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.

Receptor for advanced glycation end products contributes to postnatal pulmonary development and adult lung maintenance program in mice.

The role of the receptor for advanced glycation end products (RAGE) in promoting the inflammatory response through activation of NF-κB pathway is well established. Recent findings indicate that RAGE may also have a regulative function in apoptosis, as well as in cellular proliferation, differentiation, and adhesion. Unlike other organs, lung tissue in adulthood and during organ development shows relatively high levels of RAGE expression. Thus a role for the receptor in lung organogenesis and homeostasis may be proposed. To evaluate the role of RAGE in lung development and adult lung homeostasis, we generated hemizygous and homozygous transgenic mice overexpressing human RAGE, and analyzed their lungs from the fourth postnatal day to adulthood. Moderate RAGE hyperexpression during lung development influenced secondary septation, resulting in an impairment of alveolar morphogenesis and leading to significant changes in morphometric parameters such as airspace number and the size of alveolar ducts. An increase in alveolar cell apoptosis and a decrease in cell proliferation were demonstrated by the terminal deoxy-nucleotidyltransferase-mediated dUTP nick end labeling reaction, active caspase-3, and Ki-67 immunohistochemistry. Alterations in elastin organization and deposition and in TGF-ß expression were observed. In homozygous mice, the hyperexpression of RAGE resulted in histological changes resembling those changes characterizing human bronchopulmonary dysplasia (BPD). RAGE hyperexpression in the adult lung is associated with an increase of the alveolar destructive index and persistent inflammatory status leading to "destructive" emphysema. These results suggest an important role for RAGE in both alveolar development and lung homeostasis, and open new doors to working hypotheses on the pathogenesis of BPD and chronic obstructive pulmonary disease.

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.

A new mouse model of Peyronie's disease: an increased expression of hypoxia-inducible factor-1 target genes during the development of penile changes.

Peyronie's disease (PD) is characterized by an inflammatory response beneath the tunica albuginea with fibroblast proliferation forming a thickened fibrous plaque that may cause pain, penile curvature and erectile dysfunction. The progression of the PD plaque may eventually lead to calcification or ossification. Current therapeutic success is often unsatisfactory because of limited insight into disease mechanisms. Research has been hampered by the lack of a universally accepted animal model. We describe an animal model of spontaneous PD in tight skin (Tsk) mice, a C57Bl/6J subline that reproduces with age important features of the human disease (fibrous plaque formation, penile bending and areas of chondroid metaplasia with heterotopic ossification). Histological analysis demonstrated an evident structural disorganization of the tunica albuginea with excessive accumulation of type I collagen. At 12 months of age, fibrous plaques with areas of chondroid metaplasia and heterotopic ossification characterized Tsk penises. The up-regulation of hypoxia-inducible factor-1 (HIF-1) leads to an increased downstream expression of HIF-1 target genes, such as TGFbeta and iNOS. These factors, together with some PDGF family members, can cause collagen deposition in Tsk penises. They can also influence chondrocyte differentiation and heterotopic bone formation. In conclusion, hypoxia, HIF-1 and HIF-1 target genes appear to play an important role in the pathogenesis of PD in Tsk mice. This mouse model that is the first example of naturally occurring model of PD in laboratory animals may aid in the identification of signalling pathways crucial for PD and should facilitate the designing and testing of new therapeutic interventions.

Effect of roflumilast on inflammatory cells in the lungs of cigarette smoke-exposed mice.

