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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 485-498, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.4913

Secretory Component Is Cleaved by Neutrophil Serine Proteinases but its Epithelial Production Is Increased by Neutrophils through NF-{kappa}B– and p38 Mitogen-Activated Protein Kinase–Dependent Mechanisms

Charles Pilette, Youssef Ouadrhiri, Françoise Dimanche, Jean-Pierre Vaerman and Yves Sibille

Experimental Medicine Unit, Christian de Duve Institute of Cellular Pathology, University of Louvain, Brussels, Belgium

Address correspondence to: Yves Sibille, M.D., PhD., Unité de Médecine Expérimentale, Avenue Hippocrate, 74 BP 7430, B-1200 Bruxelles, Belgique. E-mail: sibille{at}mexp.ucl.ac.be


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously showed that expression of polymeric immunoglobulin receptor (pIgR)/secretory component (SC), the epithelial receptor assuming transport of polymeric IgA in mucosal secretions, is strongly decreased in severe chronic obstructive pulmonary disease. Here, we evaluated in vitro the effects of polymorphonuclear neutrophil (PMN) mediators on pIgR/SC. On polyacrylamide gel electrophoresis analysis, soluble SC was rapidly cleaved by supernatants from phorbol-myristate-acetate-activated PMN, through a serine proteinase activity. Moreover, purified PMN serine proteinases also cleaved SC. Similarly, polymeric IgA was rapidly cleaved in monomers by neutrophil elastase, whereas secretory immunoglobulin A was relatively resistant to neutrophil elastase. Surface pIgR on human bronchial epithelial cells was also cleaved by serine proteinases, as shown by immunofluorescence. In contrast, pIgR/SC production by cultured epithelial cells (quantified by enzyme-linked immunosorbent assay) was significantly increased by supernatants from interleukin-8/formylmethionylleucylphenylalanine-activated PMN (122.6 ± 17.3 versus 70.9 ± 9 ng/mg protein, P < 0.01). Upregulation of pIgR/SC production by bronchial epithelial cells was abolished by nuclear factor {kappa}B- and p38 mitogen-activated protein kinase (MAPK) inhibitors. Moreover, supernatants from interleukin-8/formylmethionylleucylphenylalanine-activated PMN induced the phosphorylation of I{kappa}B-{alpha} and p38 MAPK in epithelial cells, independently of serine proteinases. Thus, PMN serine proteinases cleave pIgR/SC, whereas activated PMN induce an increased pIgR/SC expression through epithelial activation of nuclear factor {kappa}B and p38 MAPK pathways.

Abbreviations: N-methoxysuccinyl-ala-ala-pro-val-chlormethylketone, APV-CMK • bovine serum albumin, BSA • cathepsin G, CathG • complete MEM, cMEM • chronic obstructive pulmonary disease, COPD • enhanced chemiluminescence, ECL • enzyme-linked immunosorbent assay, ELISA • extracellular signal-regulated kinase, ERK • fetal bovine serum, FBS • fluorescein isothiocyanate, FITC • formylmethionylleucylphenylalanine, fMLP • primary human bronchial epithelial cells, HBEC • Hanks' balanced salt solution, HBSS • N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, HEPES • leucozyme, LZ • mitogen-activated protein kinase, MAPK • modified Eagle's medium, MEM • neutrophil elastase, NE • nuclear factor {kappa}B, NF-{kappa}B • phosphate-buffered saline, PBS • Tween 20 PBS, PBST • polymeric immunoglobulin receptor, pIgR • phorbol-myristate-acetate, PMA • polymorphonuclear neutrophil, PMN • phenyl-methyl-sulfonyl-fluoride, PMSF • proteinase 3, PR3 • secretory component, SC • secretory leukocyte proteinase inhibitor, SLPI • Soybean trypsin inhibitor, STI • tumor necrosis factor, TNF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Production of secretory immunoglobulins (SIgs), i.e., SIgA and SIgM, in the respiratory mucosa is thought to protect the host against inhaled particles and micro-organisms (1). Once produced in the lamina propria by mucosal plasma cells, polymeric IgA (pIgA) is transported into secretions by transcytosis across epithelial cells via polymeric Ig receptor (pIgR)/secretory component (SC). This transcellular routing, quantitatively the most important in the body, may be the rate-limiting factor for the luminal secretion of Igs. Previously, we showed that expression of pIgR/SC was strongly decreased in the bronchial epithelium from patients with severe chronic obstructive pulmonary disease (COPD) (2). The possibility that this epithelial defect could play a role in the pathogenesis of COPD, probably by facilitating airway bacterial colonization and infections, was supported by the strong correlation between decreased SC expression and functional parameters of airflow limitation. Moreover, decreased bronchial SC expression also correlated with polymorphonuclear neutrophil (PMN) infiltration of the submucosal glands. However, the mechanisms underlying the downregulation of pIgR/SC expression in COPD airways remain unknown.

PMNs are thought to play a major role in various lung disorders, including COPD, through the release of oxygen metabolites and proteinases mediating tissue damage (reviewed in Ref. 3). Intratracheal administration of neutrophil elastase (NE) or proteinase 3 (PR3) in rodents induced lung destruction and emphysema (4–6). Moreover, subjects deficient in {alpha}1-antitrypsin, a major NE natural inhibitor, are much more susceptible to develop COPD (7). In addition, serine proteinases and defensins of PMN have important effects on several epithelial functions (reviewed in Ref. 8), including adherence, mucociliary function, cytokine release, or receptor expression. Therefore, we examined if PMN-derived proteinases could degrade pIgR/SC. To test this, we studied in vitro the proteolysis, by supernatants of activated PMN or their purified serine proteinases, on purified SC, as well as on Igs. SC production and pIgR expression by bronchial epithelial cells exposed to PMN supernatants or their purified proteinases were also evaluated, both in human bronchial carcinoma cell line Calu-3 and primary bronchial epithelial cell cultures.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
SC and SIgA were purified from human lactoserum, and myeloma sera were used to purify mIgA and pIgA, as previously described (9–11). Purified SC or Igs were dialyzed against 0.2 M Tris-HCl, pH 7.8, with 0.05 M CaCl2 and 0.2 M NaCl. Neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CathG) purified from purulent human sputum were obtained from Elastin Products Co. (Owensville, MA), as well as leucozyme, which is a crude extract from purulent sputum containing NE (5%), PR3, and traces of CathG, trypsin, and other proteins (including myeloperoxidase). The different enzymes were diluted at 1 mg/ml in 0.05 M sodium acetate, pH 5.0, with 0.5 M NaCl and filtrered (0.22 µm). Inhibitors of the different proteinases comprised: phenyl-methyl-sulfonyl-fluoride (PMSF, serine proteinase inhibitor), N-methoxysuccinyl-ala-ala-pro-val-chlormethylketone (AAPV-CMK, NE inhibitor), and soybean trypsin inhibitor (STI) from Sigma Aldrich (St. Louis, MO), secretory leukocyte proteinase inhibitor (SLPI) from R&D Systems (Minneapolis, MN), GM6001 (metalloproteinase inhibitor) from EPC, and normal human serum. Hanks' balanced salt solution (HBSS), RPMI-1640, modified Eagle's medium (MEM), and bronchial epithelial growth medium were from BioWhittaker (Walkersville, MD); MEM was supplemented with 100 IU/ml penicilline, 100 µg/ml streptomycin, 2 mM L-glutamine, 2 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 2 mM sodium pyruvate (referred as complete MEM, cMEM), and, when indicated, with 10% vol/vol decomplemented (56°C, 30 min) fetal bovine serum (FBS).

