Barbara Bartalesi

Different lung responses to cigarette smoke in two strains of mice sensitive to oxidants

The development of cigarette smoke-induced pulmonary changes in C57 Bl/6J and DBA/2 mice was investigated. Both strains are sensitive to oxidants and C57Bl/6J mice are moderately deficient in serum alpha1-proteinase inhibitor. Following chronic exposure to cigarette smoke, patchy emphysema was present in mice of both strains, but developed faster in DBA/2 mice. A positive reaction for mouse neutrophil elastase was seen on the septa of both strains. Additionally, the DBA/2 mice developed a uniform parenchymal dilation that was preceded by the appearance of apoptotic cells in areas with a low signal for vascular endothelial growth factor-receptor 2. Fibrotic areas scattered throughout the parenchyma, coupled with a positive immunohistochemical reaction for transforming growth factor-β was seen only in DBA/2 mice. Both DBA/2 and C57Bl/6J strains showed epithelial cell injury and areas of deciliation in their airways. However, the appearance of goblet cell metaplasia was common in C57Bl/6J mice but rare in DBA/2 mice. A positive immunohistochemical reaction for interleukin (IL)-4, IL-13 and MUC5AC was seen only in the airways of C57Bl/6J mice. Strain characteristics (alpha1-proteinase inhibitor levels, sensitivity to oxidants, and constitutive levels of vascular endothelial growth factor-receptor 2) and phenotypical responses (apoptosis and cytokine distribution) may condition parenchymal and airway changes to cigarette smoke.

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

BackgroundThe 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.MethodsThis 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-β (TGF-β) and transforming growth factor-α (TGF-α) 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.ResultsWe 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-β and TGF-α; 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-β and TGF-α.ConclusionThe 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.

Absence of Proteinase-Activated Receptor-1 Signaling in Mice Confers Protection from fMLP-Induced Goblet Cell Metaplasia

The morphological features of chronic obstructive pulmonary disease in man include emphysema and chronic bronchitis associated with mucus hypersecretion. These alterations can be induced in mice by a single intratracheal instillation of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), a chemoattractant and degranulating agent for neutrophils. The mechanisms underlying excessive mucus production and, in particular, goblet cell hyperplasia/metaplasia in chronic obstructive pulmonary disease remain poorly understood. The proteinase-activated receptors (PARs) are widely recognized for their modulatory properties during inflammation. In this study, we examined whether PAR-1 contributes to inflammation and lung damage induced by fMLP by comparing the response of PAR-1-deficient (PAR-1(-/-)) mice with that of wild-type (WT) mice. Mice were killed at various time points after fMLP instillation (200 microg/50 microl). WT mice developed emphysema and goblet cell metaplasia. The onset of pulmonary lesions was preceded by an increase in thrombin immunoreactivity in bronchial airways and alveolar tissue. This was followed by a decrease in PAR-1 immunoreactivity, and by an increase in IL-13 immunostaining on the luminal surface of airway epithelial cells. In PAR-1(-/-) mice, fMLP administration induced similar responses in terms of inflammation and emphysema, but these mice were protected from the development of goblet cell metaplasia. The involvement of PAR-1 in airway epithelial cell transdifferentiation was confirmed by demonstrating that intratracheal instillation of the selective PAR-1 agonist (TFLLR) induced goblet cell metaplasia in the airways of WT mice only. These data suggest that emphysema and goblet cell metaplasia occur independently, and that PAR-1 signaling through IL-13 stimulation may play an important role in inducing goblet cell metaplasia.

Genetic deficiency of α1-PI in mice influences lung responses to bleomycin

It has recently been suggested that proteinase inhibitors modulate the fibrotic response in the lung. This study investigated the development of bleomycin-induced pulmonary changes in pallid mice, deficient in serum alpha1-proteinase inhibitor, and with a lower elastase inhibitory capacity, and in congenic C57Bl/6J mice. Male pallid and C57Bl/6J mice received a single intratracheal instillation of either saline or bleomycin. The investigation was carried out by means of biochemical, morphological and morphometrical methods. In both strains, 21 and 72 h after bleomycin, the lungs showed foci of inflammatory cell infiltration associated with emphysema. Fibrosis developed with time after bleomycin. At 14 days fibrosis affected 23.46+/-9.48% (mean +/- SD) and 40.62+/-13.34% (p < 0.01) of the lungs of C57Bl/6J and pallid mice, respectively. Emphysema affected 3.68+/-3.11% and 12.57+/-4.13% (p<0.01) of lung in C57Bl/6J and pallid mice, respectively. In C57Bl/6J mice bleomycin increased lung hydroxyproline content by 34% and desmosine content by 44% (p < 0.01 for both). In pallid mice these increases were only 21% (p < 0.01) and 6% which may reflect parenchymal loss. Thus, the lung destructive response (emphysema) and the subsequent proliferative reaction (fibrosis) to bleomycin are potentiated in alpha1-proteinase inhibitor deficiency.

Synchrotron X-ray microscopy reveals early calcium and iron interaction with crocidolite fibers in the lung of exposed mice

Human exposure to asbestos can cause a wide variety of lung diseases that are still a current major health concern, even if asbestos has been banned in many countries. It has been shown in many studies that asbestos fibers, ingested by alveolar macrophages, disrupt lung iron homeostasis by sequestering iron. Calcium can also be deposited on the fibers. The pathways along which iron and above all calcium interact with fibers are still unknown. Our aim was that of investigating if the iron accumulation induced by the inhaled asbestos fibers also involves calcium ions accumulation. Lung sections of asbestos-exposed mice were analyzed using an extremely sensitive procedure available at the synchrotron facilities, that provides morphological and chemical information based on X-ray fluorescence microspectroscopy (μ-XRF). In this study we show that (1) where conventional histochemical procedures revealed only weak deposits of iron and calcium, μ-XRF analysis is able to detect significant deposits of both iron and calcium on the inhaled asbestos fibers; (2) the extent of the deposition of these ions is proportionally directly related and (3) iron and calcium deposition on inhaled asbestos fibers is concomitant with the appearance of inflammatory and hyperplastic reactions.

