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Published ahead of print on September 7, 2006, doi:10.1164/rccm.200605-632OC
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American Journal of Respiratory and Critical Care Medicine Vol 174. pp. 1189-1198, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200605-632OC

Original Article

Inflammatory Lung Secretions Inhibit Dendritic Cell Maturation and Function via Neutrophil Elastase

Ali Roghanian, Ellen M. Drost, William MacNee, Sarah E. M. Howie and Jean-Michel Sallenave

Medical Research Council (MRC) Centre for Inflammation Research (CIR), The Queen's Medical Research Institute, Edinburgh University Medical School, Edinburgh, Scotland, United Kingdom

Correspondence and requests for reprints should be addressed to Jean-Michel Sallenave, Ph.D., Université Denis Diderot-Paris 7/Institut Pasteur, Unité de Défense Innée et Inflammation, INSERM E336, Batiment Metchnikoff, Institut Pasteur 25, rue du Dr Roux, 75724 Paris Cedex, France. E-mail: jms{at}pasteur.fr


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Continuous episodes of infection are a feature of lung diseases such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). Lung antigen-presenting dendritic cells (DCs) sample inhaled antigen to initiate immune responses. Therefore, we hypothesized that inflammatory mediators, such as neutrophil elastase (NE) released into the lung, may be able to modulate their activity.

Objective: To determine whether sputum (from patients with COPD and those with CF) or NE can alter DC phenotype and function.

Method: NE and sputum samples were incubated with immature or mature murine DCs (mDCs). DC phenotype and function were studied by fluorescence-activated cell sorter and Western Blot analysis, assessing their expression of costimulatory molecules and their ability to induce T cell proliferation.

Results: COPD/CF sputum samples and human NE downregulated the expression of CD40, CD80, and CD86 (but not major histocompatibility complex II) on DCs and inhibited LPS-induced DC maturation. This effect was partially (sputa) to significantly (NE) reversed by addition of recombinant secretory leukocyte protease inhibitor. Western Blot analysis showed that purified NE degraded CD86 in mDC lysates in a time- and dose-dependent fashion, and caused shedding of CD86 into the supernatants of mDC cultures. NE treatment also inhibited the antigen-presenting ability of mDCs, as measured by their ability to induce ovalbumin-specific D011.10-transgenic T-cell proliferation.

Conclusions: Our data indicate that NE in lung inflammatory secretions of patients with COPD/CF may disable DCs and prevent them from mounting an adequate immune response. This may have implications for the infection-driven generation of disease exacerbations in these two pathologies.

Key Words: chronic obstructive pulmonary diseases • costimulatory molecules • cystic fibrosis • dendritic cells • neutrophil elastase



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Very little is known about the inflammatory factors present in the lung secretions of COPD and CF patients responsible for the modulation of dendritic cell activity.

What This Study Adds to the Field
This study provides the novel observation that COPD and CF sputum samples can adversely affect the phenotype and function of dendritic cells, through the action of neutrophil elastase.

 
Dendritic cells (DCs) are potent antigen-presenting cells. DCs are distributed throughout the lungs in the conducting airways of the tracheobronchial tree and in the parenchymal lung, and play a pivotal role in controlling the immune response to inhaled antigens (1). Respiratory tract DCs are known to be relatively immature DCs (iDCs), which can be matured ex vivo by granulocyte-macrophage colony–stimulating factor (GM-CSF) (2). A variety of factors can induce maturation after antigen uptake and processing within DCs, such as bacterial LPS, viral products, and inflammatory cytokines. The process of DC maturation results in an increased surface expression of major histocompatibility molecules, T-cell costimulatory molecules (CSMs), including CD40, CD80 (B7-1), and CD86 (B7-2), adhesion molecules, and chemokine receptors (e.g., CCR7), as well as an increase in secretion of chemokines and cytokines (3–6). Although the airways are usually sterile in healthy individuals (7), continuous infectious episodes are a feature of lung diseases, such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). The latter is associated with chronic infections with Staphylococcus aureus, Pseudomonas aeruginosa, and often, in the worst cases, with Burkholderia cepacia. Although cigarette smoking is the accepted etiology for COPD in the huge majority of cases, recent evidence suggests that the respiratory tract of patients with COPD is often colonized in 50 to 100% of cases (7), and that infections with viruses and bacterial pathogens, such as nontypeable Haemophilus influenzae, Streptococcus pneumoniae, and Branhamella catarrhalis (8), are instrumental in the generation of disease exacerbations. Both COPD and CF are characterized by intense neutrophilic inflammation and the release of high levels of cytotoxic molecules, such as neutrophil elastase (NE), that overwhelm and inactivate antiprotease and innate immune defenses (9–13). We and others have shown that NE is able to cleave important receptors from the surface of macrophages, which could potentially impair the clearing of apoptotic cells and perpetuate a proinflammatory phenotype (14–17). We therefore speculated that lung secretions from patients with COPD and CF and/or NE itself might also influence the behavior or phenotype of lung DCs in such a way as to inhibit their ability to present antigens and, thus, initiate protective immunity to pathogens. Here we present data that demonstrate that purified NE and NE in sputum samples from patients with COPD and those with CF can inhibit maturation of DCs and downregulates the ability of mature DCs (mDCs) to present antigen to T cells. The latter effect is at least in part due to NE cleavage of the CSM, CD86, from the surface of mDCs. Thus, high levels of NE in lung secretions from patients with COPD and those with CF might account, at least in part, for the continuous episodes of infection seen in these conditions. Some of the results of these studies have been previously reported in the form of an abstract (18).


