help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on October 17, 2003, doi:10.1165/rcmb.2003-0253OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2003-0253OCv1
30/4/576    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hirche, T. O.
Right arrow Articles by Belaaouaj, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hirche, T. O.
Right arrow Articles by Belaaouaj, A.
American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 576-584, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0253OC

Deficiency in Neutrophil Elastase Does Not Impair Neutrophil Recruitment to Inflamed Sites

Tim O. Hirche, Jefferey J. Atkinson, Scott Bahr and Abderrazzaq Belaaouaj

Departments of Medicine (Pulmonary Division) and Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri

Address correspondence to: Abderrazzaq Belaaouaj, Ph.D., Departments of Medicine and Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Avenue, Box 8052, St. Louis, MO 63110-1093. E-mail: Azzaq{at}im.wustl.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To reach the sites of inflammation, neutrophils traverse the endothelium, its underlying basement membrane, and other barriers depending on the localization of the insulting agent. Whether neutrophil elastase (NE) plays a role in neutrophil recruitment to inflamed sites is still debatable. By exploiting mice deficient in NE (NE-/-), we sought to address this dilemma. We recruited neutrophils to the lungs or the peritoneum of wild-type (WT) or NE-/- mice by intranasal or intraperitoneal challenge with Pseudomonas aeruginosa or its lipopolysaccharide. At designated times post-inoculation (0, 4, 24, and 48 h), groups of mice were killed to assess changes in leukocyte counts and inflammatory responses. NE-/- and WT mice had normal circulating leukocyte numbers including neutrophils and changes in the hemograms in the setting of acute inflammation were indistinguishable. Analyses of lung tissues or fluids from the lungs and peritoneum found that regardless of the inflammatory model, the leukocyte counts including neutrophils and the inflammatory response were similar in NE-/- and WT mice at all time points. In vitro, neutrophils isolated from the lungs or the peritoneum of NE-/- and WT mice had comparable chemotactic and respiratory-burst functions and migrated normally through Matrigel in response to various stimuli. Interestingly, preincubation of human peripheral blood neutrophils with NE physiologic inhibitors did not alter the migration of the cells through Matrigel. In sum, our findings present the first in vivo description that the absence of NE does not impair neutrophil recruitment to inflamed sites and that NE is not required for basement membrane transmigration of neutrophils.

Abbreviations: {alpha}1-antitrypsin, {alpha}1-AT • bronchoalveolar lavage, BAL • Evans blue dye, EBD • extracellular matrix, ECM • Hanks' balanced salt solution, HBSS • intercellular adhesion molecule-1, ICAM-1 • interleukin, IL • inferior vena cava, IVC • lactate-dehydrogenase, LDH • lipopolysaccharide, LPS • matrix metalloprotease, MMP • myeloperoxidase, MPO • neutrophil elastase, NE • phosphate-buffered saline, PBS • peritoneal lavage, PL • secretory leukocyte protease inhibitor, SLPI • tumor necrosis factor, TNF • zymosan-activated serum, ZAS


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The accumulation of neutrophils at inflamed sites represents a characteristic feature of the innate host response. In the setting of bacterial infections, the purpose of this neutrophilic infiltration is to defend the host by eliminating the invading pathogens and resolving the associated inflammation. To reach inflamed sites, neutrophils traverse various barriers including the endothelium, basement membrane/interstitium, and epithelium in response to localized inflammatory mediators. Much has been learned about the cascade of steps and molecular events during neutrophil recruitment. The adhesion and rolling of neutrophils across the endothelial lining requires the expression and exposure of various molecules on the cell surface of both endothelial cells and neutrophils such as intercellular adhesion molecule-1 (ICAM-1) and integrins (e.g., CD11/CD18) (1). For transendothelial migration, platelet-endothelial cell adhesion molecule-1 and endothelial lateral junction molecules (cadherins) are proposed to facilitate the passage of neutrophils (2, 3). Despite the progress in understanding neutrophil migration, the mechanisms underlying this process remain poorly understood. Whether neutrophil proteases are implicated in neutrophil recruitment to inflamed sites is still debatable.

Various reports have proposed a role for neutrophil elastase (NE) in neutrophil migration (4). Mature NE, a highly cationic glycoprotein, is stored in an active form in primary granules at high concentration (~ 4 µg/106 cells), making it a major component of neutrophils (5, 6). In vitro, NE cleaves ICAM-1 and cadherins (7, 8). Other studies described NE binding to CD11b/CD18 (9). NE is capable of cleaving CD14 on the surface of monocytes, resulting in decreased expression of the proinflammatory cytokines tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-8 following exposure of these cells to lipopolysaccharide (LPS) (10). The enzyme can also cleave various cytokines including TNF-{alpha} and IL-8 (11, 12). Recent studies have shown that NE cleaves vitamin D-binding protein, a molecule that binds to the surface of neutrophils and amplifies the complement C5a chemotactic activity (13). At the same time, it was shown elsewhere that NE is able to release complement receptor 1, which acts as an inhibitor of complement (14). The substrate repertoire of NE also comprises {alpha}1-antiprotease inhibitor and proteins of the extracellular matrix (ECM) such as collagen and laminin. When degraded, these proteins generate chemotactic fragments (15, 16). Although these in vitro findings suggest that NE play a role in neutrophil recruitment, it remains unclear whether the enzyme promotes or dampens neutrophil recruitment. During escape from the vasculature, neutrophils may deploy different mechanisms to emigrate from the systemic (post-capillary veins) or pulmonary (alveolar capillaries) circulation (17). Whether neutrophil-derived NE is required in these processes remains obscure as well (18, 19). More importantly, all these in vitro data do not reproduce in vivo environment, particularly in disease situations.

Together, these controversies have prompted us to investigate the role of NE in neutrophil recruitment to inflamed sites. Recently, we generated mice deficient in NE (NE-/-) (20). We subjected NE-/- mice and their wild-type (WT) littermates to intranasal or intraperitoneal challenge with Pseudomonas aeruginosa or its product LPS and analyzed them at designated time points. Our in vivo findings provide the first direct evidence that NE alters neither neutrophil recruitment to inflamed sites nor the host inflammatory responses. In addition, NE-deficient mouse neutrophils or human neutrophils pretreated with NE physiologic inhibitors migrate normally in response to different chemoattractants.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
LPS from P. aeruginosa and zymosan were purchased from Sigma-Aldrich (St. Louis, MO). Zymosan-activated serum (ZAS) was generated by incubating rat serum with zymosan (10 mg/ml) at 37°C for 1 h, followed by heat inactivation at 56°C for 30 min and centrifugation. The supernatant was collected and aliquots were stored at -80°C (21). Matrigel invasion chambers and control inserts were purchased from Becton-Dickinson (Bedford, MA). Purified NE and bovine neck ligament elastin were obtained from Elastin Products Co. (EPC, Owensville, MO). NE activity was determined spectrophotometrically using the specific chromogenic substrate N-methoxysuccinyl-Al-Al-Pro-Val-pNA (EPC) according to the manufacturer's recommendations. Purified human plasma {alpha}1-antitrypsin ({alpha}1-AT) was from Sigma-Aldrich. Recombinant human secretory leukocyte protease inhibitor (SLPI) was purchased from R&D Systems (Minneapolis, MI). All other chemicals were reagent grade and purchased from Sigma-Aldrich, unless otherwise stated.

