Loss of caveolin-1 in bronchiolization in lung fibrosis.

Bronchiolization is a key process in fibrosing lung in which the proliferative status of bronchiolar epithelium changes, leading to abnormal epithelial morphology. Within the context that caveolin-1 acts to suppress epithelial proliferation, we postulated that stimulating epithelial injury would lead to caveolin-1 downregulation and encourage proliferation. The present study evaluates the expression of caveolin-1, especially in bronchiolization, in C57BL/6J mice with bleomycin-induced lung fibrosis and in various types of re-epithelialization in human interstitial pneumonias (IPs). Immunohistochemically, levels of caveolin-1 decreased in the bronchiolar epithelium of mice treated with bleomycin. Levels of caveolin-1 mRNA in the whole lung were decreased at 7 and 14 days. Caveolin-1 mRNA was also decreased in laser-capture microdissection- retrieved bronchiolar epithelial cells at 7 days. Among patients with 12 IPs, including four usual IPs (UIPs) and eight nonspecific IPs (NSIPs), whole lung caveolin-1 was significantly decreased compared with 12 controls at both mRNA and protein levels. By scoring immunointensity, caveolin-1 was significantly reduced in bronchiolization and squamous metaplasia as well as in bronchiolar epithelium in 23 IPs (12 UIPs and 11 NSIPs) compared with bronchiolar epithelium from seven controls. These data suggested that loss of caveolin-1 is associated with abnormal re-epithelialization in lung fibrosis.     

Airway epithelial NF-kappaB activation modulates asbestos-induced inflammation and mucin production in vivo

To investigate the role of bronchiolar epithelial NF-kappaB activity in the development of inflammation and fibrogenesis in a murine model of asbestos inhalation, we used transgenic (Tg) mice expressing an IkappaBalpha mutant (IkappaBalphasr) resistant to phosphorylation-induced degradation and targeted to bronchial epithelium using the CC10 promoter. Sham and chrysotile asbestos-exposed CC10-IkappaBalphasr Tg(+) and Tg(-) mice were examined for altered epithelial cell proliferation and differentiation, cytokine profiles, lung inflammation, and fibrogenesis at 3, 9, and 40 days. KC, IL-6 and IL-1beta were increased (p < or = 0.05) in bronchoalveolar lavage fluid (BALF) from asbestos-exposed mice, but to a lesser extent (p < or = 0.05) in Tg(+) vs Tg(-) mice. Asbestos also caused increases in IL-4, MIP-1beta, and MCP-1 in BALF that were more elevated (p < or = 0.05) in Tg(+) mice at 9 days. Differential cell counts revealed eosinophils in BALF that increased (p < or = 0.05) in Tg(+) mice at 9 days, a time point corresponding with significantly increased numbers of bronchiolar epithelial cells staining positively for mucus production. At all time points, asbestos caused increased numbers of distal bronchiolar epithelial cells and peribronchiolar cells incorporating the proliferation marker, Ki-67. However, bronchiolar epithelial cell and interstitial cell labeling was diminished at 40 days (p < or = 0.05) in Tg(+) vs Tg(-) mice. Our findings demonstrate that airway epithelial NF-kappaB activity plays a role in orchestrating the inflammatory response as well as cell proliferation in response to asbestos.     

TGF-beta 1 as an enhancer of Fas-mediated apoptosis of lung epithelial cells

Transforming growth factor-beta 1 (TGF-beta 1) has important roles in lung fibrosis and the potential to induce apoptosis in several types of cells. We previously demonstrated that apoptosis of lung epithelial cells induced by Fas ligation may be involved in the development of pulmonary fibrosis. In this study, we show that TGF-beta1 induces apoptosis of primary cultured bronchiolar epithelial cells via caspase-3 activation and down-regulation of cyclin-dependent kinase inhibitor p21. Concentrations of TGF-beta 1 that were not sufficient to induce apoptosis alone could enhance agonistic anti-Fas Ab or rFas ligand-mediated apoptosis of cultured bronchiolar epithelial cells. Soluble Fas ligand in the bronchoalveolar lavage fluid (BALF) from patients with idiopathic pulmonary fibrosis (IPF) also induced apoptosis of cultured bronchiolar epithelial cells that was significantly attenuated by anti-TGF-beta Ab. Otherwise, BALF from patients with hypersensitivity pneumonitis (HP) could not induce apoptosis on bronchiolar epithelial cells, despite its comparable amounts of soluble Fas ligand. The concentrations of TGF-beta 1 in BALF from patients with IPF were significantly higher compared with those in BALF from patients with HP or controls. Furthermore, coincubation with the low concentration of TGF-beta 1 and HP BALF created proapoptotic effects comparable with the IPF BALF. In vivo, the administration of TGF-beta 1 could enhance Fas-mediated epithelial cell apoptosis and lung injury via caspase-3 activation in mice. Our results demonstrate a novel role of TGF-beta 1 in the pathophysiology of pulmonary fibrosis as an enhancer of Fas-mediated apoptosis of lung epithelial cells.     