BACKGROUND: We reported that roflumilast, a phosphodiesterase 4 inhibitor, given orally at 5 mg/kg to mice prevented the development of emphysema in a chronic model of cigarette smoke exposure, while at 1 mg/kg was ineffective. Here we investigated the effects of roflumilast on the volume density (VV) of the inflammatory cells present in the lungs after chronic cigarette smoke exposure. METHODS: Slides were obtained from blocks of the previous study and VV was assessed immunohistochemically and by point counting using a grid with 48 points, a 20x objective and a computer screen for a final magnification of 580x. Neutrophils were marked with myeloperoxidase antibody, macrophages with Mac-3, dendritic cells with fascin, B-lymphocytes with B220, CD4+ T-cells with CD4+ antibody, and CD8+T-cells with CD8-alpha. The significance of the differences was calculated using one-way analysis of variance. RESULTS: Chronic smoke exposure increased neutrophil VV by 97%, macrophage by 107%, dendritic cell by 217%, B-lymphocyte by 436%, CD4+ by 524%, and CD8+ by 417%. The higher dose of roflumilast prevented the increase in neutrophil VV by 78%, macrophage by 82%, dendritic cell by 48%, B-lymphocyte by 100%, CD4+ by 98% and CD8+ VV by 88%. The lower dose of roflumilast did not prevent the increase in neutrophil, macrophage and B-cell VV but prevented dendritic cells by 42%, CD4+ by 55%, and CD8+ by 91%. CONCLUSION: These results indicate (i) chronic exposure to cigarette smoke in mice results in a significant recruitment into the lung of inflammatory cells of both the innate and adaptive immune system; (ii) roflumilast at the higher dose exerts a protective effect against the recruitment of all these cells and at the lower dose against the recruitment of dendritic cells and T-lymphocytes; (iii) these findings underline the role of innate immunity in the development of pulmonary emphysema and (iiii) support previous results indicating that the inflammatory cells of the adaptive immune system do not play a central role in the development of cigarette smoke induced emphysema in mice.

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

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.

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.

Iron overload enhances the development of experimental liver cirrhosis in mice.

The role of iron in initiating liver fibrosis in iron overload diseases is not clearly established. Partly, this is due to the lack of suitable animal models that can produce the full liver pathology seen in genetic hemochromatosis. Recent advances in this field have demonstrated that iron may be interacting with other potential liver-damaging agents. The aim of this study was to investigate if feeding with carbonyl iron (CI) facilitates the development of carbon tetrachloride (CCl4)-induced liver fibrosis in the mouse. Mice were given a diet containing 3% CI and treated with CCl4 intraperitoneally twice weekly and 5% alcohol added to the drinking water for 12 weeks. Hepatic iron content increased 15- and 22-fold in animals receiving CI and CI + CCl4. At histological examination, iron-laden hepatocytes were found in CI treated animals, whereas these were absent in animals not exposed to CI. Mice receiving iron-enriched diet alone showed a mild fibrosis. Conversely, a marked collagen deposition was observed in CCl4 and CI + CCl4 groups. In particular, in this latter group, there was evidence of liver cirrhosis. Biochemical evaluation of collagen content substantiated histologic analysis. These results demonstrate that the addition of iron facilitates the development of cirrhosis in animals exposed to subtoxic doses of CCl4. This model may be useful in exploring the pathogenesis of liver cirrhosis. Moreover, its use in genetically altered mouse strains might provide new insight on the role of iron in fibrosis.

The HIV matrix protein p17 promotes the activation of human hepatic stellate cells through interactions with CXCR2 and Syndecan-2.

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) p17 is a matrix protein involved in virus life's cycle. CXCR2 and Syndecan-2, the two major coreceptors for the p17 protein, are expressed in hepatic stellate cells (HSCs), a key cell type involved in matrix deposition in liver fibrotic disorders. AIM: In this report we have investigated the in vitro impact of p17 on HSCs transdifferentiation and function and underlying signaling pathways involved in these processes. METHODS: LX-2 cells, a human HSC line, and primary HSC were challenged with p17 and expressions of fibrogenic markers and of p17 receptors were assessed by qRT-PCR and Western blot. Downstream intracellular signaling pathways were evaluated with qRT-PCR and Western blot as well as after pre-treatment with specific pathway inhibitors. RESULTS: Exposure of LX2 cells to p17 increases their contractile force, reshapes the cytoskeleton fibers and upregulates the expression of transdifferentiation markers including αSMA, COL1α1 and endothelin-1 through the activation of Jak/STAT and Rho signaling pathways. These effects are lost in HSCs pre-incubated with a serum from HIV positive person who underwent a vaccination with a p17 peptide. Confocal laser microscopy studies demonstrates that CXCR2 and syndecan-2 co-associate at the plasma membrane after exposure to p17. Immunostaining of HIV/HCV liver biopsies from co-infected patients reveals that the progression of liver fibrosis correlates with a reduced expression of CXCR2. CONCLUSIONS: The HIV matrix protein p17 is pro-fibrogenic through its interactions both with CXCR2 and syndecan-2 on activated HSCs.