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Since December 2019, the global burden of the COVID-19 pandemic has increased rapidly and has impacted nearly every country in the world, affecting those who are elderly or with underlying comorbidities or immunocompromised states. Aim of this systematic review is to summarize lung histopathological characteristics of COVID-19, not only for diagnostic purpose but also to evaluate changes that can reflect pathophysiological pathways that can inform clinicians of useful treatment strategies. We identified following histopathological changes among our patients:: hyaline membranes; endothelial cells/ interstitial cells involvement; alveolar cells, type I pneumocytes/ type II pneumocytes involvement; interstitial and/ or alveolar edema; evidence of hemorrhage, of inflammatory cells, evidence of microthrombi; evidence of fibrin deposition and of viral infection in the tissue samples. The scenario with proliferative cell desquamation is typical of Acute Respiratory Distress Syndrome (ARDS) that can be classified as diffuse alveolar damage (DAD) and not DAD-ARDS. The proposed pathological mechanism concerns the role of both innate and adaptive components of the immune system. COVID-19 lethal cases present themselves as a heterogeneous disease, characterized by the different simultaneous presence of different histological findings, which reflect histological phases with corresponding different pathological pathways (epithelial, vascular and fibrotic changes), in the same patient. Supplementary Information The online version contains supplementary material available at 10.1186/s13027-021-00369-0.

Rats were exposed to fluoroplast-4 pyrolysis products (sample weight 2.6 g, pyrolysis temperature 440-750°C, pyrolysis duration 4 min) containing perfluoroisobutylene over 15 min. Lung tissue samples for histological and electron microscopic examination were isolated in 3 and 30 min after intoxication and processed routinely. Histological examination revealed no structural changes in the lungs. In ultrathin sections of rat lungs, some changes in the structure of type I pneumocytes were detected in 3 min after the exposure: detachment of cytoplasmic processes and the appearance of transcytosis pores. These changes attested to impaired cell-cell interactions and their adhesion to the basement membrane, where structural disorganization and edema of the collagen matrix were observed. In 30 min following exposure, the signs of damage to type I pneumocytes became more pronounced. The increase in the equivalents of transcellular and paracellular permeability in the alveolar lining profile was observed. No changes in the pulmonary capillary endotheliocytes were detected, which suggest that type I pneumocytes are the primary target of the toxic effect of perfluoroisobutylene. The vulnerability of a particular cell population, in view of specific metabolism of these cells, can be the key to deciphering of the mechanisms of the toxic effect of pyrolysis products of fluorinated polymer materials.

Previous studies have shown that Pneumocystis binds to pneumocytes, but the proteins responsible for binding have not been well-defined. Mucins are the major glycoproteins present in mucus, which serves as the first line of defense during airway infection. MUC1 is the best characterized membrane-tethered mucin and is expressed on the surface of most airway epithelial cells. Although by electron microscopy, Pneumocystis primarily binds to type I pneumocytes, it can also bind to type II pneumocytes. We hypothesized that Pneumocystis organisms can bind to MUC1 expressed by type II pneumocytes. Over-expression of MUC1 in human embryonic kidney HEK293 cells increased Pneumocystis binding, while knock-down of MUC1 expression by siRNA in A549 cells, a human adenocarcinoma-derived alveolar type II epithelial cell line, decreased Pneumocystis binding. Immunofluorescence labeling indicated that MUC1 and Pneumocystis were co-localized in infected mouse lung tissue. Incubation of A549 cells with Pneumocystis led to phosphorylation of ERK1/2 that increased with knock down of MUC1 expression by siRNA. Pneumocystis caused increased IL-6 and IL-8 secretion by A549 cells, and knock down of MUC1 further increased their secretion in A549 cells. Taken together, these results suggest that binding of Pneumocystis to MUC1 expressed by airway epithelial cells may facilitate establishment of productive infection.

The present study examined the susceptibility of rats to the Middle East respiratory syndrome coronavirus (MERS-CoV) and determined whether this animal is a suitable model for MERS-CoV infection. Immunohistochemical analysis identified dipeptidyl peptidase 4 (DPP4), a known receptor for MERS-CoV on type I pneumocytes from infected rats. Whereas adult rats developed antibodies against MERS-CoV spike protein after intranasal inoculation, there was no evidence of viral replication in the lungs of adult, young, or neonatal rats after intranasal inoculation with MERS-CoV. In addition, human DPP4-expressing rat kidney fibroblasts, but not rat DPP4-expressing cells, were susceptible to MERS-CoV. Taken together, these results suggest that the rat is not a useful animal model for studying MERS-CoV infection.

Background and Aims: Reexpansion pulmonary edema (RPE) is a severe disorder, and its pathophysiology is not well understood. One proposed mechanism for RPE is that chemical substances such as cytokines increase alveolar permeability. Another possible mechanism is that alveolar distention during reexpansion causes physical damage. To test the hypothesis that sudden alveolar distention damages alveolar cellular structure and identify the underlying cause of RPE, we developed and evaluated the morphological characteristics of a rat RPE model. Methods: Lung from a rat model of RPE was observed by using live imaging from intravital fluorescence microscopy with fluorescein isothiocyanate–labelled albumin as tracer, light microscopy, and electron microscopy (with and without horseradish peroxidase [HRP] as tracer). Results: Intravital fluorescence microscopy and light microscopy showed that RPE developed almost immediately after lung reexpansion and that blood flow in pulmonary capillaries slowed substantially. Electron microscopy revealed pores in type I pneumocytes and overt fissures in alveolar walls. Electron microscopic observation with HRP revealed that HRP moved from capillaries to the inner surfaces of alveolar epithelia in reexpanded lungs. In addition, diaminobenzidine reaction products from the HRP enzyme reaction were visible in areas with pores. Conclusions: RPE occurred almost immediately after lung reexpansion. Pores developed in type I pneumocytes in alveolar epithelium. These pores, together with overt fissures in alveolar walls, allowed leakage of plasma components into alveoli. These findings appear to be important features of RPE development.

Recurrent primary spontaneous pneumothorax (PSP) is a troublesome problem and a major concern for the patients. This study examined whether nuclear factor erythroid 2-related factor 2 (Nrf2) expression in alveolar type I pneumocytes was associated with the clinical manifestations of PSP patients including disease recurrence. Eighty-eight PSP patients who were managed with needlescopic video-assisted thoracoscopic surgery (NVATS) were included in this study. Immunohistochemistry (IHC) was assessed to determine Nrf2 expression in resected lung tissues and the results were correlated with clinicopathological characteristics by the chi-square or the Fisher's exact test. The prognostic value of Nrf2 for overall recurrence was evaluated by univariate and multivariable Cox regression model. The expression of Nrf2 was observed in type I pneumocytes of lung tissues from PSP patients by IHC. We found that low Nrf2 expression in PSP patients, especially in young (age ≤ 20, p = 0.033) and body mass index (BMI) ≥18 kg/m2 (p = 0.019) groups, was significantly correlated with PSP recurrence. In the univariate and multivariate analyses, high Nrf2 expression was a significant protective factor for overall recurrence in PSP patients (univariate: p = 0.026; multivariate: p = 0.004). The expression level of Nrf2 in alveolar type I pneumocytes was a potential factor involved in PSP recurrence. Our findings suggest that elevated Nrf2 expression in PSP patients may be a promising way for reducing PSP recurrence.

