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Published ahead of print on January 19, 2006, doi:10.1165/rcmb.2005-0405OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 34, pp. 634-642, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0405OC

The Antimicrobial/Elastase Inhibitor Elafin Regulates Lung Dendritic Cells and Adaptive Immunity

Ali Roghanian*, Steven E. Williams*, Tara A. Sheldrake, Tom I. Brown, Karen Oberheim, Zhou Xing, Sarah E. M. Howie and Jean-Michel Sallenave

MRC Centre for Inflammation Research, The Queen's Medical Research Institute, Edinburgh University, Edinburgh; Wellcome Trust Centre for Research in Comparative Respiratory Medicine, Easter Bush Veterinary Centre, Roslin, United Kingdom; and Department of Pathology and Molecular Medicine and Division of Infectious Diseases, Centre for Gene Therapeutics, McMaster University, Hamilton, Ontario, Canada

Correspondence and requests for reprints should be addressed to Jean-Michel Sallenave, Room W2.05, MRC Centre for Inflammation Research, Queen's Medical Research Institute, Edinburgh University, 47 Little France Crescent, Edinburgh, EH16 4TJ, Scotland, UK. E-mail: J.Sallenave{at}ed.ac.uk


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Infections with bacteria and viruses such as adenovirus are a feature of chronic lung diseases such as chronic obstructive pulmonary diseases (COPD), and may be instrumental in the generation of disease exacerbations. We have previously shown in acute models that elafin (a lung natural chemotactic molecule for macrophages and neutrophils, with potent antimicrobial and neutrophil elastase inhibitor activity) is upregulated in infection and modulates innate immunity. Here we present data using two independent systems of elafin overexpression in vivo (recombinant adenovirus [Ad-elafin] and an elafin transgenic mouse line) to examine the function of elafin in adaptive immunity. We show that elafin increases the number (immunofluorescence) and activation status (flow cytometric measurement) of CD11c+/MHCII+ lung dendritic cells in vivo. Analysis of cytokines produced by spleen and lung cells, and of antibodies measured in serum and bronchoalveolar lavage fluid, shows that the immunity induced is biased toward a type 1 response (production of IL-12, IFN-{gamma}, and IgG2a). Furthermore, elafin overexpression protected mice against further challenge with Ad-LacZ, as assessed by antibody levels and neutralization titer, as well as LacZ expression in lung tissue. Thus, the pleiotropic molecule elafin has significant potential in modulating antigen-presenting cell numbers and activity, and could be beneficial in mucosal protective strategies.

Key Words: adenovirus • chronic obstructive pulmonary diseases • dendritic cells • elafin • mucosal immunity


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Infections with bacteria such as nontypeable Haemophilus influenzae (NTHI), Streptococcus pneumoniae, and Branhamella catarrhalis or with viruses such as adenovirus (Ad) are a feature of chronic lung diseases such as chronic obstructive pulmonary diseases (COPD), and may be instrumental in the generation of disease exacerbations (1–3). In normal individuals, the immune response to bacterial infection in the airways involves the concerted and sequential efforts of the innate and adaptive immune systems. The innate response to bacteria involves soluble mediators such as endogenous antimicrobial molecules (EAMs), as well as phagocytes (macrophages and neutrophils) (4–6). In addition to these phagocytes, a third phagocytic cell type, the dendritic cell (DC), is a critical cell at the interface between innate and adaptive immunity (7). DC progenitors in the bone marrow give rise to circulating precursors that home to tissues, where they reside as immature cells with high phagocytic capacity and act as critical sentinels of the mucosal surfaces, by regulating tolerance to self-antigens and responses to foreign agents. Upon infection and tissue damage, immature DCs capture antigens, maturation of DCs ensues (reduction of phagocytic activity and increase in MHC class II and co-stimulatory molecules), and the cells migrate to the lymphoid organs, where they present antigen to naïve T cells and initiate a primary adaptive immune response (7). Although little is known of the role of EAMs in COPD (8–10), their antibacterial, and more recently, antiviral activities have been established (11). Their potential importance in COPD is underscored by the finding that low levels of two EAMs (secretory leukoprotease inhibitor [SLPI] and lysozyme) are associated with increased frequency of COPD exacerbations and a decreased ability to aggregate NTHI (12, 13). In addition, data are emerging showing that EAMs such as defensins and LL-37 can be chemotactic for DCs and can contribute to their maturation program (14–16).