Cell Cultures
Calu-3, a human bronchial carcinoma cell line, was obtained from the ATCC (Rockville, MD), and cultured in cMEM. Human primary bronchial epithelial cell (HBEC) cultures were obtained from patients undergoing lung surgery for a lung tumor, as previously described (12). The protocol was approved by the Ethics Committee of the Cliniques Universitaires de Mont-Godinne. Briefly, tumor-free segments of bronchial tissues were removed from the surgical specimens and placed in cold RPMI containing 200 IU/ml penicillin, 200 µg/ml streptomycin, 2.5 µg/ml amphotericine B, 50 µg/ml gentamycin, and 2 mM HEPES. The specimens were transferred to the lab within 24 h, and bronchial fragments were carefully dissected from the lung parenchyma and peribronchial tissues. After rinsing with the same supplemented HBSS, bronchial fragments were subjected to 0.05% wt/vol Pronase E (Sigma Aldrich) overnight at 4°C in RPMI containing antibiotics and 2 mM HEPES. Primary cells were detached by vigorous shaking and washed by centrifugation (800 x g, 15 min, 4°C), once with 10% FBS to stop the enzymatic process, and twice in the same medium without FBS. After counting (Coulter counter, Harpenden, UK), HBEC were resuspended at 106 cells/ml in bronchial epithelial growth medium supplemented with the manufacturer's additives. Calu-3 and HBEC were seeded (at 0.2 x 106/ml and 106/ml, respectively) in 24-well plates on 0.4 µm insert-type filters (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ) precoated for 2 h with 2.9 µg/ml vitrogen, 10 µg/ml fibronectin, and 100 µg/ml bovine serum albumin (BSA) in RPMI containing antibiotics. Nonadherent cells were removed by gentle washing with bronchial epithelial growth medium after 24 h, and medium was further replaced every 24 h. These cultures, allowing epithelial cell polarization, were conducted in humidified atmosphere at 37°C with 5% CO2 until confluence, as examined by inverted optical microscopy and measurement of transepithelial electrical resistance using a Millicell ERS device (Millipore, Bedford, MA). Confluence was reached after 3 ± 1 (Calu-3) or 6 ± 1 d (HBEC), as confirmed by electrical resistance (> 600 {Omega}.cm2). Epithelial monolayers were then incubated for 20 h with PMN supernatants or their purified proteinases in serum-free culture medium.

PMN were obtained from whole heparinized blood from healthy donors by a one-step density gradient method using Polymorphprep (Nycomed, Oslo, Norway) following the manufacturer's protocol. After washings with calcium- and magnesium-free HBSS, PMN were resuspended in serum-free RPMI and activated by 100 ng/ml phorbol-myristate-acetate (PMA) (Sigma Aldrich) for 30 min at 37°C, or by 10 ng/ml IL-8 (R&D Systems) for 5 min followed by 1 µM fMLP (Sigma Aldrich) for 30 min. Cell-free PMN supernatants were obtained by centrifugation (400 x g, 15 min) of PMN suspensions. Cell viability was assessed by Trypan blue exlusion, and by lactate deshydrogenase measurements (Kit 500; Sigma Aldrich) in epithelial cell supernatants following the manufacturer's procedure.

Enzyme Treatment and SDS-PAGE Analysis of Purified SC and Igs
Purified SC or Ig were incubated at 37°C under gentle shaking with supernatants from resting or phorbol-myristate-acetate-activated PMN, or with purified NE, PR3, or CathG at different substrate:enzyme molar ratios. The reaction was stopped after 3 min, 1 h, or 20 h by addition of sample buffer. SC or Ig, boiled for 3 min, were then subjected (10 µg/lane) to SDS-polyacrylamide gradient (5 to 20%) gel electrophoresis (SDS-PAGE) in Laemmli's running buffer without reduction, and the gel was stained with Coomassie blue.

Western blots for NE and PR3 were performed to corroborate the results of the enzymatic assays of PMN supernatants. After SDS-PAGE, the proteins were electrotransferred onto a nitrocellulose membrane (Amersham, Pharmacia Biotech, Piscataway, NJ). After blocking with 5% wt/vol BSA in 1/1,000 vol/vol Tween 20 (Sigma Aldrich) in 0.05 M Tris-buffered saline (TBS, 0.5 M NaCl), pH 7.4, for 1 h and washings in TBS-1/1,000 Tween, the membrane was incubated with 1 µg/ml (in TBS-1/1,000 Tween-1% BSA) of monoclonal antibody to NE (Dako) or to PR3 (NeoMarkers/Labvision, Fremont, CA). After incubation with HRP-conjugated secondary antibodies to mouse IgG (1/10,000; Southern Laboratories), the membrane was revealed by enhanced chemiluminescence (ECL; New England Biolabs, Beverly, MA) and exposure to Kodak X-Omat AR films (Kodak-Pathé, Paris, France).

Cultures of Epithelial Cells with PMN Supernatants
Confluent and polarized monolayers of epithelial cells were cultured for 20 h with supernatants obtained after activation by IL-8/fMLP of 10 x 106 PMN in 0.5 ml RPMI. At the end of the 20-h culture, the required volumes of supernatant were taken to measure enzymatic activities, and 5 mM (final concentration) PMSF was then added to inhibit further serine proteinase activity. Apical and basolateral media were separately harvested and frozen at –20°C until SC titration. Epithelial cells were washed twice in PBS and lysed with 1/1,000 vol/vol Triton X-100 (Sigma Aldrich) in PBS for 15 min on ice. Cell lysates were cleared from membranes by high-speed centrifugation for 15 min at 4°C.

Determination of PMN Proteinase Activities
Activity of PMN serine proteinases was measured in PMN supernatants after phorbol-myristate-acetate or IL-8/fMLP stimulation. Although the latter stimulation was, as expected (13), less efficient than phorbol-myristate-acetate to induce the release of serine proteinases from PMN azurophilic granules, free serine proteinase activity was significantly detected in these supernatants. NE activity was {bsim} 30 nmole/liter (or 1 µg/ml) after phorbol-myristate-acetate-induced degranulation of 106 PMN or IL-8/fMLP-stimulated 10 x 106 PMN in 0.5 ml. To determine activities of PMN enzymes after culture, assays for NE, PR3, and CathG were also performed in epithelial media at the end of the 20–h culture. NE and CathG activities were determined as previously described (14, 15) with minor modifications. N-methoxysuccinyl-ala-ala-pro-val- and N-methoxysuccinyl-ala-ala-pro-met p-nitroanilides (Sigma Aldrich) were used as respective specific substrates, diluted at 1 mM in 0.1 M HEPES buffer, pH 7.5, with 0.5 M NaCl and 10% vol/vol dimethylsulfoxide. Supernatants (50 µl) were incubated with 100 µl of substrate solution at 37°C in 96-well plates, and OD increase was monitored at 405 nm using a microplate reader (Titertek Multiscan Plus MKII; Labsystems, Zellik, Belgium). Results were compared with standard curves obtained with serial dilutions of purified enzymes, and expressed in nmole/liter. Trypsin-like activity of PR3 was assessed as previously shown (6) using N{alpha}-p-tosyl-L-arginin methyl ester hydrochloride diluted at 1 mM in 0.05 M Tris, pH 8.1, with 0.01 M CaCl2. OD was recorded at 247 nm, and concentrations of PR3 (in nmole/liter) were deduced from a standard curve of the purified proteinase.