Objective: Reexpansion pulmonary edema (RPE) is a severe disorder, and its pathophysiology is not well understood. One proposed mechanism for RPE is that chemical substances such as cytokines increase alveolar permeability. Another possible mechanism is that alveolar distention during reexpansion causes physical damage. To test the hypothesis that sudden alveolar distention damages alveolar cellular structure and identify the underlying cause of RPE, we developed and evaluated the morphological characteristics of a rat RPE model. Methods: Lung from a rat model of RPE was observed by using live imaging from intravital fluorescence microscopy with fluorescein isothiocyanate labelled albumin as tracer, light microscopy, and electron microscopy (with and without horseradish peroxidase [HRP] as tracer). Results: Intravital fluorescence microscopy and light microscopy showed that RPE developed almost immediately after lung reexpansion and that blood flow in pulmonary capillaries slowed substantially. In some capillaries, however, blood flow had stopped entirely; in others, anterograde and retrograde flow alternated. Electron microscopy revealed pores in type I pneumocytes and overt fissures in alveolar walls. Electron microscopic observation with HRP revealed that HRP moved from capillaries to the inner surfaces of alveolar epithelia in reexpanded lungs. In addition, diaminobenzidine reaction products from the HRP enzyme reaction were visible in areas with pores. Conclusions: RPE occurred almost immediately after lung reexpansion. Pores developed in type I pneumocytes in alveolar epithelium. These pores, together with overt fissures in alveolar walls, allowed leakage of plasma components into alveoli. These findings appear to be important features of RPE development.

Background and Objectives: Several studies have reported that some conditions such as exercise and hypoxia induce DNA damage and dysfunction and apoptosis. Some plant foods contain numerous bioactive compounds and anti-inflammatory properties that can help fight DNA damage. Therefore, the current study evaluated the effect of supplementation of Adiantum capillus-veneris (ACV) extract on Bax/B-cell lymphoma 2 (Bcl-2) ratio apoptotic index and remodeling of pulmonary alveolar epithelial cells in lung tissue of healthy Wistar rats during stressful conditions (hypoxia). Materials and Methods: Twenty-seven Wistar male rats (four-week old, 72 &plusmn, 9 g) were randomly assigned into three groups: normoxic, sedentary, and not-supplemented (NG, n = 9), exercise and hypoxia and not-supplemented (HE, n = 9), and exercise and hypoxia and supplemented group (HS, n = 9). The NG remained sedentary in the normoxia environment for nine weeks. The HE group participated in a high-intensity (IT) program for six weeks, then remained sedentary in the hypoxia environment for three weeks. The low-pressure chamber simulated a ~2800 M altitude 24 h/d. HS participated in IT, then entered and remained sedentary in the hypoxia environment for three weeks, and they consumed 500 mg per kg of body weight ACV extract. Results: The Bax/Bcl-2 ratio of the HE group increased significantly (+50.27%, p &le, 0.05), the average number of type I pneumocytes was reduced significantly (&minus, 18.85%, p &le, 0.05), and the average number of type II pneumocytes was increased significantly (+14.69%, p &le, 0.05). Also, after three weeks of consuming the ACV extract, the HS group in comparison with the HE group had their Bax/Bcl-2 ratio reduced significantly (&minus, 24.27%, p &le, 0.05), the average number of type I pneumocytes increased significantly (+10.15%, p &le, 0.05), and the average number of type II pneumocytes reduced significantly (&minus, 7.18%, p &le, 0.05). Conclusion: The findings show that after three weeks of hypoxia following six weeks of high-intensity interval training in Wistar rats, the Bax/Bcl-2 ratio and the number of type II pneumocytes were increased and the number of type I pneumocytes was reduced significantly. These results strongly suggest that an apoptosis state was induced in the lung parenchyma, and consuming ACV extract modulated this state.

The Gram-negative bacterium Pseudomonas aeruginosa is a major cause of hospital-acquired infections and the focus of much attention due to its resistance to many conventional antibiotics. It harbors a wide range of disease-promoting virulence factors, including a type III secretion system. Here we review our recent study of ExoS, one of the effector proteins exported by this type III secretion system. Using a mouse model of pneumonia, we showed that the ADP-ribosyltransferase (ADPRT) activity of ExoS caused formation of “fields of cell injection” (FOCI) in the lungs. These FOCI represented ExoS-injected clusters of type I pneumocytes that became compromised, leading to disruption of the pulmonary-vascular barrier and subsequent bacterial dissemination from the lungs to the bloodstream. We discuss the potential mechanisms by which these processes occur as well as the novel techniques used to study ExoS function in vivo.

Although the lung is a defining feature of air-breathing animals, the pathway controlling the formation of type I pneumocytes, the cells that mediate gas exchange, is poorly understood. In contrast, the glucocorticoid receptor and its cognate ligand have long been known to promote type II pneumocyte maturation; prenatal administration of glucocorticoids is commonly used to attenuate the severity of infant respiratory distress syndrome (RDS). Here we show that knock-in mutations of the nuclear co-repressor SMRT (silencing mediator of retinoid and thyroid hormone receptors) in C57BL/6 mice (SMRTmRID) produces a previously unidentified respiratory distress syndrome caused by prematurity of the type I pneumocyte. Though unresponsive to glucocorticoids, treatment with anti-thyroid hormone drugs (propylthiouracil or methimazole) completely rescues SMRT-induced RDS, suggesting an unrecognized and essential role for the thyroid hormone receptor (TR) in lung development. We show that TR and SMRT control type I pneumocyte differentiation through Klf2, which, in turn, seems to directly activate the type I pneumocyte gene program. Conversely, mice without lung Klf2 lack mature type I pneumocytes and die shortly after birth, closely recapitulating the SMRTmRID phenotype. These results identify TR as a second nuclear receptor involved in lung development, specifically type I pneumocyte differentiation, and suggest a possible new type of therapeutic option in the treatment of RDS that is unresponsive to glucocorticoids.

Previously we determined that chronic alcohol ingestion (6 weeks) in rats increases lung epithelial permeability in vivo approximately 5-6-fold and promotes flooding of the alveolar airspaces with proteinaceous fluid in response to stresses such as sepsis. In parallel, alveolar epithelial cells isolated from alcohol-fed rats fail to form tight monolayers in vitro, even when cultured for up to 8 days in the absence of alcohol. However, the molecular mechanisms underlying alcohol-induced permeability are unknown. Claudins are key components of tight junctions that restrict the paracellular movement of water, proteins, and solutes across cellular barriers including the alveolar epithelium. In this study, we examined the expression of multiple members of the claudin protein family in the lungs of alcohol-fed versus control-fed rats (Lieber-DeCarli liquid diet with either 36% of calories as alcohol or an isocaloric substitution with maltin-dextrin for 6 weeks). We determined that chronic alcohol ingestion affected the expression of multiple claudins; most striking were decreases in claudin-1 and claudin-7, and an increase in claudin-5, in the whole lung and in alveolar epithelial monolayers derived from alcohol-fed rats. In parallel, immunocytochemistry of alveolar epithelial monolayers from alcohol-fed rats revealed abnormal intracellular accumulation of claudin-7 protein and relatively decreased localization to cell membranes. Claudin-1 and claudin-7 are relatively specific to alveolar epithelial type I pneumocytes that form the vast majority of the alveolar epithelial barrier in vivo, and increases in claudin-5 have been associated with increased epithelial permeability in other systems. Therefore, these findings suggest that changes in claudin expression in the alveolar epithelium produce a "leakier" phenotype that renders the alcoholic lung susceptible to alveolar flooding during acute inflammatory stresses.

A morphological and morphometric study of the lung of the newborn quokka wallaby (Setonix brachyurus) was undertaken to assess its morphofunctional status at birth. Additionally, skin structure and morphometry were investigated to assess the possibility of cutaneous gas exchange. The lung was at canalicular stage and comprised a few conducting airways and a parenchyma of thick-walled tubules lined by stretches of cuboidal pneumocytes alternating with squamous epithelium, with occasional portions of thin blood-gas barrier. The tubules were separated by abundant intertubular mesenchyme, aggregations of developing capillaries and mesenchymal cells. Conversion of the cuboidal pneumocytes to type I cells occurred through cell broadening and lamellar body extrusion. Superfluous cuboidal cells were lost through apoptosis and subsequent clearance by alveolar macrophages. The establishment of the thin blood-gas barrier was established through apposition of the incipient capillaries to the formative thin squamous epithelium. The absolute volume of the lung was 0.02 +/- 0.001 cm(3) with an air space surface area of 4.85 +/- 0.43 cm(2). Differentiated type I pneumocytes covered 78% of the tubular surface, the rest 22% going to long stretches of type II cells, their precursors or low cuboidal transitory cells with sparse lamellar bodies. The body weight-related diffusion capacity was 2.52 +/- 0.56 mL O(2) min(-1) kg(-1). The epidermis was poorly developed, and measured 29.97 +/- 4.88 microm in thickness, 13% of which was taken by a thin layer of stratum corneum, measuring 4.87 +/- 0.98 microm thick. Superficial capillaries were closely associated with the epidermis, showing the possibility that the skin also participated in some gaseous exchange. Qualitatively, the neonate quokka lung had the basic constituents for gas exchange but was quantitatively inadequate, implying the significance of percutaneous gas exchange.

