, no caspase-3 cleavage was detected under control conditions. In the presence of neutrophil-conditioned media or HNE, increased levels of this caspase-3 fragment were found in HASMC. {alpha}1-AT reduced the amount of cleaved caspase-3 in HASMC treated with neutrophil-conditioned media or HNE to levels detected in untreated cells, whereas MSACK was slightly less efficient (Figure 6). Because the selective HNE inhibitor MSACK was much less effective than {alpha}1-AT in reducing neutrophil-conditioned media–induced HASMC apoptosis, the capacity of other neutrophil serine proteases (cathepsin G and proteinase 3) to induce HASMC apoptosis was assessed in separate experiments. Cathepsin G and, to a lesser extent, proteinase 3 (10 µg/ml) induced cleavage of caspase 3 into its fragments, indicating that these other neutrophil serine proteases also possess apoptotic activities on HASMC (Figure 7).



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  		Figure 6. Effect of neutrophil-conditioned media (derived from 0.1–1 x 106 cells/ml; A) and HNE (3 µg/ml; B) on cleaved caspase-3 expression in HASMC in the presence and absence of the elastase inhibitors {alpha}1-AT (100 nM) and MSACK (100 µM). Cleaved caspase-3 was detected by Western blotting using a specific antibody for the large fragment (17/19 kD) of caspase-3. The membranes were stripped and reprobed using a specific antibody for GAPDH. A representative example of three identical experiments is shown. The graphs in A and B show the densitometric analysis of cleaved caspase-3 expression, normalized by GAPDH expression. Data are expressed as mean ± SEM. *P < 0.05 compared with HASMC treated with HNE only.

 


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  		Figure 7. Effect of HNE (3 µg/ml), cathepsin G (10 µg/ml), and proteinase 3 (10 µg/ml) on cleaved caspase-3 expression in HASMC. Cleaved caspase-3 was detected by Western blotting using a specific antibody for the large fragment (17/19 kD) of caspase-3. The membrane was stripped and reprobed using a specific antibody for GAPDH. A representative example of two identical experiments is shown.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In the presence of neutrophils, HASMC undergo morphologic changes of apoptosis together with cleavage of caspase-3, a biochemical hallmark of apoptosis. Apoptosis of HASMC resulted from the proteolytic activity released by neutrophils as supported by the presence of fibronectin degradation and by the inhibitory effect of a serine protease inhibitor, {alpha}1-AT. In addition, the main serine protease released by neutrophils, HNE, directly induced apoptosis in HASMC, which was inhibited by both {alpha}1-AT and the selective HNE inhibitor MSACK.

We showed that neutrophils or their conditioned media induced a rapid change of HASMC morphology with loss of cell–cell and cell–matrix contact. The observed changes in HASMC appearances are similar to the morphologic features described previously in smooth muscle cells exposed to mast cell chymase and plasmin (21, 22). These changes are examples of detachment-induced apoptosis, defined as anoikis. HASMC detachment in our study occurred within 24 h and preceded HASMC apoptosis, which was prominent at 72 h, indicating that these are consecutive events as is characteristic for anoikis. The neutrophil-induced HASMC changes were also associated with characteristic morphologic/biochemical markers of apoptosis such as nuclear condensation/fragmentation, increased annexin V binding, and cleavage of caspase-3. HASMC detachment and apoptosis were likely to be induced by elastases derived from neutrophils. Considerable elastase activity was found in conditioned media from neutrophils and blocking elastase activity by the elastase inhibitor {alpha}1-AT inhibited HASMC-apoptosis. In comparison, inhibition of elastase activity and HASMC apoptosis by the selective HNE inhibitor MSACK was incomplete even at a high concentration of 100 µM, indicating that elastases other than HNE contribute to HASMC detachment. In fact, apart from HNE, cathepsin G and to a lesser extent proteinase 3 also induced caspase-3 activation.