The Role of Catalase in Pulmonary Fibrosis

Background

Catalase is preferentially expressed in bronchiolar and alveolar epithelial cells, and acts as an endogenous antioxidant enzyme in normal lungs. We thus postulated epithelial damage would be associated with a functional deficiency of catalase during the development of lung fibrosis.

Methods

The present study evaluates the expression of catalase mRNA and protein in human interstitial pneumonias and in mouse bleomycin-induced lung injury. We examined the degree of bleomycin-induced inflammation and fibrosis in the mice with lowered catalase activity.

Results

In humans, catalase was decreased at the levels of activity, protein content and mRNA expression in fibrotic lungs (n = 12) compared to control lungs (n = 10). Immunohistochemistry revealed a decrease in catalase in bronchiolar epithelium and abnormal re-epithelialization in fibrotic areas. In C57BL/6J mice, catalase activity was suppressed along with downregulation of catalase mRNA in whole lung homogenates after bleomycin administration. In acatalasemic mice, neutrophilic inflammation was prolonged until 14 days, and there was a higher degree of lung fibrosis in association with a higher level of transforming growth factor-β expression and total collagen content following bleomycin treatment compared to wild-type mice.

Conclusions

Taken together, these findings demonstrate diminished catalase expression and activity in human pulmonary fibrosis and suggest the protective role of catalase against bleomycin-induced inflammation and subsequent fibrosis.     

Characteristics of cell structure and proliferative activity of organ cultures of normal embryonic lung tissue of mice resistant (C57BL) and predisposed (A) to lung blastomogenesis

Linear differences were discovered in the quantitative ratio and proliferative activity of the cells of the epithelium and mesenchyma of organ cultures of the normal embryonal pulmonary tissue of mice resistant (C57BL) and predisposed (A) to lung blastomogenesis. In A mouse embryos, the index of the cell labels of the bronchiolar epithelium and fibroblast-like cells of the mesenchyma which migrate on the surface of the membrane filter was several times higher than that in C57BL mice. The number of the mesenchymal cells capable of migration in A mouse explants was essentially greater, that of the mesenchymal cells being in a direct contact with the epithelium of alveoloid and bronchiolar structures was, on the contrary, lower than in C57BL mouse explants. It is suggested that the differences described, particularly epithelial-mesenchymal interactions, are of importance for realization of the genetically determined sensitivity of these mice to spontaneous and induced lung blastomogenesis, which manifests as early as during prenatal ontogenesis.     

Transgenic extracellular superoxide dismutase protects postnatal alveolar epithelial proliferation and development during hyperoxia

Transgenic (TG) human (h) extracellular superoxide dismutase (EC-SOD) targeted to type II cells protects postnatal newborn mouse lung development against hyperoxia by unknown mechanisms. Because alveolar development depends on timely proliferation of type II epithelium and differentiation to type I epithelium, we measured proliferation in bronchiolar and alveolar (surfactant protein C-positive) epithelium in air and 95% O2-exposed wild-type (WT) and TG hEC-SOD newborn mice at postnatal days 3, 5, and 7 (P3-P7), traversing the transition from saccular to alveolar stages. We found that TG hEC-SOD ameliorated the 95% O2-impaired bromodeoxyuridine uptake in alveolar and bronchiolar epithelium at P3, but not at P5 and P7, when overall epithelial proliferation rates were lower in air-exposed WT mice. Mouse EC-, CuZn-, and Mn-SOD expression were unaffected by hyperoxia or genotype. TG mice had less DNA damage than 95% O2-exposed WT mice at P3, measured by TdT-mediated dUTP nick end labeling (P < 0.05). Hyperoxia induced cell-cycle inhibitory protein p21cip/waf mRNA at P3, WT > TG, P = 0.06. 95% O2 impaired apical expression of type I cell alpha protein (T1alpha) in WT but not in TG mice at P3 and increased T1alpha in WT and TG mice at P7. Reducing the 95% O2-induced impairment of epithelial proliferation at a critical window of lung development was associated with protection against DNA damage and preservation of apical T1alpha expression at P3.     

