Cell Lamellar Bodies Research Articles

Increased expression of CHOP and LC3B in newborn rats with bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) seriously affects the health and prognosis of children, but the efficacy of treatments is poor. The present study aimed to examine the effects of C/EBP homologous protein (CHOP), activating transcription factor 4 (ATF4) and microtubule‑associated protein light chain3β (LC3B), and the interaction between CHOP and LC3B, in newborn rats with BPD. At 1, 7, 14 and 21days, the rats in the model [fraction of inspired oxygen (FiO2)=80‑85%] and control groups (FiO2=21%) were randomly sacrificed, and lung samples were collected. Alveolar development was evaluated according to the radial alveolar count (RAC) and alveolar septum thickness. Ultrastructural changes were observed by transmission electron microscopy (TEM), the expression levels of CHOP, ATF4 and LC3B were determined by immunohistochemistry, and western blot and reverse transcription‑quantitative polymerase chain reaction analyses. The co‑localization of CHOP and LC3B in lung tissues was determined by immunofluorescence. The results showed that, compared with the control group, alveolarization arrest was present in the model group. The TEM observations revealed that, at 14days, type II alveolar epithelial cell (AECII) lamellar bodies were damaged, with an apparent dilation of the endoplasmic reticulum (ER) and autophagy in cells within the model group. Between days 7 and 14, the protein levels of ATF4, CHOP and LC3B were significantly increased in the model group. The mRNA levels of CHOP and LC3B were lower at days 7‑21. CHOP and LC3B were co‑localized in the cells of the lung tissues at day 14 in the model group. Pearson's correlation analysis showed that the protein levels of CHOP and LC3B‑II were positively correlated in the model groups. As in previous studies, the present study demonstrated that BPD damaged the AECII cells, which exhibited detached and sparse microvilli and the vacuolization of lamellar bodies. In addition, it was found that the ER was dilated, with autophagosomes containing ER and other organelles in AECII cells; the expression levels of CHOP and LC3B‑II were upregulated. CHOP and LC3B‑II may have joint involvement in the occurrence and development of BPD.

Aberrant lung remodeling in a mouse model of surfactant dysregulation induced by modulation of the Abca3 gene

The lipid transporter, ATP binding cassette class A3 (ABCA3), plays a critical role in the biogenesis of alveolar type 2 (AT2) cell lamellar bodies (LBs). A relatively large number of mutations in the ABCA3 gene have been identified in association with diffuse parenchymal lung disease (DPLD), the most common of which is a missense mutation (valine substitution for lysine at residue 292 (ABCA3E292V)) that leads to functional impairment of the transporter in vitro. The consequences of ABCA3E292V gene expression in vivo are unknown. To address this question, we developed mouse models expressing ABCA3E292V knocked-in to the endogenous mouse locus. The parental (F1) mouse line (mAbca3E292V) that retained an intronic pgk-Neo selection cassette (inserted in reverse orientation) (mAbca3E292V–rNeo) demonstrated an allele dependent extracellular surfactant phospholipid (PL) deficiency. We hypothesize that this PL deficiency leads to aberrant parenchymal remodeling contributing to the pathophysiology of the DPLD phenotype. Compared to wild type littermates, baseline studies of mice homozygous for the pgk-Neo insert (mAbca3E292V–rNeo+/+) revealed nearly 50% reduction in bronchoalveolar lavage (BAL) PL content that was accompanied by quantitative reduction in AT2 LB size with a compensatory increase in LB number. The phenotypic alteration in surfactant lipid homeostasis resulted in an early macrophage predominant alveolitis which peaked at 8 weeks of age. This was followed by age-dependent development of histological DPLD characterized initially by peribronchial inflammatory cell infiltration and culminating in both an emphysema-like phenotype (which included stereologically quantifiable reductions in both alveolar septal surface area and volume of septal wall tissue) plus foci of trichrome-positive collagen deposition together with substantial proliferation of hyperplastic AT2 cells. In addition to spontaneous lung remodeling, mABCA3E292V–rNeo mice were rendered more vulnerable to exogenous injury. Three weeks following intratracheal bleomycin challenge, mAbca3–rNeo mice demonstrated allele-dependent susceptibility to bleomycin including enhanced weight loss, augmented airspace destruction, and increased fibrosis. Removal of the rNeo cassette from mAbca3 alleles resulted in restoration of BAL PL content to wild-type levels and an absence of changes in lung histology up to 32 weeks of age. These results support the importance of surfactant PL homeostasis as a susceptibility factor for both intrinsic and exogenously induced lung injury/remodeling.