Enzyme-Linked Immunosorbent Assay for SC
SC concentration was determined in culture supernatants of epithelial cells by a specific enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well microplates were coated with 1 µg/ml affinity-purified goat anti-SC antibody (developed in our laboratory, recognizing both soluble SC and membrane pIgR/SC) in 0.1 M carbonate-bicarbonate buffer, pH 9.6, overnight at 4°C. After washings with 1/1,000 vol/vol Tween 20-PBS (PBST) and blockade with 1% wt/vol BSA in PBST for 45 min at 37°C, samples and serial dilutions of purified human soluble SC were incubated for 2 h at 37°C. After washings with PBST, plates were then incubated with biotinylated goat anti-SC for 2 h and washed in TBST. Reaction was revealed by incubation for 30 min with alkaline phosphatase–conjugated streptavidin (1/20,000 in TBST) and, after washings, with 1 mg/ml paranitrophenylphosphate (Sigma Aldrich) in 10% vol/vol diethanolamine buffer, pH 9.8. After stopping the reaction with 3 M NaOH, OD were recorded at 405 nm using a microplate spectrometer. Sensitivity of the immunoassay was 0.2 ng/ml. Results were corrected for total protein concentration determined in cell lysates by the bicinchoninic acid-based method (Pierce, Rockford, IL), and expressed as ng of SC per mg of cell protein.

Immunofluorescence Analysis of Surface pIgR
Expression of pIgR was evaluated by indirect immunofluorescence at the surface of epithelial cells using confocal microscopy. Epithelial cells were cultured until confluence on insert-type filters in 24-well plates, and then incubated for 20 h with PMN (activated by IL-8/fMLP) supernatants or purified PMN proteinases in apical medium. Cells were then washed with cold HBSS, and incubated on ice with 10 µg/ml rabbit affinity-purified anti-SC antibodies for 1 h in HBSS-3% vol/vol FBS added to the apical medium. After washings, cells were incubated with 4 µg/ml fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Santa Cruz Biotech, Santa Cruz, CA) in the same medium for 1 h on ice. After washings, cells were fixed for 15 min at room temperature by 2% vol/vol formaldehyde in PBS, pH 7.4. Filters were removed from inserts and mounted on glass slides with 2.5% 1,4-diacylbicyclo 2,2,2-octane (Sigma Aldrich) in Mowiol (Calbiochem-Novabiochem, Darmstadt, Germany), and analyzed by a MRC-1024 confocal microscope (Bio-Rad Laboratories, Richmond, CA) using a x63 objective under oil immersion. Images were digitally recorded and reproduced with a photo printer.

Surface pIgR expression was also assessed on bronchial epithelial cells by flow cytometry. After culture, Calu-3 cells were detached by 0.1 M EDTA in PBS, pH 7.4 (on ice), washed twice with cold PBS, and stained for pIgR as for confocal microscopy. Fluorescence analysis was performed on a FACscan from BD Biosciences (Mountain View, CA).

IgA Transcytosis Assay
Evaluation of pIgR-mediated transepithelial transport of IgA was assessed in confluent and polarized epithelial monolayers using FITC-labeled pIgA. After incubation with NE or PMN supernatants for 20 h both in apical and basolateral media, epithelial cells were washed twice with serum-supplemented MEM and incubated with 1 mg/ml FITC-pIgA in the basolateral compartment. Both apical and basolateral media were harvested after 3 h, and fluorescence was quantified in supernatants using a computerized microplate fluorometer (Packard Instruments, Downers Grove, IL) at 494 nm excitation/518 nm emission wavelengths. Concentrations of pIgA in the apical media were deduced from a standard curve of fluorescent pIgA. FITC-conjugated mIgA used as control was not significantly transported in the apical medium.

I{kappa}B-{alpha} and p38 Mitogen-Activated Protein Kinase Phosphorylation Assays
Calu-3 cells were incubated with supernatants from IL-8/fMLP-activated PMN for 5–60 (or 240) min. When indicated, Calu-3 cells were pretreated for 1 h with 100 µM PD98059, 5 µM SB203580, or 20 µM MG132 (inhibitors of extracellular signal–regulated kinase [ERK] mitogen-activated protein kinase [MAPK], p38 MAPK, and nuclear factor (NF)-{kappa}B pathways, respectively, from New England Biolabs, Beverly, MA) before incubation with PMN supernatants. Calu-3 cells were then lyzed in ice-cold lysis buffer (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% Na deoxycholate and 0.2% SDS) containing a proteinase inhibitor mixture (Roche Diagnostics, Basel, Switzerland) including freshly added 1 mM PMSF and protein phosphatase inhibitors (25 mM NaF, 1 mM Na3VO4) from Sigma Aldrich. Cell extracts (5 µg protein, as determined by the bicinchoninic acid-based assay) were subjected to SDS-PAGE (12%), and electrotransferred onto a nitrocellulose membrane immunoblotted for both phosphorylated and total I{kappa}B-{alpha} and p38 MAPK, using specific antibodies and ECL (New England Biolabs).

Statistical Analysis
Data were obtained from experiments performed in duplicate (except for HBEC cultures) and repeated two or three times. Results are expressed as mean ± SEM or mean ± SD. The differences between the different groups were analyzed by the Student t test using InStat 2.01 statistical package (GraphPad InStat, San Diego, CA). A Bonferroni's correction was applied for multiple comparisons with the same control value, and P values < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SC Is Cleaved by Supernatants from Activated PMN
Purified soluble SC migrated as expected at {bsim} 80 kD in SDS-PAGE (Figure 1, upper panel). Soluble SC incubated from 3 min to 20 h with supernatant from resting PMN was easily distinguished from the secreted PMN proteins and appeared well preserved after as long as 20 h (Figure 1). In contrast, SDS-PAGE of soluble SC incubated for 3 min or 1 h with supernatant from phorbol-myristate-acetate-activated PMN showed a total disappearance of 80 kD native SC. At 20 h of incubation, a band of {bsim} 70–75 kD was identified in the digest (Figure 1A, last lane). However, this band was also present in the supernatant from activated PMN in the absence of SC after 20 h at 37°C, indicating that this protein did not correspond to SC. In addition, the release of the serine proteinases NE and PR3 in supernatants from activated PMN was confirmed by immunoblotting (Figure 1, lower panels). PR3 immunoreactivity was strongly reduced in PMN supernatants after 20 h of incubation, compatible with autolysis of the proteinase in these conditions.



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  		Figure 1. Cleavage of purified SC by supernatants from phorbol-myristate-acetate-activated PMN. Soluble SC was incubated at 37°C for 3 min, 1 h, or 20 h with supernatants from resting (sPMN[-]) or phorbol-myristate-acetate-activated PMN (sPMN[+]) at a molar ratio SC:NE (measured in sPMN[+]) of 4:1. Proteins (10 µg of SC per lane) were resolved by gradient SDS-PAGE and stained by Coomassie blue (upper panel), as stated in MATERIALS AND METHODS. SC and PMN supernatants alone were used as controls, and molecular weight markers are indicated on the left (kD). For Western blot analysis of NE and PR3 (lower panels), PMN supernatant proteins were run on SDS-PAGE after the incubation with SC, transferred onto nitrocellulose membrane, and blotted for PMN proteinases using specific antibodies and revealed by ECL.

 
Cleavage of SC by Activated PMN Depends on Serine Proteinase Activity
To determine the enzymatic activity responsible for SC cleavage by PMN, different proteinase inhibitors were added to supernatants from activated PMN before incubation with SC. The effects of these inhibitors on the activities of the different PMN serine proteinases is shown in Table 1.