Animals
NE-/- mice were generated by targeted mutagenesis as previously described (20) and backcrossed at least eight generations to pure 129/SvEv genetic background. NE-/- mice have normal reproduction and are not immunocompromised under normal living conditions. All mice are housed in a pathogen-free facility with food and water ad libitum, and a 12 h light/dark cycle. All procedures were approved by the Animal Studies Committee of Washington University School of Medicine.

Bacteria
P. aeruginosa (H103, kindly provided by Dr. Hancock, Vancouver, BC, Canada) was passaged twice in mice before use. An overnight culture aliquot (1 ml) was grown in Luria Bertani broth (10 ml) at 37°C to late exponential phase (3 h). Bacteria were washed twice with phosphate-buffered saline (PBS, pH 7.4), and the optical density of the culture determined at 600 nm (1 OD600 {cong} 1 x 109 bacteria/ml).

Blood Leukocyte Counts
Peripheral blood was collected from the retro-orbital venous plexus or the inferior vena cava (IVC) where indicated. Blood cell counts were determined using an ABX 9000 hematology cell counter (Biochem Immunosystems, Allentown, PA). Differential cell counts were performed on Wright stained blood smears by the Department of Comparative Medicine (Washington University School of Medicine).

Mouse Models of Neutrophil Recruitment
Intranasal model. Mice were intranasally challenged with P. aeruginosa or its purified LPS as previously described (22, 23). Briefly, mice were anesthetized by intraperitoneal injection of ketamine hydrochloride (75 mg/kg) and medotomidine hydrochloride (1 mg/kg), followed by intranasal administration of bacteria (4 x 106 cfu/mouse) or LPS (10 µg/mouse) in 50 µl PBS. At designated time points (0, 4, 24, and 48 h), mice were killed. Blood was collected from the IVC and the lungs were gently perfused with saline via the right ventricle. The trachea was exposed through a midline incision and canulated with a sterile 22-gauge catheter (Becton-Dickinson). The lungs were lavaged in situ (bronchoalveolar lavage, BAL) with a total of 2.1 ml of Hanks' balanced salt solution (HBSS, pH 7.4) in three aliquots of 0.7 ml. Cell and differential counts were immediately performed on aliquots of BAL fluids as described below. The remaining BAL samples were centrifuged for 10 min at 4°C and the supernatants aliquoted. The cell pellets were resuspended and aliquots (106 cells) were lysed by addition of Triton X-100 (0.1%). The lungs were removed, rinsed with sterile saline, and blotted dry with gauze. BAL supernatants, cell lysates, and lungs were snap-frozen and stored at -80°C until use.

Intraperitoneal model. Mice were intraperitoneally infected with 1 ml of saline containing the same inoculum size of P. aeruginosa that was used for the intranasal infection model. Saline alone was used as a control. At designated time points post-inoculation, mice were killed and underwent peritoneal lavage (PL) using 5 ml of HBSS.

The total cell numbers in BAL and PL fluids were determined by hemacytometer. For differential counts, cells were cytospun, Wright stained, and 100 cells/slide counted using standard morphologic criteria.

Protein and Albumin Concentrations
The total protein concentration in the cell-free BAL fluids was determined by the bicinchoninic acid assay (Pierce, Rockford, IL). Briefly, samples (25 µl) were incubated with bicinchoninic acid assay reagent (200 µl) in a 96-well plate at 37°C according to the manufacturer's recommendations. Bovine serum albumin was used as standard. Following incubation for 30 min, changes in the absorbances were measured using a plate reader at 562 nm and were proportional to the amounts of proteins in the sample.

The albumin concentration was determined using the bromocresol green assay (Sigma-Aldrich), according to the manufacturer's recommendations. Briefly, 100 µl of samples or standards were incubated with 900 µl of bromocresol green substrate for 60 s at room temperature. Changes in absorbances were recorded by spectrophotometer at 628 nm and were proportional to the albumin concentrations in the samples.

Lactate Dehydrogenase Activity Assay
The activity of LDH in cell-free BAL fluids was determined by the LDH determination kit according to the manufacturer's instructions (Sigma-Aldrich) and used as a marker for cytotoxicity (24). LDH catalyzes the oxidation of lactate to pyruvate. This reaction is coupled with reduction of NAD to NADH, which is followed spectrophotometrically at 340 nm. The LDH activity, which is proportional to the rate of decrease in absorbance, was expressed as mU/ml of cell-free BAL. Briefly, 100 µl of BAL samples were added to 900 µl LD-L reagent, and changes in absorbances were recorded over 3 min.

Cytokine Levels
IL-1ß and TNF-{alpha} concentrations were determined in cell-free BAL fluids using ELISA kits from R&D Systems (Minneapolis, MN). Briefly, 50 µl of BAL samples were incubated for 2 h in 96-well plates that were pre-coated with purified polyclonal antibodies specific to mouse IL-1ß and TNF-{alpha}. Recombinant mouse IL-1ß and TNF-{alpha} were used as standards. Reactions were incubated for 2 h with horseradish peroxidase–linked antibodies specific for IL-1ß or TNF-{alpha}. Following addition of chromogenic peroxidase substrate, changes in absorbances were measured using a plate reader at 450 nm and were proportional to the amounts of cytokines bound to the plates.

Myeloperoxidase Activity Assay
MPO activity was determined in perfused and lavaged lungs as previously described (20). Briefly, whole lungs of each mouse were homogenized (Tissue-Tearor; Biospec Products, Bartlessville, OK) for 30 s at 30,000 rpm in 5 ml of PBS on ice followed by centrifugation for 10 min at 4°C. The supernatant was discarded and the pellet resuspended in 1 ml PBS, containing 0.5% hexadecyl-trimethyl-ammonium-bromide (HTAB) and 5 mM EDTA. Following three freeze/thaw cycles, the suspension was centrifuged for 10 min at 15,000 rpm and the MPO activity in the supernatant determined as follows: 100 µl of sample were mixed with 1 ml HBSS, 200 µl HTAB/EDTA-PBS, 100 µl O-Dianisidine (1.25 mg/ml), and 100 µl H2O2 (0.05%). The enzymatic activity of MPO, which is proportional to changes in absorbances, was recorded over 6 min by spectrophotometer at 460 nm.