We and others have shown recently that elafin, a molecule originally characterized by its neutrophil elastase (NE)-inhibitory activity (for a review see Ref. 5), is also an EAM (17–20). It is synthesized mucosally at inflammatory loci, in response to bacterial and cytokine stimuli (21), and is able to modulate the innate immune responses of macrophages and neutrophils (22–24). Given these properties, we hypothesized that elafin may be ideally placed to act as a bridge between innate and adaptive immunity and may have modulatory activities on dendritic cells. Because of its potential as a pathogen implicated in latent infections and exacerbations in COPD (3, 25), we have tested this hypothesis using Ad as a model immunogen. To this end, we have used a dual system of elafin overexpression: (1) a gene therapy strategy (Ad-elafin [EL] construct [26]) and (2) a newly generated transgenic mouse line (23). It is noteworthy that mice do not have an elafin ortholog and therefore can be considered "knocked-out" for the elafin gene. The human elafin transgenic approaches used herein can therefore be considered as authentic overexpression approaches in a null background.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Mice
Wild-type (WT) mice (C57/Bl6, C57/Bl6/CBA) and C57/Bl6/CBA human elafin transgenic (eTg) (23) mice were used (age 6–12 wk). The latter express the elafin gene under the mouse cytomegalovirus promoter (MCMV), which we have shown to be inducible by inflammatory mediators (22). Mice were anesthesized intraperitoneally with avertin (10 µl/g body weight). For intratracheal administrations, direct visualization of the vocal cords allowed for a delivery of a 40-µl volume trans-orally through a blunted needle (18). All animal experiments were conducted in accordance with the specifications of the local regulatory committee (Edinburgh University Medical School).

Ad Vectors
The E1/E3-deleted, replication-deficient serotype 5 Ad vectors used in this study have been described previously: Ad-EL encodes human elafin (26), Ad-lacZ encodes Escherichia coli beta-galactosidase (18), and Ad-dl70/3 (the control "null vector") has no promoter nor transgene (27).

In Vivo Ad Administration and Experimental Design
Single Ad administration. Mice received intratracheal instillations of either PBS, Ad-dl70/3 (2.5 x 108 pfu), or Ad-EL (2.5 x 108 pfu). At Day 10 or 14, animals were anesthetized and blood was collected by cardiac puncture. Sera was prepared and stored at –30°C until use. Spleens were collected, and lungs, hearts and tracheae were removed en bloc. Figures 1 and 2 relate to this protocol.


Figure 1
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  		Figure 1. Splenocyte proliferation, cytokine output, and serum anti-adenoviral total IgG content after a single Ad administration. At Day 0, WT mice were treated intratracheally with PBS (n = 3), Ad-dl70/3 (n = 5), or Ad-EL (n = 5); and elafin transgenic (eTg) mice (n = 5) and their corresponding WT control mice (n = 3) were treated with PBS or Ad-dl70/3. At Day 14, mice were killed and a variety of parameters were analyzed. (A) Spleens (pooled from within each experimental group) from either WT or eTg mice treated in vivo intratracheally with either PBS, Ad-dl70/3, or Ad-EL were analyzed as described in MATERIALS AND METHODS. Briefly, splenocyte proliferation was measured in triplicate (± SD) after in vitro Ad-dl70/3 recall or no recall (medium alone). (B and C) Cytokine output (TNF-{alpha} and IFN-{gamma}) was also measured using the CBA kit (see MATERIALS AND METHODS). Open bars: mice (WT or eTg) treated in vivo with PBS; hatched bars: mice (WT or eTg) treated in vivo with Ad-dl70/3; solid bars: WT mice treated in vivo with Ad-EL. As an example, PBS/Ad-dl70/3 means: mice treated with PBS in vivo and with Ad-dl70/3 as an in vitro recall challenge. (D) Serum (pooled from each experimental group) was analyzed for anti-Ad total IgG content by ELISA (in triplicate wells; see MATERIALS AND METHODS). Numbers in parentheses represent the number of mice used. Error bars (SD) represent variability in the proliferation (A) and ELISA (D) assays.

 

Figure 2
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  		Figure 2. WT mice lung myeloid population analysis after Ad intratracheal instillation. Lungs of WT mice (three mice per group) treated at Day 0 with either PBS, Ad-dl70/3, or Ad-EL (2.5 x 108 pfu) were perfused at Day 10 and pooled cells suspensions (for sensitivity reasons) were prepared as described in MATERIALS AND METHODS. (A) We selected by FCS/SSC analysis a gate (R1). (B) This gate was analyzed for CD11c+ cells and contained more than 90% of total lung CD11c+ cells (not shown). (C) These cells were analyzed for CD11c and MHCII expression and were subdivided in four gates CD11c low (G4); CD11c+high MHCII+high (G3); CD11c+high MHCII+low (G2) and total CD11c (G1, dotted line). (D) Cells were then analyzed within G1 and G3 for CD40, CD80, CD86, and CD11b expression.

 
Priming and challenge. In experiments to assess the secondary anti-Ad immune response and study further the potential of Ad-EL, four groups of mice were used: at Day 0, the first group received PBS alone; the second group received 2.5 x 108 pfu Ad-dl70/3 (8.8 x 109 total virus particles); the third group received 0.3 x 108 pfu Ad-EL and 2.2 x 108 pfu Ad-dl70/3 (8.2 x 109 total virus particles). At Day 14, all mice received Ad-LacZ (2.5 x 108 pfu; 2.04 x 1010 particles) intratracheally, and they were culled 3 d later. Sera, bronchoalveolar lavage fluid (BALF), lungs, and spleens were obtained as explained elsewhere. For determination of Ad-derived beta-galactosidase expression, lungs were snap-frozen in liquid nitrogen and stored at –80°C. For CD11c immunohistochemistry, lungs were inflated with a 2:1 mix of OCT:30% sucrose, coated in OCT, and snap-frozen in liquid nitrogen. Table 1 and Figures 3–5 relate to this protocol.