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  		TABLE 1 Effects of different proteinase inhibitors on NE, PR-3 and CathG activity

 
Native SC was not significantly detected after incubation with supernatant from activated PMN, as compared with SC incubated with supernatant from resting PMN (Figure 2), and was easily distinguished from the smaller protein of {bsim} 70–75 kD detected in supernatants from activated PMN. PMSF (1 mM) inhibited the cleavage of SC by supernatant from activated PMN, as also observed with AAPV-CMK (Figure 2). In contrast, SLPI did not exert significant inhibitory effects on PMN-induced SC cleavage (Figure 2), as well as STI and GM6001 (or EDTA) used respectively as CathG and metalloproteinase inhibitors (not shown). The presence of serine proteinases was confirmed in supernatants from activated PMN by measurement of their proteolytic activities (see MATERIALS AND METHODS). The effects of proteinase inhibitors on SC cleavage by activated PMN supernatants suggested that degradation was mediated by a serine proteinase activity depending at least on PR3.



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  		Figure 2. Effect of proteinase inhibitors on SC cleavage by supernatants from phorbol-myristate-acetate-activated PMN. PMSF (1 mM), AAPV-CMK (0.2 mM), or SLPI (2.5 µM; diluted with Tris buffer containing 0.1% bovine serum albumin) were added to supernatants from phorbol-myristate-acetate-activated PMN (sPMN[+]) before incubation with purified SC for 1 min or 1 h. SDS-PAGE analysis was performed as for Figure 1.

 
SC Is Cleaved by Purified PMN Serine Proteinases
Experiments using purified PMN enzymes confirmed that PR3 alone induced a rapid and intense proteolysis of SC (Figure 3, upper panel) and showed a similar effect of NE. A complete disappearance of native SC associated with the induction of multiple fragments was observed as early as 3 min or 1 h with PR3 and NE (Figure 3, upper panel). In contrast, CathG cleaved SC to a much lesser extent, producing only two or three major fragments. AAPV-CMK inhibitor significantly suppressed the cleavage of SC by NE, and also to a lesser extent that mediated by PR3 (Figure 3, lower panel).



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  		Figure 3. Cleavage of purified SC by purified PMN serine proteinases (upper panel), and effect of AAPV-CMK inhibitor (lower panel). After incubation at 37°C for 3 min, 1 h, or 20 h with PR3, CathG, or NE (molar ratio 1:1), SDS-PAGE analysis was performed as for Figure 1. AAPV-CMK inhibitor (0.2 mM) was added to PR3 and NE before incubation with SC.

 
SIgA Is Resistant to NE
In contrast with SC, which appeared extremely sensitive to NE and PR3 (Figures 3 and 4A), few fragments were observed after incubation of SIgA with NE, even at high molar ratios of NE:SIgA (Figure 4B). In contrast, pIgA was rapidly cleaved into monomers (Figure 4C); afterwards these IgA monomers from cleaved pIgA exhibit the same pattern of cleavage as natural mIgA. Interestingly, mIgA was relatively resistant to NE (Figure 4D), although the size of mIgA appears slightly reduced after NE treatment. This could be due to the proteolytic cleavage of terminal fragments of mIgA or to the fact that NE modifies the migration of mIgA in SDS-PAGE. Both PR3 and CathG did not produce a significant cleavage of pIgA, nor of m- and SIgA (not shown). The sensitivity to NE of the other Ig classes, serum IgG, IgE, and IgM was also evaluated. In line with the observations of SIgA, pentameric IgM was resistant to NE-mediated cleavage; in contrast with mIgA, the monomeric immunoglobulins IgG and IgE were significantly degraded by NE treatment (not shown).



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  		Figure 4. SDS-PAGE analysis of purified SC (A), SIgA (B), pIgA (C), and mIgA (D) incubated with NE at 37°C for 1 or 20 h at indicated substrate:enzyme molar ratios, as described in MATERIALS AND METHODS (10 µg of SC or IgA per lane).

 
SC Protects pIgA from Cleavage by NE
To assess the mechanism of resistance of SIgA to NE-mediated cleavage, as compared with serum pIgA, exogenous free SC was added to pIgA (and to mIgA) in a small (1.3-fold) molar excess before incubation with NE from 3 min to 20 h (Figure 5). As expected, addition of SC had no significant effect on mIgA sensitivity to NE, and did not allow formation of covalent complexes, because free SC was strongly detected (with no band of higher molecular weight) in SDS-PAGE. In contrast, SC added to pIgA formed some covalent complexes corresponding to SIgA. Moreover, this SIgA was as much resistant to the cleavage by NE as natural milk SIgA (Figure 5). Thus, the high molecular weight protein observed after incubation of pIgA with SC and corresponding to SIgA persisted until 20 h of treatment by NE, whereas SC-unbound pIgA was rapidly cleaved in monomers.



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  		Figure 5. Protective effect of exogenous SC on pIgA cleavage by NE. When indicated*, exogenous SC was added (1.3-fold, molar excess) to mIgA or pIgA before incubation with NE at 37°C for 3 min, 1 h, or 20 h (substrate:NE molar ratio, 1:2). SDS-PAGE analysis was performed as for Figure 1.

 
SC Produced by Epithelial Cells Is Cleaved by PMN Serine Proteinases
After incubation of bronchial epithelial cells with NE (0.1–500 nM) for 20 h in serum-free culture medium, their polarized production of SC was evaluated by titrating SC in the apical media by ELISA. SC was also assessed in cell lysates and basolateral media, to evaluate the cellular pool of pIgR/SC and to confirm the functional integrity of the monolayer, respectively. SC production by Calu-3 was not significantly affected by low concentrations of NE (0.1 or 1 nM). In contrast, 10 nM NE induced a significant decrease of immunoreactive SC detected in the apical medium (35.4 ± 7.1 versus 73.6 ± 8.5 ng/mg protein, P < 0.01; Figure 6A), and no SC was detected in supernatant from Calu-3 cells incubated with 500 nM NE (Figure 6A). There was no significant cell detachment nor necrosis of Calu-3 cells during this treatment, as observed by microscopy, Trypan blue exclusion test, and lactate deshydrogenase levels (15.1 ± 3.4 versus 11.7 ± 2.8 U/ml, mean ± SD, after incubation with 500 nM NE or with medium, respectively; NS). The low levels of SC in the basolateral media also confirmed the maintenance of the monolayer integrity, even for high NE concentrations (the NE concentration inducing significant cytotoxicity in terms of cell detachment, necrosis or increase of lactate deshydrogenase release was {bsim} 2 µM, for both Calu-3 and HBEC, not shown).



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  		Figure 6. Effect of purified NE on pIgR/SC produced by cultured bronchial epithelial cells. Calu-3 (A) or HBEC (B), cultured on insert-type filters until confluence, were incubated for 20 h in the apical medium with NE (0.1 nM to 500 nM), or with sputum extract (leucozyme, LZ; diluted 1:3,000) with or without proteinase inhibitors (0.2 mM AAPV-CMK, 2.5 µM SLPI or 100 µg/ml STI). SC concentration was determined by ELISA in apical media (solid bars), cell lysates (striped bars), and basolateral media (open bars), and results were corrected for cell protein concentration. Data are means ± SEM (A), or results from one of two experiments (B). *P < 0.01, **P < 0.05 compared with cells incubated with medium.