NE Activity
The elastolytic activity in cell-free BAL fluid was assessed by zymography as previously described (20). Briefly, BAL supernatants or cell lysates (20 µl) were migrated under nonreducing conditions at 4°C on SDS-PAGE gels (12%), containing 1 mg/ml elastin. Purified NE (0.1 µg) was used as a control. Following electrophoresis, gels were soaked in 2.5% Triton X-100 for 30 min, rinsed briefly, and incubated at 37°C for 48 h in 50 mM Tris HCl (pH 8.2), containing 5 mM CaCl2. The gels were then stained in Coomassie reagent and destained in 5% acetic acid and 10% methanol. Active NE appears as a transparent lysis band at ~ 29 kD.

Lung Histology
NE-/- and WT mice were subjected to P. aeruginosa instillation or sterile saline as described above, and their lungs were processed for histology 24 h after challenge. The lungs were inflated with 10% buffered formalin at a constant pressure of 25 cm H2O for 15 min. Next, the lungs were excised, immersion-fixed with 10% buffered formalin for 24–48 h, dehydrated, embedded in paraffin, and cut into 5-µm sections. Serial lung tissue sections were processed, stained with hematoxylin and eosin (H&E), and examined by light microscopy. To grade the degree of P. aeruginosa–mediated injury, tissue sections were evaluated in a blinded fashion (25). The features for grading lung tissues were alveolar congestion, hemorrhage, infiltration of neutrophils in lung interstitium or airspaces, and thickness of the alveolar wall. Each feature received a score of 0 (none), 1 (mild), 2 (moderate), 3 (severe), or 4 (maximal). Total histology score was defined as the mean of scores of mice in each group. Each score corresponded to the sum of individual feature scores per mouse.

Lung Wet/Dry Weight Ratio
NE-/- and WT mice were subjected to P. aeruginosa or sterile saline instillation and killed 24 h after challenge as described above. To assess edema, the lung wet/dry weight ratio was determined as previously described (26). The lungs were gently perfused, excised en bloc, blotted dry on gauze, immediately weighted, and placed in a desiccating oven at 65°C for 48 h. Dried lungs were weighed and the wet/dry weight ratio calculated.

Evans Blue Dye Assay
To assess microvascular permeability related to lung injury, we used a modification of the Evans blue dye (EBD) extravasation technique as previously described (27). Mice were injected with EBD (20 mg/kg) via the left jugular vein 3 h before they were killed. Next, a heparinized sample of blood was collected and the plasma was obtained by centrifugation. Perfused lungs were homogenized in 2 ml PBS and incubated with 2 vols of deionized formamide for 4 h at 60°C, followed by centrifugation for 10 min at 2,000 rpm. Absorbances of lung tissue supernatants and plasma were measured at 620 and 740 nm and corrected for contaminating heme pigments using the formula, E620 = E620 - (1.426 x E740 + 0.03). A permeability index was defined as lung/plasma absorbance ratio.

Bacterial Clearance after Lung Infection
NE-/- and WT mice (n = 5/genotype) were intranasally infected with P. aeruginosa and killed after 24 h as described above. A blood sample was taken from the IVC. Next, the whole lungs, liver, spleen, and kidneys of each mouse were aseptically removed, rinsed with sterile PBS, and homogenized in 5 ml of PBS. Serial dilutions were immediately plated and the numbers of viable bacteria determined following overnight incubation at 37°C.

Respiratory Burst Assay
Neutrophils were recruited to the lungs of NE-/- and WT mice by instillation of LPS and harvested 24 h after challenge as described above. In a parallel experiment, mice from each genotype were intraperitoneally injected with glycogen (15%, 1 ml) and recruited cells were harvested after 4 h as previously described (20). Differential cell counts and trypan blue exclusion found that cell suspensions contained > 90% viable neutrophils. Following centrifugation for 10 min at 24°C, cells were resuspended in warm HBSS (with 1.3 mM CaCl2 and 0.4 mM MgSO4) and aliquots (1 x 106 neutrophils in 500 µl) were added to tubes containing cytochrome C (0.2 mM) or cytochrome C with 300 U/ml superoxide dismutase (SOD) in 400 µl HBSS. Next, cells were stimulated by addition of formyl-methionyl-leucyl-phenylalanine (fMLP, 1 µM) or phorbol-myristate-acetate (PMA, 100 ng/ml). Unstimulated reactions were processed as control. Following incubation for 30 min at 37°C, the reactions were stopped by placing the samples on ice. Finally, the samples were centrifuged for 5 min at 4°C and the supernatants assayed at OD550. The amount of superoxide anion production was calculated as previously described (28).

Chemotaxis and Basement Membrane Transmigration Assays
Both human and mouse neutrophils were subjected to chemotaxis or transmigration assays using a modified Boyden chamber assay as previously described (21). Briefly, glycogen-elicited neutrophils were isolated from the peritoneum of NE-/- and WT mice as described above. Human neutrophils were isolated from venous blood of healthy adults by standard density gradient centrifugation (Histopaque-1077; Sigma-Aldrich) and dextran sedimentation. Both neutrophil preparations yielded > 95% viable neutrophils, as evidenced by differential counts and trypan blue exclusion. Neutrophils were washed and resuspended at 3 x 105 cells/ml in HBSS (with 1.3 mM CaCl2 and 0.4 mM MgSO4). Mouse and human cell suspensions (500 µl each) were transferred into the upper compartments of transwell cell culture inserts (Becton-Dickinson). Where reported, SLPI (10 µg/ml) or {alpha}1-AT (30 µg/ml) were added to the cell suspension before incubation. At the indicated concentrations, SLPI and {alpha}1-AT inhibit 100 and 95% of NE activity in 106 Triton-lysed neutrophils (i.e., five times the number of neutrophils employed in the transmigration assay). The lower compartments contained 500 µl HBSS with or without addition of ZAS (2%) or 500 µl of undiluted cell-free BAL fluids from P. aeruginosa–infected WT mice. For chemotaxis and transmigration, the compartments were separated by 8-µm pore size membrane that was uncoated or Matrigel-coated, respectively. Matrigel is a solubilized basement membrane extracted from Engelbreth-Holm-Swarm mouse sarcoma that contains laminin, collagen type IV, proteoglycan, entactin, and growth factors. After 3 h incubation at 37°C in humidified 5% CO2-air, the filter membranes were stained and mounted on glass slides. The number of cells that migrated underneath the filter membranes was determined by counting five random high power fields (x400) per condition. Each experiment was performed in triplicate.

