Alveolar type II cell growth on a pulmonary endothelial extracellular matrix

1996 ◽  
Vol 270 (6) ◽  
pp. L1017-L1022 ◽  
Author(s):  
I. Y. Adamson ◽  
L. Young
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Cell Growth ◽  
Alveolar Type ◽  
Thymidine Uptake ◽  
Type I ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lamellar Bodies ◽  
Type Ii Cell

Most of the alveolar epithelium overlies a fused basement membrane produced by epithelial and endothelial cells. To determine how this type of matrix influences type II cell growth and function, we studied the effects of culturing isolated rat alveolar type II cells on an extracellular matrix (ECM) freshly produced by pulmonary vascular endothelial cells grown 5 days in culture. Type II cells from the same rats were cultured on plastic or Matrigel for comparison. A large increase in mitotic activity was seen in type II cells grown on the endothelial ECM at 2 days only; thereafter cells spread rapidly to confluence and lost their lamellar bodies. Cells grown on Matrigel remained cuboidal with lamellar bodies but grew more slowly, as judged by [3H]thymidine uptake and cell numbers. Incorporation of labeled choline into disaturated phosphatidylcholine (DSPC) was used as a marker of surfactant synthesis. After the rapid, brief burst of proliferation, type II cells on endothelial ECM showed a sudden decline in DSPC-DNA by day 4 compared with cells grown on matrigel. Binding of the lectin Bauhinia purpurea (BPA) indicated that after a phase of division, cells on endothelial ECM developed as type I epithelium by 4 days of culture, when > 70% of cells stained positively for BPA binding, whereas few cuboidal cells on Matrigel were stained. The results indicate that type II cells respond briefly to growth factors in pulmonary endothelial ECM; then this type of matrix promotes cell spreading with loss of type II function as cells subsequently resemble type I epithelium.

Enhanced alveolar type II cell growth on a pulmonary extracellular matrix over fibroblasts

1997 ◽  
Vol 272 (3) ◽  
pp. L413-L417 ◽  
Author(s):  
I. Y. Adamson ◽  
L. Young ◽  
J. Bakowska
Keyword(s):  
Extracellular Matrix ◽  
Cell Growth ◽  
Growth Effect ◽  
Lung Fibroblasts ◽  
Cell Contact ◽  
Alveolar Type ◽  
Thymidine Uptake ◽  
Type Ii Cells ◽  
Type Ii ◽  
Type Ii Cell

The growth of alveolar type II cells was studied when these cells were maintained for 2 days on a pulmonary endothelium-derived extracellular matrix (ECM) on a filter with or without lung fibroblasts in the lower chambers of culture wells. Type II cell proliferation was enhanced by the ECM compared with other substrates but was significantly higher with fibroblasts beneath. This was determined by thymidine uptake and cell numbers. The diffusing factor from fibroblasts appeared to be keratinocyte growth factor (KGF), because this cytokine increased type II cell growth in culture and the neutralizing antibody to KGF blocked the observed fibroblast-induced growth increase. None of the antibodies to various cytokines had any effect on the ECM-induced proliferation. Although the type II cells were shown to produce degradative activity for the ECM, there was little secreted enzyme activity in supernatants and there was no demonstrated autocrine-regulated growth effect. The results suggest that type II cell growth may be stimulated by both 1) a matrix-bound factor that acts through a cell contact-mediated process, and 2) a fibroblast-secreted factor that appears to be KGF.

