Loss of Rab27 function results in abnormal lung epithelium structure in mice

Rab27 small GTPases regulate secretion and movement of lysosome-related organelles such as T cell cytolytic granules and platelet-dense granules. Previous studies indicated that Rab27a and Rab27b are expressed in the murine lung suggesting that they regulate secretory processes in the lung. Consistent with those studies, we found that Rab27a and Rab27b are expressed in cell types that contain secretory granules: alveolar epithelial type II (AEII) and Clara cells. We then used Rab27a/Rab27b double knockout (DKO) mice to examine the functional consequence of loss of Rab27 proteins in the murine lung. Light and electron microscopy revealed a number of morphological changes in lungs from DKO mice when compared with those in control animals. In aged DKO mice we observed atrophy of the bronchiolar and alveolar epithelium with reduction of cells numbers, thinning of the bronchiolar epithelium and alveolar walls, and enlargement of alveolar airspaces. In these samples we also observed increased numbers of activated foamy alveolar macrophages and granulocyte containing infiltrates together with reduction in the numbers of Clara cells and AEII cells compared with control. At the ultrastructural level we observed accumulation of cytoplasmic membranes and vesicles in Clara cells. Meanwhile, AEII cells in DKO accumulated large mature lamellar bodies and lacked immature/precursor lamellar bodies. We hypothesize that the morphological changes observed at the ultrastructural level in DKO samples result from secretory defects in AEII and Clara cells and that over time these defects lead to atrophy of the epithelium.     

铜绿假单胞菌家兔肺部感染模型的建立与评价

目的:建立铜绿假单胞菌(PA,又名绿脓杆菌)肺部感染动物模型,并从病理学及细菌学、影像学等方面进行评价,研究铜绿假单胞菌对肺组织的致病作用。方法:24只健康清洁级新西兰大白兔随机分成2组,实验组采用经皮气管穿刺法,隔日注入铜绿假单胞菌反复感染家兔,对照组施以生理盐水。接种后隔日一次行胸部CT扫描。比较二组家兔的体温、血象及肺组织匀浆菌落计数;观察病理组织改变及胸部CT影像学变化。结果:注菌组家兔白细胞总数显著升高,肺组织匀浆菌落计数显著高于对照组。CT表现为双侧多发斑片状模糊影,部分可见实变及脓肿灶。大体标本可见家兔肺部结节、脓肿及出血灶,病理学镜下观察发现注菌组家兔肺组织内多个脓肿灶形成,大量中性粒细胞浸润,肺泡腔内大量渗出,部分出血,肺泡壁充血及肺水肿。随着注菌次数增多及时间推移,逐渐出现如肺泡间隔增厚、肉芽肿形成、实变等慢性肺部炎症的病理表现。结论:使用经皮气道穿刺法接种铜绿假单胞菌至新西兰兔肺部,可成功建立兔铜绿假单胞菌肺部感染模型。     

Differential and specific labeling of epithelial and vascular endothelial cells of the rat lung by Lycopersicon esculentum and Griffonia simplicifolia I lectins

In the rat lung, we found that the Lycopersicon esculentum (LEA) lectin specifically binds to the epithelium of bronchioles and alveoli whereas Griffonia simplicifolia I (GS-I) binds to the endothelium of alveolar capillaries. The differential binding affinity of these lectins was examined on semithin (approximately 0.5 microns) and thin (less than 0.1 (microns) frozen sections of rat lung lavaged to remove alveolar macrophages. On semithin frozen sections, LEA bound to epithelial cells lining bronchioles and the alveoli (type I, but not type II epithelial cells). On thin frozen sections, biotinylated Lycopersicon esculentum (bLEA)-streptavidin-gold conjugates were confined primarily to the luminal plasmalemma of type I cells. bGS-I-streptavidin-Texas Red was detected on the endothelial cells of alveolar capillaries and postcapillary venules but not on those of larger venules, veins or arterioles. By electron microscopy, GS-I-streptavidin-gold complexes were localized primarily to the luminal plasmalemma of thick and thin regions of the capillary endothelium. Neither lectin labeled type II alveolar cells, but both lectins labeled macrophages in the interstitia and in incompletely lavaged alveoli.     