Aging exacerbates damage and delays repair of alveolar epithelia following influenza viral pneumonia

Background

Influenza virus infection causes significantly higher levels of morbidity and mortality in the elderly. Studies have shown that impaired immunity in the elderly contributes to the increased susceptibility to influenza virus infection, however, how aging affects the lung tissue damage and repair has not been completely elucidated.

Methods

Aged (16–18 months old) and young (2–3 months old) mice were infected with influenza virus intratracheally. Body weight and mortality were monitored. Different days after infection, lung sections were stained to estimate the overall lung tissue damage and for club cells, pro-SPC⁺ bronchiolar epithelial cells, alveolar type I and II cells to quantify their frequencies using automated image analysis algorithms.

Results

Following influenza infection, aged mice lose more weight and die from otherwise sub-lethal influenza infection in young mice. Although there is no difference in damage and regeneration of club cells between the young and the aged mice, damage to alveolar type I and II cells (AT1s and AT2s) is exacerbated, and regeneration of AT2s and their precursors (pro-SPC-positive bronchiolar epithelial cells) is significantly delayed in the aged mice. We further show that oseltamivir treatment reduces virus load and lung damage, and promotes pulmonary recovery from infection in the aged mice.

Conclusions

These findings show that aging increases susceptibility of the distal lung epithelium to influenza infection and delays the emergence of pro-SPC positive progenitor cells during the repair process. Our findings also shed light on possible approaches to enhance the clinical management of severe influenza pneumonia in the elderly.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0116-z) contains supplementary material, which is available to authorized users.     

Effect of chronic hypoxia on proliferation, apoptosis, and HSP70 expression in mouse bronchiolar epithelial cells

Heat shock proteins (HSPs) can be induced by various stresses and play an important role in cell cycle progression. HSP70 has been shown to act as an inhibitor of apoptosis. We studied HSP70 expression in bronchial epithelial cells of C57BL/6 mice and homozygous HPS70 knockout mice (hsp70.1-/-) exposed to chronic hypoxic stress. We also investigated changes in cellular proliferation and apoptosis in relation to HSP70. Lungs were removed from mice after a three-week period of exposure to 10 % O(2). Immunoblots for HSP70 and immunohistochemical staining for HSP70 and Ki-67 were performed. Apoptosis was assessed using the TUNEL assay. The three-week period of hypoxic stress did not change HSP70 levels in total lung tissue, but a significant reduction in HSP70 expression was observed in bronchiolar epithelial cells. In wild type mice, both HSP70 and Ki-67 expression were significantly reduced in bronchiolar epithelial cells. In homozygous HPS70 knockout mice (hsp70.1-/-), apoptosis of bronchiolar epithelial cells was significantly increased. Our results suggest that HSP70 may exert anti-apoptotic effects in mouse bronchiolar epithelial cells.     

Stat3 is required for cytoprotection of the respiratory epithelium during adenoviral infection

The role of Stat3 in the maintenance of pulmonary homeostasis following adenoviral-mediated lung injury was assessed in vivo. Stat3 was selectively deleted from bronchiolar and alveolar epithelial cells in Stat3(DeltaDelta) mice. Although lung histology and function were unaltered by deletion of Stat3 in vivo, Stat3(DeltaDelta) mice were highly susceptible to lung injury caused by intratracheal administration of AV1-GFP, an early (E) region 1- and E3-deleted, nonproliferative adenovirus. Severe airspace enlargement, loss of alveolar septae, and sloughing of the bronchiolar epithelium were observed in Stat3(DeltaDelta) mice as early as 1 day after exposure to the virus. Although surfactant protein A, B, and C content and surfactant protein-B mRNA expression in Stat3(DeltaDelta) mice were similar, TUNEL staining and caspase-3 were increased in alveolar type II epithelial cells of Stat3(DeltaDelta) mice after exposure to virus. RNA microarray analysis of type II epithelial cells isolated from Stat3(DeltaDelta) mice demonstrated significant changes in expression of numerous genes, including those genes regulating apoptosis, supporting the concept that the susceptibility of Stat3-deficient mice to adenovirus was related to the role of Stat3 in the regulation of cell survival. AV1-Bcl-x(L), an E1- and E3-deleted, nonproliferative adenovirus expressing the antiapoptotic protein Bcl-x(L), protected Stat3(DeltaDelta) mice from adenoviral-induced lung injury. Adenoviral infection of the lungs of Stat3-deficient mice was associated with severe injury of the alveolar and bronchiolar epithelium. Thus, Stat3 plays a critical cytoprotective role that is required for epithelial cell survival and maintenance of alveolar structures during the early phases of pulmonary adenoviral infection.     