Sulfation of extracellular matrices modifies responses of alveolar type II cells to fibroblast growth factors

1996 ◽  
Vol 271 (5) ◽  
pp. L688-L697 ◽  
Author(s):  
P. L. Sannes ◽  
J. Khosla ◽  
P. W. Cheng
Keyword(s):  
Growth Factors ◽  
Biological Significance ◽  
Alveolar Type ◽  
Type I ◽  
Type Ii Cells ◽  
Type Ii ◽  
Fibroblast Growth ◽  
Alveolar Type Ii Cells ◽  
Proliferation And Differentiation ◽  
Type Ii Cell

The pulmonary alveolar basement membrane (BM) associated with alveolar type II cells has been shown to be significantly less sulfated than that of type I cells. To examine the biological significance of this observation, we measured the incorporation of 5-bromodeoxyuridine (BrdU) as an indicator of DNA synthesis in isolated rat type II cells cultured for 72-120 h on substrata that were naturally sulfated, not sulfated, or chemically desulfated in serum-free, hormonally defined media, with and without selected growth factors. The percentage of cells incorporating BrdU was significantly elevated by desulfated chondroitin sulfate in the presence of fibroblast growth factor-2 (FGF-2 or basic FGF) and depressed by heparin in the presence of either FGF-1 or acidic FGF or FGF-2. This depressive effect was lost by removing sulfate from the heparin. Some responses were dependent on the period of time in culture and concentration and molecular weight of the substrata. These observations support the notion that sulfation per se of certain components of BM is a key determinant of type II cell responses to select growth factors that may define patterns of proliferation and differentiation.

Secretory granule calcium loss after isolation of rat alveolar type II cells

1991 ◽  
Vol 260 (2) ◽  
pp. L129-L135 ◽  
Author(s):  
R. G. Eckenhoff ◽  
S. R. Rannels ◽  
A. B. Fisher
Keyword(s):  
Primary Culture ◽  
Sulfur Content ◽  
Lamellar Body ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lamellar Bodies ◽  
Isolated Cells ◽  
Alveolar Type Ii Cells ◽  
Type Ii Cell

Morphological change and lamellar body loss suggests that alveolar type II cells rapidly de- or redifferentiate after several days of primary culture. To determine whether type II cells or lamellar body compositional changes precede these obvious morphological changes, we examined the in situ elemental composition of lamellar bodies and type II cells from intact lung and at different times after isolation using electron probe microanalysis (EPMA). Isolated cells were prepared by standard methods and plated on either tissue culture plastic or kept in suspension with stirrer flasks. Cell pellets obtained at 0, 3, 24, and 48 h after isolation were rapidly frozen, and thin freeze-dried cryosections were prepared and examined cold in a transmission electron microscope equipped for EPMA. Eight to ten type II cells from each of three to four different preparations for each time period were analyzed. A rapid, progressive, and sustained fall in lamellar body calcium and sulfur content occurred by 48 h of primary culture, suggesting rapid alteration in calcium and protein metabolism by type II cells and/or lamellar bodies after isolation. Also, marked changes in type II cell cytoplasmic Na and K occurred in freshly isolated cells, with incomplete normalization by 48 h. Culture on laminin-enriched Matrigel for 1 wk increased both lamellar body calcium or sulfur content, but 100 nM dexamethasone had no effect. Lamellar body calcium accumulation appears to be a very sensitive index of differentiated type II cell function.

Binding and uptake of surfactant protein D by freshly isolated rat alveolar type II cells

2000 ◽  
Vol 278 (4) ◽  
pp. L830-L839 ◽  
Author(s):  
Joel F. Herbein ◽  
Jordan Savov ◽  
Jo Rae Wright
Keyword(s):  
Surfactant Protein ◽  
Surfactant Protein D ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Dependent Manner ◽  
Type Ii ◽  
Lipid Uptake ◽  
Lamellar Bodies ◽  
Alveolar Type Ii Cells ◽  
Type Ii Cell

Alveolar type II cells secrete, internalize, and recycle pulmonary surfactant, a lipid and protein complex that increases alveolar compliance and participates in pulmonary host defense. Surfactant protein (SP) D, a collagenous C-type lectin, has recently been described as a modulator of surfactant homeostasis. Mice lacking SP-D accumulate surfactant in their alveoli and type II cell lamellar bodies, organelles adapted for recycling and secretion of surfactant. The goal of current study was to characterize the interaction of SP-D with rat type II cells. Type II cells bound SP-D in a concentration-, time-, temperature-, and calcium-dependent manner. However, SP-D binding did not alter type II cell surfactant lipid uptake. Type II cells internalized SP-D into lamellar bodies and degraded a fraction of the SP-D pool. Our results also indicated that SP-D binding sites on type II cells may differ from those on alveolar macrophages. We conclude that, in vitro, type II cells bind and recycle SP-D to lamellar bodies, but SP-D may not directly modulate surfactant uptake by type II cells.

