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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Mycoplasma hyopneumoniae DNA was detected in 20 naturally infected pigs by in situ hybridization using a nonradioactive digoxigenin-labeled DNA probe. A 520-base-pair DNA probe targeting a reiterative sequence of the M. hyopneumoniae genome was generated by the polymerase chain reaction. All 20 pigs infected with M. hyopneumoniae had distinct and positive hybridization signals without background staining. A strong hybridization signal was detected mainly in the luminal surface of bronchial and bronchiolar lining epithelial cells, whereas no hybridization signal was seen in the cytoplasm of bronchial and bronchiolar lining epithelial cells. When hybridization signal was detected in the luminal surface of bronchial and bronchiolar lining epithelial cells, a given bronchus or bronchiole had peribronchiolar lymphoid hyperplastic tissues. Hybridization signals were not seen in the peribronchiolar lymphoid hyperplastic tissues. A less intense signal was detected in the interstitial and alveolar macrophages randomly scattered in the thickened alveolar septa and spaces. Hybridization signal was rarely detected in the type I pneumocytes. The in situ hybridization technique developed in this study was useful for detection of M. hyopneumoniae nucleic acids in tissues taken from naturally infected piglets and may be a valuable technique for studying the pathogenesis of M. hyopneumoniae infection.

Caveolin is a major structural protein of caveolae, also known as plasmalemmal vesicles, which are particularly abundant in type I pneumocytes and capillary endothelial cells of lung parenchyma. Here we demonstrate that caveolin expression in the alveolar epithelium of rats and mini pigs is strikingly downregulated after irradiation-induced lung injury. Indirect immunoperoxidase staining with polyclonal anti-caveolin antibodies, confirmed by double fluorescence studies with type I cell-specific monoclonal anti-cytokeratin antibodies or lectins, revealed a dramatic loss of caveolin immunoreactivity in type I pneumocytes. In contrast, caveolin expression increased in endothelial cells. Immunoblotting of lung homogenates from normal and irradiated rats using specific anti-caveolin antibodies confirmed the presence of caveolin in normal tissue and its marked decrease of expression in fibrotic tissue. The loss of caveolin as an important structural protein of caveolae in alveolar epithelial cells may be an early indicator of serious type I cell injury during fibrogenesis. The increase of caveolin immunoreactivity in endothelia of blood vessels may indicate that different types of caveolae and/or different regulatory mechanisms of caveolin expression exist.

Although immunodeficiency represents the main etiopathogenetic factor [1], the basic mechanism of Pneumocystis carinii pneumonia (PCP) pathogenesis remains poorly understood. When growing into alveoli, P. carinii interacts with cells and other components; the initial stages of PCP involve parasite attachment to alveolar epithelium, preferentially to type I pneumocytes [2–5], and produce an increase in alveolo-capillary permeability [5, 6]; this last event leads to an influx of plasma proteins into the alveolar space and thereby may alter the composition of epithelial alveolar lining fluid. Pulmonary surfactant, which covers the alveolar surface, plays an important role in the homeostasis of the alveolar environment and in the defense of the lung [7–11]. Since P. carinii comes into close contact with surfactant, it seems logical to suppose, on the one hand, that the parasite could interact with surfactant components and, on the other hand, that pulmonary surfactant may have an impact on the pathophysiology of PCP. Pulmonary surfactant, which is synthesized by alveolar type II cells, is a complex material containing lipids and proteins (Table 1) [7, 8]. Its synthesis is developmentally regulated in fetal lung and can be accelerated by glucocorticoids and other hormones [12]. Lipids are the main components (90%) of pulmonary surfactant [7, 8]. Phospholipids represent 80–90% of the lipids and phosphatidylcholines, which are mainly dipalmitoylphosphatidylcholines, constitute the major part (70–80%) of phospholipids. Cholesterol comprises the largest amount of neutral lipids. In addition to lipids, four surfactant-specific proteins (SP) with different structural properties have been identified [13, 14]: SP-B and SP-C, which represent only 1% of total surfactant, are two extremely hydrophobic proteins; they are involved in keeping surface active properties of the surfactant [14, 15]. SP-A and SP-D are hydrophilic proteins; SP-A …

