Which cells secrete surfactant

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Vavra, V. Facilitation of glutamate and GABA release by P2X receptor activation in supraoptic neurons from freshly isolated rat brain slices. Neuroscience , 1— Lung surfactant also participates in innate defense mechanisms [5] — [8]. Lack or impairment of the surfactant system is associated with some severe diseases such as respiratory distress syndrome in infants or acute respiratory distress in children and adults [9].

Much research has been carried out to study pulmonary surfactant biogenesis and secretion by primary cultures of isolated ATII cells purified mainly from mammalian lungs. However, this method is laborious, expensive, animal intensive and has a low yield of cells [10]. Thus, it does not necessarily conform with the 3-R principle of refining, reducing and replacing animal testing as defined by the Basler declaration. In addition, primary ATII cells in culture rapidly lose their original phenotypical features, differentiate into type I-like cell types and fail to proliferate further, precluding their use for long term studies [11] , [12].

On the other hand, commercially available cell lines like the L2 and the A are inherently problematic as their origin and state of differentiation are ill defined, and no study exists to unambiguously demonstrate that these lines release functionally active surfactant components after physiological stimulations.

Cells isolated from the transgenic immortal mouse models H-2K b -tsA58; 10 circumvent the problem of short-term culture to a certain extent, but are still depending on the use of laboratory animals. Thus, surfactant-producing pneumocyte-like cell lines are still not well established, preventing a proper and detailed characterization of biosynthetic and trafficking pathways involved in surfactant biogenesis. In this context, new strategies for obtaining in vitro ATII cell cultures have to be taken into account.

In the latest years, stem cells have been extensively investigated as a potential source of alveolar epithelial cells [13]. Murine [14] — [21] and human [20] , [22] , [23] embryonic stem cells, adult bone marrow-derived stem cells from rat [24] and human [25] , [26] as well as human stem cells derived from amniotic fluid [27] , amnion [28] or umbilical cord blood [29] have been derivated into cells with phenotypical features consistent with ATII cells.

Placental tissue exhibits certain advantages as a source of MSCs such as an easy isolation of cells without invasive procedures, improbability of viral infection and absence of ethical problems [31] , [32].

DMSCs are from maternal origin and display high plasticity, differentiating into derivatives of all germ layers [30] , [31] , [33]. We evaluated the capability of ATII-LCs to express genes encoding surfactant markers and whether those genes are translated into proteins, as well as the ability of ATII-LCs to generate surfactant-like assemblies, to accumulate them in specialized storage organelles and to secrete them upon a proper stimulation.

Finally, we determined the biophysical properties of the secreted material exploring its potential as a functional surface active agent. Our results, in particular those related to functional aspects, suggest that the ATII-LCs can be regarded as a novel and very promising model to study intrinsic ATII cell characteristics and functions. Human placentas were provided at the Department of Obstetrics and Gynecology from healthy mothers under written consent approved by the Ethics Committee from Hospital Universitario 12 de Octubre.

DMSCs were isolated and cultured from extraembryonic membranes as described previously [30]. Briefly, placental tissue was digested with trypsin-versene Lonza, Spain , cells were seeded at 1. The molecular species of the phophatidylcoline PC fraction of the different whole cell samples were analyzed quantitatively by HPLC coupled to mass spectrometry [34].

Supernatants of DMSCs, treated in the same way, were also collected and served as negative controls. Before use, supernatants were concentrated by ultracentrifugation at Surfactant from porcine bronchoalveolar lavages, used as a reference, was purified and separated from blood components by NaBr density-gradient centrifugation [37].

After centrifugation, clear lysates were collected and aliquoted. After washing, membranes were incubated with secondary antibodies for 1 h at RT. Images were acquired at 1. For image handling and z-stack projections we used the public domain software ImageJ v.

After orbital shaking, fluorescence coming from surface-adsorbed material was measured by the instrument, whereas fluorescence coming from non-adsorbed complexes was quenched by BB. To evaluate the inhibitory effect of plasma proteins on the adsorption of the surfactant materials, experiments were also performed in the presence of increasing concentrations of human serum.

DMSCs are a homogeneous population of fibroblast-like cells with spindle-like shape and similar characteristics to bone marrow MSCs Fig. After days of culture in pulmonary differentiation medium, morphological changes are apparent such as a slightly more epithelioid shape Fig. The morphology of these dense particles in the cytoplasm of ATII-LCs was examined by transmission electron microscopy, revealing ellipsoid and circular membranous structures with multiple, variably densely packed concentric lamellae Fig.

These structures resemble the complex internal organization of the phospholipid-rich surfactant stores lamellar bodies in ATII cells, and are also similar in morphology to the lamellar body-like granules obtained from bronchoalveolar lavage and observed under the same conditions [40]. In addition, ATII-LCs exhibit at the culture-exposed cell side the presence of structures with the appearance of incipient microvilli Fig.

B Differentiated ATII-LCs after 4 days of incubation in differentiation medium, exhibiting a more flattened shape and a highly granulated cytoplasm. A Presence of numerous electron-dense cytoplasmic organelles and cell surface associated structures, probably microvilli. We then investigate whether the morphological differentiation of DMSCs into ATII-LCs, apparently implying the assembly of lamellar body-like structures, could be associated with activation of the synthesis of genuine surfactant phospholipid species, i.

