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The Journal of Immunology, 2001, 167: 1778-1786.
Copyright © 2001 by The American Association of Immunologists

Adenoviral Augmentation of Elafin Protects the Lung Against Acute Injury Mediated by Activated Neutrophils and Bacterial Infection1

A. John Simpson*, William A. H. Wallace{ddagger}, Mark E. Marsden*, John R. W. Govan{dagger}, David J. Porteous§, Chris Haslett* and Jean-Michel Sallenave2,*

* Rayne Laboratory, Respiratory Medicine Unit, Medical Research Council Centre for Inflammation Research, and {dagger} Department of Microbiology, Medical School, University of Edinburgh, Edinburgh, United Kingdom; {ddagger} Pathology Department, Northern General Hospital, Sheffield, United Kingdom; and § Medical Genetics Section, University of Edinburgh Molecular Medicine Centre, Western General Hospital, Edinburgh, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During acute pulmonary infection, tissue injury may be secondary to the effects of bacterial products or to the effects of the host inflammatory response. An attractive strategy for tissue protection in this setting would combine antimicrobial activity with inhibition of human neutrophil elastase (HNE), a key effector of neutrophil-mediated tissue injury. We postulated that genetic augmentation of elafin (an endogenous inhibitor of HNE with intrinsic antimicrobial activity) could protect the lung against acute inflammatory injury without detriment to host defense. A replication-deficient adenovirus encoding elafin cDNA significantly protected A549 cells against the injurious effects of both HNE and whole activated human neutrophils in vitro. Intratracheal replication-deficient adenovirus encoding elafin cDNA significantly protected murine lungs against injury mediated by Pseudomonas aeruginosa in vivo. Genetic augmentation of elafin therefore has the capacity to protect the lung against the injurious effects of both bacterial pathogens resistant to conventional antibiotics and activated neutrophils.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Toxic products released by neutrophils, especially human neutrophil elastase (HNE),3 have been implicated in the pathogenesis of a variety of inflammatory disorders characterized by pulmonary neutrophilia, including cystic fibrosis (CF), non-CF bronchiectasis, emphysema, and bacterial pneumonia (1, 2, 3, 4). The therapeutic rationale for depleting circulating neutrophils in such conditions is negated by the propensity for overwhelming sepsis in neutropenic patients and by the observation that mice deficient in neutrophil elastase are predisposed to Gram-negative sepsis (5). This argument applies especially in the context of bacterial pneumonia. In such situations, inhibition of extracellular HNE by agents also possessing antimicrobial properties would be an attractive strategy in attempting to protect the lung from inflammatory injury.

Inhibitors of HNE are thought to comprise part of the human innate immune system. Three distinct antielastases have been described in the human lung; {alpha}1-protease inhibitor, secretory leukocyte protease inhibitor (SLPI), and elafin (elastase-specific inhibitor) (6). Elafin (7, 8) is a potent inhibitor of HNE and proteinase 3 produced in the skin (9, 10, 11), and in the airways (12), which is up-regulated in response to early inflammatory cytokines such as TNF and IL-1 (13). Elafin, along with SLPI, also shares characteristics with antimicrobial defensin-like molecules in being a low m.w. cationic peptide with the ability to eliminate pulmonary pathogens (14, 15, 16).

We therefore hypothesized that local augmentation of elafin by constitutive lung cells would confer protection against inflammatory injury, especially when the lung was challenged by bacterial pathogens. In these studies, we used an adenoviral gene transfer approach for a number of reasons, including the natural tropism of adenovirus for the respiratory epithelium, the potential to regulate transgene expression using carefully selected promoters (17, 18), and the well-described ability to express antielastases in the lung using adenoviral gene therapy (17, 19). Against this background, we demonstrate for the first time that A549 cells transfected with adenovirus encoding human elafin in vitro protects not only against HNE-induced damage but also against injury caused by primed and activated human neutrophils. We also show for the first time that intrachacheal (i.t.) transfer of adenovirus encoding a human gene with dual antielastase and antimicrobial properties protects murine lungs against acute inflammatory injury caused by Pseudomonas aeruginosa, a bacterial pathogen commonly resistant to conventional antibiotics (20, 21). Our findings extend the observed potential for gene therapy strategies in the management of pulmonary injury and infection (22, 23, 24, 25, 26) by demonstrating that the protection conferred in vivo was achieved using doses of adenovirus which were not themselves associated with significant vector-induced airway inflammation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Adenoviral constructs

The adenoviral constructs used have been described in detail elsewhere (17, 18). In brief, human elafin cDNA (encoding full length elafin) was cloned into pDK6 downstream of a 1.4-kb fragment of the murine CMV promoter (27). PDK6 and pBHG10 were used to cotransfect 293 cells (which generate the product of the adenoviral E1 region in trans). Homologous recombination resulted in the generation of E1-, partially E3-deleted adenovirus encoding human elafin cDNA (Ad-elafin). The second virus used (E1-, partially E3-deleted adenovirus encoding lacZ cDNA (Ad-lacZ)) was constructed in the same manner with the exception that lacZ cDNA (under the control of the same murine CMV promoter fragment) was cloned in place of elafin cDNA (17, 18).

