Is neutrophil elastase the missing link between emphysema and fibrosis? Evidence from two mouse models

The study was carried out by using strains of mice with different levels of serum antielastase screen (Balb/c, C57 Bl/6 and pallid, with normal, intermediate and low screen, respectively) [16]. Male mice, weighing 20 to 25 g (8–10 wk old) were treated under ether anaesthesia with a single intratracheal instillation of 0.1 μg BLM (Rhone-Poulenc Rorer, Milano, Italy) in saline solution (50 μl) or with the same amount of saline. At 3, 7, 14, 21, 28 and 35 days after the treatment, animals were killed by pentobarbital sodium overdose and exsanguinated by cutting the abdominal aorta. Lungs were used for immunohistochemistry of TGF-β and TGF-α and for determination of the mean linear intercept (Lm) [17] as well as the percent volume densities of fibrosis Vv(f) and emphysematous changes Vv(e). Details of the morphometric assessment are given below. Lungs were also assessed for desmosine [18] and for determination of the elastase burden by immunoelectron microscopy [19].

Two months old male mice of the strain DBA/2 were exposed to either the smoke of 3 cigarettes/day (commercial Virginia filter cigarettes: 12 mg of tar and 0.9 mg of nicotine), 5 days/week, or to room air (controls), for various periods of time (from 1 to 6 months) as previously described in detail [20]. Groups of 8 animals were sacrificed during the chronic exposure period at various time intervals. The lungs were excised and processed for histology. Histological sections were stained with hematoxylin-eosin and Masson's trichrome. Morphometric assessment of emphysema included the determination of the Lm [17] and of the internal surface area (ISA) estimated by the Lm method at postfixation lung volume [21]. Fibrosis was assessed by point counting as Vv(f) as described below. Tissue sections were also stained for TGF-β, TGF-α and NE.

To quantitate lung elastin, the lungs of each group were weighed, homogenised (1:5, w:v) and hydrolysed in 6 N HC1 before biochemical determinations. Desmosine was analysed on hydrolysates by means of an enzyme-linked immunosorbent assay essentially according to Cocci et al. [18]. Briefly, rabbit antiserum to desmosine-hemocyanin conjugate (Abl) (Elastin Product Company, Inc, Owensville, MO) was incubated with desmosine standard (0–30 ng) (Elastin Product Company, Inc, Owensville, MO) or with adequately diluted hydrolysates for 16 h at room temperature. At the same time, microtiter plates (Sigma) were incubated with 0.5 μg of desmosine-albumin conjugate (Elastin Product Company, Inc, Owensville, MO) in 0.05 M sodium carbonate buffer pH 9.6 at 4°C. After incubation, wells were washed five times with 0.05% Tween 20 in 0.15 M PBS, pH 7.2 and saturated with 0.05% Tween 20 in 0.15 M PBS, 1% BSA pH 7.2 for 1 h at room temperature. Eightfold aliquots of AbI-standard or AbI-sample solutions were then added to the wells for a 2 h incubation at room temperature. Wells were then in succession incubated with anti-rabbit IgG (1:2000) (Sigma) for 2 h at room temperature and with peroxidase-antiperoxidase complex (1:200) (Sigma) for 1 h at room temperature. 2,2'-Azino-bis (3-ethyl-benz-thiazoline-6-sulfonic acid) solution (Sigma) was then added to the wells. After incubation for 1 h at room temperature, absorbance was read at 405 nm. Data were expressed as μg/lung.

The lungs from the different groups of mice were fixed by intra-tracheal inflation with buffered formalin (5%) at a constant pressure of 20 cm H₂O at least for 24 hours. All lungs were then dehydrated, cleared in toluene, and embedded in paraffin. Sagittal sections (7 μm) of each pair of lungs were cut and stained with hematoxylin/eosin and Masson's trichrome stain. Morphometric assessment consisted of the determination by point counting, of the percent volume densities of fibrosis Vv(f) and of the emphysematous changes Vv(e) according to the stereological principle of Glagoleff and Weibel [22]: Vv = Pp, where Vv is the volume density and Pp the fraction of points superimposed a defined structural change. Point counting was performed at 100 × by determining 20 random fields per slide and using a multipurpose grid to count 45 points per field for a total of 900 points per slide. Fibrosis was defined as: "inflammatory and mesenchymal cell infiltration within the alveolar septa and alveolar spaces with deposition of extracellular matrix", and emphysematous changes were defined as: "abnormal enlargement of air spaces with loss of alveolar septa, and with or without thickening of the alveolar walls" [4].

The morphometric assessment of emphysema was also performed in all animals by determining the average inter-alveolar distance (mean linear intercept: Lm) [17]. For the determination of the Lm for each pair of lungs, 40 histological fields were evaluated both vertically and horizontally. Care was taken to avoid histological fields containing large bronchi, major vessels and areas of fibrosis.

For the immunohistochemical studies, tissue sections (8 μm) were stained for TGF-β, TGF-α and NE by an immunoperoxidase method. The sections were pre-treated with 3% hydrogen peroxide to inhibit the activity of the endogenous peroxidase. For antigen retrieval, the sections were heated in a microwave for 20 min in citrate buffer 0.01 M, pH 6.0, and allowed to cool slowly to room temperature. All the sections were incubated with 3% bovine serum albumin for 30 min at room temperature to block non-specific antibody binding. They were then incubated overnight at 4°C with the primary antibodies (Ab). The primary polyclonal Ab used were: rabbit Ab to mouse TGF-a diluted 1:50 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), rabbit Ab to mouse TGF-α diluted 1:20 (Insight Biotechnology LTD., Wembley, England). For elastase detection we used rabbit Ab to human neutrophil elastase (cross-reacting with mouse neutrophil elastase) diluted 1:500 (Calbiochem-Novabiochem, San Diego, CA). All the sections were rinsed and incubated with sheep anti-rabbit IgG for 30 min at room temperature. The staining was revealed by adding peroxidase-antiperoxidase complex, prepared from rabbit serum. Detection was accomplished by incubating in diamino-benzidine freshly dissolved in 0.03% H₂O₂ in 50 mM Tris-HCl pH 7.6. As negative controls for the immunostaining the primary antibodies were replaced by non-immunised rabbit serum. The sections were counterstained with hematoxylin.