Chronic obstructive pulmonary disease (COPD) causes significant morbidity and mortality. Cigarette smoke, the most common risk factor for COPD, induces airway and alveolar epithelial barrier permeability and initiates an innate immune response. Changes in abundance of aquaporin 5 (AQP5), a water channel, can affect epithelial permeability and immune response after cigarette smoke exposure. To determine how AQP5-derived epithelial barrier modulation affects epithelial immune response to cigarette smoke and development of emphysema, WT and AQP5(-/-) mice were exposed to cigarette smoke (CS). We measured alveolar cell counts and differentials, and assessed histology, mean-linear intercept (MLI), and surface-to-volume ratio (S/V) to determine severity of emphysema. We quantified epithelial-derived signaling proteins for neutrophil trafficking, and manipulated AQP5 levels in an alveolar epithelial cell line to determine specific effects on neutrophil transmigration after CS exposure. We assessed paracellular permeability and epithelial turnover in response to CS. In contrast to WT mice, AQP5(-/-) mice exposed to 6 months of CS did not demonstrate a significant increase in MLI or a significant decrease in S/V compared with air-exposed mice, conferring protection against emphysema. After sub-acute (4 weeks) and chronic (6 mo) CS exposure, AQP5(-/-) mice had fewer alveolar neutrophil but similar lung neutrophil numbers as WT mice. The presence of AQP5 in A549 cells, an alveolar epithelial cell line, was associated with increase neutrophil migration after CS exposure. Compared with CS-exposed WT mice, neutrophil ligand (CD11b) and epithelial receptor (ICAM-1) expression were reduced in CS-exposed AQP5(-/-) mice, as was secreted LPS-induced chemokine (LIX), an epithelial-derived neutrophil chemoattractant. CS-exposed AQP5(-/-) mice demonstrated decreased type I pneumocytes and increased type II pneumocytes compared with CS-exposed WT mice suggestive of enhanced epithelial repair. Absence of AQP5 protected against CS-induced emphysema with reduced epithelial permeability, neutrophil migration, and altered epithelial cell turnover which may enhance repair.

Putrescine is accumulated in the lungs of various species by an active uptake system that also mediates the uptake of cystamine and paraquat. We have characterized this uptake in both lung slices and type II pneumocytes isolated from hamsters by trypsin digestion, differential adherence on plastic, and centrifugation on a discontinuous Percoll gradient. The accumulation of [14C]putrescine in lung slices was shown to be temperature and energy dependent, and to obey saturation kinetics, with mean calculated values of apparent Michaelis constant (Km) 29.4 microM and maximum rate of uptake (Vmax) 637 nmol.g-1.h-1. In the presence of cystamine or paraquat, the putrescine uptake was reduced in a manner compatible with competitive inhibition. The calculated inhibitor constants (Ki) were 16 and 1,017-1,328 microM for the inhibition by cystamine and paraquat, respectively. The cellular localization of [3H]putrescine in lung slices after incubation with 2.5 microM putrescine was determined by light-microscopic autoradiography. Labeling was present in type II and possibly also in type I pneumocytes of the alveolar epithelium but not in macrophages, endothelium, or cells of the interstitium. Two days after their isolation, cultured type II pneumocytes exhibited an uptake of putrescine that had kinetic characteristics similar to those of slices (Km of 23 microM and Vmax of 3.06 mumol.g protein-1.h-1) and was also competitively inhibited by paraquat (Ki of 222-350 microM paraquat). Our data demonstrate the presence of an active uptake system for putrescine in both lung slices and cultured type II pneumocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

In an effort to develop a suitable bioassay for testing lung growth factors that might be operative during compensatory lung growth following partial pneumonectomy, a simple and inexpensive lung organ culture system was characterized. The culture employs lung tissue slices obtained by means of a device allowing thicknesses of 500 microns to be cut reproducibly. To avoid the collapse of the organ, the alveolar spaces were filled prior to culture with Noble agar-containing Eagle's-Dulbecco's modified medium. Lung tissue sections could be maintained ultrastructurally intact for at least one week. The results showed that upon culture, a part of the type II pneumocytes undergo differentiation into type I pneumocytes, thus demonstrating that the culture system may be suited for differentiation studies. One surprising feature of this culture system was the mitogenic impulse associated with culture. Radioactively labeled thymidine incorporation was strongly stimulated in the culture, mainly affecting the epithelial cells, as could be established by "back-to-back" autoradiography. With a reconstruction experiment, it was possible to demonstrate the local release of a mitogenic factor following slicing, mincing, or dissection of the lung tissue, which could be assayed by its ability to induce serum-starved Balb/c 3T3 cells to synthesize DNA in culture.