Integrin mediation of type II cell adherence to provisional matrix proteins

1996 ◽  
Vol 271 (2) ◽  
pp. L277-L286 ◽  
Author(s):  
H. J. Kim ◽  
D. H. Ingbar ◽  
C. A. Henke
Keyword(s):  
Cell Adhesion ◽  
Matrix Proteins ◽  
Alveolar Type ◽  
Type I ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Cells ◽  
Type Ii Cell ◽  
Type Ii Alveolar Cells ◽  
Alpha V Beta 3

Lung injury causes alveolar type I epithelial cell death, basement membrane denudation, and alveolar flooding with serum fibronectin and fibrinogen. For successful restoration of normal architecture, the epithelium must be regenerated from progenitor type II alveolar cells. Using adhesion assays, we examined whether type II alveolar cells adhere to the provisional matrix proteins fibronectin, fibrinogen, and fibrin, and whether integrins mediate this adherence. Rat type II cells adhered to fibronectin, vitronectin, fibrinogen, and fibrin. Synthetic RGD (arginine-glycine-aspartic acid) peptide blocked this adhesion. Flow cytometry and Western analysis indicated that type II cells expressed beta 1- and alpha v beta 3-integrins. Anti-beta 1-and anti-alpha v beta 3-integrin antibodies blocked type II cell adhesion to fibronectin and to fibronectin and fibrinogen, respectively. In summary, type II cells adhered to fibronectin, fibrinogen, and fibrin, and adhesion was partially mediated by integrins. This study provides the first evidence of type II cell adhesion to fibrin gels and vitronectin, beta 1- and alpha v beta 3-integrin mediation of type II cell adhesion, and the presence of the alpha v beta 3-integrin on type II epithelial cells.

Generation and characterization of monoclonal antibodies to alveolar type II cell lamellar body membrane

1998 ◽  
Vol 275 (1) ◽  
pp. L172-L183 ◽  
Author(s):  
K. Zen ◽  
K. Notarfrancesco ◽  
V. Oorschot ◽  
J. W. Slot ◽  
A. B. Fisher ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Membrane Protein ◽  
Lamellar Body ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Rat Lung ◽  
Lamellar Bodies ◽  
Alveolar Type Ii Cell ◽  
Type Ii Cell

Monoclonal antibodies against the limiting membrane of alveolar type II cell lamellar bodies were obtained after immunization of mice with a membrane fraction prepared from lamellar bodies isolated from rat lungs. The specificity of the antibodies was investigated with Western blot analysis, indirect immunofluorescence, and electron-microscopic immunogold studies of freshly isolated or cultured alveolar type II cells, alveolar macrophages, and rat lung tissue. One of the monoclonal antibodies identified, MAb 3C9, recognized a 180-kDa lamellar body membrane (lbm180) protein. Immunogold labeling of rat lung tissue with MAb 3C9 demonstrated that lbm180 protein is primarily localized at the lamellar body limiting membrane and is not found in the lamellar body contents. Most multivesicular bodies of type II cells were also labeled, as were some small cytoplasmic vesicles. Golgi complex labeling and plasma membrane labeling were weak. The appearance of lbm180 protein by immunofluorescence in fetal rat lung cryosections correlated with the biogenesis of lamellar bodies. The lbm180 protein decreased with time in type II cells cultured on plastic. The lbm180 protein is an integral membrane protein of lamellar bodies and was also found in the pancreas and the pancreatic βHC9 cell line but not in the rat brain, liver, kidney, stomach, or intestine. The present study provides evidence that the lbm180 protein is a lung lamellar body and/or multivesicular body membrane protein and that its antibody, MAb 3C9, will be a valuable reagent in further investigations of the biogenesis and trafficking of type II cell organelles.