Histopathological Features and Viral Antigen Distribution in the Lung of Fatal Patients with <Emphasis Type="Italic">Enterovirus</Emphasis> 71 Infection

Enterovirus 71 (EV71) infection causes hand-foot-and-mouth disease (HFMD) in children and might be accompanied by severe neurological complications. It has become one of the most important pathogens of central nervous system infection. To explore the causes of lung injury by EV71, the distribution of EV71 receptors, SCARB2 and PSGL-1, in human lung tissues was examined. Our results revealed that SCARB2 was positively distributed in the bronchial and bronchiolar epithelial cells, alveolar cells and macrophages, while PSGL-1 was positively scattered in bronchial and bronchiolar epithelial cells and macrophages, and negatively distributed in alveolar cells. The pathological changes of fatal lung with EV71 infection demonstrated intrapulmonary bronchitis and bronchiolitis, diffuse or focal infiltration of inflammatory cells, such as T cells and B cells in the wall and surrounding tissues, widened alveolar septum, capillaries in the septum with highly dilated and congested, and infiltrated inflammatory cells, showing different degrees of protein edema with fibrin exudation in the alveolar cavity, as well as obvious hyaline membrane formation in some alveolar cavities. The EV71 antigen in lung tissues was detected, and the viral antigen was positive in lung bronchial and bronchiolar epithelial cells, and positively scattered in the alveolar cells and macrophages. Therefore, in addition to the complications of central nervous system injury, the lung remains the main target organ for virus attack in severe EV71 infected patients. Lung injury was mainly caused by neurogenic damage and/or direct invasion of the virus into the lungs in critically serious children, and the lesions were mainly pulmonary edema and interstitial pneumonia.     

Essential role for the ATG4B protease and autophagy in bleomycin-induced pulmonary fibrosis

Autophagy is a critical cellular homeostatic process that controls the turnover of damaged organelles and proteins. Impaired autophagic activity is involved in a number of diseases, including idiopathic pulmonary fibrosis suggesting that altered autophagy may contribute to fibrogenesis. However, the specific role of autophagy in lung fibrosis is still undefined. In this study, we show for the first time, how autophagy disruption contributes to bleomycin-induced lung fibrosis in vivo using an Atg4b-deficient mouse as a model. Atg4b-deficient mice displayed a significantly higher inflammatory response at 7 d after bleomycin treatment associated with increased neutrophilic infiltration and significant alterations in proinflammatory cytokines. Likewise, we found that Atg4b disruption resulted in augmented apoptosis affecting predominantly alveolar and bronchiolar epithelial cells. At 28 d post-bleomycin instillation Atg4b-deficient mice exhibited more extensive and severe fibrosis with increased collagen accumulation and deregulated extracellular matrix-related gene expression. Together, our findings indicate that the ATG4B protease and autophagy play a crucial role protecting epithelial cells against bleomycin-induced stress and apoptosis, and in the regulation of the inflammatory and fibrotic responses.     

Pulmonary Multinucleate Giant Cells in Dermatosis Vegetans in Swine: Light microscopic and immunohistochemical investigations

Five pigs with dermatosis vegetans (DV), aged 1–28 days, were examined with the purpose of describing the pulmonary changes and to characterize the pulmonary multinucleate giant cells (MGC) and their possible cytogenesis. No pulmonary changes were present at birth. From 7 days of age, lung changes were characterized by proliferation of alveolar epithelial cells and formation of MGCs. Immunostaining for cytokeratin by a peroxydase–streptavidin method gave a positive reaction in MGCs, bronchial, bronchiolar and alveolar epithelium. MGCs seemed to be formed in the course of alveolar epithelial proliferations, and type-II pneumocytes were proposed as possible precursors.     