Figure 3
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  		Figure 3. Splenocyte cytokine and anti-Ad antibody output after mice priming and Ad-LacZ challenge. (A) At Day 0, WT mice were treated intratracheally with either PBS or Ad (either 0.3 x 108 pfu of Ad-dl70/3 or Ad-EL). All mice received Ad-LacZ (2.5 x 108 pfu) intratracheally at Day 14 and were killed at Day 17. Spleens were recovered, pooled from each experimental group (for sensitivity reasons), and splenocyte cytokine output was measured after Ad-dl70/3 recall as detailed in MATERIALS AND METHODS. Numbers in parentheses represent number of mice used. (B) Serum and BALF (pooled from each experimental group) were obtained from mice treated as described in A. Antibody levels were measured by ELISA (in triplicate wells) as described in MATERIALS AND METHODS, using different dilution (1:100 to 1:6,400). Numbers in parentheses represent the numbers of animals used. NB: Standard deviations are too small to appear for serum IgG2a and IgA levels.

 

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  		TABLE 1. IN VIVO ADENOVIRUS NEUTRALIZATION ACTIVITY OF BALF ANTIBODIES

 
Retrieval of BALF and Lungs and Flow Cytometry Analysis
Tracheae were intubated and BALF was obtained by instillation of 2 x 0.4 ml of sterile PBS. Typically, 0.4–0.6 ml was retrieved per animal. The cell pellet was used for cell differential analysis (performed after centrifugation of BALF at 2,000 x g for 5 min). Cytospinning (300 x g for 3 min at room temperature) was performed and slides were stained with Diff-Quick (Dade Diagnostika GmbH, Unterschleissheim, Germany). Supernatants were used for elafin and cytokine enzyme-linked immunosorbent assay (ELISA) analysis (see below). In an experimental subset, mice were killed, right heart catherization was performed, and the pulmonary circulation was perfused with PBS. After carefully dissecting the thymus and all thoracic lymph nodes, the lungs were removed and collected in ice-cold PBS. Three lungs from each experimental group were pooled and were minced using fine scissors and a scalpel, and were further incubated in PBS containing 0.7 mg/ml collagenase A (Roche Diagnostics GmbH, Mannheim, Germany) and 30 µg/ml DNase I (Sigma, Poole, UK) in a humidified incubator at 37°C and 5% CO2 for 45 min. Subsequently, the digested tissue was mechanically disrupted and passed through a 40-µm nylon cell strainer (Falcon, BD Biosciences, San Jose, CA). Finally cells were subjected to red blood lysis (1 mM NH4HCO3 and 114 mM NH4CL; Sigma), washed in complete medium (RPMI 1640 supplemented with 10% FCS, L-glutamine, penicillin/streptomycin [P/S]), and kept on ice for immunofluorescent labeling.

Monoclonal antibodies (mAbs) used to identify mouse DC populations were APC-CD11c (N418; eBiocience, San Diego, CA), and FITC-MHCII (2G9; BD Biosciences). Additional markers used for phenotyping were: PE-conjugated anti-CD11b/Mac-1{alpha} (M1/70), CD40 (1C10), CD80/B7-1 (16-10A1), CD86/B7-2 (GL1), B220/CD45R (RA3-6B2) (all from eBiocience), and F4/80 (CI-A3-1; Caltag, Burlingame, CA). Isotype-matched control antibodies to each mAb were used.

All staining reactions were performed on ice in FACS buffer (PBS containing 0.2% BSA; Sigma). Cells were pre-incubated with mouse serum (Serotec, Oxford, UK) to reduce non-specific binding of mAb. Cells were stained with mAbs for 30 min on ice and washed with FACS buffer before being analyzed. Flow cytometry data acquisition was performed on FACSVantage SE flow cytometer (Becton Dickinson, Franklin Lakes, NJ) and around 1 x 105 events were collected per sample. CellQuest software was used for data analysis (BD Biosciences).

Cytokine and Elafin ELISA
ELISA analyses to detect murine IFN-{gamma} and TNF-{alpha} were performed by using commercially available ELISA kits (R&D Systems, Abingdon, UK) in accordance with the manufacturer's instructions. Human elafin was measured by using an ELISA available in-house (28). IL-12p40 ELISA was performed on BALF using paired mAb and cytokine standards (BD Biosciences) as previously described (29).

Splenocyte Isolation and Culture, Proliferation, and Cytokine Output
Splenocytes were prepared by pushing the whole spleen through a 40-µm nylon cell strainer into 5 ml of culture medium (RPMI as above supplemented with 2-mercaptoethanol). Cells from within each group were pooled, cultured at a concentration of 4 x 105 cells/well with or without ultraviolet-inactivated Ad-dl70/3 as a recall antigen (multiplicity of infection [moi] of 100), and cytokine production was assessed in a 100-µl aliquot using Cytometric Bead Array (CBA) kit (for the measurement of TNF, IL-2, IFN-g, IL-4, IL-5), according to the manufacturer's instructions (BD Biosciences). Subsequently, to measure cellular proliferation, 18.5 kBq tritiated thymidine (Amersham, Buckinghamshire, UK) was added to each well and thymidine incorporation over 18 h was determined (assays done in triplicate). Ad-specific proliferation data are given either as absolute counts per minute or by substracting thymidine incorporation in the absence of Ad-dl70/3 from that in the presence of Ad-dl70/3.