   METHODS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sputum Samples from Patients with COPD and Those with Cystic Fibrosis
COPD sputum samples were obtained from patients attending a COPD clinic in the Edinburgh Royal Infirmary. A total of 27 patients with COPD (11 females/16 males, listed in Table 1) with an average age of 69 ± 8.29 (SD; SEM = 1.6) yr were studied. FEV1% predicted (%pred) was 39 ± 12.9 (SEM = 2.6) and FEV1/FVC %pred was 47 ± 12.9 (SEM = 2.6). Pack-year history was 55 ± 17 (SEM = 3.4). All the sputum samples were processed within 2 h of production, as described by Popov and colleagues (19). CF sputum samples were collected from a total of 18 patients with CF (9 female/9 male), with an average age of 31.5 ± 12.44 (SD) yr, attending a CF clinic at the Western General Hospital (Edinburgh). Subsequently, the samples were resuspended in Sputolysin reagent (Calbiochem, San Diego, CA) at a ratio of 1:1 and were treated as for COPD samples. All subjects were informed of the nature and purpose of the study and gave their consent. The study had the approval of the Lothian Ethics Committee.


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  		TABLE 1. CHARACTERISTICS OF PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE*

 
Mice
All animals were kept under standard conditions, with approval from the local ethics committee and the UK Home Office, in the animal facility of the College of Medicine and Veterinary Medicine, University of Edinburgh. DO11.10 ovalbumin (OVA) CD4+ T-cell receptor–transgenic mice on a BALB/c background were bred in-house, and wild-type BALB/c mice were obtained from Harlan UK (Oxon, UK).

NE Assay
The concentration of NE in COPD and CF sputum samples was determined as described previously (20). Briefly, sputum samples were diluted in NE reagent buffer (50 mM Tris, 0.5 M NaCl, 0.1% Triton X-100, pH 8.0) in a 96-well microplate, and NE activity was determined by adding the NE substrate MeOsucc-Ala-Ala-Pro-Val-p-nitroanilide (Sigma, Poole, UK) at a final concentration of 0.2 mg/ml and measuring the optical density of the released product at 450 nm on a spectrophotometer (Dynax Technologies, West Sussex, UK). Serial dilutions (0.0078–0.5000 µM) of purified human NE (Elastin Products, Owensville, MO) were performed and were used as standards in the assay.

DC Culture and Maturation
All culture reagents were obtained from Sigma, unless otherwise stated. DCs were obtained as described previously (21). Briefly, femurs from 6- to 8-wk-old female BALB/c mice were removed, dipped in 70% ethanol for 1–2 min, and then placed in DC complete medium (RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 10% heat-inactivated fetal calf serum [FCS]). DC complete medium also contained approximately 5% conditioned supernatant from a hybridoma (gift from Prof. D. Gray, Edinburgh, UK) expressing recombinant (r)GM-CSF, resulting in a final GM-CSF concentration of 20–30 ng/ml. After flushing of the femur's content, 10 ml of a single-cell suspension (2 x 105 cell/ml) was plated in non–tissue culture–grade Petri dishes. On Day 3, an additional 10 ml of fresh medium was added to the cultures. On Days 6 and 8, half of the medium was removed, and 10 ml fresh complete medium was added to the Petri dishes. On Day 10 of culture, nonadherent cells were removed, leaving strongly adherent macrophages on the plate. The nonadherent cells were centrifuged (300 x g, 5 min), resuspended in DC complete medium, and counted before use. DC cell purity, as assessed by fluorescence-activated cell sorter (FACS) analysis (CD11c cell surface expression) was typically 80 to 95%. Unless otherwise stated, mDCs were obtained after a further incubation with 100 ng/ml LPS (Escherichia coli serotype 026:B6; Sigma-Aldrich, St. Louis, MO) in DC complete medium for 24 h. For some experiments, maturation was performed in serum-free medium for a period of 4 h at 37°C, with LPS at a concentration of 2 µg/ml.