Hospital-acquired pneumonia is associated with high rates of morbidity and mortality, and dissemination to the bloodstream is a recognized risk factor for particularly poor outcomes. Yet the mechanism by which bacteria in the lungs gain access to the bloodstream remains poorly understood. In this study, we used a mouse model of Pseudomonas aeruginosa pneumonia to examine this mechanism. P. aeruginosa uses a type III secretion system to deliver effector proteins such as ExoS directly into the cytosol of eukaryotic cells. ExoS, a bi-functional GTPase activating protein (GAP) and ADP-ribosyltransferase (ADPRT), inhibits phagocytosis during pneumonia but has also been linked to a higher incidence of dissemination to the bloodstream. We used a novel imaging methodology to identify ExoS intoxicated cells during pneumonia and found that ExoS is injected into not only leukocytes but also epithelial cells. Phagocytic cells, primarily neutrophils, were targeted for injection with ExoS early during infection, but type I pneumocytes became increasingly injected at later time points. Interestingly, injection of these pneumocytes did not occur randomly but rather in discrete regions, which we designate ““fields of cell injection” (FOCI). These FOCI increased in size as the infection progressed and contained dead type I pneumocytes. Both of these phenotypes were attenuated in infections caused by bacteria secreting ADPRT-deficient ExoS, indicating that FOCI growth and type I pneumocyte death were dependent on the ADPRT activity of ExoS. During the course of infection, increased FOCI size was associated with enhanced disruption of the pulmonary-vascular barrier and increased bacterial dissemination into the blood, both of which were also dependent on the ADPRT activity of ExoS. We conclude that the ADPRT activity of ExoS acts upon type I pneumocytes to disrupt the pulmonary-vascular barrier during P. aeruginosa pneumonia, leading to bacterial dissemination., Author Summary Dissemination to the bloodstream is a poor prognostic sign in patients with hospital-acquired pneumonia, yet the mechanism by which this occurs is poorly understood. To begin to address this issue, we have used a mouse model of P. aeruginosa pneumonia to study the mechanism by which the type-III-secreted effector protein ExoS enhances bacterial dissemination. We show that intoxication of type I pneumocytes by ExoS leads to cell death and disruption of the pulmonary-vascular barrier, allowing bacterial dissemination into the bloodstream. These effects required the ADP-ribosyltransferase activity of ExoS, as strains secreting an ExoS variant lacking this activity demonstrated reduced type I pneumocytes death and pulmonary-vascular breakdown. This study indicates that inhibitors of the ADP-ribosyltransferase activity of ExoS could serve as novel therapeutics for the prevention of bacteremic pneumonia.

Pulmonary arterial hypertension (PAH) is a rare disease but with significant morbidity and high mortality. There is no specific way to diagnose PAH. Thus, an easy used with good sensitivity and specificity biomarker of PAH is highly desirable to aid in the screening, diagnosis, and follow-up. Caveolin-1 (Cav1) is the structural protein of caveolae and is highly expressed in type I pneumocytes. Lungs tissues from idiopathic PAH (IPAH) patients showed decreased expression of Cav1 in vascular endothelial cells. Therefore, we developed a direct sandwich immunoassay for the determination of Cav1 in IAPH patient’s serum. The result disclosed serum Cav1 level was significantly lower in IPAH than control groups. Using serum Cav1, 17.17 pg/mL as a cutoff value, the sensitivity was 0.59 and the specificity was 1.0. There were two major findings in our results. First, serum Cav1 might be a novel biomarker in the diagnosis of IPAH with fare sensitivity and good specificity. Second, Cav1 might be used to make differential diagnosis between COPD-PH and IPAH group.

The pulmonary surfactant synthesis is disturbed in experimentally induced asthma, as are the intracellular storage capacity and its physical activity. These alterations may also be present in chronic asthmatic patients, and therefore the dysfunction of the pulmonary surfactant system may play an important role in the pathophysiology of asthma. Some clinical reports have described favorable results with the use of diethylcarbamazine (DEC) in patients with bronchial asthma showing that DEC is effective in terminating acute attacks of bronchial asthma. The present study aimed to analyze the ultrastructural alterations of lung cells after treatment in vivo with diethylcarbamazine. After 12 days of treatment with DEC, when compared with control samples, type II pneumocytes showed active nuclei with abundant euchromatin and evident nucleoli, and a substantially greater number of mature secretion vesicle. On the other hand, type I pneumocytes showed no morphological alterations. After DEC treatment, lung macrophages also presented several characteristics of cellular activation such as nuclei with a prominence of euchromatin and central nucleoli as well as an abundance of early and late endossomes distributed throughout the cytoplasm. These results confirm that DEC exerts a role in the activation of important pulmonary cellular pathways, which are probably related to the clinical improvement of asthma symptoms after DEC treatment.

Alveolar type II pneumocytes are thought to be progenitor cells capable of self-renewal and differentiation into type I pneumocytes. Nevertheless, the existence of an alveolar stem cell has been postulated. In lungs from patients with cystic fibrosis, the alveolar epithelium is damaged with ulceration and subsequent regeneration. We characterized alveolar modifications histologically and immunohistochemically in the pulmonary tissue of a patient homozygous for the DeltaF508 mutation. Alveoli were of variable size and surrounded by an inflammatory infiltrate. They were lined by a continuous layer of cuboidal cells with very weak proliferative activity. These cells resembled type II pneumocytes. They expressed thyroid transcription factor-1, cystic fibrosis transmembrane conductance regulator, cytokeratin 7 and contained lamellar bodies. Weak expression of cytokeratin 5 considered to be a marker of progenitor cells of the bronchial and bronchiolar epithelium was detected. Explantation of this alveolar epithelium produced primary cultures and subcultures of epithelial cells that had acquired proliferative properties showing signs of dedifferentiation with a loss of lamellar bodies and a lack of expression of thyroid transcription factor-1. Persistence of the expression of cytokeratin 7 and a strong expression of cytokeratin 5 were observed. The culture conditions were thought to have circumvented the inhibition of proliferation observed in vivo due to the inflammatory peri-alveolar environment. They thus favored the multiplication of a population of cells co-expressing cytokeratin 5 and certain characteristics of type II pneumocytes. The presence of these cells of intermediate phenotype is indicative of the existence of immature precursors for type II pneumocytes.

Acute respiratory distress syndrome (ARDS) is characterized by diffuse alveolar damage, and evolves progressively with three phases: exsudative, fibroproliferative, and fibrotic. In the exudative phase, there are interstitial and alveolar edemas with hyaline membrane. The fibropro­liferative phase is characterized by exudate organization and fibroelastogenesis. There is proliferation of type II pneumocytes to cover the damaged epithelial surface, followed by differentiation into type I pneumocytes. The fibroproliferative phase starts early, and its severity is related to the patient?s prognosis. The alterations observed in the phenotype of the pulmonary parenchyma cells steer the tissue remodeling towards either progressive fibrosis or the restoration of normal alveolar architecture. The fibrotic phase is characterized by abnormal and excessive deposition of extracellular matrix proteins, mainly collagen. The dynamic control of collagen deposition and degradation is regulated by metalloproteinases and their tissular regulators. The deposition of proteoglycans in the extracellular matrix of ARDS patients needs better study. The regulation of extracellular matrix remodeling, in normal conditions or in several pulmonary diseases, such as ARDS, results from a complex mechanism that integrate the transcription of elements that destroy the matrix protein and produce activation/inhibition of several cellular types of lung tissue. This review article will analyze the ECM organization in ARDS, the different pulmonary parenchyma remodeling mechanisms, and the role of cytokines in the regulation of the different matrix components during the remodeling process.