Anti-Ad Antibody ELISA
Serum/BALF anti-Ad antibody levels were determined by ELISA. Ad-antigens were prepared by heat inactivating lysates of 293 cells previously infected with Ad (overnight infection of 293 cells with Ad-dl70/3 at an moi of 100). ELISA plates (96 well; Costar, Corning Inc., Corning, NY) were coated with 5 µg Ad-ag protein/well overnight and blocked with PBS containing 1% BSA and 0.05% Tween-20 (PBT) for 2 h at 37°C. Samples were added in serial dilutions and incubated for 2 h at 37°C. Detection antibodies (biotin conjugates of goat anti-mouse IgG and goat anti-mouse IgA; Sigma), diluted 1:10,000 in PBT was applied for 1 h at 37°C. Finally, streptavidin–horseradish peroxidase (Gibco, Invitrogen, Paisley, UK) diluted 1:20,000 in PBT was applied for 1 h at 37°C. Between each step plates were washed extensively with PBS. Plates were developed with the peroxidase substrate tetramethylbenzidine (Sigma) and the reaction was stopped with 1 M sulphuric acid. Optical density was determined at 450 nm (reference filter, 560 nm) in a microplate spectrophotometer (MRX Micoplate Reader; Dynex Technologies, Chantilly, VA).

Serum/BALF Neutralizing Anti-Ad Antibodies
Complement was inactivated by heating serum to 56°C for 30 min. Serum/BALF samples were serially diluted in culture medium (DMEM plus 10% FCS, L-glutamine and P/S) and incubated with Ad-LacZ (5.5 x 105 pfu) for 1 h at 37°C. One hundred microliters of sample/virus mixtures was added to subconfluent A549 cells in 48-well plates (~ 5 x 104 cells/well) for 1 h. Cells were cultured overnight in fresh medium before being lysed with distilled water (150 µl/well). beta-Galactosidase activity of lysates was determined with the substrate o-nitrophenol-beta-D-galactopyranoside (Sigma). Absorbance was recorded at 405 nm and beta-galactosidase activity was expressed as {Delta}OD405 nm s-1. Neutralizing titer was defined by extrapolating serum dilution: beta-galactosidase activity curves to the x-axis.

In Vivo Ad-Derived beta-Galactosidase Expression
After Ad immunizations at Day 0, protective immunity against Ad reinfection was demonstrated using Ad-LacZ (Day 14) by assessing lung beta-galactosidase expression (Day 17). Lungs were homogenized and resuspended in lysis buffer (100 mM potassium phosphate, pH 7.8, 0.1% vol/vol Triton X-100, 100mM beta-mercaptoethanol). Debris were removed by centrifugation (2,000 x g, 10 min, 4°C). Samples were extensively dialysed against distilled H2O, and beta-galactosidase content was determined by ELISA (Roche Diagnostics). Total protein concentration was determined with a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL) and beta-galactosidase expression was presented as beta-galactosidase (ng) per gram of lung tissue protein.

CD11c Immunohistochemistry
Sections (15 µm) were cut from OCT-inflated lungs using a microtome with a cabinet temperature of –30°C, mounted on polylysine-coated slides and fixed with 100% methanol. Sections were washed twice with PBS and incubated for 2 h with either PE-conjugated hamster anti-mouse CD11c (N418) or isotype control (BD Biosciences), diluted 1:20 in ChemMate Antidody Diluent (DakoCytomation, Cambridgeshire, UK). Slides were washed with PBS and briefly counterstained with TO-PRO-3 (Molecular Probes, Invitrogen). Digital images were acquired with a confocal microscope (Leica TCSNT; Leica Microsystems, Heidelberg, Germany). Cells stained with CD11c antibody were counted from three independent confocal fields taken at magnifications of x10 and x83.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Ex Vivo Studies of Elafin Enhancement of Primary Immune Responses
Peripheral lung responses. To examine the effect of Ad-mediated elafin production in mice lungs on adaptive immunity, BALF elafin levels were measured 2 wk after WT mice received PBS (n = 3), Ad-dl70/3 (n = 5), or Ad-EL (n = 5) (2.5 x 108 pfu) intratracheally; and eTg (n = 5) and their corresponding WT (n = 3) controls received either PBS or Ad-dl70/3 (2.5 x 108 pfu). WT mice do not express elafin, and as expected elafin was undetectable in WT mice receiving either PBS or Ad-dl70/3. Ad-EL treatment of WT mice induced levels of elafin (3,871.4 pg/ml ± 121), which were, expectedly, lower than those obtained in our "Day 5 acute models" (19, 22). Of note, elafin levels were not detected in the BALF of naïve eTg mice receiving PBS, but Ad-dl70/3 increased elafin secretion in these mice (2,238.6 pg/ml ± 506), probably because, as demonstrated for LPS, inflammatory stimuli are able to activate the endogenous (transgenic) elafin MCMV promoter (which contain many NF-KB sites) in eTg mice (22, 23). When BALF differential counts were performed 10 d after treatment, Ad-EL–treated mice showed a significant increase in percentage of lymphocytes compared with Ad-dl70/3 and PBS controls (36.40 [± 8.62], 8.49 [± 4.42], and 2.69 [± 1.99], respectively; Ad-EL versus Ad-dl70/3: P = 0.0012). In addition, local EL production induced an increase in BALF neutrophils (4.01 [± 2.73], 1.32 [± 1.12], and 0.13 [± 0.26], respectively) as observed previously (18), whereas it caused a reduction in percentage of macrophages (59.59 [± 9.66], 90.2 [± 4.15], and 97.31 [± 1.99], respectively; Ad-EL versus Ad-dl70/3: P = 0.0011). Notably, eTg mice also exhibited a trend toward a higher percentage of lymphocytes, compared with WT control mice, after challenge with Ad-dl70/3 (6.96 [± 3.37] and 2.36 [± 1.07], respectively) and had higher neutrophils (13.09 [± 21.7] and 1.24 [± 0.04], respectively) and lower macrophages (96.39 [± 1.11] and 80.07 [± 19.66], respectively) in their BALF.

