Cell Lamellar Bodies Research Articles

Alterations of surfactant pools in fetal and newborn rat lungs.

Preparation by the developing alveolar epithelium for the transition to air breathing and surfactant secretion at birth are critical components of neonatal survival. We combined morphometric analysis and biochemical assays of lung phospholipids to measure the amount and redistribution of lung surfactant during the perinatal period of rats. Within 10 min of the start of air breathing, there was a small increase in type II cell lamellar body content by morphometric and biochemical estimates. By 24 h, the whole lung and alveolar extracellular pool surfactant lipid had substantially increased. Subfractionation of the alveolar surfactant pool obtained at four times, from birth to 24 h of life, demonstrated a 20-fold increase in the ratio of phospholipid in a tubular myelin-rich fraction compared to a unilamellar vesicle-rich fraction. We conclude that packaging of surfactant may be very active immediately postbirth. Our results also indicate a major shift in the physical forms of extracellular surfactant during the first hours of air breathing.

Isolation of viable type II alveolar epithelial cells by flow cytometry

We developed a new method for isolating viable type II cells from fractionated and unfractionated lung cell suspensions by flow cytometry using acridine orange (AO). Fischer-344 rat lungs were dispersed into single-cell suspensions by a technique that yields a high number of cells (4-5 X 10(8) cells/lung, congruent to 85% viable), congruent to 11% of which are type II cells. Elutriated fractions from the lung cell preparation and parent, unfractionated cell suspensions were incubated with 1.0-0.02 micrograms/ml AO and analyzed by flow cytometry. Parameters analyzed included axial light loss (ALL) and red fluorescence (RF). Based on their unique RF, attributable to AO staining of type II cell lamellar bodies, and their ALL characteristics, type II pneumocytes were sorted from elutriated fractions to greater than 95% purity. Using the same approach, type II pneumocytes were sorted from unfractionated lung cell suspensions at greater than or equal to 85% purity. The viabilities of the type II alveolar epithelial cells isolated by this method range from 85% to 95%, and the ultrastructural features of the sorted cells were unaltered by AO labeling or sorting.

Pulmonary type II cell lamellar body ultrastructure preserved by rapid freezing and freeze drying.

Lamellar body ultrastructure was examined in cultured type II alveolar epithelial cells processed by a method of rapid freezing and freeze drying in the absence of both chemical fixation and solvent dehydration. This method of specimen preparation was chosen to optimize the retention of soluble substances within the type II cell. The use of cultured cell aggregates in which type II cells line the free surface facilitated the effectiveness of rapid freezing for the preservation of lamellar body fine structure. Lamellar bodies of frozen/frozen dried type II cells showed none of the often profound lipid extraction artifact produced by conventional processing. Instead they exhibited a substructure with noteworthy characteristics in common with lamellar bodies processed by resin dehydration lipid retention methods (Stratton, 1976). Importantly, the lamellae of frozen/frozen dried lamellar bodies were contiguous, with no interlamellar space, as is commonly observed in solvent-processed (extracted) specimens. The dimensions of lamellar components in frozen/frozen dried lamellar bodies were, however, different from published values for resin-dehydrated lipid-retained specimens. Lamellar width and the widths of component phospholipid head and fatty acid tail regions in frozen/frozen dried lamellar bodies were approximately 35% smaller than values reported for resin-dehydrated lamellar bodies. This difference was attributed to shrinkage of lamellar components as water was removed from the unfixed tissue during the freeze-drying process. Lamellar bodies preserved by rapid freezing/freeze drying to optimize the in situ retention of intracellular components possess closely adherent concentric membranous lamellae. This supports the contention that the widely appreciated lamellar pattern of the pulmonary lamellar body represents the in vivo molecular organization of intracellular surfactant phospholipids.

Nonrespiratory metabolic function and morphology of lung following exposure to polybrominated biphenyls in rats.

Exposure to polybrominated biphenyls (PBBs) resulted in increased activity of microsomal arylhydrocarbon hydroxylase and ethoxyresorufin-O-deethylase in rat lung. Clearance of 5-hydroxytryptamine (5-HT) and angiotensin 1 by perfused lungs was decreased by PBBs. However, PBBs had no effect on the activity of epoxide hydrolase, monoamine oxidase, or angiotensin-converting enzyme in lung. The only histopathologic change detected in lungs from PBB-treated rats was an increase in alveolar type II cell lamellar bodies. Selective accumulation of certain PBB congeners by lung was not observed in this investigation.

Precursor-product relationship between rabbit type II cell lamellar bodies and alveolar surface-active material: Surfactant turnover time

To estimate the turnover time of alveolar surfactant in New Zealand white rabbits, we injected [9,10- 3H]palmitic acid and [2- 14C]glycerol intravenously. From 1–48 h after injection, we killed the animals, lavaged the lungs for alveolar surfactant with saline, and isolated the lamellar bodies by homogenization and sucrose density gradient centrifugation. Lamellar bodies and alveolar surfactant had comparable phospholipid composition and surface activity. Lamellar bodies contained little DNA, no mitochondrial enzyme activity and less than 5% contaminating phospholipids from microsomal and Golgi-enriched fractions. We measured radioactivity of phosphatidylcholine, saturated phosphatidylcholine and phosphatidylglycerol for each isotope in lamellar bodies and surfactant at each time point. The plot of the integral with respect to time of the difference between lamellar body and surfactant specific activity against surfactant specific activity has a slope determined by the turnover time, and a shape which tests the precision of the precursor-product relationship. This analysis does not assume a pulse label and allows for possible recycling of tracer from surfactant to lamellar bodies. We obtained turnover times of 4–11 h. We detected an imprecise precursor-product relationship between lamellar bodies and alveolar surfactant, which is not due to experimental variability or to contamination of lamellar bodies by other subcellular fractions but may reflect imperfect mixing within surfactant compartments.