Cyclooxygenase-2 (COX-2) was detected and localized in 15 pigs with naturally occurring pleuropneumonia using a 437–base pair digoxigenin-labeled cDNA probe in an in situ hybridization protocol. Histopathologic changes in the acute stage were characterized by coagulative necrosis of lung parenchyma, hemorrhage, vascular thrombosis, edema, fibrin deposition, and infiltration of lung parenchyma by neutrophils and alveolar macrophages in nine pigs. In chronic lesions, a thick layer of granulation tissue surrounded foci of pulmonary necrosis in six pigs. All 15 pigs infected with Actinobacillus pleuropneumoniae, confirmed by bacterial isolation, had distinct positive hybridization signals for COX-2 in bronchial, bronchiolar epithelial cells, alveolar macrophages, neutrophils, and type I pneumocytes. COX-2 expression was detected primarily in neutrophils from pigs with acute lesions and primarily in alveolar macrophages from pigs with chronic lesions. The results suggest that a prostanoid product of COX-2 is an important component of the inflammatory response to acute and chronic A. pleuropneumoniae infection.

UNLABELLED Type II pneumocytes synthesize surfactant and differentiate into type I pneumocytes to maintain the epithelium (1). Alveolar type II cell proliferation is required for reepithelization after acute lung injury (ALI) and is thought to minimize the subsequent fibrotic response (1). Keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) are among the most potent mitogen for type II epithelial cells, but not for fibroblasts in the lung (1). These growth factors attenuate several experimental ALI models by promoting epithelial repair (2,3). Thus, KGF and HGF may be a promising therapeutic approach to ALI. Critically ill patients with ALI often receive IV anesthetics or sedatives to facilitate mechanical ventilation. Furthermore, these patients sometimes undergo bronchoscopy under local anesthesia to obtain bronchoalveolar lavage fluid or to remove respiratory secretions. Several IV and local anesthetics inhibit proliferation of various cells including epithelium (4,5). If these anesthetics impede proliferation of type II pneumocytes, this suppressive effect may be a disadvantage for alveolar reepithelization in the course of recovery from ALI. In this study, we examined the effects of midazolam, propofol, ketamine, thiopental, and lidocaine on proliferation of type II alveolar epithelial cells using in vitro culture system. Because fibroblast proliferation is a key event in late phase of ALI, inhibition of this fibroproliferation is probably beneficial. Thus, we further determined whether these anesthetics could regulate proliferation of lung fibroblasts. In the current study, rolipram was used as a positive control. In our previous preliminary experiment, we found that rolipram, a phosphodiesterase inhibitor type IV, augments spontaneous or KGF-/HGF-promoted type II cell proliferation (6). IMPLICATIONS Midazolam, ketamine, thiopental, propofol, or lidocaine did not inhibit proliferation of cultured rat type II pneumocytes. Our findings suggest that these anesthetics do not impede alveolar reepithelization after acute lung injury.

Neuroenteric cysts are benign intradural endoderm cysts lined by gastrointestinal (GI) or tracheobronchial epithelial cells. Their malignant transformation is extremely rare and only six cases have been reported. In these cases, tissue lineage of the cystic endoderm cells giving rise to carcinoma was not clearly identified either as respiratory or as GI type. Herein, we report a case of mucinous adenocarcinoma arising from the neuroenteric cyst with broncho-pulmonary differentiation in the right cerebral hemisphere of a Japanese woman in her late 50s. The cyst wall was entirely lined by the following respiratory epithelial components: stratified bronchial ciliated columnar epithelium with basal cells positive for CK5 and p63, terminal bronchiolar Clara cells positive for thyroid transcription factor (TTF)-1, surfactant B and negative for surfactant C, type I pneumocytes positive for TTF-1, negative for surfactant B and C, and type II pneumocytes positive for TTF-1 and surfactant B and C. In addition, nests of hyperplastic single layered mucinous epithelial cells with bronchial goblet cell phenotype were also demonstrated, and histologic features were almost the same as the pulmonary type I congenital adenomatoid malformation (CCAM) with mucinous epithelial proliferation. The mucinous epithelial nests of type I CCAM are liable to develop mucinous adenocarcinoma and frequently accompany K-ras mutation and expression of p16. However, K-ras mutation and p-16 expression were not detected in this case.

We assessed the capacity of plastic-adherent cultured bone marrow cells to serve as precursors of differentiated parenchymal cells of the lung. By intravenously delivering lacZ-labeled cells into wild-type recipient mice after bleomycin-induced lung injury, we detected marrow-derived cells engrafted in recipient lung parenchyma as cells with the morphological and molecular phenotype of type I pneumocytes of the alveolar epithelium. At no time after marrow cell injection, did we detect any engraftment as type II pneumocytes. In addition, we found that cultured and fresh aspirates of bone marrow cells can express the type I pneumocyte markers, T1α and aquaporin-5. These observations challenge the current belief that adult alveolar type I epithelial cells invariably arise from local precursor cells and raise the possibility of using injected marrow-derived cells for therapy of lung diseases characterized by extensive alveolar damage.

The pulmonary toxicity of sodium diethyldithiocarbamate and cadmium chloride, each separately and in combination, was compared in Sprague-Dawley rats after single intratracheal instillation in sequential experiments by chemical, immunological and morphological methods. With combined exposure, the cadmium content of the lungs increased permanently relative to that of the lungs of just cadmium-treated animals. Immunoglobulin levels of the whole blood did not change, whereas in bronchoalveolar lavage the IgA and IgG levels increased significantly. Morphological changes were characteristic of the effects of cadmium but were more extensive and more serious than in the case of cadmium administration alone: by the end of the first month, interstitial fibrosis, emphysema and injury of membranes of type I pneumocytes developed and hypertrophy and loss of microvilli in type II pneumocytes were detectable. These results showed that although dithiocarbamates as chelating agents are suitable for the removal of cadmium from organisms, they alter the redistribution of cadmium within the organism, thereby increasing the cadmium content in the lungs, and structural changes are more serious than observed upon cadmium exposure alone. Copyright © 2001 John Wiley & Sons, Ltd.

Sixteen 8- to 9-week-old Pasteurella multocida-free rabbits were divided into two equal groups. Eight rabbits in one group were inoculated intranasally with P. multocida type A:3. The other eight were inoculated intranasally with phosphate-buffered saline and used as controls. Nasal swabs taken before and after inoculation were cultured for bacterial isolation. Post-mortem nasal swabs and lung samples were cultured for bacteriological isolation. Nasal mucosa and lung samples were collected and processed for transmission electron microscopy. Pasteurella multocida was isolated from the nasal cavity of all infected rabbits and from the lungs of four infected rabbits. Degenerative ultrastructural changes in epithelial cells and endothelial cells were seen in the infected rabbits. Deciliation of the cilated epithelium and hyperplasia of the goblet cells in the nasal mucosa were noted. Thickening of the alveolar septa due to hyperplasia of type II pneumocytes, swelling of the endothelial lining of capillaries and infiltration of inflammatory cells were also observed. Intracellular invasion of the nasal epithelial cells and of type II pneumocytes by the organism was observed. Coccobacilli were observed in membrane-bound vacuoles in the cytoplasm of these cells. The vacuoles were adjacent to the host-cell mitochondria and some of these vacuoles appeared to be fused to the mitochondrial membrane. Some type I pneumocytes with intracellular membrane-bound vacuoles containing bacterial cells showed protrusions, which appeared to detach into the alveolar lumina. These results indicated that P. multocida serotype A:3 in rabbits can invade the epithelial cell and cause structural changes in the interstitium, epithelium and endothelium. Heterophils and macrophages appear to play important roles in tissue injury.