Spleen proliferative responses. Spleen cell proliferative responses after single lung Ad treatments (see above) were studied after ultraviolet-inactivated Ad-dl70/3 in vitro recall (see Figure 1A). Expectedly, Ad-dl70/3 recall of spleen cells from WT mice primed in vivo with Ad-dl70/3 (Figure 1Aa, hatched bars) showed a higher proliferative response, compared with mice primed in vivo with PBS (Figure 1Aa, open bars). Interestingly, in vivo Ad-EL treatment (Figure 1Aa, solid bars) induced a greater proliferation of splenocytes than PBS and Ad-dl70/3 treatments. Notably, eTg mice had an even higher response when treated in vivo with Ad-dl70/3 than when treated with PBS (Figure 1A). We then studied the potential immunologic bias of these spleen responses by measuring cytokines in spleen cells supernatants. Stimulated splenocytes of Ad-EL–treated WT mice and Ad-dl70/3–treated eTg mice produced markedly higher levels of TNF-{alpha} compared with their controls (Figure 1B). In addition, Figure 1C shows a clear "type 1" bias, since IFN-{gamma} levels were increased in WT mice treated with Ad-EL (solid bars) when compared to Ad-dl70/3 treatment (hatched bars) and in eTg mice treated with Ad-dl70/3, when compared to WT mice receiving the same treatment (hatched bars). IL-5 was not detected in any conditions, whereas IL-4 was observed in all treatments (without any obvious trend), at very low levels (< 13 pg/ml).

Systemic antibody responses. To assess whether Ad-EL treatment in the lungs of WT mice or the expression of elafin in eTg mice was also able to trigger an efficient antibody response against Ad antigens, we measured anti-Ad antibody levels in serum from Ad-treated mice and eTg mice, compared with controls. We detected increased serum anti-Ad total IgG in WT mice given Ad-dl70/3 in the lung (Figure 1D, hatched bars). Even higher levels of these antibodies were observed in the sera of mice given Ad-EL (solid bar) mirroring data from spleen and showing unequivocally that Ad-EL was able to functionally alter the development of a primary anti-Ad antibody response. In addition, also in agreement with data presented above, antibody levels were higher in eTg mice given Ad-dl70/3, compared with WT mice given the same regimen (hatched bars).