Statistical Analyses
Unless specified, data are expressed as means ± SD. Where appropriate, differences between single groups were tested using Student's unpaired t test. For the time course experiments, overall differences between genotypes of mice were tested by two-way analyses of variances with group and time as factors. Logarithmic transformation was performed before analyses if parameters presented a log-normal distribution. Empirically found P values were {alpha}-adjusted according to Bonferroni, and statistical significance was assumed at a predefined level of {alpha} < 0.05. Tests were performed using the Statistical Analysis System 6.12 (SAS Institute Inc., Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Differential Counts of Circulating Leukocytes
Peripheral blood from NE-/- mice and their WT littermates was collected and analyzed for circulating leukocytes. Table 1 shows that unchallenged NE-/- and WT mice have comparable total circulating leukocyte counts and percentages of neutrophils, monocytes, lymphocytes, and eosinophils (P > 0.05 for differences between groups).


View this table:
[in this window]
[in a new window]
  		TABLE 1 NE-/- and WT mice have comparable numbers of circulating leukocytes

 
Neutrophil Recruitment to the Lungs following Intranasal Infection with P. aeruginosa
Neutrophils represent the earliest phagocytes that accumulate at sites of inflammation. To determine whether NE plays a role in neutrophil recruitment, we subjected NE-/- and WT mice to a model of intranasal infection with P. aeruginosa. First, we determined a sublethal dose of P. aeruginosa that resulted in signs of distress, including lethargy and ruffled fur, but no death in both genotypes of mice within the time course of our experiments. When the ability of mice to clear bacteria 24 h after infection was examined, we found that the number of viable bacteria in the lungs (site of primary challenge) decreased markedly and was not significantly different between NE-/- and WT mice (9.4 [± 2.77] x 104 versus 8.98 [± 2.95] x 104 [mean {± SEM}, P > 0.05] for NE-/- and WT mice, respectively). At this time point, no colony forming units were detected when we plated aliquots from blood, liver, spleen, or kidneys. Using the sublethal dose, the BAL fluids from both NE-/- and WT mice had comparable low cell counts at 4 h after challenge. Past this time, there was a sharp increase of leukocyte numbers, which peaked by 24 h. These counts remained similar in both genotypes of mice (P > 0.05 for differences between groups) (Figure 1A). Differential cell counts indicated that neutrophils represented the majority of the cells (P > 0.05 for differences between groups) (Figure 1B). Analyses of NE-/- and WT lung tissue sections 24 h post-infection showed similar patchiness of cellular infiltrates (Figure 1C). Immunostaining for MPO in these infiltrates suggested the predominance of neutrophils (data not shown).



View larger version (52K):
[in this window]
[in a new window]
  		Figure 1. Neutrophil influx in response to P. aeruginosa infection is comparable in NE-/- (triangles) and WT (squares) mice. (A and B) Total leukocyte and neutrophil counts in BAL fluids from NE-/- and WT mice (n = 16/genotype) following intranasal challenge with a sublethal dose of P. aeruginosa. (C) Representative H&E-stained lung sections at 24 h after infection showed patchy neutrophil infiltrates in both groups of mice (arrowheads, x40). No infiltrates were detected in lung sections from saline control mice (n = 6/genotype). (D and E) Total leukocyte and neutrophil counts in PL fluids of NE-/- and WT mice (n = 16/genotype) in response to intraperitoneal challenge with P. aeruginosa.

 
Assessment of Lung Injury following Intranasal Infection with P. aeruginosa
Lung wet/dry weight ratio, permeability index, LDH activity, and histologic scores are shown in Table 2. These parameters were evaluated to assess the levels of inflammation and injury, associated with P. aeruginosa infection. As expected, infection of both NE-/- and WT mice resulted in significantly elevated values for all parameters when compared with saline instillation. However, there were no appreciable differences in these values between groups regardless of the genotype of mice or the type of treatment received.


View this table:
[in this window]
[in a new window]
  		TABLE 2 Assessment of lung injury 24 h after P. aeruginosa infection of NE-/- and WT mice

 
Neutrophil Recruitment to the Peritoneum following Intraperitoneal Infection with P. aeruginosa
The mechanisms of neutrophil extravasation have been shown to vary between the pulmonary and systemic circulation (17). To examine whether the role of NE in neutrophil recruitment depends on the type of vasculature beds, we subjected mice from both genotypes to intraperitoneal infection with the same P. aeruginosa dose as for intranasal infection and monitored the capacity of neutrophils to migrate to the peritoneum over time. At this dose, intraperitoneal infection resulted in less severe signs of distress than intranasal infection, but the mice still displayed decreased activity and hunched posture. Compared with the lung infection model, a marked increase of leukocytes, including neutrophils was observed as early as 4 h and reached its maximum by 24 h (Figures 1D and 1E). Notably, neutrophil recruitment to the peritoneal space appeared to plateau earlier than in the intranasal model but was comparable for both NE-/- and WT mice (P > 0.05 for differences between groups).

Recruitment of Neutrophils following Intranasal Instillation of P. aeruginosa LPS
The in vivo role of NE in neutrophil recruitment was further assessed in a different lung model where we replaced whole P. aeruginosa bacteria with its product LPS. LPS, a potent virulence factor of Gram-negative bacterial walls, has been employed in a variety of animal models to incite an acute neutrophilic response in the lung (23). At 4 h following LPS exposure, both NE-/- and WT mice had similar marked increase in circulating leukocytes (Figures 2A and 2B). By 24 h, peripheral cell counts including neutrophils declined similarly in both mice to baseline levels and increased by 48 h (P > 0.05 for differences between groups). The recruitment of neutrophils to the alveolar spaces had, however, a different dynamic. The BAL fluids from both groups of mice showed a comparable moderate increase of leukocyte numbers at 4 h, but a fulminant influx by 24 h post-challenge (Figures 2C and 2D). These cell counts outnumbered those observed in the lung infection model, and the cytospin analyses confirmed neutrophils as the dominant cells (Figure 2E). Also, there was a gradual and comparable increase of MPO activity in both genotypes up to 24 h followed by a drop in the ensuing hours (P > 0.05 for differences between groups) (Figure 2F). Of note, MPO activity reached its peak coinciding with the observed drop in the numbers of circulating leukocytes.



View larger version (41K):
[in this window]
[in a new window]
  		Figure 2. NE-/- (triangles) and WT (squares) mice show comparable neutrophil infiltration in response to LPS inhalation. (A and B) Hemograms of NE-/- and WT mice (n = 16/genotype) following intranasal challenge with P. aeruginosa LPS. (C and D) Total leukocyte and neutrophil counts in BAL fluids from mice in A. (E) Cytospins from BAL fluids at 24 h revealed that neutrophils dominated the cellular infiltrates in both groups of mice. Only a small number of resident alveolar macrophages were found in BAL fluids from mice administered saline. Shown are representative micrographs from each genotype (x200). (F) MPO activity in lung homogenates from mice in A.

 
Lung Permeability
To assess the permeability of lung alveolo-capillary barriers in the LPS-induced acute neutrophilic response, we determined the total protein and albumin concentrations in cell-free BAL fluids. Total protein levels increased markedly and similarly over time in NE-/- and WT mice. This finding was confirmed when leakage of serum albumin into the alveolar spaces was determined (P > 0.05 for differences between groups) (Figures 3A and 3B).