Scanning electron microscope study of the lung epithelium of the rabbit during ontogenesis

The development of the bronchial and alveolar epithelium was observed in rabbits from the 15th day post conception until the time of birth with the scanning electron microscope. In the pseudoglandular phase, primitive bronchi proliferate in the mesenchyme. The epithelial cells are not differentiated and have single cilia. After retraction of these single cilia cell differentiation begins. Flat cells densely populated with cytopodia can be recognized on the 22nd day, ciliated cells on the 23rd day post conception. Both are located in the bronchi near the hilus. In the canalicular phase of development, the differentiation of the mucoid cells and the Clara-cells begins. The interstitial connective tissue develops more and more capillaries. The alveolar phase begins around the 26th day p. c. The lung capillaries reach the alveolar epithelial cells and arrange themselves directly beneath the epithelial basement membrane. This "alveolarization" of the lung tissue starts in the centre of the lung lobules and proceeds to the periphery. After the 26th day post conception the alveolar epithelial cells retract their single cilium and at the same time become type I or type II pneumocytes. The undifferentiated entodermal stem cell of the alveolar epithelium is the pneumoblast.     

Sepsis impairs alveolar epithelial function by downregulating Na-K-ATPase pump

Widespread vascular endothelial injury is the major mechanism for multiorgan dysfunction in sepsis. Following this process, the permeability of the alveolar capillaries is augmented with subsequent increase in water content and acute respiratory distress syndrome (ARDS). Nevertheless, the role of alveolar epithelium is less known. Therefore, we examined alveolar fluid clearance (AFC) using isolated perfused rat lung model in septic rats without ARDS. Sepsis was induced by ligating and puncturing the cecum with a 21-gauge needle. AFC was examined 24 and 48 h later. The expression of Na-K-ATPase proteins was examined in type II alveolar epithelial cells (ATII) and basolateral membrane (BLM). The rate of AFC in control rats was 0.51 ± 0.02 ml/h (means ± SE) and decreased to 0.3 ± 0.02 and 0.33 ± 0.03 ml/h in 24 and 48 h after sepsis induction, respectively (P < 0.0001). Amiloride, significantly decreased AFC in sepsis; conversely, isoproterenol reversed the inhibitory effect of sepsis. The alveolar-capillary barrier in septic rats was intact; therefore the finding of increased extravascular lung water in early sepsis could be attributed to accumulation of protein-poor fluid. The expression of epithelial sodium channel and Na-K-ATPase proteins in whole ATII cells was not different in both cecal ligation and puncture and control groups; however, the abundance of Na-K-ATPase proteins was significantly decreased in BLMs of ATII cells in sepsis. Early decrease in AFC in remote sepsis is probably related to endocytosis of the Na-K-ATPase proteins from the cell plasma membrane into intracellular pools, with resultant inhibition of active sodium transport in ATII cells.     

In vivo exposure to hyperoxia induces DNA damage in a population of alveolar type II epithelial cells

It is well established that hyperoxia injures and kills alveolar endothelial and type I epithelial cells of the lung. Although type II epithelial cells remain morphologically intact, it remains unclear whether they are also damaged. DNA integrity was investigated in adult mice whose type II cells were identified by their endogenous expression of pro-surfactant protein C or transgenic expression of enhanced green fluorescent protein. In mice exposed to room air, punctate perinuclear 8-oxoguanine staining was detected in approximately 4% of all alveolar cells and in 30% of type II cells. After 48 or 72 h of hyperoxia, 8-oxoguanine was detected in 11% of all alveolar cells and in >60% of type II cells. 8-Oxoguanine colocalized by confocal microscopy with the mitochondrial transmembrane protein cytochrome oxidase subunit 1. Type II cells isolated from hyperoxic lungs exhibited nuclear DNA strand breaks by comet assay even though they were viable and morphologically indistinguishable from cells isolated from lungs exposed to room air. These data reveal that type II cells exposed to in vivo hyperoxia have oxidized and fragmented DNA. Because type II cells are essential for lung remodeling, our findings raise the possibility that they are proficient in DNA repair.     

