The type II epithelial cells of the lung. III. Lecithin synthesis: a comparison with pulmonary macrophages

Synthesis of lecithins in isolated type II alveolar cells was compared with that in alveolar macrophages as a means of exploring the biochemical mechanisms underlying surfactant production in the lung. Counted cell populations were suspended in a simple glucose-salt solution and 14C-labeled precursors were added singly, in physiologic concentrations, to assess the potential importance of each as a substrate for lecithin synthesis. Molar incorporation of glucose, glycerol, choline, lysolecithin, acetate, palmitate, oleate, and linoleate was determined in lecithins fractionated according to degree of saturation after 1 hour of incubation. Palmitate ws the most actively utilized substrate in type II &cells. Type II cells incorporated 6 nmoles of palmitate per 10(7) cells, of which 77% was in disaturated lecithins, and 66% at the C2 position (compared to 0.8 nmoles, 47% disaturated, in macrophages). Acetate was also incorporated mainly into disaturated lecithins in type II cells; macrophages did not utilize acetate, and no precursor specifically supported disaturated lecithin synthesis in macrophages. Type II cells and macrophages synthesized similar quantities of total lecithins and disaturated lecithins from glucose and choline. Only the type II cells, however, were capable of increasing disaturated lecithin synthesis from 14C-choline when unlabeled palmitate was added to the medium. Type II cells synthesized significantly more disaturated lecithins from lysolecithin than did macrophages (451 versus 60 pmoles per 10(7) cells). Macrophages utilized glycerol in lecithin synthesis, but type II cells did not. Our data demonstrate directly for the first time that type II cells are the site of disaturated lecithin synthesis and that acyl turnover mechanisms are important in production of disaturated lecithins by the type II cell.     

Method for the isolation of surfactant from homogenates and lavages of lung of adult, newborn, and fetal rats

A method is presented for the isolation of pulmonary surfactant from rat lung homogenates and lavages using differential discontinuous density gradient centrifugation. The isolated surfactant was characterized by electron microscopy, assay of enzyme markers for different subcellular organelles, and its chemical composition. Surfactant that was isolated from lung homogenates appeared to be free of other contaminating cellular organelles. Electron microscopic examination of surfactant isolated from whole homogenates showed the presence of lamellar bodies and tubular myelin figures and, hence, represented total lung surfactant. On the other hand, surfactant prepared from lung lavages had mostly tubular myelin figures and, thus, represented extracellular surfactant. Both preparations were rich in phospholipids, especially lecithins and phosphatidylglycerol, and showed high phospholipid to protein ratios. The method was used to study the quantity and composition of surfactant during lung development in rats. Surfactant was isolated from lung homogenates of fetuses at days 19 to 21 of gestation and from newborn and adult rats. During the period of 19 to 21 days of gestation, there is a 10- and 20-fold increase in the amount of protein- and phospholipids (milligrams per gram of wet lung), respectively. A further 2-fold increase occurs after birth. Phosphatidylcholines account for 65% of the total phospholipids at day 20, 71% on day 21, and 81% at term. A progressive increase in the phosphatidylcholine to sphingomyelin ratios and in the amount of phosphatidylglycerol occurs during this period. The amount of disaturated lecithins (expressed as percentage of total phosphatidylcholines) in the lung surfactant increases from 49% at day 20 to 53% at birth. There is no change in the amount of disaturated lecithins after the newborn stage. The present method for the isolation of lung surfactant is reproducible, is less time consuming, and can be used to isolate quantitatively surfactant from small lung aliquots.     

Lecithin Changes in Murine Mycoplasma pulmonis Respiratory Infection

We examined the lipid content of bronchoalveolar (BA) washes from both mice and rats infected with Mycoplasma pulmonis, an etiological agent of murine pneumonia. During a 30-day period after intranasal inoculation, the total lipid content from infected and control rats (in milligrams per animal) remained relatively equal and unchanged. The saturated, unsaturated, and total lecithin contents in infected rats (in milligrams per animal) all increased. The maximum lecithin values were detected at 7 to 10 days after infection; later, the levels fell to control values. There was essentially no change in any lecithin value from uninfected animals. Although in BA washes from infected animals the mass of disaturated lecithins increased, the percentage of this fraction in the total lecithin pool decreased. The fatty acids of the lecithins from BA washes of infected mice had significantly less palmitic and significantly more oleic and linoleic acids than the lecithins isolated from the BA washes of control animals. Both the relative decrease in the mass of disaturated lecithins in the BA washes and the increase in the percentage of esterified unsaturated fatty acids in the lecithins may be directly related to the reduced lung function reported to occur during the course of murine M. pulmonis pneumonia.     