The immunogold method (post-embedding technique) was used to localize elastase in thin lung sections prepared for electron microscopy using anti-mouse neutrophil elastase (MNE) antibodies obtained as previously described [19]. Lung tissue blocks (5/animal) were taken from two animals from each group. The blocks, 1 to 2 mm in thickness, were fixed for 3 hours in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), dehydrated in acetone, and embedded in epoxy resin (Araldite) without postfixation in OsO₄. Ultrathin sections (600 Å thick) were picked up on nickel grids and pretreated with phosphate-buffered saline containing 1% ovalbumin for 5 minutes. The grids were then floated on a drop of diluted anti-MNE antibodies (1:2600) for 48 h at 4°C; the grids were thoroughly rinsed for 10 min with a mild spray of phosphate-buffered saline and then distilled water and transferred onto 15 μl drops of a protein A-gold particles (15 nm) (E-Y-LABS, San Mateo, CA) solution diluted 1:8 in phosphate-buffer saline. The sections were then washed, dried, stained with uranyl acetate-lead citrate, and examined in a Philips 300 electron microscopy. Ten to 12 micrographs (final magnification, × 12,000) were taken for each grid. To exclude false-positive labeling, a series of control studies (including also the use of nonimmune rabbit serum or of BSA instead of ovalbumin) were carried out as previously described in detail [19]. The density of gold particles per square micrometer of lung tissue was determined for each of the micrographs with a superimposed quadratic lattice grid. A total of 50 micrographs was thus analyzed for each animal, and the average of gold particle density on lung connective tissue of each group was calculated.

The kinetics of the emphysematous and fibrotic changes obtained in the three strains of mice are reported in Fig 1 A and 1 B. In particular, we present the percent values of lung volume densities of fibrotic (Vv(f)) and emphysematous (Vv(e)) changes obtained at the various time points. In BLM-resistant Balbc mice with a normal antiprotease protection, negligible foci of cellular infiltration and fibrosis and no areas of air-space enlargements were detected during the period of the study. On the contrary, a progressive increase of areas of air space enlargement and fibrosis were seen in BLM-prone C57 Bl/6 and pallid mice with a mild and a marked deficiency of α1-PI, respectively.

At 3 and 7 days after BLM, C57 Bl/6 and pallid mice showed appreciable morphologic emphysema with spotty areas of inflammatory cell infiltration in the absence of fibrotic changes (Figs 2 and 3). At these times the anatomical emphysema was associated with a significant increase of the Lm (Fig. 4 A) a significant decrease in lung desmosine content (Fig. 4 B) and a high neutrophil elastase burden (Fig. 4 C).

At 14 days after BLM, air spaces enlargements affected 6,68 ± 3.11 % and 12.71 ± 4.17 % (p < 0.01) of lung in C57Bl/6 and pallid mice, respectively (Fig. 1 A). At this time, the lungs of C57 Bl/6 and pallid mice showed also large areas of fibrosis which involved 24.11 ± 8.81 and 36.84 ± 11.47 % of the lungs, respectively (Fig. 1 B). Both lesions were widely spread and intermixed (Fig. 5 A). Nevertheless, areas of emphysema could also be detected in lung lobes without any fibrotic reaction. Additionally, in several areas the emphysematous changes were distant from the fibrotic foci (Fig. 5 B and 5 C).

At later times (21 days onward), the volume density of emphysematous and fibrotic changes markedly increased (Fig. 1 A and 1 B). From a morphological point of view, the emphysematous lesions appeared to be mainly of paracicatricial type (Fig. 6 A and 6 B). Nevertheless, large areas of emphysema could also be found in lung lobes without obvious fibrosis or situated adjacent to the fibrotic lesions (Fig. 6 C).

Lungs of mice from the three strains were also analysed for cytokine expression by immuno-histochemistry. A significant change of some cytokines related to the NE activity was observed in mice with a mild (C57 Bl/6) and a marked deficiency (pallid) of αl-PI, after BLM administration. In particular, TGF-β was detected in subpleural foci of cellular proliferation at 7 days (Fig. 7 A) when an increase of the elastase burden could also be demonstrated. Also at 7 days, an evident staining for TGF-α was observed in subpleural and peribronchiolar areas (Fig. 7 B).

The treatment of animals with 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, a serine-proteinase inhibitor, significantly prevented the BLM-induced lesions in C57 Bl/6 mice. In particular, treated animals showed 14 days after BLM treatment no areas of emphysema and only trivial foci of fibrotic reaction (Fig. 8 A, and 8 B). The Lm values (40.75 ± 0.91 μm) and the lung (Vv(e)) (0.93 ± 0.89 %) in these mice were not significantly different from those observed in control mice (Lm: 39.71 ± 0.80 μm; Vv(e): 0.26 ± 0.74 %). In addition the lung (Vv(f)) (14.13 ± 6.21 %) in mice receiving BLM plus proteinase inhibitor was significantly lower (p < 0.01) than that observed in mice receiving only BLM (24.11 ± 9.41 %).

No immunological reaction for TGF-α and a faint positive staining TGF-β was found 7 days after BLM administration in animals treated with 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (data not shown).