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.     

Angiotensin II mediates glutathione depletion, transforming growth factor-beta1 expression, and epithelial barrier dysfunction in the alcoholic rat lung

Alcohol abuse markedly increases the risk of sepsis-mediated acute lung injury. In a rat model, ethanol ingestion alone (in the absence of any other stress) causes pulmonary glutathione depletion, increased expression of transforming growth factor-beta1 (TGF-beta1), and alveolar epithelial barrier dysfunction, even though the lung appears grossly normal. However, during endotoxemia, ethanol-fed rats release more activated TGF-beta1 into the alveolar space where it can exacerbate epithelial barrier dysfunction and lung edema. Ethanol ingestion activates the renin-angiotensin system, and angiotensin II is capable of inducing oxidative stress and TGF-beta1 expression. We determined that lisinopril, an angiotensin-converting enzyme inhibitor that decreases angiotensin II formation, limited lung glutathione depletion, and treatment with either lisinopril or losartan, a selective angiotensin II type 1 receptor blocker, normalized TGF-beta1 expression. The glutathione precursor procysteine also prevented TGF-beta1 expression, suggesting that TGF-beta1 may be induced indirectly by angiotensin II-mediated oxidative stress and glutathione depletion. Importantly, lisinopril treatment normalized barrier function in alveolar epithelial cell monolayers from ethanol-fed rats, and treatment with either lisinopril or losartan normalized alveolar epithelial barrier function in ethanol-fed rats in vivo, as reflected by lung liquid clearance of an intratracheal saline challenge, even during endotoxemia. In parallel, lisinopril treatment limited TGF-beta1 protein release into the alveolar space during endotoxemia. Together, these results suggest that angiotensin II mediates oxidative stress and the consequent TGF-beta1 expression and alveolar epithelial barrier dysfunction that characterize the alcoholic lung.     

Depletion of resident alveolar macrophages does not prevent Fas-mediated lung injury in mice

Activation of the Fas/Fas ligand (FasL) system in the lungs results in a form of injury characterized by alveolar epithelial apoptosis and neutrophilic inflammation. Studies in vitro show that Fas activation induces apoptosis in alveolar epithelial cells and cytokine production in alveolar macrophages. The main goal of this study was to determine the contribution of alveolar macrophages to Fas-induced lung inflammation in mice, by depleting alveolar macrophages using clodronate-containing liposomes. Liposomes containing clodronate or PBS were instilled by intratracheal instillation. After 24 h, the mice received intratracheal instillations of the Fas-activating monoclonal antibody Jo2 or an isotype control antibody and were studied 18 h later. The Jo2 MAb induced increases in bronchoalveolar lavage fluid (BALF) total neutrophils, lung caspase-3 activity, and BALF total protein and worsened histological lung injury in the macrophage-depleted mice. Studies in vitro showed that Fas activation induced the release of the cytokine KC in a mouse lung epithelial cell line, MLE-12. These results suggest that the lung inflammatory response to Fas activation is not primarily dependent on resident alveolar macrophages and may instead depend on cytokine release by alveolar epithelial cells.     