The type II epithelial cells of the lung. II. Chemical composition and phospholipid synthesis.

A method to isolate 93 per cent viable (trypan blue), 95 plus or minus 2 per cent pure type II alveolar epithelial cells (Kikkawa, Y., and Yoneda, K. Lab. Invest. 30: 76, 1974) was slightly modified and was used to isolate type II cells of similar purity and viability from New Zealand White male rabbits. The modification as described in this report now permits the regular harvest of 20 to 30 times 10-6 cells from a rabbit weighing 2 to 4 kg. Lipid composition of type II cell pools thus collected was compared with that of macrophages collected by alveolar washing. Per milligram of protein, type II cells contained 19 mug. of lipid phosphorus, compared with 11 mug. for macrophages. Per 1 times 10-8 cells, type II cells contained 110 mug. of lipid phosphorus compared with 155 mug. for macrophages; Phosphatidylcholine comprised 48 per cent of total phospholipid in type II cells and 34 per cent in macrophages. Forty-nine per cent of the phosphatidylcholine in type II cells was disaturated, compared with 29 per cent in macrophages. These data indicate that, per cell, type II cells contain almost twice as much disaturated phosphatidycholine as do alveolar macrophages. Radiolabeled precursor studies suggest that isolated type II cells synthesize disaturated lecithin predominantly through the cytidine diphosphocholine pathway. -14C-choline incorporation into disaturated lecithin was 3 times as active in type II cells as in macrophages. No evidence was found for a significant contribution to synthesis of disaturated lecithin by the transmethylation pathway in isolated type II cell preparations. These results offer the most direct evidence to date of the significant role of the type II alveolar epithelial cell in the synthesis of disaturated lecithin, a major constituent of pulmonary surfactant.     

Iprindole reverses the lamellar body deficiency of cultured L-2 cells. Possible implications in the reversal of surfactant deficiency.

Type II alveolar epithelial cells in long-term culture typically lose the ability to synthesize surfactant together with a loss of the characteristic lamellar bodies in the cytoplasm of the cells. Iprindole, a cationic amphiphilic drug, induces lamellar body formation in cultured L-2 cells, a cell line derived from rat Type II cells, but devoid of lamellar bodies. With concentrations of 10(-7)-10(-5) M iprindole, which approximate therapeutic plasma concentrations in human subjects, the drug induced the formation of lamellar bodies within 8 hours of incubation. This effect on cell morphology was rapidly lost after withdrawal of the drug. At concentrations of iprindole which induced lamellar body formation, there was a significant increase in phospholipid content of the L-2 cells as well as an increase in disaturated phosphatidylcholine, an important constituent of surfactant. These studies suggest that the use of drugs such as iprindole may represent a novel approach in the augmentation of phospholipid (and possibly surfactant) content of Type II cells in the lung.     

Isolated lamellar bodies from rat lung: correlated ultrastructural and biochemical studies.