 
In addition, a significant decrease of SC was also observed in Calu-3 cell lysates (Figure 6A), although this was to a lesser extent than in apical media. A similar effect of PR3 was observed on SC in Calu-3 cell cultures, in contrast with CathG, which had no significant effect (not shown). When compared with the SC:NE molar ratio required to observe a cleavage of SC in the acellular system (Figure 4A), a much higher concentration of NE was required to observe a decrease of SC in cell cultures, as quantified by ELISA, because 10 nM of NE corresponded to an approximated SC:NE molar ratio of 1:80. This difference was not due to an active production of natural proteinase inhibitors by epithelial cells treated with NE, because NE activity was not significantly reduced in Calu-3 media as compared with the control medium incubated at 37°C for 20 h (Table 2), but was due to the relative maintenance of SC immunoreactivity after fragmentation by NE. Thus, a SC:NE ratio of {bsim} 1:80 was necessary to observe a significant decrease of immunoreactive SC in ELISA (not shown).


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  		TABLE 2 NE activity after epithelial cell culture

 
Cleavage of SC in Cell Culture by PMN Proteinases Is Mainly Due to NE
To clarify the respective role of the different PMN serine proteinases, the effects of PMN proteinase inhibitors were evaluated on SC production by Calu-3 cells after incubation with sputum extract. Similarly to the effect of activated PMN on purified SC, SC produced by cultured epithelial cells was strongly reduced after treatment with sputum extract (diluted 1/3,000; containing {bsim} 50 nM NE). In addition, AAPV-CMK and SLPI inhibitors almost abrogated the effect of sputum extract on SC produced by Calu-3 cells, in contrast with STI (Figure 6A), suggesting that in contrast with the effect of SLPI in the acellular system, cleavage of SC produced in cell culture is mainly due to NE. This difference of sensitivity of SC to PR3 and NE could be related to differences in the molecular form of SC in cell culture, such as the formation of complexes with other secreted proteins. Another possibility could be that the dose of PR3 contained in sputum extract in lower concentration than NE is not sufficient to mediate the cleavage of SC in cell culture.

A decrease in SC level was also observed in primary cultures of bronchial epithelial cells exposed to NE (Figure 6B). Thus, less SC was detected in apical media (14.1 versus 21.8 ng/mg protein) and lysates (6.9 versus 10.4 ng/mg protein) from HBEC treated by 10 nM NE (Figure 6B). However, the effect of NE on SC produced by HBEC was less pronounced than on Calu-3 cells. This could be explained by the fact that NE activity was significantly decreased after incubation of HBEC with NE, in contrast with Calu-3 cells (Table 2).

SC Production by Epithelial Cells Is Upregulated by Activated PMN
To evaluate the effect of whole supernatants from PMN on epithelial SC production, PMN were activated by IL-8/fMLP rather than phorbol-myristate-acetate because the latter is known to upregulate SC production (16). Supernatants from PMN activated by IL-8/fMLP were confirmed to cleave purified SC (not shown), as observed with phorbol-myristate-acetate-activated PMN. In contrast with the effects of purified PMN proteinases (NE and PR3) and of sputum extract on SC, both in the acellular system and cell cultures, SC concentrations in apical media from Calu-3 cells incubated with supernatants from IL-8/fMLP-activated PMN was significantly increased, as compared with Calu-3 cells treated with culture medium (122.6 ± 17.3 versus 70.9 ± 9 ng/mg protein, P < 0.01; Figure 7A) or with IL-8/fMLP (Figure 7A). SC concentration in lysates of Calu-3 cells exposed to supernatants from activated PMN was also upregulated (Figure 7A). Moreover, addition of the NE inhibitor AAPV-CMK induced an additional increase in apical SC production by Calu-3 cells incubated with supernatants from activated PMN (168.9 ± 13.4 ng/mg protein; Figure 7A). This observation suggested that upregulation of epithelial SC production induced by activated PMN was mediated by a mechanism independent from NE or PR3 proteolytic activity. Interestingly, similar results were obtained with two HBEC cultures (39.7 versus 18.7 ng/mg protein for one of these HBEC cultures; Figure 7B).



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  		Figure 7. Increased epithelial pIgR/SC production induced by supernatant from activated PMN. Calu-3 cells (A) or HBEC (B) cultured on insert-type filters were incubated for 20 h in the apical medium with supernatant from IL-8/fMLP-activated PMN (0.5 ml sPMN[+], from 10 x 106 PMN), with or without AAPV-CMK inhibitor (0.2 mM). SC concentration was determined by ELISA in apical media (solid bars), basolateral media (open bars), and cell lysates (striped bars), and results were corrected for cell protein concentration. Data are means ± SEM (A), or results from one of two experiments (B). *P < 0.01 compared with cells incubated with medium.

 
Cell-Surface pIgR Is Cleaved by NE
Surface presence of pIgR on Calu-3 cells, evaluated by confocal microscopy after specific immunofluorescence staining (Figures 8B and 8C), was strongly decreased after incubation with NE at 10 nM and 100 nM for 20 h (Figures 8E and 8F); it was also decreased after PR3 treatment, but to a much lower extent than with NE (not shown). In contrast, no effect was observed with CathG (Figures 8G–8I). A similar effect of NE on pIgR expression was observed on HBEC treated for 20 h by 100 nM NE (Figure 8L).



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  		Figure 8. Effect of purified NE on surface pIgR expression by epithelial cells. Calu-3 (A–I) or HBEC (J–L) were cultured on insert-type filters and incubated at 37°C for 20 h with medium (A–C, J–K), NE (1 nM, D; 10 nM, E; 100 nM, F and L) or CathG (1 nM, G; 10 nM, H; 100 nM, I). Immunofluorescence analysis of pIgR expression was performed using confocal microscopy, as detailed in MATERIALS AND METHODS. Transmission images are also shown (A, J), and scale bars are in micrometers (all panels correspond to x2 zoom, except A, B, J, and K).

 
Similarly to cleavage of soluble SC, pIgR staining on Calu-3 cells was strongly reduced by sputum extract (diluted 1/3,000; Figure 9), and this effect was significantly reversed by the PMN serine proteinase inhibitors PMSF, AAPV-CMK, and SLPI, as well as by normal human serum (Figure 9), but not by STI (not shown). Interestingly, supernatants from IL-8/fMLP-activated PMN did not decrease pIgR expression on Calu-3 cells (Figure 9). Similar results were obtained by flow cytometric analysis of surface pIgR expression on Calu-3 cells exposed to activated PMN-supernatants, as compared with NE or PR3 (not shown).



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  		Figure 9. Effect of sputum extract (leucozyme, LZ) and supernatant from IL-8/fMLP-activated PMN on surface pIgR expression by Calu-3 cells. Calu-3 cells were incubated for 20 h with supernatant from IL-8/fMLP-activated PMN (sPMN[+]), or with sputum extract (LZ). Different proteinase inhibitors (PMSF, AAPV-CMK, SLPI) or human serum were added to sputum extract before incubation of Calu-3 cells. Immunofluorescence analysis was performed as for Figure 8. Scale is in micrometers.

 
Receptor-Mediated Transepithelial Transport of pIgA Is Decreased by NE but Upregulated by Supernatants from Activated PMN
The functional consequence of the PMN proteinase-induced pIgR downregulation was assessed considering the role of transport of pIgR for pIgA and IgM. In line with the effect of PMN proteinases on pIgR/SC expression, transport of fluorescent pIgA by Calu-3 cells exposed to NE (10 or 100 nM for 20 h) was strongly decreased (Table 3). In contrast, transcytosis of pIgA was increased in Calu-3 cells preincubated with supernatant from IL-8/fMLP-activated PMN (Table 3). This last observation was in line with the observations of upregulation of pIgR/SC production and persistent surface pIgR in Calu-3 cells exposed to such activated PMN-supernatants (Figures 7 and 9).