Neutrophils represent a rich source of elastases in the human lung (18). The degradation of ECM by neutrophil elastases is believed to contribute to decreased airway stability in COPD. Elastic fiber disruption has also been observed in the bronchi of patients with asthma (23), indicating that an imbalance between proteases and antiproteases is also relevant in this disease. HNE is known to be the most destructive neutrophil enzyme due to its ability to break down not only elastin, but also collagen (types I–IV) and fibronectin (14). Increased levels of HNE were found in induced sputum of patients with asthma and COPD (24) and correlated with the severity of these diseases. In these patients, total elastase levels reached up to 50 µg/ml sputum. In addition, HNE can reproduce many of the features found in patients with COPD and asthma, including mucous gland hyperplasia, excess mucus secretion, epithelial damage, and connective tissue destruction (25), further supporting a role for HNE in asthma and COPD. In this study, the apoptotic changes induced by neutrophils and their conditioned media were reproduced in separate experiments where HASMC were treated with HNE alone and were attributed to its proteolytic activity.

Further support for the hypothesis that neutrophil-derived enzymes induced HASMC apoptosis is provided by the increased fibronectin degradation in the presence of neutrophil-conditioned media or HNE. Smooth muscle cells are anchorage-dependent cells that receive important survival signals from the surrounding ECM. These signals are mediated by the transmembrane glycoprotein receptor integrins (26). In the airways, HASMC produce their own ECM, including fibronectin, elastin, and collagen I and IV (6). In particular, fibronectin provides essential proliferative and antiapoptotic signals to the muscle (8, 27). The disruption of integrin-mediated signals by enzymes capable of degrading ECM proteins, such as neutrophil elastases, could therefore trigger the execution of cell death programs leading to HASMC apoptosis.

A fine-tuned balance between cell proliferation and death tightly controls the number of structural cells in the tissue under normal conditions. Recent studies have focused on the identification of factors controlling HASMC proliferation and accumulation in asthmatic airways. Thus, a number of mitogenic stimuli for HASMC have been defined, among those thrombin, PDGF, EGF, histamine, and mast cell tryptase (28). However, little is known about processes that may counterbalance HASMC proliferation such as apoptosis. Until now it has been very difficult to induce apoptosis in HASMC (29, 30). Resistance to apoptosis caused by changes in the cellular microenvironment such as altered ECM composition may be an important factor contributing to an increased mass of smooth muscle in asthmatic airways.

Chronic inflammation is a feature of asthma and COPD, together with the repair process, may result in long-term structural changes of the lung. At present, it is not known how the neutrophil participates in the repair or remodeling process of the airway. However, because neutrophils are known to produce enzymes, oxygen active radicals, and cytokines (18), which affect cell proliferation and matrix production, it is reasonable to believe that they play an important role in remodeling. A recent study showed increased neutrophil infiltration of the smooth muscle layer of patients with COPD (17), supporting the hypothesis that neutrophil and smooth muscle interactions may be involved in airway smooth muscle changes. An interesting finding in our study was that neutrophil apoptosis was strongly inhibited in the presence of HASMC-conditioned medium, which may result in prolonged interactions between the neutrophil and HASMC. Circulating neutrophils normally have a short half-life of ~ 6 h (18), which can be significantly increased with production of factors that suppress neutrophil apoptosis, such as GM-CSF and G-CSF (27). HASMC are known to produce GM-CSF and G-CSF, in particular when stimulated with proinflammatory cytokines (31). Thus, the interactions between neutrophils and HASMC are complex, with HASMC prolonging neutrophil survival and neutrophils inducing HASMC apoptosis. The effects of these interactions in disease are unclear.

We have shown that neutrophil serine proteases have the potential to influence HASMC survival by triggering detachment-induced apoptosis. The local release of neutrophil elastases in areas of airway inflammation with neutrophil infiltration may serve to limit HASMC hyperplasia and ECM deposition.


   Acknowledgments
 
The authors are grateful to Drs. Susan Smith and Tricia Finney-Hayward for their help with the isolation of neutrophils from whole blood.


   Footnotes
 
This work was supported by a Welcome Trust (UK) grant. Dr. Oltmanns was supported by a European Respiratory Society Fellowship and by a Merck-Sharpe-Dohme (Germany) research grant.

Conflict of Interest Statement: U.O. has no declared conflicts of interest; M.B.S. has no declared conflicts of interest; S.X. has no declared conflicts of interest; M.J. has no declared conflicts of interest; and K.F.C. has no declared conflicts of interest.

Received in original form October 8, 2004

Received in final form January 6, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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