Preparation of human neutrophils

Fresh citrated whole blood was obtained from healthy volunteers. Neutrophils were prepared by dextran sedimentation and Percoll gradient extraction using a standard technique described elsewhere (28). BSA (Sigma, Poole, U.K.) at a final concentration of 1% was added to cell supernatants before addition of neutrophils. Neutrophils were activated by the sequential addition to cell supernatants of platelet-activating factor (PAF) (Calbiochem, Nottingham, U.K.) at a final concentration of 10-9 M, and fMLP (Calbiochem), final concentration 10-7 M.

In vitro transfection experiments

A549 cells were used in in vitro transfection experiments. A549 cells are derived from bronchioloalveolar cell carcinoma (29) and share several phenotypic characteristics with type II alveolar epithelial cells (29, 30). Indeed, several features of A549 cells have been reproduced in primary cell lines; in particular elafin is expressed by pulmonary epithelial cell lines and up-regulated in response to IL-1 and TNF, features characteristic of A549 cells (13, 30).

A549 cells were incubated at 37°C (and 5% CO2) in DMEM (Sigma) containing 10% FCS (Sigma), penicillin G (final concentration, 100 U/ml; Life Technologies, Paisley, U.K.), streptomycin sulfate (final concentration, 100 µg/ml; Life Technologies), and 1% L-glutamine (final concentration, 2 mM; Life Technologies) and grown to confluence in 24-well plates (Corning Costar, High Wycombe, U.K.). Cells were washed with PBS (Sigma) and then incubated for 30 min at 37°C with one of three treatments; Ad-elafin in DMEM containing 5% FCS; Ad-lacZ in DMEM containing 5% FCS; or DMEM containing 5% FCS with no virus added. Adenovirus was applied at a multiplicity of infection (moi) of 50 PFU (except in dose-response experiments where adenovirus was applied over the dose range 0–50 PFU). Cells were washed with PBS, and the medium was changed to DMEM containing 10% FCS, before incubation overnight at 37°C. Cells were washed extensively using PBS and incubated at 37°C in serum-free DMEM before the addition of either purified HNE at a final concentration of 4 µg/ml (Elastin Products, Owensville, MO) or 2 x 106 human neutrophils (activated by PAF and fMLP as above). After 16 h, the damage to the monolayer was assessed morphologically by light microscopy or by counting the number of A549 cells liberated into the supernatant.

In a variation of these experiments, A549 cells were radiolabeled with 111In, using a variation on methods described previously (31). 111In (DuPont, NEN Life Sciences, Brussels, Belgium) was incubated with 4 x 10-3 M tropolone (Sigma) for 1 min. 111In (3 µCi) was added to A549 cells at the end of the overnight incubation in DMEM containing 10% FCS (final concentration of tropolone, 4 x 10-4 M). Damage to the monolayer after addition of human neutrophils was assessed 16 h later by measuring radioactivity in the supernatant using a scintillation analyzer (model 1900TR; Canberra Packard, Pangbourne, U.K.). It is well established that neutrophils from different healthy individuals may respond very differently to the effects of priming agents. In preliminary experiments, 2 x 106 neutrophils, activated with 10-9 M PAF and 10-7 M fMLP, caused 30–70% damage to untransfected A549 cells at 16 h in the significant majority of individuals. A protocol was therefore devised whereby neutrophils causing <30% or >70% damage to untransfected monolayers were excluded from analysis. In subsequent experiments, 10 healthy volunteers donated neutrophils. In eight of these the protocol was satisfied (in one of the remaining cases, 100% of untransfected A549 cells were damaged at 16 h, and in the other no untransfected A549 cells were morphologically damaged at 16 h).

To correct for the theoretical risk of nonspecific leakage of 111In from A549 cells, radioactivity in supernatants from cells that did not have neutrophils applied was subtracted from the count in neutrophil-treated cells for all conditions studied.

Preparation of P. aeruginosa PAO1

P. aeruginosa PAO1, a well-characterized type strain used in many genetic (21) and animal studies, and the first strain of P. aeruginosa to be fully sequenced (32), was inoculated into nutrient broth (Oxoid, Basingstoke, U.K.) containing 0.5% yeast extract (Difco, Detroit, MI) and incubated overnight at 37°C in an orbital incubator at 200 rpm (3). The culture was centrifuged at 4500 rpm at room temperature for 15 min, and the bacteria were washed in 0.01 M phosphate buffer, pH 7.0. The bacterial suspension used in in vivo experiments was prepared in the same buffer to provide a population density of ~2.2 x 1011 CFU/ml when cultured on Pseudomonas Isolation Agar (Difco).

In vivo experiments

Female C57BL/6 mice between 6 and 8 wk old were from Harlan Olac (Bicester, U.K.). Mice were anesthetized using i.p. avertin (~10 µl/g body weight; avertin comprised 1.25% 2,2,2-tribromoethanol (Aldrich, Gillingham, U.K.) and 2.5% 2-methyl-2-butanol (Sigma)). In all experiments involving i.t. administration, the vocal cords were viewed directly, and a blunted catheter was passed beyond them according to methods previously described by our group (33). Treatments of known volume were instilled directly, and body fluids or organs extracted as described below. Preliminary experiments in which trypan blue was administered i.t. consistently revealed a similar distribution of dye to all lobes of the lung macroscopically (data not shown). All mice were euthanized under anesthesia (using avertin) by transection of the abdominal aorta.