Background: Leptospirosis is a re-emerging zoonosis with protean clinical manifestations. Recently, the importance of pulmonary hemorrhage as a lethal complication of this disease has been recognized. In the present study, five human necropsies of leptospirosis (Weil‘s syndrome) with extensive pulmonary manifestations were analysed, and the antibodies expressed in blood vessels and cells involved in ion and water transport were used, seeking to better understand the pathophysiology of the lung injury associated with this disease. Principal Findings: Prominent vascular damage was present in the lung microcirculation, with decreased CD34 and preserved aquaporin 1 expression. At the periphery and even inside the extensive areas of edema and intraalveolar hemorrhage, enlarged, apparently hypertrophic type I pneumocytes (PI) were detected and interpreted as a non-specific attempt of clearence of the intraalveolar fluid, in which ionic transport, particularly of sodium, plays a predominant role, as suggested by the apparently increased ENaC and aquaporin 5 expression. Connexin 43 was present in most pneumocytes, and in the cytoplasm of the more preserved endothelial cells. The number of type II pneumocytes (PII) was slightly decreased when compared to normal lungs and those of patients with septicemia from other causes, a fact that may contribute to the progressively low PI count, resulting in deficient restoration after damage to the alveolar epithelial integrity and, consequently, a poor outcome of the pulmonary edema and hemorrhage. Conclusions: Pathogenesis of lung injury in human leptospirosis was discussed, and the possibility of primary noninflammatory vascular damage was considered, so far of undefinite etiopathogenesis, as the initial pathological manifestation of the disease.

The cellular and subcellular localizations of annexins I, IV, and VI in the rabbit tracheal and alveolar epithelia were studied by performing immunofluorescence labeling on thin frozen sections of these tissues, using specific monoclonal antibodies. Annexin I was highly expressed by ciliated cells, where it was concentrated in the cilia but was also present along the basolateral domain of the plasma membrane and in the nuclei. It was also abundant in the cytoplasm of type II pneumocytes and alveolar macrophages. Either one or both of these alveolar cells might be capable of secreting a very small amount of annexin I, which was found to be associated with the surfactant layer. Annexin IV was synthesized by all the lung epithelial cells. It was associated with the plasma membrane basolateral domain in ciliated cells and with either the apical or basal domain of plasma membrane in type I pneumocytes, whereas it was cytoplasmic in goblet cells and type II pneumocytes. Annexin VI was expressed only by alveolar endothelial cells, where it was probably cytoplasmic. None of these three annexins seem to be expressed in the nondifferentiated tracheal basal epithelial cells.

The blood-air barrier is a most important functional element of the lung but little information is available about the cells constituting this barrier in vivo. The aim of the present study was to create an in vitro model of the blood-air barrier that would allow investigation of cellular interactions and alveolar metabolism, and would be suitable for in vitro drug screening. Rat pneumocytes and bovine microvascular endothelial cells were grown on opposite sides of microporous polycarbonate filters, as immersion, perfusion and liquid-air interface (LAI) cultures. The effects of culture conditions on cell morphology were examined by light and transmission electron microscopy. For immersion and perfusion co-cultures, both compartments were supplied with culture medium. In contrast, for liquid-air interface studies, only the endothelial cell compartment was continuously supplied with serum-free medium, whilst the type II pneumocytes were ventilated with air. The pneumocytes lost their morphological characteristics when using immersion or perfusion co-cultures. Under liquid-air interface conditions, they retained most of their characteristic morphological features when compared to the intact blood-air barrier. A subset of primary type II pneumocytes retained its differentiated phenotype, with cuboidal morphology, lamellar bodies and apical microvilli. These type II pneumocytes appeared to be connected by tight junctions to cells expressing morphological characteristics of type I pneumocytes. As shown herein, the liquid-air interface co-culture possesses many morphological characteristics of the intact blood-air barrier. In summary, this article describes the design of an artificial blood-air barrier, in which rat pneumocytes were cultivated with bovine microvascular endothelial lung cells on opposing sides of a microporous polycarbonate filter. We conclude that it might be a promising in vitro model for studies of molecular transport via the blood-air barrier, the investigation of repair mechanisms after alveolar injury, or as an in vitro screening system.

Background The aim of this morphological investigation was to obtain more information about the structural and cellular mechanisms of interalveolar pore formation in postnatal lung development. Assuming that alveolar pore formation is related to the general thinning of interalveolar walls observed in the postnatal period, we have focused our attention on the topographical relationship between epithelial cells and connective tissue in the septum. Thereby we tried to formulate a uniform concept of pore formation. Methods After fixation with glutaraldehyde and osmiumtetroxide, tissue blocks of rat lungs aged 44 days were embedded in Epon. Serial sections were obtained in order to analyse precisely pores and supposed sites of pore formation (type II cells and thin spots in transsections of interalveolar walls). Results We made the following observations: there are pores with or without type II cells in the neighbourhood, and “pre-pores” with either fully transseptal granular pneumocytes, or thin spots in the interalveolar wall consisting of one or two layers of type I cell epithelium or of type II and type I cells without intervening connective tissue. Conclusions From these findings we deduce that there is a general principle of interalveolar pore formation which consists in the formation of transseptal interepithelial cell contacts (i.e., between cells of type II and type I or type I and type I), promoted by the thinning of interalveolar walls in the stage of microvascular maturation. Within the zone of contact the cells thin out and give way to form an interalveolar opening. © 1996 Wiley-Liss, Inc.

Cellulose after a single intratracheal dose (15 mg per animal) brought about fibrosing granulomatous alveobronchiolitis and an increase of IgA production in the bronchoalveolar lavage. Fibrosing alveolitis showed moderate progression as a function of time. With different morphological methods, injury of type I pneumocytes and the incomplete repair of type II pneumocytes were detected. The damage of the alveolar epithelium initiated and activated a series of processes that led to definite pulmonary alterations: pulmonary fibrosis leading to the disintegration of the alveolo-capillary morphological functional unit.

Histopathological examination and cytological analyses in bronchial alveolar lavage fluids (BALF) were performed to clarify the acute toxicity of diesel exhaust particles (DEP) introduced into the lung of ICR mice by intratracheal instillation. Activated charcoal (Norit) was intratracheally administered as a control for non-oedemagenic carbon particles. After administration of two doses (0.4 mg or 0.8 mg per mouse) of DEP, lung water contents increased with instillation dose and with time and increased 1.9 and 2.7-fold, respectively, compared to control animals 24 h after the administration of DEP. In contrast, the instillation of Norit had no effect on the increase in water contents. An inflammatory response in lungs was observed by an increase of inflammatory cells in BALF from mice instilled with DEP. The degree of increase in neutrophils of BALF from mice treated with DEP was much greater than in mice treated with Norit. An intense color of MB-pigment, which showed the extent and degree of endothelial cell injury, was found up to 4 h after administration of DEP. Histopathologically, the disruption of capillary endothelial cells, the detachment from their basement membrane and necrosis, disruption and desquamation of type I pneumocytes were observed, 6 h after the injection of DEP, by electron microscopy. An influx of neutrophils into alveoli, intra-alveolar hemorrhage, perivascular oedema and bronchiolar cell hypertrophy were detected between 18 and 24 h after DEP administration. However, the magnitude of these appearances was greater in mice treated with 0.8 mg of DEP than in mice treated with 0.4 mg. The administration of Norit caused an increase of alveolar macrophages and slight infiltration of neutrophils into the alveolar air spaces and alveolar septa in the animals and had no effects on the bronchioles. These results may suggest that damage of capillary endothelial cells and type I pneumocytes are the earliest changes of lung toxicities by DEP and these cell injuries lead to alveolar oedema and the subsequent inflammatory response.