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  		TABLE 3 NE and PMN effects on transcytosis of pIgA across Calu-3 cells

 
PMN-Mediated Upregulation of SC Production Depends on NF-{kappa}B and p38 MAPK Pathways
To assess the mechanisms of SC upregulation in epithelial cells incubated with activated PMN-supernatants, specific inhibitors of different intracellular signaling pathways were used. Interestingly, the stimulatory effect of activated PMN on SC production by Calu-3 cells was abrogated by pretreatment of these cells with MG132 or SB203580 (Figure 10A), suggesting the requirement of both NF-{kappa}B and p38 MAPK pathways for the PMN-induced pIgR/SC upregulation. In contrast, ERK MAPK activation was not necessary because its inhibitor, PD98059, had no significant effect on PMN-induced SC upregulation in Calu-3 cells (Figure 10A).



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  		Figure 10. Effect of NF-{kappa}B, ERK, and p38 MAPK inhibitors on PMN-induced SC upregulation (A) and PMN-induced phosphorylation of I{kappa}B-{alpha} (B) and p38 MAPK (C) in Calu-3 cells. (A) Confluent Calu-3 monolayers were pretreated with MG132 (20 µM), SB203580 (5 µM) or PD98059 (100 µM) 1 h before incubation for 20 h with supernatant from IL-8/fMLP-activated PMN (sPMN[+]). SC concentration was determined in media and cell lysates by ELISA, and results were corrected for cell protein concentration. Data are mean ± SEM. *P < 0.01 compared with cells incubated with supernatant from activated PMN. (B, C). Calu-3 cells were incubated for 5 to 60 or 240 min with supernatant from IL-8/fMLP-activated PMN. When indicated, Calu-3 cells were pretreated with inhibitors (PMSF [1 mM], SB203580, MG132) 1 h before incubation with PMN supernatant for 15 min; Calu-3 incubated for 15 min with IL-8/fMLP, PMSF, or supernatant from resting PMN (sPMN[-]) were used as additional controls. Both phosphorylated and total I{kappa}B-{alpha} and p38 MAPK proteins were detected by specific western blots and ECL, as described in MATERIALS AND METHODS.

 
NF-{kappa}B and p38 MAPK Pathways Are Activated in Epithelial Cells Incubated with Stimulated PMN Supernatants
The phosphorylated form of I{kappa}B-{alpha} protein was induced in Calu-3 cells after 5 min of incubation with supernatants from IL-8/fMLP-activated PMN, as compared with the lack of effect of IL-8/fMLP or resting PMN (Figure 10B). No significant effect on I{kappa}B-{alpha} phosphorylation was observed with MG132 and SB203580 inhibitors (not shown), nor with PMSF alone (Figure 10B). In addition, p38 MAPK was also rapidly phosphorylated in Calu-3 cells exposed to supernatants from such activated PMN, in contrast with resting PMN (Figure 10C). Induction of p38 MAPK phosphorylation was abrogated in Calu-3 cells pretreated by SB203580 before incubation with PMN supernatants, and was not significantly changed by MG132 (Figure 10C). Although their levels were slightly changed (small reduction for I{kappa}B-{alpha}, and modest increase for p38 MAPK), the phosphorylated forms of I{kappa}B-{alpha} and p38 MAPK induced by activated PMN were still observed in the presence of PMSF, indicating that PMN-induced epithelial cell activation can occur independently from PMN serine proteinases (Figures 10B and 10C).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We previously observed that bronchial pIgR/SC expression is strongly decreased in severe COPD (2). In addition, decreased SC expression in COPD correlated with airway obstruction and PMN infiltration, both considered as hallmarks of the disease. Here, we show that PMN products exert important effects on pIgR/SC expression in cultured human bronchial epithelial cells, as well as on soluble SC. Thus, purified soluble SC appeared extremely sensitive to the PMN serine proteinases released by activated PMN, and PR3 was suggested to mostly mediate this effect, although NE is also able to cleave soluble SC. Interestingly, increased concentrations of NE are found in airway secretions from patients with COPD (17). Moreover, among PMN serine proteinases, which are major mediators from these leukocytes (reviewed in Ref. 18), both NE and PR3 are able to induce emphysema in vivo in rodents (4–6), and this effect is thought to be mediated at least in part by the cleavage of lung matrix proteins such as elastin. Previous studies showed that NE can also cleave IgG (19) and IgA (20, 21), and it was suggested that this cleavage can occur in vivo, as shown in patients with chronic tracheostomy (21). We also observed that NE (and not PR3) degrades immunoglobulins including IgA, although SIgA appears partly protected from cleavage by its binding to SC, which probably limits the accessibility of cleavage sites for NE. Thus, the major soluble effectors of secretory immunity, SC and pIgA, may be digested by NE and PR3, but this study also highlights the protective role of SC on degradation of SIgA by PMN proteinases.

In addition to the direct effects of serine proteinases on SC and IgA isoforms, the expression of pIgR/SC was evaluated both in cell lysates and on cell surface of cultured bronchial epithelial cells exposed to PMN mediators. In contrast with Calu-3 cells, NE activity decreased significantly in HBEC cultures, in agreement with previous studies showing induction of cell-associated SLPI and elafin release in HBEC exposed to NE (22). The poor NE inhibition by Calu-3 cells, as compared with HBEC, is in contrast with other airway or lung epithelial cell lines such as A549 or NCI-322 (23). However, the cellular pool of pIgR/SC and surface pIgR were decreased in both Calu-3 and HBEC incubated with NE, and to a lesser extent with PR3, suggesting that in contrast with the cleavage of soluble SC, which appeared mainly mediated by PR3, cleavage of cell pIgR and soluble SC produced in epithelial cell cultures could be mostly mediated by NE. However, the apparent difference of sensitivity of purified SC and SC produced in cell culture to PR3 might also be relating at least partly to the lower concentration of PR3 present in the sputum extract as compared with PMN supernatants. Previous studies reported that NE can cleave various membrane receptors, such as complement receptor-1 on PMN (24); CD23 on B cells (25); CD2, CD4, and CD8 on T cells (26); CD14 and tumor necrosis factor (TNF) receptors on monocytes (27) and fibroblasts; as well as ICAM-1 on epithelial cells (28). Although different effects may result from the cleavage of soluble or membrane molecules, we show that pIgR cleavage by NE does affect its role in pIgA transepithelial transport. Moreover, in addition to its anti-infective role (reviewed in Ref. 29), it was recently reported that SC inhibits IL-8–mediated PMN chemotaxis through the formation of inactive covalent complexes with this chemokine (30). Thus, it is suggested that SC degradation by PMN serine proteinases could further favor PMN mucosal infiltration as observed in COPD airways.