FACS Analysis of Cell Surface Markers and Viability of DCs
Flow cytometric analysis was performed using a FACSCalibur (Becton Dickinson, San Jose, CA), gating on a live cell gate based on forward and side scatter and propidium iodide (Invitrogen Ltd., Paisley, UK) staining. CellQuest software was used for data analysis (BD Biosciences, San Jose, CA). The following antibodies (Abs) were used (all from BD Biosciences): FITC-I-Ad/I-Ed (major histocompatibility complex [MHC] II), PE-CD40, PE-CD86, and allophycocyanin-CD11c. The CD surface expression levels of the cells were assessed and quantified in arbitrary units as mean fluorescence intensity (MFI). The MFI of a population of cells labeled with a fluorochrome conjugated to a ligand or monoclonal Ab (mAb) is linearly related to the mean number of receptors/cell or mean number of mAb binding sites/cell.

Treatment of DCs with either NE or Sputum Samples from Patients with COPD/CF
LPS-mDCs were washed with phosphate-buffered saline (PBS) and checked for viability by Trypan blue staining. Cells were resuspended in 300 µl PBS containing 0.1% (wt/vol) bovine serum albumin (BSA; Sigma), with active NE (0.5–3.0 µM) or with sputum, and incubated at 37°C for 90 min on a shaker, as described previously (22). The percentage reduction in CSM expression was determined by flow cytometry and calculated as:

100 – [MFI NE/sputum-treated cells)/(MFI PBS-treated cells) x 100]

and plotted against the concentration of active NE in sputa. When NE inhibition was assessed, purified active NE (2 µM) or a pool of COPD or CF sputa (containing 0.2 µM and 3.39 µM active NE, respectively) were preincubated at 37°C for 90 min on a shaker, either with excess concentrations (8 µM) of recombinant murine secretory leukocyte protease inhibitor (SLPI, a kind gift from Dr. C. Wright, Amgen, Thousand Oaks, CA), or with a 1:25 dilution of an EDTA-containing broad-spectrum protease inhibitor (PI) cocktail, inhibiting serine, cysteine, metalloproteinases, as well as calpains (Roche Diagnostics GmbH, Mannheim, Germany).

In some experiments, mDCs (106/ml) were incubated at 37°C in 24-well Costar plates in serum-free medium with or without 2 µM purified active NE for 90 min. FCS, which is a rich source of antiproteases, was then added (final concentration, 15%) to inhibit NE activity. Cells were taken at various time points and tested for expression of surface markers by FACS.

Murine CD86 Western Blot Analysis
mDC cell culture supernatants.
After NE treatment, mDC culture supernatants were concentrated 10 times (with cold methanol). Pellets were resuspended in reducing sample buffer (NuPAGE; Invitrogen Ltd.) and were immediately heat-denatured at 70°C for 10 min before electrophoresis on a NuPAGE 4–12% Bis-Tris polyacrylamide gel at 200 V (constant voltage) for 35 min.

mDC lysates.
mDCs were washed with PBS and lysed (106 cells/ml) in 1:1 ratio of PBS:RIPA buffer [150 mM NaCl, 50 mM Tris-HCl, 0.5% sodium deoxycholate (DOC), 0.1% SDS, and 1% Nonidet P-40]. The cell lysate was heated at 95°C for 10 min and centrifuged at 13,000 rpm at room temperature for 5 min. Supernatants were concentrated by methanol precipitation, as described previously here; pellets were resuspended in PBS and frozen at –20°C until further use. For CD86 digestion assays, the mDC lysate was incubated with various concentrations of purified NE on a shaker at 37°C for various times. For NE inhibition studies, SLPI was preincubated with NE at 37°C for 30 min. mDC lysates were added to electrophoresis reducing sample buffer, and SDS-PAGE gels were run as described previously here.

Western blotting.
Transfer to Hybond enhanced chemiluminescence (ECL) nitrocellulose membranes (in an X Cell II Blot module; Invitrogen Ltd.) was performed according to the manufacturer's instructions. Membranes were then blocked in Tris-buffered saline (TBS) containing 0.05% Tween-20 (TBS-T) and 5% dry skimmed milk for 2 h. After blocking, membranes were probed overnight at 4°C with 0.1 µg/ml goat polyclonal anti-CD86 (B7-2) (R&D Systems, Oxon, UK) in TBS-T. Membranes were washed three times for 5 min with TBS-T and incubated with rabbit anti-goat IgG horseradish peroxidase–conjugated secondary Ab (1:50,000; Dako, Cambridgeshire, UK) for 1 h at room temperature. Membranes were incubated with Hybond ECL Plus reagent and exposed to Hyperfilm ECL (Amersham Biosciences, Buckinghamshire, UK).