Effect of elafin expression on cytokine production and dendritic cells phenotype in vivo. In order to reduce the number of parameters involved and to concentrate our study on the potential of elafin in the context of genetic vaccination, the rest of the experiments described herein were performed with Ad vectors on WT mice. Lungs from WT mice given either PBS, Ad-dl70/3, Ad-EL intratracheally were lavaged with PBS and perfused 10 d after treatment, minced, and digested, and the total lung cells were studied using flow cytometry analysis. When levels of BALF proinflammatory cytokines were analyzed, Ad-EL–treated mice, compared with Ad-dl70/3 and PBS-treated mice (n = 4 mice in each group), showed significant increased levels of IL-12p40 (8,950 [± 2,640], 880 [± 410], and 170 [± 140] pg/ml, respectively; Ad-EL versus Ad-dl70/3: P = 0.0009), IFN-{gamma} (6.1 [± 2.1], 0.8 [± 0.7], and 0.7 [± 1.3] pg/ml, respectively; Ad-EL versus Ad-dl70/3: P = 0.0148), and TNF-{alpha} (13 [± 3.9], 1.3 [± 1.8], and 2.9 [± 0.9] pg/ml ,respectively; Ad-EL versus Ad-dl70/3: P = 0.0001). In addition, whole lung single-cell suspensions (n = 3 mice per group; pooled samples) were stained with mAbs to CD11c and MHC class II. More than 90% of CD11c+ cells were found within the R1 region (Figure 2A) by back-gating analysis (data not shown). Ad-EL gene transfer increased the total number of CD11c+ lung cells compared with those treated with Ad-dl70/3 and PBS (Figure 2B). The cells from the R1 region were further divided into four subpopulations (G1–G4) on the basis of CD11c and MHCII expression. Dual labeling of cells with CD11c and MHCII allowed us to identify two main CD11c+ "high" cell populations (G1), one being MHCII+ "low" (G2) and the other MHCII+ "high" (G3) (Figure 2C). We considered the CD11c+high MHCII+high G3 gate to represent "bona fide" dendritic cells and CD11c+high MHCII+low to be macrophages/monocytic cells for the following reasons: "naïve" PBS-treated mice had a very low percentage of CD11c+high MHCII+high cells (~ 1% of total lung cells, not shown), consistent with them being lung dendritic cells (25, 30, 31). In addition, this population expanded significantly upon Ad treatment (1.7% and 7.6% of total lung cells, after Ad-dl70/3 and Ad-EL treatments, respectively, compared with 0.8% in PBS-treated mice, not shown). Furthermore, in PBS-treated mice, when cells were labeled with antibodies to F4/80, a macrophage marker, about 32% of CD11c+high MHCII+low cells were labeled, whereas only 15% of CD11c+high MHCII+high cells were positive for F4/80 (data not shown). Our analysis shows that the percentage of CD11c+high MHCII+high cells (G3) was markedly increased in WT mice treated intratracheally with Ad-EL (Figure 2C). Interestingly, Ad-EL also induced a small increase in the number of cells in G4 compared with Ad-dl70/3, which may be B lymphocytes (Figure 2C) (31). In addition, when co-stimulatory molecules were analyzed, increased levels of CD80 and CD86 were observed in Ad-EL–treated mice (Figure 2D), particularly in the double-positive "‘DC G3." Of note, the level of CD40 was low in PBS-treated mice, compared with levels of CD80 and CD86, and was not induced by Ad treatments.

Ad-EL Overexpression for Genetic Vaccination
Having demonstrated an enhanced in vivo primary response (involving an increase in dendritic cell numbers and activation) to Ad in WT mice receiving Ad-EL and in eTg mice receiving Ad-dl70/3 (first part of the study), we investigated further the role of elafin on a secondary immune response. We administered PBS, Ad-dl70/3, and Ad-EL intratracheally at Day 0 and challenged the mice at Day 14 with Ad-LacZ (by the same route), to allow us to assess the efficiency of Ad-EL vaccination by measuring beta-galactosidase expression. Three days later (Day 17 of experiment) mice were killed and samples analyzed. The data relating to this experiment are shown in Table 1 and Figures 3–5GoGo.

Spleen cytokine responses. After in vitro antigen recall, splenocyte IFN-{gamma} output from mice treated in vivo with Ad-EL was higher than in PBS- and Ad-dl70/3–treated mice (Figure 3A), in keeping with the "single dose" protocol, which also showed increased levels of IFN-{gamma} (Figure 1). The type 2 cytokines IL-4 and IL-5 were undetectable.

Generation of neutralizing anti-Ad5 antibodies. Anti-Ad total IgG, IgG2a, and IgA levels were measured in sera and BALF, as previously described. While the main anti-Ad antibody class detected in serum after Ad-dl70/3 priming was IgG, both IgA and IgG were found in BALF. The levels of all classes of antibody were increased in Ad-EL–treated mice (Figure 3B). Interestingly, although the latter two treatments were equally effective in generating anti-Ad IgG and IgG2a, Ad-EL was the best inducer of IgA in the lungs. BALF antibodies were shown, expectedly, to be neutralizing in the Ad-treated group, but more significantly, the neutralizing titer was higher in BALF from Ad-EL–treated mice compared with that of mice treated with Ad-dl70/3 (Figure 4).


Figure 4
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  		Figure 4. In vitro Ad neutralization activity of BALF antibodies. BALF recovered from mice (see legend of Figure 3 for protocol) were pooled and used to neutralize Ad-LacZ expression in vitro. BALF was incubated with Ad-LacZ before infection of A549 cells (see MATERIALS AND METHODS). After overnight infection, cells were lysed and beta-galactosidase expression was measured by ELISA (pool of samples within each group). The endpoint titer was determined by extrapolating the dilution curves to the x-axis. Numbers in parentheses represent the number of mice used.

 
In vivo Ad neutralization. Three days (Day 17) after Ad-LacZ intratracheal administration (Day 14), LacZ expression was assessed in lung homogenates. In keeping with the in vitro neutralization data, where Ad-dl70/3 treatment had a small effect in the induction of neutralization antibodies (Figure 4), in vivo Lac Z expression was only modestly reduced (statistically nonsignificant) in the Ad-dl70/3–treated group (Table 1) compared with the PBS group. By contrast, the Ad-EL–treated group showed significant reduction in LacZ expression (Table 1), establishing that elafin had a beneficial role in the anti-Ad immunization protocol.