Extracellular matrix synthesis and turnover by type II pulmonary epithelial cells

1992 ◽  
Vol 262 (5) ◽  
pp. L582-L589 ◽  
Author(s):  
D. E. Rannels ◽  
S. E. Dunsmore ◽  
R. N. Grove
Keyword(s):  
Extracellular Matrix ◽  
Steady State ◽  
Epithelial Cells ◽  
Primary Cultures ◽  
Calf Serum ◽  
Type I ◽  
Type Ii Cells ◽  
Type Ii ◽  
Pulmonary Epithelial Cells ◽  
Type Ii Cell

Both type I and type II pulmonary epithelial cells contact the extracellular matrix (ECM). Type II cell-ECM interactions are bidirectional; they involve matrix-mediated modulation of type II cell differentiation, as well as cellular synthesis and deposition of ECM components. The present experiments examine the kinetics of accumulation of newly synthesized proteins in cell and matrix fractions from primary cultures of type II pneumocytes. Cycloheximide-sensitive incorporation of [3H]leucine into total protein of both the cell and ECM fractions was linear for 24–30 h, when steady-state labeling was reached and maintained to at least day 8. Over this interval, the cells enlarged but did not divide. Newly synthesized proteins recovered in the matrix fraction averaged 1–2% of those in the cells. Relative rates of radiolabeling of matrix proteins peaked at culture day 2 and increased in the absence of serum. In short-pulse studies, initial rates of protein synthesis were equal on culture days 1 and 3; this suggested that the steady-state labeling kinetics above reflected protein turnover. This was supported by rapid loss of radioactivity from the ECM after fresh type II cells were seeded on a prelabeled, cell-free matrix surface. Fresh or conditioned Dulbecco's modified Eagle's medium containing 10% fetal calf serum had little effect on matrix stability. These results demonstrate regulated deposition and turnover of a complex ECM by type II cells and provide a basis for further investigations of factors that control these processes.

scholarly journals Role of Phosphocholine Cytidylyltransferase α in Lung Development

2006 ◽  
Vol 27 (3) ◽  
pp. 975-982 ◽  
Author(s):  
Yong Tian ◽  
Ruobing Zhou ◽  
Jerold E. Rehg ◽  
Suzanne Jackowski
Keyword(s):  
Epithelial Cell ◽  
Lung Development ◽  
Cultured Cells ◽  
Lung Surfactant ◽  
Surfactant Protein ◽  
Alveolar Type ◽  
Type I ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lamellar Bodies

ABSTRACT Lung development depends upon the differentiation and expansion of a variety of specialized epithelial cell types, including distal type I and type II pneumocytes in the late term. Previous studies have shown a strict dependence on the choline cytidylyltransferase α isoform (CCTα) to mediate membrane phospholipid formation in cultured cells and during preimplantation embryogenesis. CCTα expression is highest in lung, and there has long been speculation about its precise role, due to the dual requirement for phospholipid in proliferating cell membranes and for lung surfactant production from alveolar type II cells. We investigated the function of CCTα in lung development, using an inducible, epithelial cell-specific CCTα knockout mouse line. Deletion of CCTα beginning at embryonic day 7.5 did not restrict lung development but resulted in severe respiratory failure at birth. Alveolar lavage and lung lipid analyses showed significant decreases in the major surfactant phospholipid, dipalmitoyl-phosphatidylcholine. The fatty acids destined for the surfactant phospholipid were redirected to an expanded triglyceride pool. Transcripts encoding type II cell-specific markers were expressed in the knockout mice, indicating the expected progression of differentiation in lung epithelia. However, surfactant protein levels were reduced, with the exception of that for surfactant protein B, which was elevated. Ultrastructural analysis of the type II cells showed Golgi complex abnormalities and aberrant lamellar bodies, which deliver surfactant lipid and protein to the alveolar lumen. Thus, CCTα was not required for the proliferation or differentiation of lung epithelia but was essential for the secretory component of phospholipid synthesis and critical for the proper formation of lamellar bodies and surfactant protein homeostasis.