Lymphocyte Proliferation Assays
Single-cell splenocyte suspensions were made from spleens of 6- to 8-wk-old female OVA CD4 T-cell receptor–transgenic DO11.10 mice (BALB/c background, two to four animals per experiment) by passing the spleen through a 40-µm pore-size cell strainer (BD Biosciences). Debris and red cells were removed by density gradient sedimentation through Lympholyte-M (Cedarlane Laboratories, Hornby, ON, Canada) according to the manufacturer's instructions. Day 10 wild-type BALB/c bone marrow-derived DCs were matured with 100 ng/ml LPS overnight, washed with PBS, and incubated with or without 3 µM NE for 90 min, as described previously here. The treated mDCs were then pulsed with 0.1 µM OVA peptide (OVA323–339; Peptides International, Louisville, KY) for 90 min at 37°C and washed thoroughly. A total of 104 OVA-pulsed mDCs were added to 105 DO11.10 lymphocytes in 96-well tissue culture plates in a final volume of 200 µl and incubated in a humidified 5% CO2 incubator. Activation of the transgenic DO11.10 CD4 T cells was assessed after 48 h by removing 100 µl of the supernatants (for cytokine analysis), and proliferation was determined by adding Alamarblue (Serotec, Oxford, UK) to the cells, to yield a final concentration of 20% in the incubation mixture. Cell proliferation was measured by fluorimetry (545-nm excitation and 590-nm emission wavelengths) using a Fluoroskan Ascent FL plate reader (MTX Lab Systems, Vienna, VA).

Cytokine Analysis
Cytokine secretion was assessed either by using the mouse T helper (Th) cell type 1/Th2 Cytometric Bead Array (CBA) kit (BD Biosciences) or by use of ELISA kits (R&D Systems) in accordance with the manufacturers' instructions. The detection limit for CBA kit cytokines was 20 pg/ml, and 15 pg/ml for interleukin (IL)-12p40 and tumor necrosis factor (TNF)-{alpha} in the ELISA kits.

Statistical Analysis
Statistical significance was analyzed by Student's unpaired t test. Statistical significance was assigned to data returning a p value less than 0.05.


   RESULTS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Reduction of DC Expression of CSMs by COPD, CF Sputum Samples, and Purified NE
When iDCs were matured with LPS, the levels of the CSMs CD40, CD80, and CD86 typically increased three- to fourfold (see below). In preliminary experiments, to determine the effects of inflamed lung secretions on CSM expression, mDCs were incubated with pooled samples of COPD and CF sputa. Treated mDCs were shown to express significantly lower levels of CD80 (~ 60 and 20%, respectively; p < 0.0001) and CD86 (~ 40 and 15%, respectively; p < 0.0001), as assessed by mean fluorescence intensity. A comprehensive study was then performed with the latter marker of DC activation (CD86). Figure 1A shows that CD86 levels were lower after treatment with CF sputum samples (mean NE concentration, 4.7 µM ± 0.73; n = 18; p < 0.0001) than after treatment with COPD sputum samples (mean NE concentration, 0.11 µM ± 0.05; n = 7; p = 0.0015), and for each type of sputum, there was a strong, statistically significant correlation between the NE concentration in individual sputum samples and reduction of CD86 levels (CF: r = 0.94; COPD: r = 0.76). Interestingly, MHCII levels were unchanged, showing that the sputa did not affect all DC surface receptors equally and, importantly, the reduction in expression of CSMs in mDCs was not due to cell apoptosis and/or necrosis, as measured by annexin V and propidium iodide staining (data not shown).


Figure 1
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  		Figure 1. Reduction of CD86 by chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) sputa (A) is inhibited by secretory leukocyte protease inhibitor (SLPI) and by protease inhibitor (PI) cocktail (B and C). (A) Mature DCs (mDCs; matured with LPS in serum-containing medium for 24 h) were washed to remove serum and incubated with individual sputa from patients with COPD (open circles; n = 7; Patients 1, 5, 6, 8, 12, 13, and 14) and from those with CF (filled diamonds; n = 18; Patients 1–18), or an equivalent volume of phosphate-buffered saline (PBS) for 90 min at 37°C. R = equation of straight line. (B and C) Pooled COPD (n = 18) and CF (n = 9) sputa (containing 0.2 µM and 3.39 µM active neutrophil elastase [NE], respectively) were preincubated with either SLPI (8 µM) or a cocktail of PIs before incubation with mDCs and CD86 expression was assessed. (B) Representative flow plots; (C) data from three experiments. Values represent means ± SD; *significant difference (p < 0.05) compared with DCs incubated with sputa alone (not pretreated with SLPI or PI).