In one set of preliminary experiments, four mice that had not received any form of i.t. treatment were anesthetized as above and euthanized. The lungs and trachea were removed en bloc and bronchoalveolar lavage fluid (BALF) was obtained by instillation of two separate aliquots of 250 µl sterile PBS. BALF was serially diluted, inoculated onto Pseudomonas Isolation Agar, and incubated at 37°C overnight. The remaining BALF was centrifuged at 2000 rpm for 10 min at 4°C. The pellet was resuspended in PBS; the total cell count was established and cytospins were prepared to estimate differential cell count. The supernatant was stored at -40°C before further use.

In a further set of preliminary experiments mice were anesthetized as above and received an i.t. instillation of either Ad-elafin (3 x 107 PFU) suspended in PBS, Ad-lacZ (3 x 107 PFU) suspended in PBS, or PBS alone (n = 4 in each group). Five days later, mice were anesthetized in the same way and given i.t. PBS. After 24 h, mice were euthanized as above. BALF was prepared to establish P. aeruginosa PAO1 colony counts and differential cell counts as described above (with the exception that BALF was prepared using aliquots of 250 µl and then 200 µl).4

In the ensuing experiments, Ad-elafin (3 x 107 PFU) suspended in PBS, Ad-lacZ (3 x 107 PFU) suspended in PBS, or PBS alone were instilled by i.t. injection in a final volume of 40 µl (PBS group n = 18, Ad-lacZ group n = 20, Ad-elafin group n = 16). Five days later, mice were anesthetized in the same way, and a direct i.t. instillation of P. aeruginosa PAO1 was administered (~2.2 x 1011 CFU/ml suspended in 0.01 M phosphate buffer; final volume, 34 µl). In preliminary experiments, we found that P. aeruginosa PAO1 at this predetermined dose consistently produced a sublethal pneumonia. Twenty-four hours after administration of P. aeruginosa PAO1, mice were anesthetized and euthanized by transection of the abdominal aorta. Whole blood was retrieved; an aliquot of 100 µl was inoculated onto Pseudomonas Isolation Agar and incubated at 37°C overnight. The spleen was stored in 0.01 M phosphate buffer and homogenized for 30 s using an Omni hand-held homogenizer (Camlab, Cambridge, U.K.); an aliquot of 100 µl was inoculated onto Pseudomonas Isolation Agar and incubated at 37°C overnight. The lungs and trachea were removed en bloc, and BALF was obtained as described above. P. aeruginosa colony counts and differential cell counts in BALF were established as described above. The left lung was weighed, snap frozen in liquid nitrogen, and stored at -80°C. Immediately upon thawing, the lung was placed in homogenization buffer (100 mM sodium acetate, 20 mM EDTA, 1% hexadecyltrimethylammonium bromide (all Sigma)) as described elsewhere (34), and homogenized for 1 min. The homogenate was centrifuged at 10,000 rpm for 30 min at room temperature. The supernatant was used immediately to determine the concentration of myeloperoxidase (MPO) in the homogenized lung (as an index of the number of neutrophils in both pulmonary parenchyma and circulation) (34).

In separate experiments, three additional mice from each group (i.e., PBS then P. aeruginosa PAO1; Ad-lacZ then P. aeruginosa PAO1; Ad-elafin then P. aeruginosa PAO1) were euthanized 24 h after administration of P. aeruginosa PAO1, and the lungs were removed en bloc before fixing in formalin and embedding in paraffin wax. Sections 3 µm thick were cut and stained using hematoxylin and eosin.

Assays

Levels of human elafin Ag were measured using a sandwich ELISA available in-house, performed on 96-well plates (Linbro; Flow Laboratories, McLean, VA). In brief, the primary Ab was polyclonal rabbit anti-human elafin Ig, gelatin (BDH, Poole, U.K.) was used as a blocking agent, the sample (or purified recombinant elafin as standard) was applied, and the secondary Ab was biotinylated polyclonal rabbit anti-human elafin Ig (biotin was from Pierce, Rockford, IL). Streptavidin-HPO complex (Sigma) and then chromogenic substrate (2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid); Sigma) were added sequentially and the OD550 of the product was quantified using a Dynatech M5000 Plate Reader (Dynex, Billingham, U.K.).

Elastase activity was measured by applying the elastase-specific chromogenic substrate N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Sigma), and measuring change of OD405. Elastase-inhibitory activity (EIA) was measured by adding sample (serially diluted in buffer (50 mM Tris, 0.1% Triton, 0.5 M sodium chloride, pH 8); final volume, 10 µl), or buffer alone to a known quantity of purified HNE (50 ng in 10 µl) and incubating for 30 min at 37°C before adding 50 µl N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide and measuring change of OD405. Plots of OD (reflecting HNE activity) against concentration of sample were constructed. As described elsewhere (35), EIA was derived from extrapolation of the curve to the abscissa.