The biochemical and morphological aspects of BALB/c mice with many features of the Niemann-Pick disease type C in man (NP-C mouse) have been studied extensively. However, the pulmonary pathology has not been studied extensively and we describe here some unique ultrastructural features of the lung in the NP-C mouse. Ultrastructurally, macrophages in younger mice contained osmiophilic dense granules and annulolamellar structures, but larger multilamellar concentric structures increased in the macrophages of older mice. In contrast, endothelial cells and type I pneumocytes showed membrane-bound bodies with dense granules and vesicular or vesiculogranular structures as well as amorphous materials. Type II pneumocytes were unremarkable throughout. Our study suggests that endothelial cells and type I pneumocytes are the major site of metabolic derangement resulting in pronounced morphological changes with granular and round membranous structures in the lungs of NP-C mouse. Alveolar macrophages with multilamellar concentric structures may be a result of disturbed disposal of surfactant material from type II pneumocytes rather than that from storage material of type I pneumocyte.

Lung contains many highly specialized cells. Type I pneumocytes are responsible for gas exchange, while type II pneumocytes secrete surfactant which prevents alveoli from collapsing. Airways are lined by non-ciliated secretory (Clara) and ciliated cells that eliminate excess mucus and harmful particles, and rare pulmonary neuroendocrine cells (PNECs) which regulate breathing and contribute to carcinogenesis. PNECs have unknown histogenesis but differentiate prior to the other cells. The neural transcription factor achaete-scute homolog 1 (Ascl1) is critical for PNEC development and is associated with stem cell marker expression in normal and neoplastic lung. Hence, we used in vivo lineage and fate tracing strategy to investigate Ascl1-defined cells (ASDCs) during lung development and injury repair. R26R-stop-lacZ (Rosa) reporter mice were crossed with Ascl1-Cre and Ascl1-CreERTM mice, in which the Ascl1 promoter drives the expression of Cre or inducible Cre recombinase, respectively (Battiste et al., Development (2007) 134:285-93). Following recombination, ASDCs and their descendants will be permanently labeled and were further characterized by immunohistochemistry using cellular differentiation markers. Labeled cells in Ascl1-Cre/Rosa mice gave rise not only to PNECs but also to neural, Clara, ciliated, alveolar type I and type II cells. Interestingly, tamoxifen (TM) administration to Ascl1-CreERTM/Rosa dams at embryonic day E9.5 labeled both airway and alveolar cells, while TM injected at E11.5 only labeled airway cells. Moreover, adult Ascl1-CreERTM/Rosa mice were administered TM followed by naphthalene (Nap), which kills Clara cells. It is notable that in addition to PNECs which are resistant to Nap, regenerating Clara cells were now labeled during the injury repair. We conclude that ASDCs are not limited to giving rise to neural cells or PNECs during pulmonary development, but they also contribute to a range of non-neuroendocrine cells such as Clara and ciliated cells as well as alveolar pneumocytes. Further, the lineage participates in injury repair of the airway lining in mature lung. The current Ascl1 lineage study provides a basis for recent studies on mouse small cell lung cancer (Sutherland et al., Cancer Cell (2011) 19:754-64) which suggested that both PNECs and non-neuroendocrine cells such as alveolar type II pneumocytes may serve as progenitors for this very virulent lung cancer type. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1308. doi:1538-7445.AM2012-1308

Polyclonal and monoclonal antibodies to the beta subunit of prolyl 4-hydroxylase, the protein disulphide isomerase, were used to compare the pulmonary cells in 13 normal and in 20 fibrotic rat and mini-pig lungs made fibrotic by X-ray irradiation, using the ABC immunoperoxidase technique. In normal lungs, prominent staining of Clara cells and type II pneumocytes and weaker reactivity with alveolar macrophages, fibroblasts, endothelial and smooth muscle cells were detectable. In pulmonary disease, in which interstitial fibrosis was the characteristic feature, the immunoreactivity was increased in both the epithelial and interstitial cells. Type I pneumocytes remained negative. In the early stages of disease (3 to 4 weeks after irradiation) when little morphological alteration was seen, capillary endothelial cells had already become immunoreactive. These results underline the complex involvement and interaction of different lung cell populations in the process of pulmonary fibrogenesis.

In the rat lung, we found that the Bauhinia purpurea lectin (BPA) specifically binds to the type I alveolar epithelial cells and to the alveolar macrophages. Double label fluorescence employing FITC-coupled Maclura pomifera lectin (MPA) and bBPA-avidin-Texas Red showed that BPA binding was confined to type I cells. In addition, a minor staining of the luminal border of the terminal bronchiolar epithelium was found. The sequence of tissue injury following X-radiation was examined in rats. BPA is a suitable marker which indicates epithelial changes during the early stage of pneumonitis and the subsequent development of fibrosis.