Lung dendritic cells immunohistochemistry. The presence of lung dendritic cells in this second protocol was studied by immunohistochemistry (Figure 5). Many more CD11c+ cells (labeled "grey" with PE; Figure 5, open arrow) were evident in the airways and in the lung interstitium of Ad-EL–treated mice, as compared with the PBS and Ad-dl70/3 groups (Ad-EL versus Ad-dl70/3: P = 0.0328). Furthermore, these antigen-presenting cells seem to localize around the airways and show the presence of numerous typical dendrites (as indicated with white arrowheads in Figure 5), suggesting an activated phenotype.


Figure 5
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  		Figure 5. Histologic analysis of CD11c+ cells in lungs given Ad constructs. Lungs were obtained at Day 17 after intratracheal Ad instillation (see Figure 3A legend). Sections (15 µm) were cut from OCT-inflamed lungs using a microtome with a cabinet temperature at –30°C, mounted on polylysine-coated slides, and fixed with 100% methanol (see MATERIALS AND METHODS). Cells were labeled with PE-conjugated hamster anti-mouse CD11c (open arrow) and the nuclei were counterstained with TO-PRO-3 (white arrow). White arrowheads indicate extended dendrites in lungs treated with Ad-EL. Digital images were acquired with a confocal microscope using an 83x magnification. Quantification of CD11c+ cells was done by counting three fields per experimental group taken using a 10x magnification.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
We have previously shown that the antimicrobial/neutrophil elastase (NE) inhibitor elafin, when overexpressed in an Ad vector, is able in an acute model to induce an inflammatory cell influx after lung LPS treatment (22). In that study, we showed that a very small dose of Ad-EL (0.3 x 108 pfu), which was apparently noninflammatory by itself, was able, in conjunction with LPS, to induce the influx of neutrophils and macrophages in the lung 24 h after LPS treatment (5 d after Ad treatment). This increase was shown to be due to the LPS-mediated upregulation of the MCMV promoter in the Ad-EL construct (which contains many NF-{kappa}B sites), resulting in increased secretion of this antimicrobial molecule in the lung. The activity of elafin on innate immune cells was confirmed in vitro, using Ad vectors (22), as well as in vivo, using a nonviral approach in which newly characterized eTg were given LPS intratracheally and lung parameters were studied 24 h later (23). Altogether, these results suggested that in addition to its role in an acute model of innate immunity, elafin, a molecule also upregulated by bacterial LPS and early wave cytokines such as IL-1 and TNF in human epithelial cells (21), may be involved in the initiation of mucosal events leading to adaptive immunity. As a model system, we chose in the present study to examine immunity against Ad, a well-researched pathogen since the emergence of adenovirus as a potential tool in gene therapy in the last decade (32–35) and itself a potential agent implicated in chronic lung diseases such as COPD and emphysema (3, 25). Using the dual system of elafin expression described above (Ad-EL and eTg mice), we demonstrated here in the first part of our study that elafin induces a type 1–biased inflammatory and immunological response (cellular and humoral) in the lungs and spleens of mice over-expressing elafin (see above, p. 637, and Figure 1). We then set out to demonstrate whether the effect described for elafin was likely to be mediated through the increase in numbers and/or activation status of lung antigen-presenting cells. We first defined phenotypically the lung populations and studied more particularly CD11c+ and MHCII+ cells. In agreement with von Garnier and coworkers (31), we defined CD11c+high MHCII+high cells as bona fide lung dendritic cells (DC) and CD11c+high MHCII+low cells as lung monocytes/macrophages, the latter population expressing higher levels of the F4/80 marker than CD11c+high MHCII+high cells (see RESULTS). Using the Ad-EL system of delivery (Figure 2), we showed that elafin overexpressers exhibited higher numbers of total lung CD11c+high cells and CD11c+high MHCII+high cells (DCs). In addition, Ad-EL treatment induced higher levels of CD80 and CD86 in the latter cell population, showing that DCs were activated, in accordance with the increased levels of lung IL-12 (a cytokine produced abundantly by activated DCs [7]) observed in BALF (see above, p. 639). Interestingly, as reported by Dodge and colleagues (36) we found that pulmonary DCs had low levels of CD40 expression.

Murine DCs have been traditionally identified by their expression of CD11c and, in addition, DC subsets have been described, based on the expression of CD11b, CD8-{alpha}, and CD45R/B220 (37–39). Murine myeloid DCs co-express CD11c and CD11b and lack CD45R/B220, whereas lymphoid DCs do not express the latter two markers, but express CD8-{alpha}. Plasmacytoid DCs express both CD11c and CD45R/B220, but not CD11b. We have also measured in the present study the levels of the myeloid marker CD11b on our different cell populations and shown that it was elevated in Ad-EL–treated mice, in particular in CD11c+high MHCII+high cells (Figure 2D). We also investigated the presence of plasmacytoid DCs (CD45R/B220) and found that more than 90% of CD45R/B220 cells were present in the CD11c+high MHCII+high cells population (data not shown). However, there was no difference in the % expression of this marker on CD11c+high MHCII+high cells between the experimental treatments, PBS, Ad-dl70/3, and Ad-EL (12.95, 12.7, and 11.46, respectively).