MPO activity was calculated by addition of chromogenic substrate (0.1 mg/ml tetramethylbenzidine (DAKO, Denmark), 0.03% hydrogen peroxide (Sigma) in 0.1 M sodium acetate, pH 4.9) and measurement of the change of OD630. To measure MPO activity in homogenized lung supernatants, samples were diluted in homogenization buffer, and 100-µl aliquots (in triplicate) added to 100 µl chromogenic substrate. To measure MPO activity in BALF, 50-µl aliquots of BALF were treated with 10 µl sodium acetate buffer, pH 4.2, before addition of 100 µl chromogenic substrate.

Protein was measured using the bicinchoninic acid method (Pierce) using purified albumin (Pierce) as standard.

Commercial ELISA kits were used to measure concentrations of murine albumin (Bethyl Laboratories, Montgomery, TX) and human IL-8 (R&D Systems, Abingdon, U.K.).

Measurements of murine keratinocyte-derived chemokine (mKC) concentration in BALF, using an ELISA, were kindly performed by Professor T. Standiford (University of Michigan, Ann Arbor, MI).

Statistics

The parameters studied were not normally distributed, and nonparametric tests were applied. For comparisons involving three groups, the Kruskal-Wallis test was used, and comparisons between pairs of groups were performed using the Mann-Whitney U test. Paired data were studied using Wilcoxon’s rank sum test. Nominal data were compared using the {chi}2 test. Correlations were studied using Spearman’s rank correlation test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of adenoviral augmentation of elafin on damage mediated by HNE and activated human neutrophils in vitro

Transfection of A549 cells with Ad-elafin resulted in a dose-dependent increase in both secretion of elafin Ag and antielastase activity, indicating that elafin production was efficient and that the elafin produced was functionally active (Fig. 1Go). Adenoviral transfection did not increase secretion of IL-8 above that from untransfected cells. Indeed Ad-lacZ and Ad-elafin transfection (each at an moi of 50 PFU) resulted in IL-8 levels that were, respectively, 54 and 63% of those from untransfected cells (n = 3; p = 0.05 comparing the three groups).



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  		FIGURE 1. Transfection of pulmonary epithelial cells with Ad-elafin results in dose-dependent production of elafin and anti-elastase activity. A549 cells were treated with Ad-elafin (moi 0–50 PFU) or Ad-lacZ (moi 0–50 PFU) for 30 min at 37°C. Cells were washed with PBS to remove residual virus and then incubated overnight at 37°C in medium containing serum. Cells were extensively washed with PBS to remove serum and incubated at 37°C for 24 h in serum-free medium. Supernatants were retrieved 16 h later. Two aliquots of supernatant were prepared. A known quantity of HNE was added to the first aliquot, and residual HNE activity measured using a chromogenic substrate assay specific for HNE. In the second aliquot, elafin concentration was determined by ELISA. Bottom, Dose-dependent increase in elafin secretion after Ad-elafin transfection (heavy line) but not after Ad-lacZ transfection (broken line); top, corresponding dose-dependent generation of antielastase activity after Ad-elafin (heavy line), but not Ad-lacZ (broken line) transfection.

 
Ad-elafin significantly protected A549 cells against injury induced by HNE as assessed morphologically (Fig. 2Go, a–c). This observation was supported by a significant reduction in the number of cells released from pulmonary epithelial monolayers after treatment with HNE (Fig. 2God). The protection conferred was associated with significant and almost complete inhibition of HNE (Fig. 2Goe).



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  		FIGURE 2. Ad-elafin transfection protects pulmonary epithelial cells against HNE in vitro. A549 cells were treated with Ad-elafin (moi 50 PFU), Ad-lacZ (moi 50 PFU), or medium alone (untransfected cells) for 30 min at 37°C. Cells were washed with PBS to remove residual virus and then incubated overnight at 37°C in medium containing serum. Cells were extensively washed with PBS to remove serum and incubated at 37°C for 48 h in serum-free medium. HNE was then added directly to the supernatant (final concentration, 4 µg/ml) and incubated at 37°C for 16 h. Photomicrographs of cell layers were taken (using a blue filter to optimize contrast), cells in the supernatant were counted, and the residual HNE activity of supernatants was measured using a chromogenic substrate assay specific for HNE. a, Untransfected cells, with no HNE added; b, Ad-lacZ-treated cells, after addition of HNE (appearances of untransfected cells treated with HNE were almost identical); c, Ad-elafin-treated cells, after addition of HNE; d, cell counts in supernatants retrieved after addition of HNE (n = 6). Results were expressed relative to the count in supernatants from untransfected cells, which was regarded as 100% in each experiment. Values represent medians (and interquartile ranges) from the six experiments performed. **, Significantly lower compared with either of the other treatments, p < 0.01. e, Residual HNE activity in supernatants (n = 6). Results were expressed relative to the HNE activity in supernatants from untransfected cells, which was regarded as 100% in each experiment. The values shown represent medians (and interquartile ranges) from the six experiments performed. ***, Significantly lower compared with each of the other treatments, p < 0.005.

