Is neutrophil elastase the missing link between emphysema and fibrosis? Evidence from two mouse models - PubMed (original) (raw)
Comparative Study
Is neutrophil elastase the missing link between emphysema and fibrosis? Evidence from two mouse models
Monica Lucattelli et al. Respir Res. 2005.
Abstract
Background: The separation of emphysema from fibrosis is not as clear-cut as it was thought in early studies. These two pathologies may be present at the same time in human lungs and in mice either instilled with elastolytic enzymes or bleomycin or exposed to cigarette-smoke. According to a current view, emphysema originates from a protease/antiprotease imbalance, and a role for antiproteases has also been suggested in the modulation of the fibrotic process. In this study we investigate in experimental animal models of emphysema and fibrosis whether neutrophil elastase may constitute a pathogenic link between these two pathologies.
Methods: This study was done in two animal models in which emphysema and fibrosis were induced either by bleomycin (BLM) or by chronic exposure to cigarette-smoke. In order to assess the protease-dependence of the BLM-induced lesion, a group mice was treated with 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, a serine proteinase inhibitor active toward neutrophil elastase. Lungs from each experimental group were used for the immunohistochemical assessment of transforming growth factor-beta (TGF-beta) and transforming growth factor-alpha (TGF-alpha) and for determination of the mean linear intercept as well as the percent volume densities of fibrosis and of emphysematous changes. Additionally, the lungs were also assessed for desmosine content and for the determination of elastase levels in the pulmonary interstitium by means of immunoelectron microscopy.
Results: We demonstrate that in BLM-treated mice (i) the development of elastolytic emphysema precedes that of fibrosis; (ii) significant amount of elastase in alveolar interstitium is associated with an increased expression of TGF-beta and TGF-alpha; and finally, (iii) emphysematous and fibrotic lesions can be significantly attenuated by using a protease inhibitor active against neutrophil elastase. Also, in a strain of mice that develop both emphysema and fibrosis after chronic cigarette-smoke exposure, the presence of elastase in alveolar structures is associated with a positive immunohistochemical reaction for reaction for both TGF-beta and TGF-alpha.
Conclusion: The results of the present study strongly suggest that neutrophil elastase may represent a common pathogenic link between emphysema and fibrosis. Proteases and in particular neutrophil elastase could act as regulatory factors in the generation of soluble cytokines with mitogenic activity for mesenchymal cells resulting either in emphysema or in fibrosis or both.
Figures
Figure 1
Emphysema and fibrosis after bleomycin challenge. The volume densities of emphysematous changes (Vv(e)) (A) and fibrosis (Vv(f)) (B) were quantitated by morphometry (point counting) on hematoxylin/eosin or Masson's trichrome stained lung sections, at various times after bleomycin. Data from 10 animals for each time point are given as mean ± SEM of per cent lung volume densities. *: p < 0.01 versus respective untreated controls (0 days).
Figure 2
Histological appearance of C57 BI/6 mouse lungs 3 and 7 days following bleomycin challenge. (A) Histologic section from the lung of a C57 Bl/6 mouse treated with saline showing a normal parenchyma. Representative histologic sections of C57 Bl/6 mice at 3 (B) and 7 (C and D) days after bleomycin treatment showing appreciable morphologic emphysema but not fibrosis. Scattered inflammatory cells are present through lung parenchyma (E). (E) Shows a higher magnification of (D). (A-C): Hematoxylin-eosin stain, scale bar represents 400 μm. (D) and (E): Masson's trichrome stain, original magnification × 40 and × 100, respectively. Scale bars represent 400 μm and 100 μm, respectively.
Figure 3
Histological appearance of pallid mouse lungs 7 days following bleomycin challenge. (A) Histologic section from the lung of a pallid mouse treated with saline showing a normal parenchyma. Lung sections of pallid mice at 7 days after bleomycin showing appreciable emphysema (B) and spotty areas of inflammatory cell infiltration without fibrosis (C). (A) and (B): Hematoxylin-eosin stain, scale bar represents 400 μm. (C): Masson's trichrome stain, scale bar represents 100 μm.