Lamellar bodies isolated from rat lung homogenates were prepared for electron microscopy by two methods: OsO4 fixation followed by dehydration either with acetone or ethanol. Electron microscopy revealed that the acetone-dehydrated fraction contained many well preserved lamellar bodies with intact outer membrane, and the high retention of a material of low electron density between the fine and closely spaced concentric lamellae. This appearance was contrasted by the increased loss of this interlamellar material and coarse and irregularly spaced lamellae in the ethanol-dehydrated fraction. The supernatants from each respective fixation and dehydration were collected for lipid and protein analyses in an attempt to correlate any detected biochemical difference with a correspondingly modified ultrastructural appearance. Thin layer chromatography on the supernatant from the ethanol-dehydrated fraction revealed the extraction of 40 per cent lecithin and 4 per cent phosphatidylglycerol as compared to 1 per cent lecithin and an undeterminably small percentage of phosphatidylglycerol extracted by acetone dehydration. The effect of fixation and dehydration upon the extraction of chemical components of the lamellar bodies was compared to extraction by those of the following treatments: (1) freezing and thawing (a loss of 7 per cent lecithin, undetectably small losses of phosphatidylglycerol and protein); (2) incubation at 37 degrees C. with magnetic stirring (losses of 35 per cent lecithin, 5 per cent phosphatidylglycerol, and 4 percent protein, respectively); (3) sonication (loss of 57 per cent lecithin, 31 per cent phosphatidylglycerol, and 27 per cent protein, respectively). Further lipid analyses of the supernatants from dehydration and those obtained from the mechanical treatments revealed dipalmitoyl lecithin as the most readily extracted lipid. These data indicate that OsO4 fixation followed by acetone dehydration improves the preservation of the lamellar body ultrastructure by retaining an amorphous material between the lamellae, thus preserving the spatial relationship between the lamellae, and further that this improved morphologic preservation correlates well with retention of disaturated lecithin.     

A simple method for obtaining cultures enriched for Type II pneumonocytes from fetal rabbit

Summary A differential plating method permitted preparation of cultures significantly enriched for Type II pneumocytes. These cells were maintained in a differentiated state for at least 12 d, identifiable morphologically (by presence of osmiophilic lamellar inclusion bodies) and bio-chemically (by demonstration of synthesis of phosphatidyl choline and production of disaturated lecithin).     

Isolation and characterization of hypertrophic type II cells from the lungs of silica-treated rats

Type II cells isolated from the lungs of rats exposed to silica can be separated into two populations by using centrifugal elutriation. One population, designated type IIA, appears similar to type II cells isolated from control lungs. The second population, designated type IIB, consists of type II cells that are larger than type IIA cells or control type II cells. In addition, the type IIB (or hypertrophic) cells contain lamellar bodies which are larger and more numerous than lamellar bodies in type IIA or control type II cells. After centrifugal elutriation, the type II cell populations were purified by differential adherence on rat IgG-coated culture dishes for biochemical and metabolic studies. Type IIB cells contained elevated amounts of protein and total RNA in comparison to type IIA and control type II cells. Type IIB cells contained approximately 2.5-fold more phospholipid than control type II cells (51.5 +/- 3.0 micrograms/10(6) cells versus 20.5 +/- 4.1 micrograms/10(6) cells). Incorporation of the phospholipid precursors [14C]choline and [3H]palmitate into cellular phosphatidylcholines was also increased in type IIB cells. After 120 minutes of incubation, incorporation of [14C]choline into total phosphatidylcholine by type IIB cells was 250% greater than in controls; at this same time point, incorporation of [3H]palmitate was approximately 150% greater than in controls. Incorporation of [14C]choline into disaturated phosphatidylcholine by type IIB cells was 200% greater than in controls. However, no increased incorporation of [3H]-palmitate into disaturated phosphatidylcholine by type IIB cells was seen. These results suggest that the hypertrophic type II cell is responsible for the increases in surfactant-associated phospholipids in the lungs of rats exposed to silica.     

Temporal segregation of surfactant secretion and lamellar body biogenesis in primary cultures of rat alveolar type II cells