 
When human neutrophils, activated with a combination of priming agents (PAF) and secretagogues (fMLP), were used in place of purified HNE, morphological evidence of protection was again conferred by Ad-elafin (Fig. 3Go, a and b). This was associated with a reduction in release of 111In from radiolabeled epithelial cells (Fig. 3Goc). The protection conferred by Ad-elafin was associated with a significant reduction in measured HNE activity (Fig. 3God).



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  		FIGURE 3. Ad-elafin transfection protects pulmonary epithelial cells against activated human neutrophils in vitro. A549 cells were treated as described in Fig. 2Go with the exception that incubation in serum-free medium was for 24 h, at which point 1% BSA (final concentration) was added. Freshly prepared human neutrophils (2 x 106) were added directly to the supernatant along with 10-9 M PAF (final concentration). At 1 h, 10-7 M fMLP was added (final concentration). Cells were incubated at 37°C for 16 h; photomicrographs were taken (with a blue filter to optimize contrast), and the residual HNE activity of supernatants was measured using a chromogenic substrate assay specific for HNE. In a variation of this experiment, before being washed to remove serum, cells were incubated with 3 µCi 111In in 4 x 10-4 M tropolone (final concentration). Cells were extensively washed to remove unincorporated radiolabel and serum and then incubated in serum-free medium for 24 h at 37°C. Neutrophils were added as above, and radiolabel was counted in supernatants at 16 h. a, Ad-lacZ-treated cells, after addition of activated human neutrophils (arrows). Discontinuation of epithelium is seen adjacent to clusters of neutrophils, indicating epithelial injury (appearances of untransfected cells after addition of neutrophils were almost identical). b, Ad-elafin treated cells, after addition of activated human neutrophils (arrows). Epithelium adjacent to clusters of neutrophils is intact, indicating protection against injury. c, Radiolabel in supernatants after addition of activated human neutrophils (n = 8). Results were expressed relative to the radiolabel retrieved in supernatants from untransfected cells, which was regarded as 100% in each experiment. Values represent medians (and interquartile ranges) from the eight experiments performed. §, Significant difference comparing Ad-elafin with Ad-lacZ, p < 0.05. d, Residual HNE activity in supernatants (n = 4). Results were expressed relative to the HNE activity in supernatants from untransfected cells, which was regarded as 100% in each experiment. Values represent medians (and interquartile ranges) from the four experiments performed. *, Significantly lower compared with each of the other treatments, p < 0.05.

 
Preliminary in vivo experiments

In mice receiving no i.t. treatments (i.e., normal mice), the median total cell count in BALF was 1.84 x 105, with macrophages comprising >95% of the count in all animals. The remaining cells in BALF were almost exclusively neutrophils, as is characteristic of BALF from normal mice. Median protein concentration in BALF was 0.45 g/L (range, 0.43–0.53 g/L). In mice receiving i.t. vector and/or PBS, very similar trends were observed (Table IGo).Total cell counts were similar, and the alveolar macrophage was the predominant cell type, with the neutrophil comprising a median of 0.7% of cells in BALF among PBS/PBS mice, 3.4% in Ad-lacZ/PBS mice, and 0.6% of Ad-elafin/PBS mice (no significant difference). Similarly, adenovirus administration itself did not significantly influence the concentration of protein in BALF characteristic of untreated animals or those treated sequentially with vehicle alone (Table IGo). Therefore, adenovirus administration per se was not associated with significant airway inflammation as assessed by cellular content and protein concentration of BALF. No bacterial growth was obtained on Pseudomonas Isolation Agar plates inoculated with BALF (Table IGo), blood, homogenized spleen, or homogenized lung from untreated, vehicle-treated, or virus-treated mice, reflecting the absence of either commensal or contaminating P. aeruginosa in our laboratory.


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  		Table I. Ad-elafin administration is not associated with a significant rise in neutrophil count or protein concentration in BALF1

 
Effects of Ad-elafin transfection on lung injury mediated by P. aeruginosa in vivo

Delivery of Ad-elafin i.t. followed by P. aeruginosa PAO1 resulted in a median human elafin concentration in BALF of 8.5 ng/ml (interquartile range, 5.9–10.4 ng/ml). No human elafin was detected in any of the mice given Ad-lacZ (as viral control) or PBS (as nonviral control) and then P. aeruginosa PAO1, confirming that antielafin IgG does not cross-react with murine or bacterial proteins. Histologically, the administration of P. aeruginosa PAO1 in control mice was associated with a patchy, multilobar pneumonia, associated with extravasation of neutrophils into alveolar airspaces (Fig. 4Go, a and c) with associated protein leak. These effects were much less pronounced in mice receiving Ad-elafin (Fig. 4Gob) in which there was a significant reduction in the concentrations of protein and albumin in BALF (Fig. 4God).