Figure 4
Mean linear intercepts, lung desmosine and elastase burden in various strains of mice following bleomycin challenge. (A) Mean linear intercepts (Lm) in Balb/C, C57 Bl/6 and pallid mice after bleomycin challenge. Data are from 10 animals for each time point and are given as mean ± SD. *: p < 0.01 versus respective saline-treated group. (B) Lung desmosine content in Balb/C, C57 Bl/6 and pallid mice after bleomycin treatment. Data from 10 animals for each time point are given as mean ± SD and represent per cent change over respective saline-treated controls (Balb/C: 2.50 ± 0.28 μg/lung; C57 BI/6: 2.48 ± 0.30 μg/lung; pallid: 2.44 ± 0.32 μg/lung). *: p < 0.01 versus respective saline-treated group. (C) Lung elastase burden in Balb/C, C57 Bl/6 and pallid mice at 7 days after bleomycin treatment. Data are given as mean ± SD of the number of gold particles per μm2. *: p < 0.01 versus respective saline-treated group.
Figure 5
Histological appearance of pallid and C57 Bl/6 mouse lungs 14 days following bleomycin challenge. Representative lung histologic sections of a pallid (A) and a C57BI/6 (B) mouse at 14 days after bleomycin. Fibrotic and emphysematous areas are widely spread and intermixed. Emphysema is often located quite distant from the fibrotic reaction (A-B).(C) shows a higher magnification of (B). (A): Hematoxylin-eosin stain, scale bar represents 400 μm. (B) and (C): Masson's trichrome stain, scale bars represent 400 μm and 100 μm, respectively.
Figure 6
Histological appearance of C57 BI/6 and pallid mouse lungs 21 days following bleomycin challenge. Histologic sections from lungs of a C57BI/6 (A and C) and pallid (B) mouse at 21 days after bleomycin. The emphysematous lesions within or adjacent to the fibrotic areas appear to be mainly of the paracicatricial type (A and B). Nevertheless, several areas of emphysema can be detected quite distant from the fibrotic zones (C). (A) and (C): Hematoxylin-eosin stain, scale bar represents 400 μm. (B): Masson's trichrome stain, scale bar represents 400 μm.
Figure 7
Immunohistochemical reaction for TGF-β and TGF-α 7 days following bleomycin challenge. Lung parenchyma of a C57BI/6 mouse at 7 days after bleomycin treatment. (A) Immunohistochemical reaction for TGF-β. Counterstained with hematoxylin, scale bar represents 40 μm. (B) Immunohistochemical reaction for TGF-α. Counterstained with hematoxylin, scale bar represents 25 μm.
Figure 8
Histological appearance of C57 BI/6 lung receiving 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride 21 days after bleomycin challenge. Representative histological section of a C57BI/6 mouse, receiving 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride and bleomycin, at 14 days after the treatment. No appreciable areas of emphysema are detectable in the lung parenchyma (A). Few trivial foci of fibrosis can be appreciated in some areas (B). (B) shows a higher magnification of (A). (A) and (B): Masson's trichrome stain, scale bars represent 400 μm and 50 μm, respectively.
Figure 9
Mean linear intercepts and lung internal surface areas of DBA/2 mice at various time-points during chronic cigarette smoke exposure. Mean linear intercept (LM) (A) and internal surface area /ISA) (B) of the lungs of DBA/2 mice at various time-intervals during chronic exposure to cigarette smoke. Data from 8 animals for each time point are given as mean ± SD. * p < 0.05 versus air-exposed controls.
Figure 10
Lung histology and immunohistochemical reaction for neutrophil elastase in DBA/2 mice 3 months after chronic cigarette smoke exposure. Lung parenchyma from an air-exposed (A) and a smoke-exposed DBA/2 (B) mouse, at 3 months. Hematoxylin-eosin stain, scale bar represents 400 μm. (C) and (D): Immunohistochemical reaction for neutrophil elastase on alveolar septa of DBA/2 mouse at 3 months after cigarette smoke exposure in absence (C) or in presence of the primary antibody (D). Scale bar represents 25 μm.
Figure 11
Histological appearance of DBA/2 lungs 4 and 6 months after chronic cigarette smoke exposure. Lung parenchyma from smoke-exposed DBA/2 mice, at 4 (A) and 6 months (B). The first foci of subpleural fibrosis are seen from 4 months of smoke exposure (A). After 6 months of cigarette smoke exposure, disseminated foci of severe emphysema and evident areas of subpleural fibrosis are present (B). (A) and (B): Hematoxylin-eosin stain, Scale bars represent 25 μm and 400 μm. respectively.
Figure 12
Immunohistochemical reaction for TGF-β and TGF-α 3 months after chronic cigarette smoke exposure. Lung parenchyma of a smoke-exposed DBA/2 mouse, at 3 months. (A) Immunohistochemical reaction for TGF-β. Counterstained with hematoxylin, scale bar represents 40 μm. (B) Immunohistochemical reaction for TGF-α. Counterstained with hematoxylin, Scale bar represents 15 μm.
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