In contrast with the effects of purified PMN proteinases, alone or in combination such as in sputum extract, supernatants from activated PMN induced an increased pIgR/SC production by bronchial epithelial cells. The increased transcytosis of pIgA across Calu-3 cells pretreated with supernatant from activated PMN supported this upregulation of pIgR expression. SC upregulation by activated PMN appeared independent of serine proteinase activity, and was even enhanced in the presence of an inhibitor of NE and PR3. This appeared in contrast with NE-induced upregulation of IL-8 or MUC5AC gene transcription in epithelial cells, which depends on active NE (31, 32). The in vivo induction of mucous metaplasia (33) and hypersecretion (34) by NE also required an active catalytic site. Thus, our observations on pIgR/SC suggested another mechanism of PMN-induced epithelial activation. Using synthetic inhibitors of different signaling pathways mediating inflammatory responses, i.e., NF-{kappa}B and MAPK (p38 and ERK), we observed that PMN-induced pIgR/SC upregulation in Calu-3 cells was quite completely abolished by an inhibitor of the NF-{kappa}B pathway, MG132. Interestingly, NF-{kappa}B pathway has been shown to mediate pIgR/SC upregulation in epithelial cells stimulated by proinflammatory mediators such as TNF-{alpha} (35, 36). In addition, we show here that p38 MAPK activation was also required to observe the pIgR/SC increase in epithelial cells cultured with supernatant from activated PMN, in contrast with ERK MAPK. Moreover, supernatants from activated PMN induced the phosphorylation of p38 MAPK and that of I{kappa}B-{alpha}, a crucial step generally required to allow NF-{kappa}B translocation into the cell nucleus. Interestingly, it has been recently shown that cytokine-induced synthesis of elafin (an SLPI-related proteinase inhibitor) in A549 lung epithelial cells is mediated by NF-{kappa}B activation (37). In keratinocytes, induction of elafin by TNF-{alpha} is also dependent on p38 MAPK (38). We previously observed that IL-1ß may upregulate pIgR/SC production by bronchial epithelial cells (not shown). Interestingly, IL-1ß is significantly released by PMN upon stimulation, and could therefore represent one candidate among PMN-derived products able to activate pIgR expression. Thus, activation of NF-{kappa}B and p38 MAPK pathways appears critical to mucosal protective mechanisms elicited at the level of the airway epithelium, including pIgR/SC expression.

In conclusion, this study highlights a dual effect of blood PMN on secretory immunity, and provides new information concerning interactions between PMN and epithelial cells. Thus, the PMN-derived serine proteinases PR3 and NE mediate a proteolytic cleavage of soluble SC and cell-surface pIgR, as well as serum pIgA. On the other hand, activated PMN induce an increased epithelial expression of pIgR/SC through cell activation mediated by NF-{kappa}B– and p38 MAPK-dependent intracellular mechanisms. In normal bronchial epithelial cells, the stimulatory effect of activated PMN on pIgR/SC largely overcomes the PR3- and NE-mediated proteolysis of SC, because SC was strongly increased in culture media despite the presence of active proteinases. In severe COPD, decreased pIgR/SC expression might be related to a PMN serine proteinase effect overcoming epithelial cell activation of pIgR/SC production, due either to differences in the products released after degranulation of PMN in COPD airways (as compared with blood PMN from healthy subjects) or changes in epithelial cell reactivity to PMN mediators. This hypothesis will be further investigated in another study evaluating airway epithelial cells (and blood and/or airway PMN) from patients with COPD.


   Acknowledgments
 
C.P. is currently Aspirant of the Fonds National de la Recherche Scientifique (Belgium, Grant no. 3.4590.99), and Y.O. is supported by an FSR Grant (University of Louvain, Brussels, Belgium). The authors thank Dr. M. Delos, P. Thurion, and P. Eucher (Laboratory of Histopathology and Department of Thoracic Surgery, Cliniques Universitaires de Mont-Godinne, University of Louvain) for providing the bronchial tissues from lung surgical specimens. The authors also thank Professor P. Courtoy (Cell Unit, Institute of Cellular Pathology, University of Louvain) for access to the confocal microscope, and P. Staquet (Laboratory of Haematology, Cliniques Universitaires de Mont-Godinne, University of Louvain) for help in flow cytometric analysis.