Pathogenetic process of lung tumors induced by inhalation exposures of rats to plutonium dioxide aerosols

Sequential examinations were done on the pulmonary cytokinetics and pulmonary lesions in rats after inhalation exposure to (239)PuO(2) aerosols to investigate the pathogenesis of lung tumors. Total cell yields of lavaged bronchoalveolar cells as well as the estimated numbers of pulmonary alveolar macrophages were significantly reduced from 1 to 3 months after exposure but recovered thereafter to the control levels. The proportions of multinucleated or micronucleated pulmonary alveolar macrophages increased significantly in lavaged cells from 1 month, and the increase was sustained up to 18 months after exposure. Both tumor necrosis factor and nitric oxide were shown to be differentially released from stimulated cultures of pulmonary alveolar macrophages during the period from 6 to 18 months after exposure. The labeling indices of alveolar and bronchiolar epithelial cells treated with 5-bromo-2'-deoxyuridine increased significantly in lungs from 3 months and were sustained up to 18 months after exposure. Histopathological examinations revealed that after the early inflammation, hyperplasia and metaplasia of the lining of the bronchioloalveolar epithelium were predominant from 3 to 6 months, while adenomatous or adenocarcinomatous lesions appeared and developed from 12 months after exposure. The appearance of primary lung tumors, almost all of which were adenomas and adenocarcinomas, was found in the dose range of 1 to 2 Gy from 12 months after exposures. These results indicate that the pathogenetic process initiated by early cellular damage and alterations associated with inflammation is followed by the proliferative and metaplastic lesions of pulmonary epithelium, leading to the appearance and development of pulmonary neoplasms from 1 year after the inhalation exposures in rats that received a minimum lung dose of more than 1 Gy.     

Changes of colon epithelium proliferation due to individual aging with cyclin proliferating cell nuclear antigen (PCNA/cyclin) immunostaining compared to [3H]-thymidine radioautography

We report a change in the proliferative activity of mouse colonic epithelium due to development and aging. In order to measure the proliferative activity, colonic epithelium was immunostained for cyclin proliferating cell nuclear antigen (PCNA/cyclin), which appears from the Gl to the S phase of the cell cycle, and compared with labeling obtained by [³H]-thymidine radioautography. Litter mice of six age groups from the fetal period (embryonic day 19), newborn period (postnatal day 1), suckling period (postnatal day 5), weaning period (postnatal dy 21), adult period (2 month old) to the senescent period (11 month old) were examined by immunohistochemistry. The descending colons were fixed in methacarn (method-Carnoy) and embedded in paraffin. Sections were stained for PCNA/cyclin activity using 19A2 monoclonal antibody and the avidin-biotin peroxidase complex (ABC) technique. For radioautography, litter mice of nine age groups using in vivo intraperitoneal administration of [³H]-thymidine. The labeling indices of colonic epithelial cells in the proliferative zone were then analyzed and compared between the two investigative methods. Our results show that the prliferative activity of mice colon was high in the fetal and newborn periods and almost constant from the suckling period to senescence, as demonstrated by both PCNA/cyclin immunohistochemistry and [³H]-thymidine radioautography. The labeling index seen by PCNA/cyclin immunohistochemistry was, however, higher than that seen by [³H]-thymidine radioautography.     

NF-κB acts downstream of EGFR in regulating low dose cadmium induced primary lung cell proliferation

Apart from cytotoxicity cadmium has no special attributes towards cell’s physiological function. The role of cadmium with respect to cell growth is still under debate. Mitogen activated protein kinase and Ca²⁺/calmodulin dependent protein kinase dependent pathways are the two elaborately studied concerning cadmium induced cell proliferation. Low concentration of cadmium chloride (2.5 μM) was applied to mice primary lung epithelial cells and cell proliferation was measured both by cell cycle analysis and Brdu incorporation assay. Effects of differential dose of cadmium chloride on lung epithelial cells were evaluated morphologically by atomic force microscopy. RT-PCR and western blot altogether corroborated the specific signalling pathways concerning cadmium induced lung cell proliferation. Cadmium induced lung epithelial cells which over-expressed EGFR, were transfected with siEGFR, revealed downstream molecules and RNAi induced EGFR silencing. Use of siEGFR effectively prevents expression of proinflammatory and cell proliferative markers. Moreover N-acetyl cysteine and ascorbic acid mediated inhibition of EGFR and downstream signalling molecules indicate the involvement of reactive oxygen species. Exposure to low concentration of cadmium promotes the growth of primary mice lung epithelial cell by EGFR signalling. We have also transfected the primary lung epithelial cell with siRNA against the regulatory subunit of nuclear factor-κB (NF-κB) and the data shows that cadmium induced lung cell proliferation is the effect of EGFR mediated NF-κB activation.