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  		FIGURE 4. Ad-elafin transfection confers protection against acute lung injury induced by P. aeruginosa in vivo. C57BL/6 mice were given an i.t. dose of one of the following: Ad-elafin (3 x 107 PFU); Ad-lacZ (3 x 107 PFU); or PBS. Five days later, P. aeruginosa PAO1 was given i.t. (34 µl of a suspension of ~2.2 x 1011 CFU/ml). After 24 h, the mice were sacrificed, and BALF was retrieved. One aliquot of BALF was cultured overnight to determine the number of P. aeruginosa PAO1 colonies, and another was centrifuged and used to determine protein and albumin concentrations. In a variation of this experiment, lungs were removed en bloc, fixed in formalin, and examined histologically. a, Histological appearance of lung from a representative mouse treated with PBS and then P. aeruginosa PAO1; b, histological appearance of lung from a representative mouse treated with Ad-elafin and then P. aeruginosa PAO1; c, histological appearance of lung from a representative mouse treated with Ad-lacZ then P. aeruginosa PAO1; d, protein concentration in BALF, albumin concentration in BALF, and P. aeruginosa PAO1 colonies in BALF (results represent medians and interquartile ranges). *, Significant difference by Kruskal-Wallis test, p < 0.05: when comparing groups directly, for each parameter, the difference between Ad-elafin/PAO1 and PBS/PAO1 was significant, p < 0.05; Ad-elafin/PAO1 was significantly different from Ad-lacZ/PAO1 with respect to protein and PAO1 colonies (p < 0.05) but not albumin concentration; no significant differences were observed comparing Ad-lacZ/PAO1 and PBS/PAO1.

 
The protection conferred by Ad-elafin was associated with a significant reduction in the quantity of bacteria retrieved from BALF (Fig. 4God). Indeed, among the mice receiving Ad-elafin, the concentration of elafin in BALF significantly and inversely correlated with the number of P. aeruginosa PAO1 colonies (log transformed) in BALF (r = -0.75; p = 0.02). Administration of Ad-elafin was also associated with a significantly lower incidence of bacterial growth in blood (colonies detected in 31% of Ad-elafin mice, 44% of Ad-lacZ mice, and 61% of PBS mice, p < 0.05) and spleen (colonies detected in 19% of Ad-elafin mice, 40% of Ad-lacZ mice, and 44% of PBS mice, p < 0.05).

In none of the mice studied was elastase activity detected in BALF, reflecting a relative excess of elastase inhibitors in BALF from all animals. However, the total EIA in BALF was lower in the Ad-elafin group than in either of the control groups (Fig. 5Go).



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  		FIGURE 5. Protection against P. aeruginosa PAO1 conferred by Ad-elafin is associated with a reduction in EIA. BALF obtained from the mice described in Fig. 4Go was analyzed for EIA. EIA was determined by adding sample (serially diluted in buffer (Tris 50 mM, 0.1% Triton, 0.5 M sodium chloride, pH 8)) or buffer alone to a known quantity of purified HNE then adding N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide and measuring change of OD405. Plots of OD (reflecting HNE activity) against concentration of sample were constructed. Extrapolation of the curve to the abscissa determined the EIA of each sample. Values represent medians, with bars indicating interquartile ranges. {wedge}, Significant difference comparing treatment with Ad-elafin then P. aeruginosa PAO1, and treatment with PBS then P. aeruginosa PAO1, p < 0.05.

 
In addition, BALF from mice receiving Ad-elafin contained significantly lower concentrations of the murine neutrophil chemokine mKC than did BALF from mice receiving control virus (Table IIGo). In keeping with this finding, a trend was observed toward lower concentrations of MPO in homogenized lung (reflecting lung neutrophil count) in mice receiving Ad-elafin (Table IIGo). Indeed, considering all mice together, there was a significant correlation between the concentration of mKC in BALF and the concentration of MPO in homogenized lung (r = 0.43, p < 0.005). A trend toward lower levels of free MPO in BALF (reflecting neutrophil degranulation products) was also found in mice receiving Ad-elafin (Table IIGo).


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  		Table II. Administration i.t. of Ad-elafin is associated with a trend toward reduced pulmonary neutrophilia1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We believe these data represent the first description of protection against acute lung injury conferred by genetic augmentation of a human pulmonary antielastase/antimicrobial peptide. The findings support and advance the concept that gene therapy may be used to obviate acute lung injury generally and that caused by bacterial infection specifically by manipulation of host defense molecules (22, 23, 24, 25, 26).

Our data indicate that Ad-elafin is capable of protecting A549 cells against acute injury mediated by HNE (Fig. 2Go). Untransfected A549 cells constitutively produce small quantities of antielastases, including elafin (36), but these were overwhelmed by the concentrations of HNE applied here (Fig. 2Gob), resulting in cell damage. In this context, it is interesting both that a similar concentration of HNE may be found in sputum from patients with CF (3) and that high concentrations of HNE down-regulate constitutive expression of elafin (37). In contrast Ad-elafin transfection augmented elafin secretion sufficiently to protect A549 cells significantly (Fig. 2Goc).