Distribution of actin and fodrin, a nonerythroid analogue of spectrin, was studied in cytocentrifuge preparations and in tissue sections of normal and pathologic respiratory epithelium by using immunofluorescence and immunoelectron microscopy. In ciliated epithelial cells and in goblet cells of normal bronchial epithelium, fodrin and actin were located in the apical parts and along the lateral walls of the cells. In basal cells, fodrin and actin were also seen diffusely in the cytoplasm. Immunoelectron microscopy showed fodrin in close association with the basal bodies and rootlets of the cilia and microvilli in the ciliated cells. In alveolar epithelium, fodrin and actin were located at the apical membrane in type I pneumocytes and along the apical and lateral membranes in type II pneumocytes. In type II pneumocytes, fodrin was also seen in close association with the secretory vacuoles. In metaplastic and dysplastic bronchial epithelium, a diffuse cytoplasmic and a circumferential, membrane-associated staining for fodrin and actin was seen. In all types of carcinomas, fodrin was seen along the lateral walls and diffusely in the cytoplasm. The staining was more intense than in the normal cells. In immunoblotting of the normal bronchial epithelium, and peripheral lung and lung carcinomas, a single 240 kD band was recognized with antibodies to fodrin. The results show distinct differences in the distribution of fodrin in the various cell types of the respiratory epithelium. In ciliated cells, the close relationship with cytoskeleton suggests a role of fodrin in the establishment of the elaborate structural architecture of the apical compartment. In type II pneumocytes, on the other hand, fodrin probably plays a role in secretion of the surfactant. In basal cells, the diffuse distribution of fodrin probably reflects the high proliferative capacity of this cell compartment. Interestingly, a similar distribution was also seen in premalignant and malignant cells.

Sixteen adult sheep (ten females, six males obtained from a closed flock at National Animal Disease Center, Ames, IA) were experimentally infected with bovine respiratory syncytial virus strain 375 (BRSV), and lung tissues were stained for viral antigen. Two infected sheep were euthanatized at each of the following post-inoculation times: 12, 24, 36, 48, 72, 96, 144, and 192 hours. Lung, nasal turbinates, trachea, right cranial bronchial and mediastinal lymph nodes, liver, and spleen were collected for histologic evaluation. An indirect immunoperoxidase technique was performed on routine paraffin-embedded sections of lung tissue, trachea, turbinates, and bronchial and mediastinal lymph nodes to determine the location of the BRSV antigen. For lung tissue from each sheep 400 light microscopic fields at 160x magnification were examined for staining for BRSV antigen. Lung tissue was also collected for virus and bacterial isolation. Daily serum samples were taken for determination of anti-BRSV titers. Severe respiratory disease was not produced in any sheep. Bovine respiratory syncytial virus was isolated from lung tissue collected from all sheep up through 144 hours postinoculation. At 12 hours post-inoculation (case No. 2) respiratory syncytial virus antigen was detected in bronchiolar epithelium and a mononuclear cell within an alveolar space. Lung tissue from the sheep necropsied between 24 and 144 hours postinoculattion (case Nos. 3–14) contained BRSV antigen in bronchiolar epithelium, type I pneumocytes, type II pneumocytes. alveolar macrophages, and mononuclear cells within alveolar spaces. Macrophages staining for viral antigen were rare. Bronchiolar and type I epithelial cells comprised the majority of infected cells. In a separate experiment, lung slices inoculated in vitro with either BRSV or ovine adenovirus did not stain for the respective antigens. Slices inoculated with parainfluenzavirus-3 did stain for that viral antigen.