Pulmonary alveolar type II cells synthesize and secrete the phospholipids of surfactant. However, type II cells isolated from adult rat lungs rapidly lose their characteristic morphology and differentiated functions (such as surfactant-specific phospholipid and protein biosynthesis) when maintained on tissue culture plastic. In this study, phospholipid secretion and its regulation by type II cells grown on tissue culture plastic were examined up to 8 days after isolation. Type II cells were preincubated with [3H]choline for varying 24-h periods during culture prior to examining phosphatidylcholine ([3H]PtdCho) secretion. Type II cells cultured for 4 days and incubated with [3H]choline 24 h before the secretion experiment failed to show significant basal and tetradecanoyl phorbol acetate (TPA, 100 nM)-stimulated [3H]PtdCho secretion (basal, 0.29 +/- 0.01%; TPA, 0.48 +/- 0.04%). In contrast, type II cells incubated with [3H]choline for the first 24 h during culture and then cultured for 3 more days showed significant [3H]PtdCho secretion (basal, 1.27 +/- 0.19%; TPA, 6.24 +/- 0.82%). Subcellular fractionation of type II cells revealed that [3H]choline was incorporated into phosphatidylcholines in a lamellar body-enriched fraction during the first 24 h of culture but that the assimilation of phosphatidylcholine into the lamellar body fraction progressively declined with increasing time in culture. Radiolabel incorporated into the lamellar body fraction labeled during the first 24 h of culture was detectable for up to 8 days in culture. The [3H]PtdCho incorporated into the lamellar body during the first 24 h of culture was lost gradually over 8 days, suggesting the continuous secretion or turnover of the lamellar bodies during culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbon stripping extracts serum free fatty acids: implications for media supplementation of cultured type II pneumocytes

Carbon stripping is a process that is widely used to remove hormones from serum. Because addition of serum to culture media also provides exogenous fatty acids that influence lipid metabolism of cultured cells, we investigated the effects of carbon stripping on the composition of the phospholipid and free fatty acid fractions in fetal bovine serum and the effects of these changes on phosphatidylcholine synthesis by cultured adult alveolar type II cells. Carbon stripping resulted in quantitative and qualitative changes in serum free fatty acids. The process effectively extracted greater than or equal to 99% free fatty acids and, to a lesser extent, phospholipids. There were also qualitative changes in the relative composition of the remaining free fatty acids with a selective loss of oleic and linoleic free fatty acids. However, the relative composition of the serum phospholipid fatty acid fraction was unaffected. Type II cells isolated from adult male rat lung and cultured in Dulbecco's modified Eagle's medium supplemented with 10% carbon-stripped fetal bovine serum (FBS-CS) incorporated [3H]choline into phosphatidylcholine at a rate 36% less than the rate of control cells cultured with unstripped FBS. Addition of oleic acid to FBS-CS supplemented media increased total phosphatidylcholine synthesis by adult type II cells by 67-71%. In contrast, addition of palmitic acid inhibited PC synthesis 51-67%. The combination of oleic and palmitic acids resulted in a rate of [3H]choline incorporation into phosphatidylcholine similar to the rate for control cells cultured in FBS-CS-supplemented media alone. Although synthesis of disaturated phosphatidylcholine was unaffected by exogenous fatty acids, addition of fatty acids altered the proportion of disaturated phosphatidylcholine synthesis relative to total phosphatidylcholine synthesis. The presence or absence of the hormones, dexamethasone and triiodothyronine, did not explain the difference in rate of phospholipid synthesis by type II cells cultured in untreated versus carbon-stripped serum supplemented media. These results suggest that the removal of serum free fatty acids by carbon stripping can influence phospholipid metabolism of cultured type II cells. Because serum free fatty acids influence cellular lipid composition and potentially cell metabolic functions, carbon-stripped serum may not be the optimal choice for media supplementation of cultured cells.     

Recirculation and reutilization of micellar bile lecithin.

Bile lecithins, solubilized in micellar bile salt and radiolabeled in the 1-acyl fatty acid, phosphorus, and choline positions, were infused in the small bowel of fasted rats. Absorption of each label was virtually complete after 24 h. However, these lecithins were extensively hydrolyzed in the bowel lumen as well as after absorption, and neither the fatty acid nor phosphorus was significantly retained in the enterohepatic circulation or reutilized for biliary lecithin synthesis. In contrast, while choline was also dissociated from absorbed lecithin, choline was instead retained in the liver, reincorporated into newly synthesized hepatic lecithin, and sercreted in biliary lecithin in 10-fold greater amounts than either the fatty acid or phosphorus. However, the extent of choline incorporation into bile lecithin was limited and was not further increased when free choline was directly injected into the portal vein. The data therefore suggest that although only choline of absorbed lecithin is retained in the enterohepatic circulation and preserved for new biliary lecithin synthesis, exogenous choline utilization is regulated by the size of the available hepatic pool.     