View larger version (24K):
[in this window]
[in a new window]
  		Figure 3. NE-/- (triangles) and WT (squares) mice show comparable protein and cytokine levels following LPS inhalation. (A and B) Total protein and albumin concentrations in cell-free BAL fluids from mice in Figure 2 (n = 16/genotype) in response to LPS inhalation. (C and D) Levels of proinflammatory cytokines IL-1ß and TNF-{alpha} in cell-free BAL fluids from mice in Figure 2 in response to LPS inhalation.

 
Cytokine Levels
The local productions of the proinflammatory cytokines IL-1ß and TNF-{alpha} associated with LPS-mediated inflammation were measured. Although the levels of IL-1ß gradually increased over the observed period, the TNF-{alpha} response attained its peak by 4 h and declined considerably thereafter (Figures 3C and 3D). These changes in cytokine levels were comparable in both groups of mice (P > 0.05 for differences between groups).

NE Activity
As shown above, high numbers of neutrophils accumulate in the alveolar spaces by 24 h following challenge with P. aeruginosa or LPS. Using cell-free BAL fluids from both models, we aimed to determine the activity of released NE by elastin zymography (Figure 4). At equal volumes, a single lysis band that migrated similarly to control NE was detected in both P. aeruginosa and LPS cell-free BAL fluids from WT mice. However, this band was more intense in the P. aeruginosa BAL samples than the LPS samples. No NE lysis bands were detected in cell-free BAL fluids from either NE-/- or saline control mice. This indicates that while NE-/- mice do not express NE, saline instillation does not cause recruitment of neutrophils and/or release of NE. Of note, the observed NE activity was inhibited by PMSF (1 mM), but not EDTA (10 mM), excluding the contribution of P. aeruginosa metalloelastase (data not shown).



View larger version (60K):
[in this window]
[in a new window]
  		Figure 4. Detection of NE activity in cell-free BAL of P. aeruginosa or LPS-challenged mice. Active NE is detected as a single lysis band at ~ 29 kD in cell-free BAL fluids of LPS- and P. aeruginosa (P.a.)-challenged WT mice at 24 h (lanes 4 and 5). No lysis band (thus, no activity) was detected in cell-free BAL fluids from either saline control mice (lane 2) or NE-/- mice following LPS- or P. aeruginosa challenge (lanes 3 and 6). Purified NE (lane 1) or neutrophil lysates from LPS-BAL pellets (lane 7) were used as positive controls. The lysis band at ~ 105 kD (arrow) corresponds to MMP-9 (21). Molecular mass standards are shown on the left.

 
Respiratory Burst, Chemotaxis, and Migration
NE-/- and WT neutrophils isolated from the peritoneum or the lungs had normal and similar respiratory burst as determined by superoxide anion productions in response to fMLP or PMA stimulation. There was a ~ 2-fold and a > 7-fold increase upon stimulation with fMLP or PMA, respectively (P > 0.05 for differences between groups) (Figures 5A and 5B). In a parallel experiment, the chemotaxis of both NE-/- and WT neutrophils was assessed in a modified Boyden chamber assay. The number of NE-/- and WT neutrophils that responded to the chemoattractant ZAS was similar (Figure 6A). The ability of neutrophils to migrate through Matrigel, a prototype of a basement membrane, was also assessed. Isolated neutrophils from NE-/- and WT mice traversed Matrigel-coated membranes with equal efficiency in response to ZAS or cell-free BAL fluids from P. aeruginosa-infected WT mice (P > 0.05 for differences between groups) (Figure 6B). Thus, NE was required for neither chemotaxis nor migration through artificial basement membrane-like barriers. To further examine whether NE contributes to neutrophil migration, we tested the capacity of human neutrophils to cross Matrigel-coated membranes in the absence or presence of NE physiologic inhibitors, SLPI and {alpha}1-AT. Under our experimental conditions, neither SLPI nor {alpha}1-AT altered the transmembrane migration of neutrophils (P > 0.05 for differences between groups) (Figure 6C).



View larger version (21K):
[in this window]
[in a new window]
  		Figure 5. Oxidative burst of recruited NE-/- (filled squares) and WT (open squares) neutrophils. Superoxide anion production (oxidative burst) in neutrophils isolated from the peritoneum (A) or the lungs (B) of NE-/- or WT mice in response to fMLP or PMA stimulation was determined by cytochrome C assay. Data represent the mean ± SD of three independent experiments.

 


View larger version (23K):
[in this window]
[in a new window]
  		Figure 6. The absence of NE does not impair chemotaxis and transmembrane migration of isolated neutrophils. (A) Chemotaxis of neutrophils from NE-/- and WT mice in response to ZAS (2%). (B) Neutrophils from both genotypes of mice traversed Matrigel in a similar fashion when ZAS or cell-free BAL fluids from P. aeruginosa infected mice were used as chemoattractants. (C) The presence or absence of the serine proteinase inhibitors SLPI (10 µg/ml) or {alpha}1-AT (30 µg/ml) did not affect the ability of isolated human neutrophils to traverse Matrigel in response to chemoattractants as in (B). Data represent the mean ± SD of three independent experiments. A and B: open squares, WT; filled squares, NE-/-. C: open squares, control; filled squares, +SLPI; shaded squares, +{alpha}1-AT.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Because of its ability to degrade various structural proteins (e.g., ECM proteins and cadherins) NE was proposed to clear the path for neutrophil recruitment to inflamed tissues (29). This is an attractive possibility, particularly that NE was shown to bind to the surfaces of neutrophils and is catalytically active and resistant to inhibition by circulating antiproteases (30, 31). Furthermore, a recent study reported that NE is preferentially localized at the leading front of migrating neutrophils (32). By comparing NE-/- mice to their WT littermates, we found that unstressed NE-/- mice have normal circulating leukocyte numbers and differential cell counts when compared with WT mice. Also, changes in the hemograms of both types of mice were indistinguishable in the setting of acute inflammation. Next, to examine whether NE could play a role in neutrophil recruitment to inflamed sites, we chose bacterial infection to induce an innate neutrophilic response in mouse lungs or peritoneum. Under our experimental conditions, we show that regardless of the sites of infections, neutrophils are recruited normally in the absence of NE. Also, macroscopic analyses of infected lung tissues from NE-/- and WT mice revealed similar neutrophilic infiltration. Together, these data suggest that neither the presence of NE nor its translocation to the plasma membrane are required for neutrophil egress from the bone marrow or the blood.