The pathology of aerosolized staphylococcal enterotoxin B (SEB) was studied in the nonhuman primate. Six juvenile rhesus monkeys that received multiple lethal inhaled doses of SEB developed diarrhea and vomiting within 24 hr followed by depression, dyspnea, and shock. Three of 6 animals died by 52 hr. The most striking gross lesion in all 6 monkeys was diffuse severe pulmonary edema. Histologically, edema fluid was present within the peribronchiolar, peribronchial, and perivascular interstitium, alveolar septa, and alveoli. The adventitia of pulmonary vessels was infiltrated by lymphocytes, macrophages, and fewer neutrophils. Numerous large lymphocytes with occasional mitotic figures were within pulmonary vessels, often occluding alveolar capillaries. These cells were strongly immunoreactive with monoclonal antibodies against CD3, establishing them as T cells. Ultrastructurally, endothelial cell junctions were intact, and endothelial cells and type I pneumocytes contained numerous pinocytotic vesicles. Alveolar septal interstitial spaces were expanded by edema. The mechanism of these SEB-induced pulmonary lesions was not determined. We hypothesize that cytokine production by activated T cells may have caused vascular permeability changes leading to widespread pulmonary edema and shock.

Summary The expression of pan-cadherin was investigated immunohistochemically in normal and irradiated mini pig lungs. Using a double labelling immunofluorescence technique, pancadherin expression was also compared with that of cytokeratin 18, which is selectively present in type II cells. In untreated animals pan-cadherin was detected in type I pneumocytes, in alveolar macrophages and in endothelial cells of larger blood vessels. Radiation caused increased pan-cadherin immunoreactivity of bronchial epithelial cells and interstitial cells. In addition, pan-cadherin expression was induced in type II pneumocytes. These changes in the distribution of epithelial cadherin molecules may implicate a role of cadherins for epithelial remodeling in response to radiation-induced lung injury

Papillary pneumocytoma, so-called sclerosing hemangioma of the lung, is a benign pulmonary neoplasm. In the present study, clinical aspects, immunohistochemical characteristics, and ultrastructural findings of two cases of such tumors are described. Using a panel of various antibodies, the authors found that cytokeratin, Leu-M1 and Ber-EP4 were expressed only in papillary areas. Similarly, apocrine epithelial antigen was positive in cells lining papillary stalks. In contrast, epithelial membrane antigen (EMA) stained cells both in solid and papillary areas. CD 31, Ulex 1 and factor VIII-related antigen were positive only in stromal vessels, as desmin and muscle actin. Vimentin was positive in stromal cells and in papillary areas in one case. KP 1 staining was negative in neoplastic cells. Ultrastructurally, both neoplastic cell types showed features of epithelial cells, and the cells lining papillary stalks contained lamellar bodies characteristic to type II pneumocytes. The evidence obtained clearly shows that the neoplastic cells of this tumor are of epithelial type; one cell type represents neoplastic type II pneumocytes, and another cell type represents the heterogeneous group of epithelial cells, also sharing features of type I pneumocytes.

It has been recently shown that epithelial cell monolayers of rat type II pneumocytes cultivated on tissue culture-treated polycarbonate Transwell filters are tight (> 2,000 ohm-cm2) and exhibit morphological and phenotypic characteristics of in vivo type I pneumocytes. We studied, utilizing these tight monolayers, the transepithelial transport of thyrotropin-releasing hormone (TRH). Either [125I]TRH or [3H]TRH was used to measure the transalveolar epithelial radiolabel fluxes across the monolayer. Radiochromatography was performed, utilizing reverse-phase HPLC techniques, to determine the presence of TRH and its subspecies in dosing, donor and receiver fluids. The apparent permeability coefficient (Papp) estimated from 125I-radiolabel fluxes was approximately 1.7 x 10(-7) cm/sec in both the apical-to-basolateral (AB) and basolateral-to-apical (BA) directions. In contrast, the Papp for 3H-radiolabels was approximately 4.2 x 10(-7) cm/sec in both directions. Radiochromatography results indicated that neither apical nor basolateral receiver fluid collected at the end of 4 h flux studies contained metabolites of [125I]TRH or [3H]TRH. In the presence of 1,000-fold excess of unlabeled TRH in the donor fluid, the Papp of neither [125I]- or [3H]-TRH in either direction was altered. These data taken together provide evidence for restricted diffusional transport of intact TRH across the rat alveolar epithelial cell monolayer, most likely via paracellular pathways. Thus, it appears that TRH delivery via pulmonary alveolar epithelium in the distal airspaces of the mammalian lung may be feasible without significant interference from peptidase activities.

Localization of non-specific esterases, Cu-Zn superoxide dismutase and dehydropeptidase-I, in rat lung was investigated enzymecytochemically or immunohistochemically. Esterase was demonstrated in Clara cells, type II pneumocytes, and septal cells (or vitamin A-storing lung cells), to a somewhat lesser extent in type I pneumocytes and ciliated epithelial cells of the bronchioles, and to a minor extent in interstitial fibroblasts of the alveolar septum. Large amounts of esterase reaction product were deposited in the rough endoplasmic reticulum and the nuclear envelope in Clara cells, type II pneumocytes, and septal cells, in addition to smaller amounts in other organelles. No reaction product was found in macrophages (histiocytes) in alveolar septi and alveolar macrophages, except for the primary lysosomes or phagolysosomes and trace amounts in the Golgi vesicles, and none in endothelial cells of alveolar blood capillaries, except for primary lysosomes. Immunolocalization of Cu-Zn superoxide dismutase was generally limited to a particular area of Clara cells. A constriction occurred in the apical cytoplasm of Clara cells between an immunoreactive dome-like protrusion and the non-immunoreactive cytoplasm of the supranuclear area, and the dome-like protrusion appereared to be pinched off in a ball-like or oval from. Immunolocalization of dehydropeptidase-I was demonstrated in a dome-like protrusion or supranuclear area of Clara cells or throughout the cytoplasm and in the surface plasma membrane of mesothelial cells. The presence of these enzymes in Clara cells suggests a contribution to the detoxification system of the lung, together with cytochrome p-450-dependent monooxygenase systems. © 1994 Wiley-Liss, Inc.

Objective: To describe the lung pathological changes in influenza A (H1N1) viral pneumonia. We studied morphological changes, nitro-oxidative stress and the presence of viral proteins in lung tissue. Methods and patients: Light microscopy was used to examine lung tissue from 6 fatal cases of pandemic influenza A (H1N1) viral pneumonia. Fluorescence for oxidized dihydroethydium, nitrotyrosine, inducible NO synthase (NOS2) and human influenza A nucleoprotein (NP) (for analysis under confocal microscopy) was also studied in lung tissue specimens. Results: Age ranged from 15 to 50 years. Three patients were women, and 5 had preexisting medical conditions. Diffuse alveolar damage (DAD) was present in 5 cases (as evidenced by hyaline membrane formation, alveolo-capillary wall thickening and PMN infiltrates), and interstitial fibrosis in one case. In the fluorescence studies there were signs of oxygen radical generation, increased NOS2 protein and protein nitration in lung tissue samples, regardless of the duration of ICU admission. Viral NP was found in lung tissue samples from three patients. Type I pneumocytes and macrophages harbored viral NP, as evidenced by confocal immunofluorescence microscopy. Conclusions: Lung tissue from patients with pandemic influenza A (H1N1) viral pneumonia shows histological findings consistent with DAD. Prolonged nitro-oxidative stress is present despite antiviral treatment. Viral proteins may remain in lung tissue for prolonged periods of time, lodged in macrophages and type I pneumocytes.

The mesodermal capsule of the fetal lung plays a role in differentiation of the respiratory region. It has been proposed for other epithelial organs that the mesodermal capsule influences development by modifying the basal lamina or the extended extracellular matrix. The effect could be on deposition or turnover of collagens, proteoglycans, and/or glycoproteins. This study tests the role of glycoproteins in differentiation of respiratory endings by inhibiting their synthesis with the antibiotic tunicamycin (TM). Lungs at 16 and 18 days gestation and 3 days after birth were cultured with TM and examined for morphological and biochemical differences from normal controls. With TM, alveolar regions did not expand properly and formed fewer type I pneumocytes, although type II pneumocytes were unaffected. The epithelium of untreated respiratory regions showed greater incorporation of radioactive mannose than the airways region or mesenchyme. This incorporation was diminished in TM, but the pattern persisted. Comparison with the results obtained with beta-xyloside suggested that differentiation of type I and type II pneumocytes is under separate control.