Smoking p66Shc knocked out mice develop respiratory bronchiolitis with fibrosis but not emphysema.

The adaptor protein p66Shc regulates intracellular oxidant levels through the modulation of a forkhead-related transcription factor (FOXO3a). The genetic ablation of p66Shc (p66Shc–/–) renders mice resistant to oxidative stress and p53-dependent apoptosis. We investigated whether p66Shc ablation in mice modifies lung cellular and molecular responses to cigarette smoke (CS) exposure. No differences between wild type (WT) and p66Shc–/– mice were observed in terms of inflammation and oxidant burden after acute CS exposure; however,p66Shc ablation modifies specific features of chronic inflammation induced by repeated exposure to CS. Unlike WT mice, p66Shc–/– mice did not develop emphysema, showing protection toward oxidative damage to DNA and apoptosis as revealed by a trivial 8-hydroxyguanosine staining and faint TUNEL and caspase-3 positivity on alveolar epithelial cells. Unexpectedly, CS exposure in p66Shc–/– mice resulted in respiratory bronchiolitis with fibrosis in surrounded alveoli. Respiratory bronchiolitis was characterized by peribronchiolar infiltrates of lymphocytes and histiocytes, accumulation of ageing pigmented macrophages within and around bronchioles, and peribronchiolar fibrosis. The blockage of apoptosis interferes with the macrophage “clearance” from alveolar spaces, favouring the accumulation of aging macrophages into alveoli and the progressive accumulation of iron pigment in long-lived senescent cells. The presence of areas of interstitial and alveolar fibrosis in peripheral parenchyma often accompanied the bronchiolar changes. Macrophages from smoking p66Shc–/– mice elaborate M2 cytokines (i.e., IL-4 and IL-13) and enzymes (i.e., chitinase and arginase I), which can promote TGF-beta expression, collagen deposition, and fibrosis in the surrounding areas. We demonstrate here that resistance to oxidative stress and p53-dependent apoptosis can modify tissue responses to CS caused by chronic inflammation without influencing early inflammatory response to CS exposure.

Histopathological data of iron and calcium in the mouse lung after asbestos exposure

This data article contains data related to the research article entitled, “Synchrotron X-ray microscopy reveals early calcium and iron interaction with crocidolite fibers in the lung of exposed mice” [1]. Asbestos fibers disrupt iron homeostasis in the human and mouse lung, leading to the deposition of iron (Fe) onto longer asbestos fibers which forms asbestos bodies (AB) [2]. Similar to Fe, calcium (Ca) is also deposited in the coats of the AB. This article presents data on iron and calcium in the mouse lung after asbestos exposure detected by histochemical evaluation.

Severe Reduction in Number and Function of Peripheral T Cells Does Not Afford Protection toward Emphysema and Bronchial Remodeling Induced in Mice by Cigarette Smoke

The protein Lck (p56(Lck)) is a Src family tyrosine kinase expressed at all stages of thymocyte development and is required for maturation of T cells. The targeted disruption of Lck gene in mice results in severe block in thymocyte maturation with substantial reduction in the development of CD4(+)CD8(+) thymocytes, severe reduction of peripheral T cells, and disruption of T-cell receptor signaling with defective function of T-cell responses. To investigate the role of T lymphocyte in the development of cigarette smoke-induced pulmonary changes, Lck(-/-) mice and corresponding congenic wild-type mice were chronically exposed to cigarette smoke, and their lungs were analyzed by biochemical, immunologic, and morphometric methods. Smoking mice from both genotypes showed disseminated foci of emphysema and large areas of goblet cell metaplasia in bronchial and bronchiolar epithelium. Morphometric evaluation of lung changes and lung elastin determination confirmed that mice from both genotypes showed the same degree of emphysematous lesions. Thus, cigarette smoke exposure in the presence of severe reduction in number and function of peripheral T cells does not influence the development of pulmonary changes induced by cigarette smoke. The data obtained suggest that innate immunity is a leading actor in the early development of pulmonary changes in smoking mice and that the adaptive immune response may play a role at later stages.

Ongoing Lung Inflammation and Disease Progression in Mice after Smoking Cessation: Beneficial Effects of Formyl-Peptide Receptor Blockade

The most important risk factor for chronic obstructive pulmonary disease (COPD) is cigarette smoking. Until now, smoking cessation (SC) is the only treatment effective in slowing down the progression of the disease. However, in many cases SC may only relieve the airflow obstruction and inflammatory response. Consequently, a persistent lung inflammation in ex-smokers is associated with progressive deterioration of respiratory functions. This is an increasingly important clinical problem whose mechanistic basis remains poorly understood. Available therapies do not adequately suppress inflammation and are not able to stop the vicious cycle that is at the basis of persistent inflammation. In addition, in mice after SC an ongoing inflammation and progressive lung deterioration is observed. After 4 months of smoke exposure mice show mild emphysematous changes. Lung inflammation is still present after SC, and emphysema progresses during the next 6-month period of observation. Destruction of alveolar walls is associated with airways remodeling (goblet cell metaplasia and peribronchiolar fibrosis). Modulation of formyl-peptide receptor signaling with antagonists mitigates inflammation and prevents deterioration of lung structures. This study suggests an important role for N-formylated peptides in the progression and exacerbation of COPD. Modulating formyl-peptide receptor signal should be explored as a potential new therapy for COPD.