Neutrophils contain at least four classes of proteases, and it could be that the role of NE is redundant or masked by other proteases. However, deficiency in cathepsin G, a neutrophil serine proteinase stored with NE, does not affect neutrophil recruitment (28) and neutrophils of mice deficient in the matrix metalloprotease-9 (MMP-9) migrate normally in response to various stimuli (21). It was also suggested that NE activates other proteolytic enzymes such as MMP-9, which degrade ECM proteins and facilitate neutrophil escape (18). In other reports, NE was proposed to generate chemotactic factors for leukocytes by degrading various proteins, including those of the ECM, and to activate proinflammatory mediators, both of which mediate neutrophil recruitment (16, 33). However, our data indicate that the expression of MMP-9 and the counts of attracted neutrophils to inflamed sites were similar in both NE-/- and WT mice. Our findings also contrast studies that suggested NE involvement in adhesion of neutrophils to the endothelial lining and their transmigration (e.g., NE degradation of ICAM-1 and cadherins leads to de-adhesion and transmigration of neutrophils) (7, 8). A support for our in vivo data comes from a study by Luscinskas and his group. Using an in vitro flow model, they observed no differences in the ability of WT and NE-/- or MMP-9-/- neutrophils to adhere to or migrate through the endothelial monolayer (34). Another possibility that merits consideration is that the serine proteinase inhibitors block NE (free or cell-surface bound) abrogating its role in cell migration. In vitro, pretreatment of human neutrophils with SLPI or {alpha}1-AT did not, however, hamper the cell's migration through Matrigel in response to different chemoattractants. Altogether, these findings suggest that other proteases secreted by neutrophils or cells in the vicinity (e.g., endothelial cells and/or macrophages) might be involved in neutrophil emigration (35). Alternatively, neutrophils egress to the sites of inflammation without the need of any proteolytic activity (36). For example, depending on the tissue and stimulus, activated neutrophils could signal changes in their biomechanical and adhesion properties and/or those of endothelial cells for migration (37).

Although our findings demonstrate that NE may not be required for neutrophil recruitment to inflamed sites, its role in host defense against invading pathogens is well established (38). Indeed, we and others have shown that the presence of NE is required for maximal neutrophil intracellular killing of Gram-negative, but not Gram-positive, bacteria (20, 39). Our data from P. aeruginosa– and LPS-induced acute inflammation shows that neutrophil influx, lung injury scores, biochemical markers, and antigenic levels of the proinflammatory mediators were similar in NE-/- and WT mice. These findings suggest that NE is not required for cell migration and does not cause changes in the host inflammatory response, but its role in the development of lung injury still cannot be ruled out when taking various points into consideration. Our evaluation of mouse lung injury was performed following intranasal infection with a small inoculum size and at one time point (24 h after infection). Qualitative analyses of cell-free BAL fluids by elastin zymography indicated the presence of free active NE in WT lungs following intranasal challenge with both LPS and P. aeruginosa. Other lung studies particularly in humans demonstrated the presence of NE (active or antigen) as well (40–42). Because of its extracellular release and its large substrate repertoire, NE has been implicated in the pathogenesis of various tissue-destructive diseases, including acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) (43). Also, much interest has focused on the development of a variety of inhibitors to block NE proteolytic activity (44). Studies that focus on whether NE contributes to lung injury in the setting of acute inflammation are underway.


   Acknowledgments
 
The authors thank D. P. Schuster and S. L. Brody for critical review of the manuscript and helpful discussions, and D. Kelly for excellent technical assistance. This work was supported by grants from the Barnes-Jewish Hospital Foundation and HL-66415 to A.B. and the German Academic Exchange Service (DAAD) to T.O.H.