Rats were exposed to Ludox colloidal amorphous silica at concentrations of 0, 10, 50 and 150 mg/m3 for 6 h/day, 5 days/week for 4 weeks. The rats were killed after 4 weeks exposure and at 10 days or 3 months post-exposure (PE). Dose-related pulmonary lesions were observed at 50 and 150 mg/m3, but not at 10 mg/m3. Inhaled particles were mostly phagocytized by alveolar macrophages (AMs) in the alveolar duct region and a few free particles were found in Type I pneumocytes in the alveoli. Particle-laden AMs directly penetrated into the bronchiolar interstitium from alveoli and accumulated in bronchus-associated lymphoid tissue (BALT), peribronchiolar, or perivascular interstitium. The particle-laden AMs in the interstitium further migrated into the peribronchial or perivascular lymphatics and accumulated in the tracheo-bronchial lymph nodes (TBLN). Some particle-laden AMs in the BALT transmigrated directly into bronchial lumen through the epithelium. The transmigrated particle laden-AMs in the TBLN were indistinguishable from those seen in the alveoli. They were characterized by slender cytoplasmic processes, phagosomes, myelin figures (alveolar surfactant), cholesterol clefts and lipid droplets. Many migrated particle-laden AMs were necrotic and released particles in the TBLN. The released particles were phagocytized by histiocytes and formed histiocytic granulomas. Silicotic granulomas were initially formed in alveoli with particle-laden AMs and proliferating epithelioid cells. Subsequently, the granulomas were incorporated into the interstitium. However, perivascular silicotic granulomas were developed with accumulation of transmigrated particle-laden AMs and minimal collagenized fibers at 3 months PE. There was no alveolar lipo-proteinosis as seen in crystalline silica exposure.

Lanthanum (La) is one of the rare earths used in diverse high technology fields for which sufficient data for assessing its health effects have been lacking. The biological effects and metabolic behaviors of La were studied by instilling lanthanum chloride intratracheally into male Wistar rats. The distribution of La among tissues revealed that the metal remains mostly in the lung with a biological half-time of 244 days. The subcellular localization by transmission electron microscopy with an X-ray microanalyzer indicated that La localizes in macrophages as high electron-dense granular inclusions in lysosomes and on the cell surface and basement membranes of type I pneumocytes among lung cells. The pulmonary health effects were examined by biological indices of the bronchoalveolar lavage fluid (BALF) and lung tissue. The acute toxicity estimated by lactate dehydrogenase activity in BALF was comparable to those of yttrium and copper that had been determined under the same protocol. Microscopic examination of the lung indicated a characteristic increase in the number of eosinophils.

Using immunoelectron microscopy, we examined the distribution of cytochrome P-450 in rat lungs and determined the cell types in which P-450 was localized. To demonstrate P-450, a rabbit polyclonal antibody against P-450b (P-450 IIB1), the major constitutive isozyme of rat pulmonary P-450, was used. Strong reactivity for anti-P-450b antibody was observed in nonciliated bronchial and bronchiolar epithelial cells (Clara cells) and type I pneumocytes. Type II pneumocytes and Kultschitzky cells showed no reactivity. Most ciliated cells were negative, although a small number in peripheral bronchioles showed a weakly positive reaction. Endothelial cells and other mesenchymal cells were P-450b negative. Many studies have suggested that the Clara cell was metabolically very active and had P-450 containing mixed function oxidase systems. Our study shows that both Clara cells and type I pneumocytes have P-450 and may play an important role in the metabolism of xenobiotics. The metabolic significance of type I pneumocytes was not recognized, but it is reasonable to postulate that type I pneumocytes may have a xenobiotic metabolic function because they comprise most of the surface area of the lungs and are thus extensively exposed to the environment.

The capacity of different lung parenchymal cells to accumulate putrescine was investigated by incubating slices of rat lung in a medium containing the tritiated compound. Quantitative examination of autoradiographs, by electron microscopy, indicated that accumulation of putrescine occurred in both the Type I and Type II cells of the alveolar epithelium. Putrescine uptake was abolished by the addition of spermidine to the medium or by incubating at 0 degrees C. Lung samples from rats dosed with the pneumotoxin O,S,S-trimethyl phosphorodithioate (OSSMeO), which selectively damages Type I pneumocytes, showed a large reduction in the uptake of label by both Type I and Type II cells. This treatment also resulted in an increase in the labeling of alveolar macrophages. Control samples, from undosed rats, were incubated in medium containing tritiated 5-hydroxytryptamine; this compound did not accumulate in epithelial cells but it was concentrated in the endothelium of the alveolar capillaries and in the blood cells within these vessels. The demonstration of putrescine uptake by both Type I and Type II pneumocytes, together with its reduction by dosing with OSSMeO, has vindicated the use of this activity, in lung slices, as an index of damage to the alveolar epithelium.

We describe eight patients who developed interstitial pulmonary fibrosis following BCNU (carmustine) therapy for cerebral tumours. The fibrosis presented 12-17 (mean 14) years after exposure to the drug. A distinctive pattern of pulmonary fibrosis with involvement of the apices and subpleural areas was seen in one patient dying of the disease. Light microscopy showed interstitial elastosis and intra-alveolar fibrosis which was often focal with an associated mild lymphoplasmacytic infiltrate, intra-alveolar oedema, macrophages, and some neutrophils. Ultrastructural studies showed electron lucency of type I pneumocytes, with breaks in the cytoplasmic membranes leaving a bare basement membrane. Degenerative change was also seen in endothelial cell cytoplasm along with lipofuscin deposition. While BCNU pulmonary fibrosis has been described up to 2 years after treatment, this complication so late after therapy, though rare, has important implications for the follow-up of patients receiving this drug.

Rabbits and rats were infected intratracheally with extracellular enzyme of Streptomyces thermohygroscopicus (H9-4) by only one exposure, and lesions of the lung developed including mononuclear macrophage infiltration as well as bronchitis and vasculitis. The obvious damages in type I pneumocytes, endothelial cells of capillaries and arterioles in the lung were observed by electron microscopy. Immunofluorescent histochemistry examination revealed exudation of plasma fibronectin which might play an important role during the process of lesion repairing in lung. The experiment also confirmed that extracellular enzyme of Streptomyces thermohygroscopicus might directly damage the lung tissue. These experimental data may serve as valuable reference for studying the etiology and pathogenesis of farmer's lung disease.

The rat cell line R3/1 displays several phenotypical features of alveolar epithelial type I cells. In order to evaluate this cell line as potential in vitro model for drug disposition studies, R3/1 cells were cultured on Transwell filters and the transepithelial electrical resistance (TEER) was measured to test the integrity of cell layers. The mRNA expression of cell junctional components including E-cadherin, occludin, ZO-1 and ZO-2 was studied using reverse transcriptase-polymerase chain reaction (RT-PCR) and the corresponding proteins by immunofluorescence microscopy (IFM). Moreover, the expression pattern of catabolic peptidases, carboxypeptidase M, aminopeptidases (AP): A, B, N and P, gamma-glutamyltransferase (GGT), dipeptidylpeptidase IV, angiotensin-converting enzyme (ACE), and endopeptidases (EP) 24.11 and 24.15 was analysed in R3/1 cells and compared to rat alveolar epithelial I-like cells in primary culture. TEER peaked at 99+/-17Omegacm(2) after 5 days in culture. Addition of 0.1muM dexamethasone (DEX) with 20% foetal bovine serum further increased TEER by 65%. However, none of the culture conditions used in our study yielded monolayers with TEER values comparable to those of primary cultures of rat pneumocytes. No transcripts encoding for E-cadherin and occludin were detected by RT-PCR. However, ZO-1 and -2 mRNA transcripts were found. IFM using a monoclonal antibody against occludin confirmed the absence of the protein in R3/1 cells. Of the investigated proteolytic enzymes, mRNA transcripts encoding APA and APB as well as EP 24.11 and EP 24.15 were detected; a pattern similar to that of rat alveolar epithelial I-like cells in primary culture. Thus, although R3/1 cells express certain markers typical for type I pneumocytes (e.g., T1alpha, ICAM-1, connexin-43, caveolins-1 and -2) they do not form electrically tight monolayers. This excludes R3/1 cells from being used as an in vitro model for alveolar absorption. However, the cell line may be suitable to study stability of inhaled and endogenous proteins.