 
The finding that the reduction in CD86 expression correlated with NE concentration in sputa prompted us to examine whether PIs of NE were able to reverse these effects. Recombinant murine SLPI (8 µM), a well characterized inhibitor of NE (23), and a cocktail of PIs were incubated with pooled COPD (18 samples) and CF (9 samples) sputa (containing 0.2 µM and 3.39 µM active NE, respectively) to inactivate NE before addition to mDCs. Indeed, SLPI and PI significantly rescued CD86 expression in DCs treated with COPD and CF sputa (Figures 1B and 1C). Because the level of inhibition is similar with SLPI and PI, and because NE levels are by far the highest compared to other enzymes present in sputum secretions, these data strongly suggest (but do not irrefutably prove) that both SLPI and PI act as "NE inhibitors" in these experiments.

These results prompted us to determine whether NE in isolation was also able to affect the levels of mDC CSM expression. Indeed, Figure 2 shows that purified NE was also able to down-regulate CD80, CD86, and CD40 expression (whereas MHCII levels were unchanged). This effect was shown to be dose-dependent (Figure 2B). As for the sputum sample experiments, we showed that preincubating NE and PIs before stimulation of mDCs rescued, to a large degree, CD86 expression (Figure 3). Also, as mentioned for sputum samples, there was no increase in annexin V/propidium iodide staining after NE treatment (data not shown).


Figure 2
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  		Figure 2. Reduction of CD40, CD80, and CD86 costimulatory molecules, but not major histocompatibility complex (MHC) II by purified NE. (A) mDCs were washed, resuspended in PBS containing 0.1% bovine serum albumin (BSA), and exposed to NE (2 µM) for 90 min at 37°C. NE-treated mDCs were then washed, stained with antibodies (Abs) to CD40, CD80, CD86, and MHCII, and analyzed by flow cytometry (gray line, mDCs; dark line, NE-treated mDCs; filled line, isotype control cells). (B) mDCs were exposed to a range of NE concentrations (0.5–3.0 µM), and expression of CD40, CD80, and CD86 was analyzed. The percentage reduction was calculated as described in METHODS. Values were pooled from three to six independent experiments and represent means ± SD compared to untreated control cells.

 

Figure 3
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  		Figure 3. The reduction of mDC CD86 by purified NE is prevented by SLPI and PI. Purified NE (2 µM) was preincubated with either SLPI (8 µM) or a cocktail of PI before incubation with mDCs for 90 min at 37°C. (A) Representative flow plots; (B) pooled data from three experiments. Values represent means ± SD; *significant difference (p < 0.001) compared to NE-treated mDCs.

 
Enzymatic Cleavage of CD86 by NE
The findings that NE and sputa were able to affect the expression of CSMs and that PIs were able to alleviate this effect prompted us to determine whether NE enzymatic activity was responsible for their shedding. Western blot analysis for murine CD86 (Figure 4) showed that mDC lysate solutions contained a protein reacting with the polyclonal Ab to murine CD86, and had a molecular weight consistent with that of the CD86 protein (~ 34 kD before glycosylation and 65–100 kD after glycosylation) (24, 25). Indeed, the band around 38 kD likely represents the nonglycosylated native protein, whereas proteins migrating between 38 and 62 kD probably represent glycosylation intermediates (Figure 4A, lane 1).


Figure 4
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  		Figure 4. NE cleaves CD86 in lysates of and supernatants from mDCs. (A) mDC cell lysate was incubated with 2 µM NE at 37°C for 5–40 min. Samples were concentrated 10x and run (10 µl) on a NuPAGE 4–12% Bis-Tris polyacrylamide gel, transferred onto a nitrocellulose membrane, and probed with anti-mCD86 polyclonal Ab (as described in METHODS). Lane 1 represents untreated mDC lysates showing CD86 staining; lanes 2–6 indicate that the majority of the protein is degraded within 5 min of NE treatment (*nonspecific labeling). (B) mDC cell lysate (20 µl) was incubated with 0.5–3.0 µM NE, as described previously here, for 10 min at 37°C. Lane 1 represents untreated mDC lysates showing CD86 staining; lanes 2–5 show a dose-dependent degradation of CD86 by NE. (C) NE (2 µM) was preincubated with SLPI (8 µM) for 30 min before treatment of mDCs for 20 min. Lane 1 represents untreated mDC lysates showing CD86 staining; lane 2 is mDC lysate treated with SLPI-inhibited NE; and lane 3 represents mDC lysate treated with NE only. (D) mDCs were resuspended in PBS containing 0.1% BSA and exposed to 2 µM NE or PBS for 90 min at 37°C. Supernatants were concentrated 10x and lysates were prepared. Lanes 1 and 2 are samples of the same mDC lysate loaded neat (lane 1) or at 50% in PBS (lane 2), showing strong staining with the anti-CD86 Ab. Lane 3 was concentrated 10x supernatant from untreated mDCs showing no evidence of CD86. Lane 4 represents supernatant from NE-treated mDCs showing staining with the anti-CD86 Ab.