Type I cell-like morphology in tight alveolar epithelial monolayers

The pulmonary alveolar epithelium separates air spaces from a fluid-filled interstitium and might be expected to exhibit high resistance to fluid and solute movement. Previous studies of alveolar epithelial barrier properties have been limited due to the complex anatomy of adult mammalian lung. In this study, we characterized a model of isolated alveolar epithelium with respect to barrier transport properties and cell morphology. Alveolar epithelial cells were isolated from rat lungs and grown as monolayers on tissue culture-treated Nuclepore filters. On Days 2-6 in primary culture, monolayers were analyzed for transepithelial resistance (Rt) and processed for electron microscopy. Mean cell surface area and arithmetic mean thickness (AMT) were determined using morphometric techniques. By Day 5, alveolar epithelial cells in vitro exhibited morphologic characteristics of type I alveolar pneumocytes, with thin cytoplasmic extensions and protruding nuclei. Morphometric data demonstrated that alveolar pneumocytes in vitro develop increased surface area and decreased cytoplasmic AMT similar to young type I cells in vivo. Concurrent with the appearance of type I cell-like morphology, monolayers exhibited high Rt (greater than 1000 omega.cm2), consistent with the development of tight barrier properties. These monolayers of isolated alveolar epithelial cells may reflect the physiological and morphological properties of the alveolar epithelium in vivo.     

Ultrastructural and morphometric study of the lung of the European salamander,Salamandra salamandra L.

Ultrastructural and morphometric investigations were performed on the lung of the European salamander, Salamandra salamandra L. Folds of first and second order are covered with a ciliated epithelium containing goblet cells. The respiratory surface of the lung is lined by a single type of cell which, in amphibians, combines features of type I and type II alveolar cells of the mammalian lung. In the salamander the respiratory and ciliated epithelial cells as well as goblet cells possess electron dense and lucent vesicles in their cytoplasm as well as lamellar bodies. A small amount of surfactant, composed most probably of phospholipids and mucopolysaccharides, was observed covering the entire inner surface of the lung. Morphometric methods were used to determine the dimensions of the perinuclear region of pneumocytes, the thickness of the air-blood barrier and lung wall, and also the diameter of capillaries. The thickness of the respiratory air-blood barrier was found to be considerably higher than that of the corresponding barrier in mammals.     

Essential contribution of monocyte chemoattractant protein-1/C-C chemokine ligand-2 to resolution and repair processes in acute bacterial pneumonia

Neutrophil infiltration is the first step in eradication of bacterial infection, but neutrophils rapidly die after killing bacteria. Subsequent accumulation of macrophage lineage cells, such as alveolar macrophages (AMs), is essential to remove dying neutrophils, which are a source of injurious substances. Macrophage lineage cells can promote tissue repair, by producing potential growth factors including hepatocyte growth factor (HGF). However, it remains elusive which factor activates macrophage in these processes. Intratracheal instillation of Pseudomonas aeruginosa caused neutrophil infiltration in the airspace; subsequently, the numbers of total AMs and neutrophil ingested AMs were increased. Bronchoalveolar lavage (BAL) fluid levels of monocyte chemoattractant protein (MCP)-1/CC chemokine ligand-2 (CCL2), a potent macrophage-activating factor, were increased before the increases in the number of AM ingesting neutrophils and HGF levels in BAL fluid. Immunoreactive MCP-1 proteins were detected in alveolar type II epithelial cells and AMs only after P. aeruginosa infection. The administration of anti-MCP-1/CCL2 Abs reduced the increases in the number of AM-ingesting neutrophils and HGF levels in BAL fluid, and eventually aggravated lung tissue injury. In contrast, the administration of MCP-1/CCL2 enhanced the increases in the number of AM ingesting neutrophils and HGF levels in BAL fluid, and eventually attenuated lung tissue injury. Furthermore, MCP-1/CCL2 enhanced the ingestion of apoptotic neutrophils and HGF production by a mouse macrophage cell line, RAW 267.4, in a dose-dependent manner. Collectively, MCP-1/CCL2 has a crucial role in the resolution and repair processes of acute bacterial pneumonia by enhancing the removal of dying neutrophils and HGF production by AMs.     