Extending these findings, we found that Ad-elafin protected A549 cells against human neutrophils maximally activated by a combination of a priming agent and a secretagogue (Fig. 3Go). Despite the capability of neutrophils to liberate a plethora of potentially cytotoxic agents during activation, the selective inhibition of HNE (and proteinase 3) by elafin resulted in a marked reduction in epithelial injury. This extends our previous observation that HNE is specifically important in neutrophil-mediated injury of endothelial cells (31) and considerably adds to the weight of evidence that HNE is centrally important in neutrophil-mediated tissue injury. Furthermore, the finding that the protection conferred by elafin was associated with functional antielastase activity suggests that, under the experimental conditions described, the methionine in the antielastase site of elafin (38, 39) is resistant to the effects of oxidants released by activated neutrophils.

The epithelial protection associated with Ad-elafin treatment in vitro provided the necessary proof of principle to proceed with gene therapy studies using Ad-elafin in vivo. P. aeruginosa PAO1 was used in these experiments because it is arguably the most studied strain of P. aeruginosa with respect to genetic background (32), virulence, and animal models. Furthermore we have shown that elafin has antimicrobial activity against P. aeruginosa PAO1 in vitro (16). The experimental approach used in this study is worthy of discussion. Firstly the i.t. route used was simple, effective, and minimally invasive (33). This approach efficiently generated human elafin in murine airways; previous immunohistochemical studies have demonstrated human elafin expression in surface epithelia from conducting airways and in alveolar epithelium (data not shown). Secondly, significant human elafin was attained using a dose of adenovirus that produced little, if any, overt airway inflammation (Table IGo). We cannot exclude a low grade inflammation of the airways induced by adenovirus (indeed, a small rise in relative neutrophil count was observed in mice treated with Ad-lacZ). However, the variations in neutrophil count after vector administration were small, and the effect on epithelial integrity minimal given that BALF protein concentrations in vector-treated mice were similar to those in normal and vehicle-treated mice. The small fluctuations in neutrophil count observed in vector-treated mice may be considered negligible when set against the characteristically marked neutrophilia seen when such mice are exposed to low doses of inflammatory mediators such as LPS. Importantly, in the context of this study, we can be confident that adenoviral vector per se did not significantly influence outcome in mice receiving bacteria, given that PBS-PAO1 mice had a neutrophil count in BALF of 8.2 x 106 (~200 times the number in mice treated with vector then PBS (Table IGo)) and a protein concentration of 5.7 g/L (~15 times the concentration in mice treated with vector then PBS (Table IGo and Fig. 4Go)). We are not aware of studies demonstrating significant protective effects after doses of adenovirus as low as those used here. Therefore, it appears that gene therapy protocols using sufficiently powerful promoters (17, 18) and low concentrations of vector may, at least in part, be able to circumvent important concerns relating to adenovirus-mediated immune responses (40, 41) and inefficiency of transfection of surface epithelium in the lung (42). Recent advances in vectorology are also likely to improve adenoviral gene delivery to the airways (43).

In our model of murine P. aeruginosa lung injury, lungs from control mice showed patchy multilobar consolidation, in keeping with histological appearances in human pneumonia associated with this pathogen (Fig. 4Go). The appearances were associated with a rise in BALF protein, in turn reflecting disruption of the alveolar-capillary membrane (44, 45). The significant protection against lung injury consequent on Ad-elafin transfection, as demonstrated by BALF protein levels, was also associated with significantly enhanced elimination of bacteria from the airways (Fig. 4Go) and with a significantly lower incidence of hematogenous bacterial dissemination. Elafin therefore appears to be part of a network of endogenous pulmonary antibiotics which includes the defensins (14, 16, 46).

The neutrophil may potentially have advantageous or detrimental effects in pulmonary inflammation (Ref. 47 ; reviewed in Ref. 48). Excessive activation of neutrophils certainly appears to be associated with the potential for tissue damage (48). In the mice studied here, the presence of MPO in BALF provided evidence of neutrophil degranulation and by implication neutrophil activation (Table IIGo). Elafin augmentation was associated with a modest reduction in BALF MPO, potentially supporting the inhibition of neutrophil-mediated damage seen in vitro (Table IIGo and Fig. 3Go). Elafin augmentation was also associated with a reduction in the neutrophil chemokine mKC, and with a corresponding reduction in total lung neutrophils (Table IIGo). Importantly, the relative reduction in pulmonary neutrophilia associated with elafin augmentation did not preclude tissue protection, despite the observation that neutrophilia correlates with reduced mortality in pulmonary infection caused by Nocardia (47).

Interestingly, no free murine elastase activity was detected in BALF from any of the mice studied. This can be attributed to the characteristic elevation of EIA consequent on severe lung injury, comprising part of the acute phase response (49). We calculate that human elafin contributed less than 1% of the EIA of BALF in mice receiving Ad-elafin. Taken together, these findings suggest that a direct anti-neutrophil elastase effect does not explain the protective effect of Ad-elafin, in keeping with the growing body of evidence that pulmonary anti-neutrophil elastases harbor several functions in addition to inhibition of neutrophil elastase (14, 15, 16, 50, 51, 52, 53). Furthermore, inhibition of Pseudomonas elastase (a metalloelastase) does not explain the effects of Ad-elafin in this study, because elafin is an inhibitor of serine proteinases and not metalloproteinases. Against this background, the reduction in EIA associated with Ad-elafin administration (Fig. 5Go) reflects a significant and appropriate down-regulation in the acute phase response as a consequence of tissue protection conferred by Ad-elafin. Thus, the protective anti-inflammatory effect of elafin is associated with antimicrobial activity, with a reduction in neutrophils in lung tissue, and with a reduction in the characteristic host response to tissue injury.