Highly purified populations of alveolar epithelial cells (type II pneumocytes) were isolated from human lung specimens. These cells were characterised histochemically, by demonstrating the presence of intracellular alkaline phosphatase, and morphologically, by electron microscopic demonstration of lamellar bodies and microvilli. Expression of the epithelial glycoprotein HEA-125, of MHC class I and class II (HLA-DR, -DP and -DQ) antigens and of the intercellular adhesion molecules ICAM-1, VCAM-1, LFA-3 and B7 was quantified by flow cytometry. Comparison was made between the expression of these molecules by isolated type II cells and by alveolar epithelium in normal human lung tissue after immunocytochemical staining of frozen sections of donor lung. Isolated type II pneumocytes expressed HEA-125 and class I MHC molecules and the class II MHC molecules HLA-DR and -DP; HLA-DQ was not detected. The intercellular adhesion molecule ICAM-1 was expressed constitutively at low levels but there was minimal expression of VCAM-1, LFA-3 and B7. It was not possible to differentiate type II cells from the predominant type I pneumocytes on frozen sections. Alveolar epithelium expressed HEA-125, class I MHC antigens, the class II molecules HLA-DR, and -DP and the intercellular adhesion molecule LFA-3. Expression of the adhesion molecules ICAM-1, VCAM-1 and B7 was variable. As with the isolates, HLA-DQ was not observed on alveolar epithelium. In conclusion, a reproducible method for the isolation of pure populations of human type II pneumocytes has been developed. These cells were not damaged by the isolation procedure. It is not known whether alveolar epithelium can present antigens to T lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Pneumocystis carinii, an extracellular parasite thriving in the lungs of immunosuppressed mammals, is a major cause of death in AIDS patients in the USA. As a prelude to growth, the parasite adheres mostly to type I pneumocytes lining the alveolar spaces. The mechanism of adherence remains unknown, largely because of difficulties in isolating type I pneumocytes and maintaining them in vitro. As a first step to understand P. carinii adherence to its natural substrate, we developed an in situ method to directly study parasite binding to lung alveolar cells. We used formaldehyde-fixed paraffin-embedded sections of normal rat lung as substrate for adhesion. As in its binding to the lungs in vivo, P. carinii adhered preferentially to type I pneumocytes. Adherence was saturable, time and dose dependent, and selectively blocked by glycoconjugates, in particular bovine submaxillary mucin, fetuin, and asialofetuin, suggesting that it may be mediated by a lectin type of interaction. Further, IgG of rats with P. carinii pneumonia inhibited adherence, suggesting that it may react with parasite ligands involved in the recognition of type I cell receptors. Our results demonstrate the usefulness of the in situ model for studying the mechanisms of P. carinii adherence to alveolar cells. In addition, this method may be valuable for identifying neutralizing antibodies and drugs potentially useful for controlling the infection in vivo.

Summary A spontaneous infection with Pneumocystis carinii ( P. c. ) caused enzootic fatal pneumonia in a long-term experiment with athymic Rowett Nude (Han:RNU rnu/rnu ) and New Zealand Nude (Han:NZNU rnu N / rnu N ) rats. In order to reproduce the infection and to characterize the early pathogenesis of lung lesions, 13 young athyrnic Han:RNU and Han:NZNU rats from P. c. -tree breeding colonies were housed together in one room with chronically P. c. -infected rats. They were killed after 8, 12, 16, 20 or 24 weeks, and their lungs were examined for P. c. infection by means of light microscopy, immunohistochemistry and transmission electron microscopy. Lung lesions progressed from a mild interstitial pneumonia with scattered alveolar macrophages (8 weeks) to a severe diffuse interstitial pneumonia with widespread areas of distended alveoli, filled with foamy material. In all lungs, P. c. antigen was detected immunohistochemically, developing from small intra-alveolar aggregations of organisms into large multifocal clusters. Ultrastructurally, P. c. trophozoites and cysts were seen attached to type I pneumocytes and lying free in the alveolar lumen, Chronic severe P. c. pneumonia (20 and 24 weeks) was characterized by masses of P. c. trophozoites in the alveoli and alveolar walls, extensive proliferation of type II pneumocytes and interstitial fibrosis. The easy and consistent horizontal transmission of spontaneous P. c. pneumonia to previously non-infected athymic Han:RNU and Han:NZNU rats and the similarity of the disease to human infection demonstrate both rat strains to be excellent models for studying pulmonary pneumocystosis of immunodeficient human patients.

Relatively little is known about the histogenesis and the underlying mechanisms of lung cancer; in particular about the target cell type(s) of carcinogens (Schuller 1987). The same can be said about the mechanisms of epithelial regeneration in the lung after injury by toxic chemicals. One of the morphologically appreciable responses of the lung to such injury is a hyperplastic growth of bronchiolo-alveolar epithelium. There is speculation that type II pneumocytes or Clara cells may give rise to the hyperplasia. Neuroendocrine (APUD) cells and type I pneumocytes are supposed to be less capable of initiating hyperplasia because of their low activity of cell division, although hyperplasia of the former cell type is experimentally inducible (Schuller et al. 1988). Mutual transformation has even been postulated between type II pneumocytes and Clara cells in certain cases (Schuller 1987). There is another cell type which, occurring in a fetal as well as regenerating adult airway epithelium (McDowell et al. 1984; Emura 1985; Strum et al. 1990), closely resembles the small mucus granule cells (SMGC) postulated to be one of the carcinogen targets in the lung (McDowell and Trump 1983). In our cell culture study (Emura et al. 1990) a large population of cells possessing SMGC characteristics are inducible in vitro from an undifferentiated population of airway epithelial cells.