大鼠低氧肺损伤的超微结构及肺泡液体清除功能的变化

目的研究肺损伤大鼠肺泡液体清除率（alveolar flui dclearance,AFC）及肺脏超微结构的变化规律,并探讨二者的内在联系。方法雄性Wistar大鼠120只,体重300～380g,随机分成12组,低氧大鼠暴露于10％的氧浓度,分别于24、48和72h测定基础AFC、特布他林作用后AFC、总肺水量（totallung water,TLW）,并取病理切片观察肺脏超微结构的变化。结果大鼠暴露于10％的氧浓度24h后AFC（18．7±3．19％）与正常大鼠AFC（18．9±2．26％）比较没有明显变化,内皮细胞出现损伤改变,而上皮细胞并没有明显的结构变化;48h时AFC（13．6±2．60％）明显降低,但肺泡上皮细胞的结构和上皮细胞紧密连接并无明显损伤;72h AFC（9．93±1．95％）进一步降低,且经特布他林作用后AFC（12．7±2．64％）仍低于对照组基础AFC,肺泡上皮细胞的结构破坏。结论当轻、中度损伤时,虽AFC降低,但肺泡上皮屏障的结构可以没有明显改变,对药物有较好的反应性。     

Electron microscopy of lung rapidly frozen under controlled physiological conditions

Light microscopy of lung rapidly frozen under controlled physiological conditions has been very successful in correlating pulmonary structure and function. However, to study some aspects of pulmonary capillary morphology, the higher resolution of electron microscopy (EM) is necessary. To date, most EM of lung has involed the instillation of a fixative through the airways or vascular system, techniques that probably alter the normal pressure relationships of the capillaries and therefore their morphology. We describe here a technique for rapidly freezing lung to a depth of 1--2 mm below the pleural surface and preparing sections for EM. Lungs from open-chest rats were frozen at various transpulmonary pressures with cold (--80 degrees C) 70% ethylene glycol. Small pieces were then fixed with a solution containing glutaraldehyde and paraformaldehyde for 24 h at --50 degrees C. Staining was with osmium tetroxide and uranyl acetate. Lung frozen at high volumes showed marked stretching of the alveolar septa with severe deformation of the capillaries. Lung frozen at low inflation pressures revealed open capillaries containing numerous red blood cells; in addition, infolding of the alveolar wall was frequently seen. We conclude that this technique gives a level of preservation of rapidly frozen lung suitable for electron microscopy.     

Lung infection with gamma-herpesvirus induces progressive pulmonary fibrosis in Th2-biased mice

Idiopathic pulmonary fibrosis (IPF) is a progressive, fibrotic lung disease of unknown etiology. A viral pathogenesis in IPF has been suggested since >95% of IPF patients have evidence of chronic pulmonary infection with one or more herpesviruses. To determine whether pulmonary infection with herpesvirus can cause lung fibrosis, we infected mice with the murine gamma-herpesvirus 68 (MHV68). Because IPF patients have a T helper type 2 (Th2) pulmonary phenotype, we used IFN-gammaR-/-, a strain of mice biased to develop Th2 responses. Chronic MHV68 infection of IFN-gammaR-/- mice resulted in progressive deposition of interstitial collagen as shown by light and electron microscopy. A significant decrease in tidal volume paralleled the collagen deposition. Five features typically seen in IPF, increased transforming growth factor-beta expression, myofibroblast transformation, production of Th2 cytokines, hyperplasia of type II cells, and increased expression of matrix metalloproteinase-7, were also present in chronically infected IFN-gammaR-/- mice. There also was altered synthesis of surfactant proteins, which is seen in some patients with familial IPF. MHV68 viral protein was found in type II alveolar epithelial cells, especially in lung areas with extensive alveolar remodeling. In summary, chronic herpesvirus pulmonary infection in IFN-gammaR-/- mice causes progressive pulmonary fibrosis and many of the pathological features seen in IPF.     