Chlorphentermine-induced neonatal and maternal pulmonary phospholipidosis in rats

Pregnant rats were treated on Days 16–20 of pregnancy with 30 mg/kg chlorphentermine hydrochloride (CP) or an equal volume of 0.9% NaCl. Females delivered on Day 21 and neonates were examined 12 hr postpartum. Electron micrographs showed the CP neonates to have lamellar inclusions, characteristic of drug-induced phospholipidosis in Type I and Type II cells, macrophages, and nonciliated bronchiolar epithelial cells of the lung. These inclusions were either unicentric, multicentric, or consisted of whorls of membranous material. The content of total phospholipids was increased in neonatal lung as a result of CP treatment. A decrease in the percentage of total phospholipid present as phosphatidylinositol plus phosphatidylserine and an increase in the content of disaturated phospphatidylcholine were found in neonatal lung as a result of maternal chlorphentermine treatment. In maternal lung, increases were seen in the content of total phospholipids, total phosphatidylcholine, disaturated phosphatidylcholine, and phosphatidylinositol plus phosphatidylserine. A decrease in the percentage of sphingomyelin was also found. The results of this study indicate that following prepartum maternal administration of CP morphological changes are accompanied by biochemical alterations which are consistent with the induction of phospholipidosis as observed in neonatal lung. These biochemical changes were different than those associated with the induction of this disorder in the maternal lung.     

Morphologic and biochemical study of pulmonary changes induced by bleomycin in mice.

Pulmonary changes induced by bleomycin were investigated in mice by using light and electron microscopy and by phospholipid analysis of the alveolar washings. The damage began in the endothelium of capillaries and was accompanied by interstitial edema and an appearance of enlarged macrophages along with hypertrophy of type II epithelial cells. This condition was followed by degeneration of type I cells. The denuded epithelium appeared to be repaired by two different mechanisms. In areas away from the bronchioles, division of existing type II cells and subsequent transformation into type I cells appeared to be the pattern of epithelial repair. In areas near the bronchiole, downgrowth of bronchiolar undifferentiated cells and subsequent maturation of these cells to type II and type I cells appeared to be prominent. Biochemically, total phospholipids and disaturated lecithin in the alveolar wash increased along with the increase of the alveolar lining layer and the hypertrophy of type II cells. This was considered to be consistent with the view that the hyperactive type II cells secreted more surfactant in the early phase of the experiment.     

Keratinocyte growth factor enhances maturation of fetal rat lung type II cells

Keratinocyte growth factor (KGF) or fibroblast growth factor (FGF)-7, a peptide produced by stromal cells and in particular by lung mesenchyme, has recently been shown to influence early lung morphogenesis and to be a mitogen for fetal and adult alveolar type II cells. Although contradictory findings have been reported regarding its effects on surfactant protein expression, its effects on surfactant phospholipids have not been studied. We investigated the effects of KGF on the synthesis of surfactant components by cultured fetal rat type II cells isolated during the late gestational period, when surfactant accumulates in preparation for extrauterine life. We show that KGF is a potent stimulus of surfactant phospholipid synthesis, particularly for the major component of surfactant, disaturated phosphatidylcholine (DSPC). KGF increased choline incorporation into DSPC in a dose-dependent manner up to 25 ng/ml (1.3 x 10(-9) M), and this effect was greater for surfactant than for nonsurfactant DSPC. KGF was several times more potent in this respect than acidic FGF at the same molar concentration. KGF, similar to epidermal growth factor, also stimulated acetate incorporation and increased the surfactant phospholipid and DSPC content of cultured cells twofold. These effects correlated with increased choline phosphate cytidylyltransferase activity and increased fatty acid synthase activity and gene expression. KGF also induced a dose-dependent stimulation of surfactant protein-A, -B, and -C gene expression, leading to a 2- to 3-fold increase in their messenger RNAs. KGF therefore stimulates the synthesis of all surfactant components in developing type II cells at the time of surfactant accumulation. Its secretion by lung fibroblasts may thus be an important factor in promoting the maturation of fetal lung epithelium and the synthesis of sufficient surfactant. The results suggest that KGF could provide a new therapeutic agent for the management of the immature or injured lung.     