Received in original form July 3, 2003

Received in final form September 30, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wagner, J. G., and R. A. Roth. 2000. Neutrophil migration mechanisms, with an emphasis on the pulmonary vasculature. Pharmacol. Rev. 52:349–374.[Abstract/Free Full Text]
  2. Newman, P. J. 1994. The role of PECAM-1 in vascular cell biology. Ann. N. Y. Acad. Sci. 714:165–174.[Medline]
  3. Luscinskas, F. W., S. Ma, A. Nusrat, C. A. Parkos, and S. K. Shaw. 2002. Leukocyte transendothelial migration: a junctional affair. Semin. Immunol. 14:105–113.[CrossRef][Medline]
  4. Lee, W. L., and G. P. Downey. 2001. Leukocyte elastase: physiological functions and role in acute lung injury. Am. J. Respir. Crit. Care Med. 164:896–904.[Free Full Text]
  5. Bieth, J. G. 1986. Elastases: catalytic and biological properties. In Regulation of Matrix Accumulation. R. Mecham, editor. Academic Press, New York. 2228–2232.
  6. Liou, T. G., and E. J. Campbell. 1995. Nonisotropic enzyme–inhibitor interactions: a novel nonoxidative mechanism for quantum proteolysis by human neutrophils. Biochemistry 34:16171–16177.[CrossRef][Medline]
  7. Champagne, B., P. Tremblay, A. Cantin, and Y. St Pierre. 1998. Proteolytic cleavage of ICAM-1 by human neutrophil elastase. J. Immunol. 161:6398–6405.[Abstract/Free Full Text]
  8. Carden, D., F. Xiao, C. Moak, B. H. Willis, S. Robinson-Jackson, and S. Alexander. 1998. Neutrophil elastase promotes lung microvascular injury and proteolysis of endothelial cadherins. Am. J. Physiol. 275:H385–H392.
  9. Cai, T. Q., and S. D. Wright. 1996. Human leukocyte elastase is an endogenous ligand for the integrin CR3 (CD11b/CD18, Mac-1, alpha M beta 2) and modulates polymorphonuclear leukocyte adhesion. J. Exp. Med. 184:1213–1223.[Abstract/Free Full Text]
  10. Le Barillec, K., M. Si-Tahar, V. Balloy, and M. Chignard. 1999. Proteolysis of monocyte CD14 by human leukocyte elastase inhibits lipopolysaccharide-mediated cell activation. J. Clin. Invest. 103:1039–1046.[Medline]
  11. Scuderi, P., P. A. Nez, M. L. Duerr, B. J. Wong, and C. M. Valdez. 1991. Cathepsin-G and leukocyte elastase inactivate human tumor necrosis factor and lymphotoxin. Cell. Immunol. 135:299–313.[CrossRef][Medline]
  12. Leavell, K. J., M. W. Peterson, and T. J. Gross. 1997. Human neutrophil elastase abolishes interleukin-8 chemotactic activity. J. Leukoc. Biol. 61:361–366.[Abstract]
  13. DiMartino, S. J., A. B. Shah, G. Trujillo, and R. R. Kew. 2001. Elastase controls the binding of the vitamin D-binding protein (Gc-globulin) to neutrophils: a potential role in the regulation of C5a co-chemotactic activity. J. Immunol. 166:2688–2694.[Abstract/Free Full Text]
  14. Sadallah, S., C. Hess, S. Miot, O. Spertini, H. Lutz, and J. A. Schifferli. 1999. Elastase and metalloproteinase activities regulate soluble complement receptor 1 release. Eur. J. Immunol. 29:3754–3761.[CrossRef][Medline]
  15. Banda, M. J., A. G. Rice, G. L. Griffin, and R. M. Senior. 1988. Alpha 1-proteinase inhibitor is a neutrophil chemoattractant after proteolytic inactivation by macrophage elastase. J. Biol. Chem. 263:4481–4484.[Abstract/Free Full Text]
  16. Senior, R. M., G. L. Griffin, and R. P. Mecham. 1980. Chemotactic activity of elastin-derived peptides. J. Clin. Invest. 66:859–862.
  17. Doerschuk, C. M. 2001. Mechanisms of leukocyte sequestration in inflamed lungs. Microcirculation 8:71–88.[CrossRef][Medline]
  18. Delclaux, C., C. Delacourt, M. P. D'Ortho, V. Boyer, C. Lafuma, and A. Harf. 1996. Role of gelatinase B and elastase in human polymorphonuclear neutrophil migration across basement membrane. Am. J. Respir. Cell Mol. Biol. 14:288–295.[Abstract]
  19. Mackarel, A. J., D. C. Cottell, K. J. Russell, M. X. FitzGerald, and C. M. O'Connor. 1999. Migration of neutrophils across human pulmonary endothelial cells is not blocked by matrix metalloproteinase or serine protease inhibitors. Am. J. Respir. Cell Mol. Biol. 20:1209–1219.[Abstract/Free Full Text]
  20. Belaaouaj, A., R. McCarthy, M. Baumann, Z. Gao, T. J. Ley, S. N. Abraham, and S. D. Shapiro. 1998. Mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis. Nat. Med. 4:615–618.[CrossRef][Medline]
  21. Betsuyaku, T., J. M. Shipley, Z. Liu, and R. M. Senior. 1999. Neutrophil emigration in the lungs, peritoneum, and skin does not require gelatinase B. Am. J. Respir. Cell Mol. Biol. 20:1303–1309.[Abstract/Free Full Text]
  22. Schultz, M. J., A. W. Rijneveld, S. Florquin, P. Speelman, S. J. Van Deventer, and T. van der Poll. 2001. Impairment of host defence by exotoxin A in Pseudomonas aeruginosa pneumonia in mice. J. Med. Microbiol. 50:822–827.[Abstract/Free Full Text]
  23. Szarka, R. J., N. Wang, L. Gordon, P. N. Nation, and R. H. Smith. 1997. A murine model of pulmonary damage induced by lipopolysaccharide via intranasal instillation. J. Immunol. Methods 202:49–57.[CrossRef][Medline]
  24. Denis, M., L. Guojian, M. Widmer, and A. Cantin. 1994. A mouse model of lung injury induced by microbial products: implication of tumor necrosis factor. Am. J. Respir. Cell Mol. Biol. 10:658–664.[Abstract]
  25. Belperio, J. A., M. P. Keane, M. D. Burdick, V. Londhe, Y. Y. Xue, K. Li, R. J. Phillips, and R. M. Strieter. 2002. Critical role for CXCR2 and CXCR2 ligands during the pathogenesis of ventilator-induced lung injury. J. Clin. Invest. 110:1703–1716.[CrossRef][Medline]
  26. Kaner, R. J., J. V. Ladetto, R. Singh, N. Fukuda, M. A. Matthay, and R. G. Crystal. 2000. Lung overexpression of the vascular endothelial growth factor gene induces pulmonary edema. Am. J. Respir. Cell Mol. Biol. 22:657–664.[Abstract/Free Full Text]
  27. Green, T. P., D. E. Johnson, and C. W. Gatto. 1988. Transvascular flux and tissue accrual of Evans blue: effects of endotoxin and histamine. J. Lab. Clin. Med. 111:173–183.[Medline]
  28. MacIvor, D. M., S. D. Shapiro, C. T. Pham, A. Belaaouaj, S. N. Abraham, and T. J. Ley. 1999. Normal neutrophil function in cathepsin G-deficient mice. Blood 94:4282–4293.[Abstract/Free Full Text]
  29. Shapiro, S. D. 2002. Neutrophil elastase: path clearer, pathogen killer, or just pathologic? Am. J. Respir. Cell Mol. Biol. 26:266–268.[Free Full Text]
  30. Owen, C. A., M. A. Campbell, P. L. Sannes, S. S. Boukedes, and E. J. Campbell. 1995. Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases. J. Cell Biol. 131:775–789.[Abstract/Free Full Text]
  31. Owen, C. A., M. A. Campbell, S. S. Boukedes, and E. J. Campbell. 1997. Cytokines regulate membrane-bound leukocyte elastase on neutrophils: a novel mechanism for effector activity. Am. J. Physiol. 272:L385–L393.
  32. Cepinskas, G., M. Sandig, and P. R. Kvietys. 1999. PAF-induced elastase-dependent neutrophil transendothelial migration is associated with the mobilization of elastase to the neutrophil surface and localization to the migrating front. J. Cell Sci. 112:1937–1945.[Abstract]
  33. Bank, U., and S. Ansorge. 2001. More than destructive: neutrophil-derived serine proteases in cytokine bioactivity control. J. Leukoc. Biol. 69:197–206.[Abstract/Free Full Text]
  34. Allport, J. R., Y. C. Lim, J. M. Shipley, R. M. Senior, S. D. Shapiro, N. Matsuyoshi, D. Vestweber, and F. W. Luscinskas. 2002. Neutrophils from MMP-9- or neutrophil elastase-deficient mice show no defect in transendothelial migration under flow in vitro. J. Leukoc. Biol. 71:821–828.[Abstract/Free Full Text]
  35. Allport, J. R., H. Ding, T. Collins, M. E. Gerritsen, and F. W. Luscinskas. 1997. Endothelial-dependent mechanisms regulate leukocyte transmigration: a process involving the proteasome and disruption of the vascular endothelial-cadherin complex at endothelial cell-to-cell junctions. J. Exp. Med. 186:517–527.[Abstract/Free Full Text]
  36. Walker, D. C., A. R. Behzad, and F. Chu. 1995. Neutrophil migration through preexisting holes in the basal laminae of alveolar capillaries and epithelium during streptococcal pneumonia. Microvasc. Res. 50:397–416.[CrossRef][Medline]
  37. Wang, Q., and C. M. Doerschuk. 2002. The signaling pathways induced by neutrophil-endothelial cell adhesion. Antioxid. Redox Signal. 4:39–47.[CrossRef][Medline]
  38. Belaaouaj, A. 2002. Neutrophil elastase-mediated killing of bacteria: lessons from targeted mutagenesis. Microbes Infect. 4:1259–1264.[CrossRef][Medline]
  39. Tkalcevic, J., M. Novelli, M. Phylactides, J. P. Iredale, A. W. Segal, and J. Roes. 2000. Impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G. Immunity 12:201–210.[CrossRef][Medline]
  40. Cochrane, C. G., R. G. Spragg, S. D. Revak, A. B. Cohen, and W. W. McGuire. 1983. The presence of neutrophil elastase and evidence of oxidation activity in bronchoalveolar lavage fluid of patients with adult respiratory distress syndrome. Am. Rev. Respir. Dis. 127:S25–S27.[Medline]
  41. Suter, P. M., S. Suter, E. Girardin, P. Roux-Lombard, G. E. Grau, and J. M. Dayer. 1992. High bronchoalveolar levels of tumor necrosis factor and its inhibitors, interleukin-1, interferon, and elastase, in patients with adult respiratory distress syndrome after trauma, shock, or sepsis. Am. Rev. Respir. Dis. 145:1016–1022.[Medline]
  42. Delacourt, C., S. Herigault, C. Delclaux, A. Poncin, M. Levame, A. Harf, F. Saudubray, and C. Lafuma. 2002. Protection against acute lung injury by intravenous or intratracheal pretreatment with EPI-HNE-4, a new potent neutrophil elastase inhibitor. Am. J. Respir. Cell Mol. Biol. 26:290–297.[Abstract/Free Full Text]
  43. Moraes, T. J., C. W. Chow, and G. P. Downey. 2003. Proteases and lung injury. Crit. Care Med. 31:S189–S194.[CrossRef][Medline]
  44. Zeiher, B. G., S. Matsuoka, K. Kawabata, and J. E. Repine. 2002. Neutrophil elastase and acute lung injury: prospects for sivelestat and other neutrophil elastase inhibitors as therapeutics. Crit. Care Med. 30:S281–S287.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am. J. Respir. Cell Mol. Bio.Home page
R. L. Zemans, S. P. Colgan, and G. P. Downey
Transepithelial Migration of Neutrophils: Mechanisms and Implications for Acute Lung Injury
Am. J. Respir. Cell Mol. Biol., May 1, 2009; 40(5): 519 - 535.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
E. Kolaczkowska, W. Grzybek, N. van Rooijen, H. Piccard, B. Plytycz, B. Arnold, and G. Opdenakker
Neutrophil elastase activity compensates for a genetic lack of matrix metalloproteinase-9 (MMP-9) in leukocyte infiltration in a model of experimental peritonitis
J. Leukoc. Biol., March 1, 2009; 85(3): 374 - 381.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. A. Bergin, C. M. Greene, E. E. Sterchi, C. Kenna, P. Geraghty, A. Belaaouaj, C. C. Taggart, S. J. O'Neill, and N. G. McElvaney
Activation of the Epidermal Growth Factor Receptor (EGFR) by a Novel Metalloprotease Pathway
J. Biol. Chem., November 14, 2008; 283(46): 31736 - 31744.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
T. O. Hirche, R. Benabid, G. Deslee, S. Gangloff, S. Achilefu, M. Guenounou, F. Lebargy, R. E. Hancock, and A. Belaaouaj
Neutrophil Elastase Mediates Innate Host Protection against Pseudomonas aeruginosa
J. Immunol., October 1, 2008; 181(7): 4945 - 4954.
[Abstract] [Full Text] [PDF]