In the Absence of Effector Proteins,the Pseudomonas aeruginosa Type Three Secretion System Needle Tip Complex Contributes to Lung Injury and Systemic Inflammatory Responses

Herein we describe a pathogenic role for the Pseudomonas aeruginosa type three secretion system (T3SS) needle tip complex protein, PcrV, in causing lung endothelial injury. We first established a model in which P. aeruginosa wild type strain PA103 caused pneumonia-induced sepsis and distal organ dysfunction. Interestingly, a PA103 derivative strain lacking its two known secreted effectors, ExoU and ExoT [denoted PA103 (ΔU/ΔT)], also caused sepsis and modest distal organ injury whereas an isogenic PA103 strain lacking the T3SS needle tip complex assembly protein [denoted PA103 (ΔPcrV)] did not. PA103 (ΔU/ΔT) infection caused neutrophil influx into the lung parenchyma, lung endothelial injury, and distal organ injury (reminiscent of sepsis). In contrast, PA103 (ΔPcrV) infection caused nominal neutrophil infiltration and lung endothelial injury, but no distal organ injury. We further examined pathogenic mechanisms of the T3SS needle tip complex using cultured rat pulmonary microvascular endothelial cells (PMVECs) and revealed a two-phase, temporal nature of infection. At 5-hours post-inoculation (early phase infection), PA103 (ΔU/ΔT) elicited PMVEC barrier disruption via perturbation of the actin cytoskeleton and did so in a cell death-independent manner. Conversely, PA103 (ΔPcrV) infection did not elicit early phase PMVEC barrier disruption. At 24-hours post-inoculation (late phase infection), PA103 (ΔU/ΔT) induced PMVEC damage and death that displayed an apoptotic component. Although PA103 (ΔPcrV) infection induced late phase PMVEC damage and death, it did so to an attenuated extent. The PA103 (ΔU/ΔT) and PA103 (ΔPcrV) mutants grew at similar rates and were able to adhere equally to PMVECs post-inoculation indicating that the observed differences in damage and barrier disruption are likely attributable to T3SS needle tip complex-mediated pathogenic differences post host cell attachment. Together, these infection data suggest that the T3SS needle tip complex and/or another undefined secreted effector(s) are important determinants of P. aeruginosa pneumonia-induced lung endothelial barrier disruption.     

Estrogen aggravates inflammation in Pseudomonas aeruginosa pneumonia in cystic fibrosis mice

Background

Among patients with cystic fibrosis (CF), females have worse pulmonary function and survival than males, primarily due to chronic lung inflammation and infection with Pseudomonas aeruginosa (P. aeruginosa). A role for gender hormones in the causation of the CF "gender gap" has been proposed. The female gender hormone 17β-estradiol (E2) plays a complex immunomodulatory role in humans and in animal models of disease, suppressing inflammation in some situations while enhancing it in others. Helper T-cells were long thought to belong exclusively to either T helper type 1 (Th1) or type 2 (Th2) lineages. However, a distinct lineage named Th17 is now recognized that is induced by interleukin (IL)-23 to produce IL-17 and other pro-inflammatory Th17 effector molecules. Recent evidence suggests a central role for the IL-23/IL-17 pathway in the pathogenesis of CF lung inflammation. We used a mouse model to test the hypothesis that E2 aggravates the CF lung inflammation that occurs in response to airway infection with P. aeruginosa by a Th17-mediated mechanism.

Results

Exogenous E2 caused adult male CF mice with pneumonia due to a mucoid CF clinical isolate, the P. aeruginosa strain PA508 (PA508), to develop more severe manifestations of inflammation in both lung tissue and in bronchial alveolar lavage (BAL) fluid, with increased total white blood cell counts and differential and absolute cell counts of polymorphonuclear leukocytes (neutrophils). Inflammatory infiltrates and mucin production were increased on histology. Increased lung tissue mRNA levels for IL-23 and IL-17 were accompanied by elevated protein levels of Th17-associated pro-inflammatory mediators in BAL fluid. The burden of PA508 bacteria was increased in lung tissue homogenate and in BAL fluid, and there was a virtual elimination in lung tissue of mRNA for lactoferrin, an antimicrobial peptide active against P. aeruginosa in vitro.

Conclusions

Our data show that E2 increases the severity of PA508 pneumonia in adult CF male mice, and suggest two potential mechanisms: enhancement of Th17-regulated inflammation and suppression of innate antibacterial defences. Although this animal model does not recapitulate all aspects of human CF lung disease, our present findings argue for further investigation of the effects of E2 on inflammation and infection with P. aeruginosa in the CF lung.