Using an Ad-EL vaccination and Ad-LacZ challenge protocol as a convenient read-out, we showed that Ad-EL treatment was able to induce blocking antibodies against Ad-LacZ in vitro (Figure 4) and to prevent Ad-LacZ infection in vivo (Table 1). Biragyn and colleagues were recently also able to immunize mice with fusion proteins consisting of murine active beta-defensins linked to a nonimmunogenic lymphoma antigen (40), suggesting that fusion constructs with inflammatory mediators targeting immature DCs could elicit protective antitumor immunity. The mechanism of action of elafin in our experiments is likely to be different since elafin was not a fusion construct (adjuvant-immunogen) but was instead given either as an independent Ad-EL construct, or secreted as soluble protein in the eTg mice, concomitantly with the Ad-dl70/3 immunizing dose. However, the increase in lungs CD11c+ cell numbers after Ad-EL treatment suggests that elafin may be a chemoattractant and/or activator of lung DCs. Alternatively, locally produced elafin may act via other indirect mechanisms—for example, through the inhibition of neutrophil elastase (NE). Indeed, we have recent evidence showing that NE, is able to downregulate DC co- stimulatory molecules, and affects negatively their ability to present antigen to splenocytes (41, and A. Roghanian and coworkers, unpublished data). Hence, overexpression of elafin may rescue DC function by restoring DC activation and or prevent NE-mediated DC inactivation. Irrespective of the potential mechanism of action of Ad-EL on lung DCs (currently under investigation in our laboratory), we cannot rule out a concomitant effect of elafin in peripheral lymphoid organs. Although Ad-derived elafin protein was not detected in spleen or blood of WT mice during our study (data not shown), elafin protein (at a level below the threshold of detection) may have "spilled over" from the lung into the circulation and into the spleen and activated immune cells in situ. Alternatively, Ad-EL may have migrated from the lung to the regional lymph nodes and spleen and have directed transgene expression at these immune sites. Although this has not to our knowledge been investigated with lung delivery of Ad vectors, they have been shown, when delivered intramuscularly (42) and in hind footpads, to migrate to proximal lymph nodes as well as (in a minor way) to the spleen (42–44), resulting in transgene expression at these sites. Although it is difficult, when WT mice are used, to ascertain whether Ad-EL–mediated elafin expression in the spleen may be in part responsible for the effect demonstrated here, that may be the case in the eTg mice studied in the first protocol. Indeed, since we have previously shown that the elafin message is present in the spleen of these mice (23), it is possible that its secretion in the spleen milieu may increase lymphocyte responses, either directly or through activation of splenic DCs. Relatedly, we have demonstrated that WT mice splenocytes infected with Ad-EL exhibited higher proliferation than PBS- or Ad-dl70/3–treated splenocytes, when nonspecifically stimulated with anti-CD3 antibodies (data not shown). Similar results have also recently been obtained with incubation and activation of splenocytes with human neutrophil defensins (45). Even though the two models of elafin overexpression (transient Ad-EL and constitutive transgenic mice) were similarly useful for the demonstration of the adjuvant effect of elafin, the different characteristics of these models should allow us, in the future, to dissect further the molecular mechanisms (e.g., to elucidate whether the elafin effect is restricted to the lung mucosal surfaces or is also operative in peripheral lymphoid organs).

In summary, we have demonstrated here, using a dual approach of gene transfer that the antimicrobial/NE inhibitor molecule elafin is an efficient mucosal agent for Ad immunization, a pathogen implicated in chronic pathologies such as chronic obstructive pulmonary diseases (COPD and emphysema [3, 25]). Since innate immune products such as NE (41) and exogenous stimuli such as cigarette smoke have been shown both in vitro (46–48) and in vivo (25; S. D. Shapiro, personal communication) to cause a reduction in DC numbers and maturation as well as a decrease in their antigen-presenting capacity and/or to skew immunity toward type 2 responses (46), the type 1 responses elicited by overexpressing elafin (concomitantly with a very effective IgA response) may be beneficial in the context of COPD infections and exacerbations.


   Acknowledgments
 
The authors are grateful to Drs. Georgia Perona-Wright and Andrew S. MacDonald (Institute for Immunology & Infection Research, University of Edinburgh) for their help with the IL-12 ELISA, and to Drs. Donald J. Davison and Jason A. King (CIR, Edinburgh) for useful discussions. Extended thanks also go to Mrs. Shonna M. Johnston for excellent technical help with FACS analysis, Mrs. Lesley A. Farrell for helping with in vivo experiments, Drs. Robert A. Benson and Julia V. Marley for helping us with splenocytes proliferation assays, and to Prof. Christopher Haslett (all from CIR, Edinburgh) for continuous support.


   Footnotes
 
* These authors contributed equally to this publication. Back

This work was supported by the MRC (Ph.D. studentship to A.R.) and the Wolfson Trust (grant to S.E.W.).

Originally Published in Press as DOI: 10.1165/rcmb.2005-0405OC on January 19, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form October 31, 2005

Accepted in final form December 28, 2005


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 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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