Received in original form June 17, 2002

Received in final form October 14, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Underdown, B. J., and J. M. Schiff. 1986. Immunoglobulin A: strategic defense initiative at the mucosal surface. Annu. Rev. Immunol. 4:389–417.[CrossRef][Medline]
  2. Pilette, C., V. Godding, R. Kiss, M. Delos, E. Verbeken, C. Decaestecker, K. De Paepe, J.-P. Vaerman, M. Decramer, and Y. Sibille. 2001. Reduced epithelial expression of secretory component in small airways correlates with airflow obstruction in chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 163:185–194.[Abstract/Free Full Text]
  3. Sibille, Y., and F. X. Marchandise. 1993. Pulmonary immune cells in health and disease: polymorphonuclear neutrophils. Eur. Respir. J. 6:1529–1543.[Abstract]
  4. Janoff, A., B. Sloan, G. Weinbaum, V. Damiano, R. A. Sandhaus, J. Elias, and P. Kimbel. 1980. Experimental emphysema induced with purified human neutrophil elastase: tissue localization of the instilled protease. Am. Rev. Respir. Dis. 115:461–478.
  5. Senior, R. M., H. Tegner, C. Kuhn, K. Ohlsson, B. C. Starcher, and J. A. Pierce. 1977. The induction of pulmonary emphysema with human leukocyte elastase. Am. Rev. Respir. Dis. 116:469–475.[Medline]
  6. Kao, R. C., N. G. Wehner, K. M. Skubitz, B. H. Gray, and J. R. Hoidal. 1988. Proteinase 3. A distinct human polymorphonuclear leukocyte proteinase that produces emphysema in hamsters. J. Clin. Invest. 82:1963–1973.
  7. Carrell, R. W., J. O. Jeppsson, C. B. Laurell, S. O. Brennan, M. C. Owen, L. Vaughan, and D. R. Boswell. 1982. Structure and variation of human alpha 1-antitrypsin. Nature 298:329–334.[CrossRef][Medline]
  8. Hiemstra, P. S., S. van Wetering, and J. Stolk. 1998. Neutrophil serine proteinases and defensins in chronic obstructive pulmonary disease: effects on pulmonary epithelium. Eur. Respir. J. 12:1200–1208.[Abstract]
  9. Altamirano, G. A., C. Barranco-Acosta, E. van Roost, and J.-P. Vaerman. 1980. Isolation and characterization of secretory IgA (sIgA) and free secretory component (FSC) from rat bile. Mol. Immunol. 17:1525–1537.[CrossRef][Medline]
  10. Delacroix, D. L., and J.-P. Vaerman. 1981. A solid phase, direct competition, radioimmunoassay for quantitation of secretory IgA in human serum. J. Immunol. Methods 40:345–358.[CrossRef][Medline]
  11. Vaerman, J.-P., A. Langendries, and C. Vander Maelen. 1995. Homogenous IgA monomers, dimers, trimers and tetramers from the same IgA myeloma serum. Immunol. Invest. 24:631–641.[Medline]
  12. Godding, V., Y. Sibille, P. P. Massion, M. Delos, C. Sibille, P. Thurion, D. Giffroy, A. Langendries, and J.-P. Vaerman. 1998. Secretory component production by human bronchial epithelial cells is upregulated by interferon gamma. Eur. Respir. J. 11:1043–1052.[Abstract]
  13. 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.[Abstract/Free Full Text]
  14. Nakajima, K., J. C. Powers, B. M. Ashe, and M. Zimmerman. 1979. Mapping the extended substrate binding site of cathepsin G and human leukocyte elastase: studies with peptide substrates related to the alpha 1-protease inhibitor reactive site. J. Biol. Chem. 254:4027–4032.[Free Full Text]
  15. Barrett, A.J. 1981. Cathepsin G. Methods Enzymol. 80:561–565.
  16. Cardone, M. H., B. L. Smith, W. Song, D. Mochly-Rosen, and K. E. Mostov. 1994. Phorbol myristate acetate-mediated stimulation of transcytosis and apical recycling in MDCK cells. J. Cell Biol. 124:717–727.[Abstract/Free Full Text]
  17. Fujita, J., N. L. Nelson, D. M. Daughton, C. A. Dobry, J. R. Spurzem, S. Irino, and S. I. Rennard. 1990. Evaluation of elastase and antielastase balance in patients with chronic bronchitis and pulmonary emphysema. Am. Rev. Respir. Dis. 142:57–62.[Medline]
  18. Owen, C. A., and E. J. Campbell. 1999. The cell biology of leukocyte-mediated proteolysis. J. Leukoc. Biol. 65:137–150.[Abstract]
  19. Folds, J. D., H. Prince, and J. K. Spitznagel. 1978. Limited cleavage of human immunoglobulins by elastase of human neutrophil polymorphonuclear granulocytes: possible modulator of immune complex disease. Lab. Invest. 39:313–321.[Medline]
  20. Tax, A., and L. Korngold. 1971. Comparison of the effect of elastase on human secretory IgA and serum IgA. J. Immunol. 107:1189–1191.[Abstract/Free Full Text]
  21. Niederman, M. S., W. W. Merrill, L. M. Polomski, H. Y. Reynolds, and J. B. Gee. 1986. Influence of sputum IgA and elastase on tracheal cell bacterial adherence. Am. Rev. Respir. Dis. 133:255–260.[Medline]
  22. van Wetering, S., A. C. van der Linden, M. A. van Sterkenburg, K. F. Rabe, J. Schalkwijk, and P. S. Hiemstra. 2000. Regulation of secretory leukocyte proteinase inhibitor (SLPI) production by human bronchial epithelial cells: increase of cell-associated SLPI by neutrophil elastase. J. Investig. Med. 48:359–366.[Medline]
  23. Sallenave, J. M., J. Shulmann, J. Crossley, M. Jordana, and J. Gauldie. 1994. Regulation of secretory leukocyte proteinase inhibitor (SLPI) and elastase-specific inhibitor (ESI/elafin) in human airway epithelial cells by cytokines and neutrophilic enzymes. Am. J. Respir. Cell Mol. Biol. 11:733–741.[Abstract]
  24. Tosi, M. F., H. Zakem, and M. Berger. 1990. Neutrophil elastase cleaves C3bi on opsonized pseudomonas as well as CR1 on neutrophils to create a functionally important opsonin receptor mismatch. J. Clin. Invest. 86:300–308.
  25. Brignone, C., O. Munoz, M. Batoz, A. Rouquette-Jazdanian, and J. L. Cousin. 2001. Proteases produced by activated neutrophils are able to release soluble CD23 fragments endowed with proinflammatory effects. FASEB J. 15:2027–2029.[Abstract/Free Full Text]
  26. Doring, G., F. Frank, C. Boudier, S. Herbert, B. Fleischer, and G. Bellon. 1995. Cleavage of lymphocyte surface antigens CD2, CD4, and CD8 by polymorphonuclear leukocyte elastase and cathepsin G in patients with cystic fibrosis. J. Immunol. 154:4842–4850.[Abstract]
  27. 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]
  28. 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]
  29. Pilette, C., Y. Ouadrhiri, V. Godding, J.-P. Vaerman, and Y. Sibille. 2001. Lung mucosal immunity: immunoglobulin-A revisited. Eur. Respir. J. 18:571–588.[Abstract/Free Full Text]
  30. Marshall, L. J., B. Perks, T. Ferkol, and J. K. Shute. 2001. IL-8 released constitutively by primary bronchial epithelial cells in culture forms an inactive complex with secretory component. J. Immunol. 167:2816–2823.[Abstract/Free Full Text]
  31. Nakamura, H., K. Yoshimura, N. G. McElvaney, and R. G. Crystal. 1992. Neutrophil elastase in respiratory epithelial lining fluid of individuals with cystic fibrosis induces interleukin-8 gene expression in a human bronchial epithelial cell line. J. Clin. Invest. 89:1478–1484.
  32. Voynow, J. A., L. R. Young, Y. Wang, T. Horger, M. C. Rose, and B. M. Fischer. 1999. Neutrophil elastase increases MUC5AC mRNA and protein expression in respiratory epithelial cells. Am. J. Physiol. 276:L835–L843.[Abstract/Free Full Text]
  33. Breuer, R., T. G. Christensen, E. C. Lucey, P. J. Stone, and G. L. Snider. 1987. An ultrastructural morphometric analysis of elastase-treated hamster bronchi shows discharge followed by progressive accumulation of secretory granules. Am. Rev. Respir. Dis. 136:698–703.[Medline]
  34. Takeyama, K., C. Agusti, I. Ueki, J. Lausier, L. O. Cardell, and J. A. Nadel. 1998. Neutrophil-dependent goblet cell degranulation: role of membrane-bound elastase and adhesion molecules. Am. J. Physiol. 275:L294–L302.[Abstract/Free Full Text]
  35. Takenouchi-Ohkubo, N., T. Takahashi, M. Tsuchiya, J. Mestecky, Z. Moldoveanu, and I. Moro. 2000. Role of nuclear factor-kappaB in the expression by tumor necrosis factor-alpha of the human polymeric immunoglobulin receptor (plgR) gene. Immunogenetics 51:289–295.[CrossRef][Medline]
  36. Schjerven, H., P. Brandtzaeg, and F. E. Johansen. 2001. A novel NF-kappa B/Rel site in intron 1 cooperates with proximal promoter elements to mediate TNF-alpha-induced transcription of the human polymeric Ig receptor. J. Immunol. 167:6412–6420.[Abstract/Free Full Text]
  37. Bingle, L., T. D. Tetley, and C. D. Bingle. 2001. Cytokine-mediated induction of the human elafin gene in pulmonary epithelial cells is regulated by nuclear factor-kappaB. Am. J. Respir. Cell Mol. Biol. 25:84–91.[Abstract/Free Full Text]
  38. Pfundt, R., M. Wingens, M. Bergers, M. Zweers, M. Frenken, and J. Schalkwijk. 2000. TNF-alpha and serum induce SKALP/elafin gene expression in human keratinocytes by a p38 MAP kinase-dependent pathway. Arch. Dermatol. Res. 292:180–187.[CrossRef][Medline]



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Low Levels of Tissue Inhibitors of Metalloproteinases With a High Matrix Metalloproteinase-9/Tissue Inhibitor of Metalloproteinase-1 Ratio Are Present in Tracheal Aspirate Fluids of Infants Who Develop Chronic Lung Disease
Pediatrics, June 1, 2004; 113(6): 1709 - 1714.
[Abstract] [Full Text] [PDF]

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 Proc Am Thorac SocHome page
C. Pilette, S. R. Durham, J.-P. Vaerman, and Y. Sibille
Mucosal Immunity in Asthma and Chronic Obstructive Pulmonary Disease: A Role for Immunoglobulin A?
Proceedings of the ATS, April 1, 2004; 1(2): 125 - 135.
[Abstract] [Full Text] [PDF]

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 Am. J. Respir. Crit. Care Med.Home page
L. J. Marshall, B. Perks, K. Bodey, R. Suri, A. Bush, and J. K. Shute
Free Secretory Component from Cystic Fibrosis Sputa Displays the Cystic Fibrosis Glycosylation Phenotype
Am. J. Respir. Crit. Care Med., February 1, 2004; 169(3): 399 - 406.
[Abstract] [Full Text] [PDF]