In the "merge" panel of Fig 9, row B, the authors inadvertently inserted the incorrect image. The corrected version of this figure is provided here. Fig 9 FOCI injected with ExoS contain many dead type I pneumocytes, whereas FOCI injected with ADPRT-deficient ExoS contain only a few dead phagocytic cells.

Previous studies on double deficient mice for leukemia inhibitory factor (LIF) and insulin-like growth factor I (IGF-I) reported that they died of respiratory failure, with abnormal lung histology and altered expression of pulmonary markers. Here we analyzed prenatal Lif/Igf-I double mutant mouse embryos to characterize LIF and IGF-I cooperative roles in distal lung epithelium and vascular maturation. Lungs of IGF-I-deficient embryos displayed a higher proportion of type II pneumocytes, less differentiated type I pneumocytes, and failure in alveolar capillary remodeling compared to wild type and LIF-deficient mice. Lif/Igf-I double knockout lungs showed aggravated pulmonary hypoplasia, lower airway volume, increased proliferation, and elevated levels of ERK1/2 activation. In addition, their alveoli were collapsed and lined by type II cells. The differentiation of type I cells barely occurred and capillaries remained in the abundant mesenchyme. These results indicate that LIF collaborates with IGF-I in lung alveolar epithelium and vascular maturation. © 2006 Wiley-Liss, Inc., This work was funded in part by grants BMC 2002-01680 (to J.G.P.) and BFU 2004-02352 (to F. de P.) from the Spanish Ministerio de Educación y Ciencia (MEC)

In the current study, the role of contractile activity of respiratory muscles in fetal lung growth and cell differentiation was examined using Myf5-/-:MyoD-/- mouse embryos. As previously found, Myf5-/-:MyoD-/- mouse embryos had no respiratory musculature. Consequently, they suffered from pulmonary hypoplasia and died shortly after birth. The hypoplastic lung had decreased proliferation and increased apoptotic index as early as embryonic day 14.5. By contrast, only at the last gestational day, the number of lung cells expressing platelet derived growth factor B and insulin growth factor I was decreased, while the gradient of the thyroid transcription factor 1 was not maintained. Type II pneumocytes had a failure in glycogen utilization and surfactant storage and secretion but were able to synthesize the surfactant-associated proteins. Type I pneumocytes were readily detectable using an early differentiation marker (i.e., Gp38). However, the late differentiation of type I pneumocytes never occurred, as revealed by transmission electron microscopy. Together, our findings suggest that pulmonary distension due to fetal breathing-like movements plays an important role not only in lung growth but also in lung cell differentiation.

This study aimed to identify and specify the glycotypes of cell populations in normal human lung including types I and II pneumocytes, alveolar macrophages and mast cells, and also in the larger tissue structures of lung, including blood vessels and bronchi/bronchioles, using lectin- and immuno-histochemistry on paraffin-embedded tissue from 11 normal cases. The alveolar macrophages were anti-CD68 positive whereas the cells lining the alveolar walls were positive for cytokeratins. The alveolar macrophages in normal lung tissues showed a broad spectrum of staining for different subsets of N-linked saccharides, N-acetylgalactosamine, N-acetylglucosamine, terminal beta-D-galactose and sialyl groups. This study showed that some lectins could be used as specific markers for some cell types i.e. Galanthus nivalis and Narcissus pseudonarcissus lectins for macrophages, Psophocarpus tetragonolobus lectin-II for capillary endothelium, Dolichos biflorus agglutinin for bronchial epithelial cells, Lycopersicon esculentum, Phytolacca americana or Triticum vulgaris (succinylated) for type I pneumocytes and Hippeastrum hybrid or Maclura pomifera lectins for type II pneumocytes. Patchy staining of type I pneumocytes by peanut agglutinin indicated the possibility of two distinct populations of these cells or a pattern of differentiation that is unapparent morphologically.

The chronologic localization of Mycoplasma hyopneumoniae was examined by in situ hybridization in experimentally infected pigs for a period of 35 days after intratracheal inoculation. M. hyopneumoniae DNA was detected in bronchial and bronchiolar epithelial cells from infected pigs at 7, 14, 21, and 28 days postinoculation (DPI) and in alveolar and interstitial macrophages and type I pneumocytes from infected pigs at 14, 21, 28, and 35 DPI. Strong hybridization signals for M. hyopneumoniae were detected mainly at the luminal surface of bronchial and bronchiolar lining epithelial cells. When a hybridization signal was detected at the luminal surface of bronchial and bronchiolar lining epithelial cells, a given bronchus or bronchiole also exhibited peribronchiolar lymphoid cuffing. These observations suggested that the presence of M. hyopneumoniae in different tissues could be due to a difference in the duration of the infection.

In mammalian lung, type II pneumocytes are especially critical in normal alveolar functioning, as they are the major source of surfactant and the progenitors of type I alveolar cells. Moreover, they undergo proliferation and transformation into type I cells in most types of cellular injury, where flattened type I pneumocytes are selectively destroyed. Hyperplasia of alveolar type II cells has also been described in some human chronic lung diseases. In lung, type II pneumocytes and non-ciliated bronchiolar cells are the unique cell types that contain a considerable amount of peroxisomes. Due to the presence of dihydroxyacetone phosphate acyltransferase and non-specific lipid-transfer protein, these organelles have been suggested to be involved in the synthesis and/or transport of the lipid moiety of surfactant. In the present research, the peroxisomal marker enzyme catalase was immunolocalised at the light microscopic level, utilising the avidin-biotin complex method, in lung specimens excised from newborn, adult and aged rats. In all the examined stages the immunoreactivity was so selective for type II pneumocytes it allowed quantitation of these cells by an automated detection system. This was accomplished on specimens from newborn rat lung, in which labelled alveolar cells were counted by a grey level-based procedure and their main morphometric parameters were determined.

Background Activation of intracellular signalling cascades such as mitogen-activated protein (MAP) kinase pathways has been implicated in various animal models of acute lung injury. However, such data rely almost exclusively on measurements within whole lung homogenate samples, so little information is available regarding cell type specific processes. In order to address this, we have developed a flow cytometric technique to identify discrete pulmonary cell populations and examine their early activation in terms of phosphorylation of intracellular MAPkinase pathway intermediates (ERK, p38 and its downstream target MK2). Methods Anaesthetised C57BL6 mice were given an intratracheal 20 μg dose of lipopolysaccharide (LPS). Mice were sacrificed at various time points, their lungs were removed, immersed in a buffer to fix and permeabilise cells, and a single cell suspension was produced by mechanical disruption. After incubation at 37°C the cell suspension was washed and centrifuged. Samples were stained at room temperature in the dark with antibodies to identify alveolar macrophages (AM) and epithelial cells (AEC), and to measure the intracellular levels of active phosphorylated forms of ERK, p38 and MK2. Finally the cells were washed and resuspended for flow cytometric analysis. Results AM showed a rapid increase in levels of phosphorylated MK2 (see Abstract P251 Figure 1) as well as p38 and ERK on stimulation by intraalveolar LPS. As MK2 showed the most robust response, we determined its phosphorylation in AEC. Those AEC expressing low levels of surface ICAM-1 (likely type II pneumocytes) showed a pattern of increased MK-2 phosphorylation, while ICAM-1 high expressing AEC (type I pneumocytes) did not show a clear response to LPS stimulation. Conclusion We have developed a method of investigating activation of individual cells within the lung using flow cytometry, in terms of intracellular MAPkinase phosphorylation. Our data show that AM are rapidly activated upon stimulation by intraalveolar LPS, whereas AEC show signs of delayed activation with type II pneumocytes more responsive than type I cells. Applying this technique to other models, in which alternative cell types and/or pathways may be involved, will enhance our understanding of cellular activation and interactions during acute lung injury.