Ultrastructural changes in type I and type II alveolar epithelial cells in isolated perfused dog lungs after production of moderate and severe hydrostatic edema.

Volume densities of type-I and type-II alveolar cell cytoplasmic organelles were estimated by established stereologic procedures. The measurements were obtained from isolated-perfused dog lungs after the onset of acute hydrostatic edema. The isolated lungs provided three successive lobar preparations: control, moderately edematous, and severely edematous. The type-II cell lamellar body volume densities were decreased in both stages of edema. Volume density of the granular endoplasmic reticulum (including polyribosome clusters) was increased after progression to severely edematous conditions. Concomitant increases in vesicular volume densities were recorded in type-I cells after both moderate and severe edema. In addition, the relative percentages of vesicles directly attached to the luminal and abluminal plasma membranes were increased in the edematous lungs. These marked ultrastructural changes in the alveolar epithelium are consistent with the interpretation that both the release of lamellar bodies from type-II cells and the number of type-I cell pinocytotic vesicles are increased in isolated dog lungs after production of edema.

Organotypic cultures of diploid type II alveolar pneumonocytes: surfactant associated esterase activity.

Organotypic cultures, established from enzymatically dispersed day 19 fetal rat lung, are comprised primarily of cells which are morphologically similar to type II alveolar pneumonocytes, the cells involved in surfactant synthesis. To further characterize these cultures, the nonspecific esterase pool was examined to determine if these cultures contained certain nonspecific esterases previously shown to be enzyme markers for the surfactant system. The results of biochemical, electrophoretic and cytochemical studies indicate that these organotypic cultures contain the same nonspecific esterases already demonstrated in surface active fractions derived from rat and mouse lung homogenates and pulmonary lavage fluid. As in whole lung, the major site of esterase activity in the organotypic cultures is the type II cell lamellar body, the putative site of surfactant synthesis and storage. These findings support the concept that the organotypic cultures derived from fetal rat lung are comprised predominantly of type II cells which retain surfactant associated functions in vitro.

The freeze‐fracture study of alveolar type II cells and alveolar content in fetal rabbit lung

Freeze-fracture replication technique was utilized to study the morphology of type II alveolar epithelial cells and alveolar contents in the late gestation of rabbit fetuses. It was shown that the lamellar inclusion bodies of type II cells were enveloped by the usual type of unit membrane with membrane-associated particles of 15 nm diameter. The interior of the inclusion bodies was composed of multiple stacks and/or whorls of membranes which were devoid of membrane-associated particles. Small vesicles within the inclusion were found more frequently with this technique than in chemically fixed thin-sectioned preparations. The intra-alveolar contents were comprised of two components; sperical bodies, which were identical to the internal contents of the lamellar bodies of type II cells, and tubular elements. These tubules most often appeared rectangular on cross-fractured faces. Triangular hexagonal fracture faces were also noted. The tubules were seen to rest on the surfaces of the spherical components. Our observations suggest that the tubular element of alveolar contents is formed through the interaction between the discharged lamellar body content and the alveolar fluid, and further suggest that at least the major constituent of type II cell lamellar bodies is lipid not bound to protein. Three new observations were made in this study; the absence of membrane-associated particles on the interior of the lamellae of the inclusions, the cross-fractured profiles of tubular elements of the alveolar contents, and the occasional multicompartmental nature of type II cell inclusions.

A morphometric examination of type II alveolar epithelial cells in normal and isolated-perfused dog lungs.

The volume densities of type II alveolar cell cytoplasmic organelles and alveolar surface densities were estimated by established stereologic procedures. The morphometric measurements were obtained from normal dog lungs (in situ) and isolated dog lungs perfused for 30-minute, 1-hour, and 2-hour periods. The type II cell lamellar body volume densities and the alveolar surface densities progressively decreased as the times of perfusion were increased. The volume densities of the granular and agranular endoplasmic reticulum progressively increased during the periods of perfusion. These morphometric parameters from lungs in situ and isolated lungs suggest that changes occur in pulmonary surfactant synthesis and activity during perfusion. It is further postulated that progressive increases in the rates of surfactant removal and/or inactivation during perfusion may contribute to spontaneous edema in lungs isolated for periods exceeding two hours. The morphologic and physiologic integrity of isolated perfused lung preparations, widely used as models of lungs in vivo, in situ requires further evaluation.

Selective Staining of Pulmonary Type II Alveolar Cell Lamellar Bodies in “I-Micron” Plastic Sections

Schlipköter in 1954 described a concentric lamellated osmiophilic body contained within the cytoplasm of certain pulmonary alveolar epithelial cells. The occurrence of these bodies and microvilli on the free cell border is the ultrastructural trademark of the type II alveolar epithelial cell. Numerous reports have been made concerning the histogenesis of this cell type and the composition and function of its lamellated inclusions. Though the significance of these bodies remains undetermined, their faithful occurrence facilitates the identification of the type II cell in fine structure studies.In paraffin sections of lung tissue, vacuoles are found in those cytoplasmic areas of the type II cell which are normally occupied by the lamellar inclusions. Presumably a chemical extraction has occurred during the embedding procedure. Thus, the identification of the type II cell in these sections is based mainly upon its shape and location. When 1-2 micron-thick sections of plastic-embedded lung tissue are stained with methylene blue and azure II, then counterstained with basic fuchsin according to the method of Mackay and Mead, the lamellar bodies of the type II cell show an affinity for the basic fuchsin.