 
NE treatment of mDC lysates induced a loss of CD86 immunoreactivity in a time- (Figure 4A) and dose- (Figure 4B) dependent manner. In addition, this loss was largely prevented by adding the NE inhibitor, SLPI (Figure 4C). Because NE was able to digest CD86 in mDC lysates, we also studied whether NE could cleave CD86 from mDC cell surface, as suggested by the FACS experiments. Figure 4D shows that mDCs incubated with NE released few proteins in the supernatant, the main protein having a similar molecular weight to that of the one found in lysates (60 kD), suggesting that this is indeed a product of CD86 cleavage by NE. The slight difference between the lysate and the supernatant-derived molecule (Figure 4D, lanes 1–2 and 4, respectively) likely reflects the presence of the intracellular portion (7 kD) in the lysate-derived molecule absent in the species cleaved from the cell surface.

Kinetics of CD40, CD80, and CD86 Re-expression after NE Treatment of mDCs
To determine whether NE treatments induced a durable change in the mDC phenotype, mDCs were exposed for 90 min to 2 µM purified NE. The cells were then washed and resuspended in medium containing 15% FCS. Figure 5 shows that expression of CD80 and CD86 on mDC increased gradually over the 8-h recovery period, although not to pretreatment levels. In contrast, CD40 expression did not recover at all during this period.


Figure 5
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  		Figure 5. Re-expression of CD40, CD80, and CD86 after NE treatment of mDCs. mDCs were plated in serum-free medium and treated with NE (2 µM) for 90 min at 37°C. FCS (15%) was then added to the wells (time 0) to inhibit NE, and re-expression of CD40 (hatched bars), CD80 (open bars), and CD86 (closed bars) analyzed every 2 h up to 8 h after inhibition. Expression on immature DCs (iDCs) is given for comparison. Values represent means ± SD of three independent experiments, each performed in triplicate (*p < 0.01 and **p < 0.001, significant differences compared to corresponding control mDCs).

 
NE Interferes with Normal LPS-induced Maturation of iDCs
Because iDCs are exposed to bacterial or host-derived maturation signals in the context of high NE concentration in inflammatory conditions, such as COPD and CF, we exposed iDCs to LPS (2 µg/ml) and NE (2 µM) simultaneously for 4 h in serum-free conditions. Figure 6A shows that NE treatment of iDCs resulted in lower expression of CSMs, indicating that iDCs, as well as mDCs (as shown in Figures 1–3), are also susceptible to NE alone. In contrast, LPS- and LPS/NE-stimulated iDCs both significantly up-regulated cell surface MHCII levels (p = 0.003 and p = 0.01, respectively). iDCs exposed to NE alone also had slightly increased levels of MHCII, but this was not statistically significant.


Figure 6
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  		Figure 6. iDCs fail to mature normally when simultaneously stimulated with LPS and NE. iDCs were plated in serum-free medium and treated with either PBS (control), LPS (2 µg/ml) alone, NE (2 µM) alone, or LPS and NE together, for 4 h at 37°C. After treatment, supernatants were collected and cells were washed, stained, and fixed for fluorescence-activated cell sorter (FACS) analysis. (A) Flow cytometry data were analyzed and expressed as percentage change in mean fluorescence intensity (MFI) relative to iDC + PBS controls (mean ± SD; n = 3; open bars, iDC; gray bars, iDC + LPS; closed bars, iDC + LPS + NE; hatched bars, iDC + NE). (B) Supernatants were analyzed for proinflammatory cytokines IL-12p40 (closed bars) and tumor necrosis factor-{alpha} (gray bars) by ELISA. Values represent means ± SD of three independent experiments performed in triplicate wells (*p < 0.01 compared with corresponding controls; ND = nondetected).

 
In addition, and most significantly, these results show that NE interferes with normal iDC maturation upon stimulation with LPS by inhibiting cell surface expression of CSMs (Figure 6A) and reducing levels of IL-12p40 and TNF-{alpha} cytokines in the supernatant (Figure 6B).