The relative level of protection provided by Ad-elafin in our in vivo studies is worthy of note. Ad-elafin effected a 40% reduction in BALF protein concentrations as compared with control mice (Fig. 4Go). Given both the severity of injury in the control mice and the remarkable complexity and redundancy in pulmonary anti-inflammatory responses (6), the degree of protection after one administration of a single human gene is encouraging. Interestingly, a nonsignificant trend also emerged potentially suggesting a weak protective effect for adenovirus over vehicle alone (indeed the Ad-lacZ group was included specifically to control for confounding effects of adenovirus). This was manifest as a trend toward lower levels of IL-8 secretion and greater with cytoprotection (Fig. 2God) in vitro and reduction in albumin concentration and EIA in vivo (Figs. 4Go and 5Go). However, the effect seemed most pronounced with regard to bacterial loads, with strikingly (although not statistically significantly) lower levels of bacteria in BALF (Fig. 4Go) and blood in Ad-lacZ/PAO1 mice as compared with PBS/PAO1 mice. Although it must be emphasized that these trends did not reach statistical significance (whereas differences between Ad-elafin/PAO1 mice and Ad-lacZ/PAO1 mice generally did attain significance), it is tempting to speculate that adenoviral treatment per se may have contributed in some way to bacterial eradication by priming innate immunity. A similar trend has been observed in studies in which rats received sequential i.t. administrations of adenovirus encoding Fc{gamma}R and P. aeruginosa PAO1; delivery of control adenovirus (a null adenovirus, expressing no foreign transgene) resulted in greater clearance of Pseudomonas than did PBS, thus further suggesting stimulation of innate immunity by adenovirus per se (54). Interestingly, stimulation of adaptive immunity by recombinant null adenoviruses has also been recognized (55), and priming of immune mechanisms has also been attributed to Ad-lacZ in gene therapy protocols directed at lung tumors (56).

Overall, our data provide proof of principle for the prevention of inflammatory lung injury using augmentation of elafin, particularly in the context of infection with antibiotic-resistant pathogens. P. aeruginosa PAO1 is classically associated with antibiotic resistance, and the mechanisms responsible have been extensively characterized (21, 32). More specifically, the approach we describe may potentially find application in preventing colonization with P. aeruginosa in CF and in the setting of patients at risk of pneumonia attributable to P. aeruginosa in intensive care units (57). Nosocomial Pseudomonas pneumonia is associated with a high mortality despite conventional treatment, due in part to antibiotic resistance (21, 58). Current opinion suggests that endogenous cationic peptide antibiotics, such as elafin, may be less susceptible to resistance than are conventional antibiotics (59).

In summary, genetic augmentation of elafin proved effective at protecting pulmonary epithelium against neutrophil-mediated injury in vitro and against acute injury induced by P. aeruginosa in vivo. These data support the concept that endogenous defense molecules can contribute to innate immunity by protecting tissue against microbial damage in vivo, and in so doing they suggest novel therapeutic strategies.


   Acknowledgments
 
We thank Professor T. Standiford for help in performing mKC ELISAs. We are grateful to Prof. J. Gauldie and D. Chong (both McMaster University, Hamilton, Ontario, Canada) for suppling Ad-laZ. We also thank many colleagues for helpful advice in preparation of this work, particularly Dr. G. Cunningham, Dr. D. Davidson, Dr. G. McLachlan, Dr J. Murray, and Dr. T. Walker (all at the University of Edinburgh, Edinburgh, U.K.).


   Footnotes
 
1 This work was supported by the Wellcome Trust (Clinical Training Fellowship to A.J.S.) and the Norman Salvesen Emphysema Research Trust (grant to J.-M.S.). Back

2 Address correspondence and reprint requests to Dr. Jean-Michel Sallenave, Rayne Laboratory, Respiratory Medicine Unit, Medical School, University of Edinburgh, Edinburgh EH8 9AG, U.K. E-mail address: j.sallenave{at}ed.ac.uk Back

3 Abbreviations used in this paper: HNE, human neutrophil elastase; Ad-elafin, E1-, partially E3-deleted adenovirus encoding human elafin cDNA; Ad-lacZ, E1-, partially E3-deleted adenovirus encoding lacZ cDNA; BALF, bronchoalveolar lavage fluid; CF, cystic fibrosis; EIA, elastase-inhibitory activity; i.t, intratracheal(ly); mKC, murine keratinocyte-derived chemokine; moi, multiplicity of infection; MPO, myeloperoxidase; PAF, platelet-activating factor; SLPI, secretory leukocyte protease inhibitor. Back

4 Other data relating to these control mice have been submitted elsewhere, but the data presented in this article do not appear elsewhere. Back

Received for publication February 6, 2001. Accepted for publication May 21, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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