Table 1 summarizes data on the lipidomic analysis, by means of mass spectrometry, of the whole PC fraction from full membranes obtained from different pelleted cells for the complet set of data regarding the content in single PC molecular species, see Table S1.

Although the small number of samples analyzed precludes a proper statistical analysis of differences, it seems clear that the content in total disaturated PCs and DPPC of differentiated ATII-LCs approach that determined for a cell population enriched in true type II pneumocytes, as purified from lung tissue. Expression of SP-D was barely detectable. The expression of these two genes decreased at 48 h and this level was maintained until 96 h. Error bars indicate standard deviation.

Human adult lung was used as a calibration control. The results were normalized to TBP values used as a housekeeping gene. We also examined the gene expression pattern of the surfactant lipid importer ABCA3, which plays a critical role in the biogenesis of surfactant complexes in ATII cells. Since regulated secretion of lung surfactant is considered to be the most important functional characteristic in ATII cells, we aimed to explore the ability of ATII-LCs to exocytose their surfactant-like stores upon stimulation.

For this purpose, we used a method previously described to monitor the exocytotic activity of primary cultures of ATII cells, taking advantage of the special properties of the fluorescent probes LTG and FM 1— LTG permeates through the plasma membrane and accumulates into acidic compartments such as surfactant-accumulating lamellar bodies and lysosomes. We stained cells with LTG to firstly demonstrate the presence of acidic compartments, secondly to visualize the cells and to get the LBs in the focal plane, and lastly to make sure that always approximately the same amount of LTG-positive vesicles and cells are in the respective field of measurement.

At this point, FM 1—43, a membrane-impermeable dye that has no fluorescence in aqueous solution but exhibits fluorescence once intercalated into lipid membranes, was added to the cells. FM 1—43 enters through the fusion pore of the exocytosed vesicles, staining their lipid content and thus emitting a conspicuous yellow fluorescence. Stimulation of ATII-LCs cultures resulted in a progressive appearance of secreted lipid material visualized as bright yellow spots forming clusters at the periphery of the cells.

In contrast, DMSCs were practically unresponsive to secretagogues. Asthma is another chronic obstructive respiratory disease, but in contrast to COPD, it is reversible. This disease characteristically demonstrates airway hyperreactivity and inflammation. Alveolar macrophages are responsible for regulating pro- and anti-inflammatory responses in the lungs, so it may be possible that these cells play a role in asthma. CF is an autosomal recessive disorder characterized by the dysfunction of the cystic fibrosis transmembrane conductance regulator CFTR located on type II pneumocytes and other body areas.

The CFTR protein is responsible for the production of sweat production, digestive fluids, and mucous. Specifically, in the lungs, loss of function of the CFTR protein causes mucous lining the alveoli to become thick instead of thin. Additionally, there is an increased number of alveolar macrophages leading to an upregulation of inflammation. Thick mucus and inflammation clog the airways and can cause difficulty breathing, coughing, and pulmonary infections.

Pulmonary fibrosis is a restrictive disease of the lower airway characterized by fibrosis and inflammation of the alveoli. Autoimmune or allergic reactions, environmental particulates, infection, or mechanical damage can initiate this disease process, starting with pulmonary injury.

After the initial insult, inflammation in the airways results. Subsequent tissue repair and contraction leads to fibrosis and reduced ability to expand alveoli. In an attempt to replace damaged alveolar epithelium, type II pneumocytes become hyperplastic and proliferate and differentiate chaotically. Pneumoconioses are interstitial lung diseases caused by the inhalation of organic and inorganic particles that are phagocytized by alveolar macrophages.

Subsequently, they secrete cytokines that cause inflammation in the lungs. When alveolar macrophages engulf asbestosis, it is referred to as ferruginous bodies within the cytoplasm, which appear dumbbell-shaped. TB occurs when alveolar macrophages phagocytize the bacteria Mycobacterium tuberculosis. Also, alveolar macrophages attempt to wall off the infection by encircling it, forming multinucleated giant cells or Langerhans giant cells.

It is important to note that damage to these alveolar macrophages caused by another infection can trigger the release of M. Sarcoid is a condition that forms non-caseating granulomas in the lungs of patients by the joining of alveolar macrophages in an attempt to wall off the infectious process.

Alveolar macrophages in sarcoidosis also secrete vitamin D, which contributes to the hypercalcemia seen in this disease. ARDS is characterized by non-cardiogenic pulmonary edema caused by some inflammatory condition that causes capillary endothelial leakage. The fluid eventually resolves, but some patients may develop residual fibrous tissue. In addition to bronchial mucous cells and Clara cells, alveolar carcinoma can arise from type II pneumocytes as well.

Left-sided congestive heart failure leads to the backup of blood within the pulmonary vasculature. When this happens, erythrocytes can pass into the alveolar septum, where they are taken up by alveolar macrophages, which become filled with hemosiderin.

When looking at these cells under the microscope, they take on a brown granule appearance due to the hemosiderin within the cytoplasm of these cells. The name for these cells is hemosiderin-laden macrophages.

Influenza pneumonia results from the influenza virus directly infecting alveoli epithelial cells, causing inflammation and alveoli damage.