NE Impairs the Antigen-presenting Function of DCs
To assess whether NE impaired the antigen-presenting function of DCs, control or NE-treated mDCs were pulsed with OVA peptide and incubated with D011.10-derived transgenic spleen lymphocytes. Figure 7 shows that untreated antigen-pulsed mDCs induced proliferation, whereas NE-treated mDCs did not (p = 0.042). In parallel, the cytokine profile of supernatants 48 h after DC:T-cell coculture showed that TNF-{alpha}, IFN-{gamma}, and IL-2 levels were drastically reduced in cultures containing NE-treated mDCs. Whether NE affected type 2 cytokines, such as IL-4 and IL-5, is difficult to ascertain in this protocol, as their levels were always below the detection limit of the assay.


Figure 7
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  		Figure 7. NE-treated mDCs have impaired antigen-presenting ability. mDCs (from BALB/c wild-type mice) were treated with PBS or NE (3 µM), washed, pulsed with ovalbumin (OVA) peptide (0.1 µM) for 90 min at 37°C, and washed three times with PBS. Spleen lymphocytes were purified from two to four D011.10 transgenic mice and incubated with NE-treated, OVA-pulsed mDCs at a ratio of 1:10 (mDC [104/well]:lymphoctye [105/well]) in a 96-well cell culture plate, and incubated at 37°C. Concanavalin A (ConA) was used at a concentration of 2 µg/ml as a positive control. Half of the supernatant (100 µl) was removed and saved for cytokine analysis after 24 h, and Alamarblue solution was added at a 20% final concentration to each well. (A) Lymphocyte proliferation measured after 48 h. Values represent means ± SD of three independent experiments. *Statistical significance (p < 0.05). (B) Cytokine output after the coculture was analyzed using a Th1/Th2 CBA kit (BD Biosciences). A representative experiment is shown. Dark gray bars, mDC + lymphocyte; filled bars, mDC ConA + lymphocyte; open bars, OVA mDC + lymphocyte; light gray bars, OVA mDC + NE + lymphocyte.

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
COPD and CF affect millions of people worldwide. The infectious component of CF has been established for decades (with chronic infection of patients' lungs with pathogens such as P. aeruginosa and B. cepacia) (26), whereas it has only been recently fully recognized that COPD is worsened by lung infections (with, e.g., H. influenzae) (7, 8, 27–32), which lead to debilitating clinical exacerbations. Both conditions are characterized by an activation of the innate immune system, involving mostly neutrophils in CF, and both neutrophils and macrophages in COPD. Because of an excessive and maladaptive activation of neutrophils, very high amounts of NE are released into the lung fluid of patients with COPD and CF (9–13). As the innate and acquired immune systems are linked by the function of DCs, and because of the abundance of NE in the sputa of patients with COPD and those with CF, we hypothesized that lung secretions from these patients might interfere with the initiation of acquired immunity by inactivating or biasing DC function.

We report here the novel findings that NE concentration correlated with the ability of sputa to reduce DC CD86 CSM expression, and that CF sputa were more potent than COPD sputum samples (Figure 1A). This led us to test whether NE alone could influence mDC CSM expression. We found that NE treatment of mDCs did not affect MHCII expression, but significantly reduced CD40, CD80, and CD86 expression (Figure 2). Comparison of Figures 1A and 2B (bottom panel) indicate that, for the same concentration of NE, sputa were slightly more efficient at downregulating CD86 expression than was purified NE, suggesting that other factors may also be operative.

Furthermore, the reduction in CD86 caused by NE was abrogated by the use of both the NE inhibitor, SLPI, and that of a broad-spectrum PI cocktail (Figure 3; 74 and 83% inhibition, respectively). By contrast, SLPI and the PI cocktail were not fully effective at reversing the effect of CF sputum samples on CSM expression (Figure 1C; 36% inhibition in both cases). This may reflect the fact that other proteases (not inhibited by SLPI and the antiproteases present in the PI cocktail) may also be involved in affecting CSM levels in sputum secretions, or that the antiproteases may be less active in the complex mix of inflammatory sputum. Indeed, although present in high amounts in these secretions, it has been shown that endogenous SLPI and {alpha}1-antiprotease are inactivated in these secretions by proteolytic cleavage and oxidation (33–35).