Opioids accelerate fetal rat lung maturation in vitro

Infants born to heroin- and cocaine-addicted mothers have been reported to have a lower incidence of respiratory distress syndrome (RDS) compared with nonaddicted infants. However, it is not known whether these are direct drug-mediated effects or secondary phenomena. We therefore investigated the effect of opioids and cocaine on fetal rat lung maturation in vitro. Using 18- to 20-d fetal rat lung explants and 20-d fetal type II cells, we measured the effect of varying concentrations (1 x 10(-8) to 1 x 10(-3) M) of heroin, morphine, methadone, and the nonopioid cocaine on the rate of choline incorporation into phosphatidylcholine (PC) and disaturated PC. We also analyzed the morphology of 19-d explants after exposure to opioids. Significant increases in rate of choline incorporation were noted in 19- and 20-d explants using 1 x 10(-3) M heroin, 1 x 10(-3) M morphine, and 1 x 10(-4) M methadone (P < 0. 005). No acceleratory effect was seen with cocaine. Morphologic analysis of the three opioid-treated groups revealed a significant (192 to 251%) increase in type II pneumocytes and lamellar bodies per alveolar lining cell (P < 0.01). Choline incorporation into PC by type II cells was also significantly increased by opioids (P < 0. 01); lactate dehydrogenase release and cell viability were not affected by opioid treatment. These data indicate that high-dose opioids have an acceleratory effect on biochemical and morphologic parameters of fetal lung maturation in vitro. The lack of in vitro acceleration with cocaine suggests that any cocaine-related reduction in the incidence of RDS is a secondary effect.     

Major phospholipids and phosphatidylcholine synthesis in adult Schistosoma mansoni

The major phospholipids present in the phospholipid extract of Schistosoma mansoni were phosphatidylcholine (28%), phosphatidylethanolamine (25%), phosphatidylserine (15%) and phosphatidylglycerol (8%). The synthesis of phosphatidylcholine in S. mansoni adults occurred by the choline to phosphatidylcholine or Kennedy pathway. Incorporation of CDPcholine and choline into the phosphatidylcholine of worm slices appeared linear over time with no demonstrable sex differences in choline incorporation. A slight difference in the incorporation of CDPcholine by separate sexes was evident. Methylation of phosphatidylethanolamine to phosphatidylcholine could not be demonstrated.     

Phospholipid profile of Pneumocystis carinii and its interaction with alveolar type II epithelial cells.

Pneumocystis carinii is an obligate parasite of mammalian lungs, attaching to but not invading the alveolar epithelium. The alveolar air spaces are rich in phospholipids, which are secreted by steroid-responsive alveolar type II epithelial cells. P. carinii isolated from rat lungs was found to contain the expected structural phospholipids as well as a large amount of firmly attached disaturated phosphatidylcholine, the characteristic phospholipid of alveolar surfactant. In vitro, P. carinii cells synthesized phospholipids from simple radiolabeled precursors; disaturated phosphatidylcholine was not formed. However, washed P. carinii cells avidly adsorbed radiolabeled rat surfactant, a process that appeared to be saturable, not dependent on viability of the organisms, and abolished by incubation at 4 degrees C. The surfactant was neither harmful nor beneficial to in vitro survival of the organisms. With the exception of high concentrations of arachidonic acid, fatty acids found in rat alveolar lining material were also not toxic. In addition, cultures consisting primarily of rat type II alveolar epithelial cells were toxic to P. carinii when the organisms were added to monolayers of type II cells at less than or equal to 10:1 multiplicity. At higher multiplicities, the parasite survived (but did not increase in numbers), and the type II cells deteriorated. The mechanism for this effect has not been determined.     

Localization of surfactant-associated proteins SP-A and SP-B mRNA in rabbit fetal lung tissue by in situ hybridization.