Expression ofN-deacetylase/sulfotransferase and 3-O-sulfotransferase in rat alveolar type II cells

2000 ◽  
Vol 279 (2) ◽  
pp. L292-L301 ◽  
Author(s):  
Zhong-Yuan Li ◽  
Kazunori Hirayoshi ◽  
Yasuhiro Suzuki
Keyword(s):  
Heparan Sulfate ◽  
Protein A ◽  
Sodium Chlorate ◽  
Alveolar Type ◽  
Type I ◽  
Type Ii Cells ◽  
Type Ii ◽  
Type I Cells ◽  
Type Ii Cell ◽  
I Cells

Basal laminae beneath alveolar type I cells are suggested to contain highly sulfated heparan sulfate-containing proteoglycans (PGs), and cultured type II cells accumulate highly sulfated matrices. To characterize the regulation of PG synthesis during the transition from type II cells to type I cells, we examined mRNA expression of N-deacetylase/sulfotransferase (NST) and 3- O-sulfotransferase (3-OST), two enzymes specific for heparan sulfate synthesis. We found that both freshly isolated and cultured type II cells expressed NST and 3-OST as shown by in situ hybridization. Expression of surfactant-associated protein A, B, and C mRNAs, determined by semiquantitative PCR, decreased during culture. Expression of type I cell marker T1α mRNA increased except in cells cultured on an Engelbrecht-Holm-Swarm gel. Expression of NST was dependent on cell density and matrix and was intense in conditions where cells spread fully, whereas 3-OST expression was unchanged in the conditions examined. The PG sulfation inhibitor sodium chlorate significantly inhibited cultured type II cell spreading, and this inhibition was reversed by sodium sulfate. These results suggest that highly sulfated PGs modified by NST are necessary for the spreading of cells during transdifferentiation of type II cells to mature type I cells.

Alterations of type II cell microvilli following ozone-induced alveolar injury

1988 ◽  
Vol 46 ◽  
pp. 326-327
Author(s):  
E. M. Haller ◽  
J. F. Paterson ◽  
J. M. Lundh ◽  
J. U. Balis
Keyword(s):  
Left Lobe ◽  
Ozone Exposure ◽  
Male Rats ◽  
Alveolar Type ◽  
Type I ◽  
Type Ii Cells ◽  
Type Ii ◽  
Type Ii Cell ◽  
High Concentrations ◽  
Injury And Repair

Ozone is a major oxidizing constituent in photochemical smog, and it is known to cause, at high concentrations, bronchiolo-al veoar injury with early loss of cilia and desquamation of alveolar type I cells, followed by proliferation of type II cells. In the present study, we investigated the sequential sur face alterations of alveolar type II cells using an established model for ozone-induced alveolar injury and repair.Fisher 344 male rats weighing 260±10g were exposed to 3 ppm ozone for 8 h. The animals were killed at various time intervals, inclduing zero time, 24, 48, and 96 h after termination of ozone exposure. The lungs were inflated at 20cm pressure with 2.5% glutaraldehyde in 0.1M Sorensen's phosphate buffer, ph 7.2, at 37 C for 4 h. Lung slices, 10 x 4 mm x 500 μm, were obtained by random selection from the left lobe, osmicated in 1% phosphate buffered OsO4, for 1 h at 4 C, dehydrated in a graded series of acetone, infiltrated with Freon 113, and critical-point dried using carbon dioxide.

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