We demonstrated that NE was able to degrade CD86 protein in mDC lysates in a time- and dose-dependent manner (Figures 4A and 4B), and that this was prevented by SLPI (Figure 4C). NE was also able to release the extracellular portion of CD86 in mDC supernatants (Figure 4D). These observations are further strengthened by recent data by Löfdahl and colleagues showing that alveolar macrophages isolated from bronchoalveolar lavage fluid of COPD patients had a lower expression of CD86 molecule compared with "healthy" smokers (36). We then examined whether the effect of NE on CSM expression was short-lived, or whether it may impair the function of mDCs more durably. Figure 5 shows that up to 8 h after NE treatment (a time-frame relevant to DC activation and migration to local lymph nodes) (37), CSM re-expression was only partial; in particular, CD40, an essential molecule for providing an activation/antiapoptotic signal to the cells (38–40), did not show any increased level of expression over the 8-h time period. This suggests that chronic NE overexpression in the lung, as in COPD or CF, may have a durable effect on DC antigen-presenting ability.

In addition to reducing mDC CSM expression, NE also prevented the LPS-induced maturation of iDCs (Figure 6A) and the concomitant release of IL-12p40 and TNF-{alpha} by the activated DCs (Figure 6B). In these experiments, NE could potentially be acting by either reducing the levels of LPS-induced CSMs or by interfering with the LPS signaling pathway (e.g., by cleaving CD14 receptor molecules) (14, 15, 17). DCs simultaneously treated with LPS and NE up-regulated MHCII levels equally, which indicates that LPS was able to signal intracellularly, and that NE is unlikely, in this model, to interfere with the LPS transduction machinery. We prefer the explanation that NE acts by cleaving susceptible CSMs when they arrive at the cell surface. The long-term (4 h) effect of NE on MHCII expression on DCs is interesting: NE alone slightly upregulated MHCII levels on iDCs, but this did not reach significance (Figure 6A), demonstrating that, as shown by us and others previously in other cell types (41–44), NE can signal through receptors at cellular surfaces. Whether this differential effect of NE on iDCs (up-regulating MHCII while down-regulating CD40, CD80, and CD86 levels) is physiologically important is unclear.

Ultimately, it was important to demonstrate whether the NE-differential effect on DCs CSM levels had functional consequences on DC–lymphocyte interactions. Figure 7 shows that, indeed, NE treatment of mDCs followed by OVA pulsing significantly reduced lymphocyte proliferation and TNF-{alpha}, IFN-{gamma}, and IL-2 cytokine output. Whether NE also interferes with type-2 cytokine output (IL-4 and IL-5) was difficult to determine here, as their levels were very low.

In conclusion, our data show that NE and NE-containing secretions from patients with COPD or CF can disable DC function by interfering both with the ability of immature DCs to mature in response to bacterial LPS stimulation, and by reducing the antigen-presenting activity of mDCs. Although the in vivo effects of these changes remain to be investigated, the reduced Th1 cytokine levels induced by NE treatment of DCs may, in part, explain the reported Th2 imbalance in the lung immune response to bacteria in patients with CF (45, 46) and the reported loss of IFN-{gamma}–secreting cells in patients with COPD (47). This local Th2-biased phenotype may be instrumental in the inability of these patients to clear semifacultative intracellular pathogens in the lung, such as P. aeruginosa, B. cepacia, and H. influenzae, as suggested recently by a variety of animal and human studies (48–57). In that context, our strategy of overexpression of the elastase inhibitor, elafin, could be dually advantageous by inhibiting NE and, as shown recently, by providing a Th1-biasing signal in the lungs (58).


   Acknowledgments
 
The authors thank Dr. Donald J. Davison (CIR, Edinburgh) for useful discussions. Extended thanks also go to Dr. Catherine J. Doherty and Prof. John R. W. Govan (both from the Cystic Fibrosis Laboratory, Medical Microbiology, The University of Edinburgh) for providing the CF sputum samples, and to Prof. Christopher Haslett (CIR, Edinburgh) for continuous support.


   FOOTNOTES
 
Supported by the Medical Research Council (Ph.D. Studentship to A.R.) and the Wellcome Trust.

Current address for Ali Roghanian: Division of Immunology, Department of Pathology, University of Cambridge, Cambridge, CB2 1QP, U.K.

Originally Published in Press as DOI: 10.1164/rccm.200605-632OC on September 7, 2006

Conflict of Interest Statement: A.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; E.M.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; W.M. has been reimbursed for travel by GlaxoSmithKline (GSK), Zambon, AstraZeneca (AZ), Boehringer Ingelheim, Pfizer, and Micromet for attending conferences. He has received honoraria from GSK, AZ, Zambon, and Pfizer for participating as a speaker in scientific meetings. He served on advisory boards for Pfizer, Almirall, Amgen, Bayer, and Micromet, and serves on an expert panel for GSK. He serves as a consultant for Pfizer and GSK. Research grants to support work carried out in his laboratory come from Pfizer and Unilever; S.E.M.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.-M.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form May 9, 2006; accepted in final form September 5, 2006


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
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