Pulmonary surfactant is a lipoprotein substance, comprised of approximately 80% phospholipid and approximately 10% protein, that lowers surface tension at the air-alveolar aqueous interface. Surfactant is synthesized and secreted by alveolar type II epithelial cells where it is stored intracellularly in lamellar bodies. In the present study, we used the technique of in situ hybridization to localize the mRNA for two surfactant-associated proteins, SP-A and SP-B, in developing rabbit fetal lung tissue. We found that SP-A mRNA was first localized in rabbit fetal lung alveolar type II cells on day 26 of gestation, the time at which lamellar bodies are first observed within fetal lung type II cells. On day 28 of gestation, a very small amount of SP-A mRNA was also detectable in the epithelial cells of some bronchioles. In neonatal and adult rabbit lung tissue, SP-A mRNA was primarily restricted to alveolar type II cells; however, the epithelial cells of some bronchioles contained small amounts of SP-A mRNA. SP-B mRNA was first detected in cuboidal epithelial cells in the prealveolar region of the rabbit fetal lung tissue on day 24 of gestation, i.e., at least 2 days before the appearance of SP-A mRNA and lamellar bodies within differentiated alveolar type II cells. SP-B mRNA was detected in most bronchiolar epithelial cells of the rabbit fetal lung tissue at day 28 of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ozone-induced lamellar body responses in a rat model for alveolar injury and repair.

Exposure of adult rats to 3 ppm ozone for 8 hours results in diffuse alveolar damage with well-defined sequential stages of bronchiolo-alveolar injury and repair. This model is characterized by acute pulmonary edema showing high concentration of lavage fluid protein that is maximally elevated at 24 hours with return to control level at recovery (96 hours). Using techniques that enable optimal preservation of lamellar body ultrastructure, it was demonstrated morphometrically that expansion of the vacuolated lamellar body (LB) compartment is an early, transient LB response of the type II cell to acute injury. This change appears to be initiated by increased LB secretion. The reparative stage, 24-48 hours postexposure, begins with hypertrophy rather than hyperplasia of many type II cells, resulting in a 3-fold increase of mean type II cell volume at 48 hours. During this stage there is also significant expansion of the total LB compartment with corresponding increased LB storage of surfactant disaturated phosphatidylcholine (DSPC) per type II cell. At recovery, 96 hours, the lungs contained twice the normal numbers of type II cells, but the total size of lamellar body compartment per type II cell as well as the DSPC content of the isolated lamellar body pool returned to normal levels. In contrast, accumulating surfactant DSPC in lavage fluid increased progressively throughout the reparative and recovery stages presumably due in part to parallel increase in type II cell numbers at 48 and 96 hours. Additional changes of surfactant included abnormal secretion of densely coiled lamellar bodies that accumulated in alveolar spaces at the expense of tubular myelin. These observations indicate that acute oxidant injury to alveoli initiates progressive hypertrophy followed by hyperplasia of type II cells, in association with sequential development of characteristic lamellar body changes leading to increased storage and secretion of surfactant with reduced ability to form tubular myelin.     

Recovery from chlorphentermine-induced phospholipidosis in rat alveolar macrophages: Morphological features

Rats were treated for 4 weeks with the amphiphilic, anorexic drug chlorphentermine resulting in the induction of a phospholipid storage disorder in alveolar macrophages. When examined with the electron microscope, the cells were found to be filled with lamellarshaped and membranous inclusions often associated with amorphous granular and membranous material. These features presumably represent the accumulated phospholipid which occurs with this disorder. We have previously reported that following withdrawal of the rats from chlorphentermine treatment, the phospholipid content of the macrophages returned to the control level by 14 days of recovery (Reasor and Koshut, Toxicol. Appl. Pharmacol.55, 334–341, 1980). Morphological features of the cells were examined at various times after termination of drug treatment in order to monitor the recovery process from this perspective. After 7 days of recovery, the macrophages were generally free of the amorphous material, however, they contained prominent lamellar inclusions. At 14 days of recovery, most of the cells contained large pleomorphic bodies, which were probably autophagic vacuoles. These structures appeared to be involved in degrading the lamellar material and converting it into residual bodies. At 21 days of recovery, most of the macrophages appeared normal, although some contained structures similar to those present at 14 days of recovery. By 28 days of recovery, essentially all of the macrophages were normal in appearance. Therefore, while the phospholipid content of the cells returned to the control level by 14 days of recovery, the cells still had an altered morphological appearance. Thus, assessment of recovery from phospholipidosis should not be based on phospholipid content alone.