Home page
 ChestHome page
A. S. Cowburn, A. M. Condliffe, N. Farahi, C. Summers, and E. R. Chilvers
Advances in Neutrophil Biology: Clinical Implications
Chest, September 1, 2008; 134(3): 606 - 612.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
A. M. Kaynar, A. M. Houghton, E. H. Lum, B. R. Pitt, and S. D. Shapiro
Neutrophil Elastase Is Needed for Neutrophil Emigration into Lungs in Ventilator-Induced Lung Injury
Am. J. Respir. Cell Mol. Biol., July 1, 2008; 39(1): 53 - 60.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. Mydel, J. M. Shipley, T. L. Adair-Kirk, D. G. Kelley, T. J. Broekelmann, R. P. Mecham, and R. M. Senior
Neutrophil Elastase Cleaves Laminin-332 (Laminin-5) Generating Peptides That Are Chemotactic for Neutrophils
J. Biol. Chem., April 11, 2008; 283(15): 9513 - 9522.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
P. Geraghty, M. P. Rogan, C. M. Greene, R. M. M. Boxio, T. Poiriert, M. O'Mahony, A. Belaaouaj, S. J. O'Neill, C. C. Taggart, and N. G. McElvaney
Neutrophil Elastase Up-Regulates Cathepsin B and Matrix Metalloprotease-2 Expression
J. Immunol., May 1, 2007; 178(9): 5871 - 5878.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
C. C. Taggart, C. M. Greene, T. P. Carroll, S. J. O'Neill, and N. G. McElvaney
Elastolytic Proteases: Inflammation Resolution and Dysregulation in Chronic Infective Lung Disease
Am. J. Respir. Crit. Care Med., May 15, 2005; 171(10): 1070 - 1076.
[Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. de Garavilla, M. N. Greco, N. Sukumar, Z.-W. Chen, A. O. Pineda, F. S. Mathews, E. Di Cera, E. C. Giardino, G. I. Wells, B. J. Haertlein, et al.
A Novel, Potent Dual Inhibitor of the Leukocyte Proteases Cathepsin G and Chymase: MOLECULAR MECHANISMS AND ANTI-INFLAMMATORY ACTIVITY IN VIVO
J. Biol. Chem., May 6, 2005; 280(18): 18001 - 18007.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
T. O. Hirche, J. P. Gaut, J. W. Heinecke, and A. Belaaouaj
Myeloperoxidase Plays Critical Roles in Killing Klebsiella pneumoniae and Inactivating Neutrophil Elastase: Effects on Host Defense
J. Immunol., February 1, 2005; 174(3): 1557 - 1565.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
T. L. Adair-Kirk, J. J. Atkinson, D. G. Kelley, R. H. Arch, J. H. Miner, and R. M. Senior
A Chemotactic Peptide from Laminin {alpha}5 Functions as a Regulator of Inflammatory Immune Responses via TNF{alpha}-mediated Signaling
J. Immunol., February 1, 2005; 174(3): 1621 - 1629.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. O. Hirche, E. C. Crouch, M. Espinola, T. J. Brokelman, R. P. Mecham, N. DeSilva, J. Cooley, E. Remold-O'Donnell, and A. Belaaouaj
Neutrophil Serine Proteinases Inactivate Surfactant Protein D by Cleaving within a Conserved Subregion of the Carbohydrate Recognition Domain
J. Biol. Chem., June 25, 2004; 279(26): 27688 - 27698.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2003-0253OCv1
30/4/576    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hirche, T. O.
Right arrow Articles by Belaaouaj, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hirche, T. O.
Right arrow Articles by Belaaouaj, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2004 American Thoracic Society.