Lethal H1N1 influenza A virus infection alters the murine alveolar type II cell surfactant lipidome.

Alveolar type II (ATII) epithelial cells are the primary site of influenza virus replication in the distal lung. Development of acute respiratory distress syndrome in influenza-infected mice correlates with significant alterations in ATII cell function. However, the impact of infection on ATII cell surfactant lipid metabolism has not been explored. C57BL/6 mice were inoculated intranasally with influenza A/WSN/33 (H1N1) virus (10,000 plaque-forming units/mouse) or mock-infected with virus diluent. ATII cells were isolated by a standard lung digestion protocol at 2 and 6 days postinfection. Levels of 77 surfactant lipid-related compounds of known identity in each ATII cell sample were measured by ultra-high-performance liquid chromatography-mass spectrometry. In other mice, bronchoalveolar lavage fluid was collected to measure lipid and protein content using commercial assay kits. Relative to mock-infected animals, ATII cells from influenza-infected mice contained reduced levels of major surfactant phospholipids (phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine) but increased levels of minor phospholipids (phosphatidylserine, phosphatidylinositol, and sphingomyelin), cholesterol, and diacylglycerol. These changes were accompanied by reductions in cytidine 5'-diphosphocholine and 5'-diphosphoethanolamine (liponucleotide precursors for ATII cell phosphatidylcholine and phosphatidylethanolamine synthesis, respectively). ATII cell lamellar bodies were ultrastructurally abnormal after infection. Changes in ATII cell phospholipids were reflected in the composition of bronchoalveolar lavage fluid, which contained reduced amounts of phosphatidylcholine and phosphatidylglycerol but increased amounts of sphingomyelin, cholesterol, and protein. Influenza infection significantly alters ATII cell surfactant lipid metabolism, which may contribute to surfactant dysfunction and development of acute respiratory distress syndrome in influenza-infected mice.

The Application of Paraphenylenediamine Staining for Assessment of Phospholipidosis.

Drug-induced phospholipidosis is characterized by intracellular accumulation of phospholipids with lamellar bodies in cells exposed to xenobiotics. Demonstration of the lamellar bodies by transmission electron microscopy (TEM) is the hallmark for a definitive diagnosis of phospholipidosis. However, the preparation of tissue samples for TEM and their ultrastructural evaluation are technically challenging and time consuming. Paraphenylenediamine (PPD) is essentially a fat stain, and the staining mechanism is based upon the osmication of unsaturated lipids. Thus, the application of PPD staining to osmicated tissue samples is considered an optimal way to identify lipids. We evaluated the potential of PPD staining to localize phospholipid accumulations on osmium-fixed semi-thin tissue sections of the lung, kidney, and liver, which were affected with phospholipidosis, under a light microscope. PPD staining revealed the presence of PPD positive dark fine granular material in the cytoplasm for all affected tissues examined, which correlated ultrastructurally with lamellar bodies as well as a light microscopic finding of cytoplasmic vacuolation. The great advantage of PPD is that it can be incorporated into the protocol for standard TEM tissue preparation and significantly improve the efficiency of TEM work. In conclusion, PPD provides a simple, sensitive, and reliable method for visualizing phospholipid accumulation on light microscopy and represents an easy tool to study drug-induced phospholipidosis.

Alveolar Type II Cells from Mice Expressing a Lung Disease‐Associated ABCA3 E292V Mutation Exhibit Alterations in Organellar Homeostasis and Autophagy

The lipid transporter, ATP binding cassette class A3, ABCA3, plays a critical role in the biogenesis of alveolar type II (AT2) cell lamellar bodies (LBs). A relatively large number of mutations in the ABCA3 gene have been identified in association with interstitial lung disease (ILD). The most common mutation is a valine for glutamate missense mutation at residue 292 (E292V) which results in a functional impairment of lipid transport in vitro. To characterize the cellular consequences induced by this mutation in vivo, a novel knockin (KI) mouse model carrying allelic ABCA3 E292V mutations was produced. Lung ultrastructure from E292V homozygous mice revealed smaller size but increased numbers of LBs accompanied by increased AT2 cell mitochondrial counts. Examination of 16 wk and 32 wk homozygous E292V mice showed age‐dependent increase in bronchoalveolar lavage cellularity as well as reduction in alveolar wall thickness coupled with enlarged alveolar air spaces. While isolated E292V AT2 cells demonstrated no alterations in the unfolded protein response, an age‐dependent increase in autophagy components LC3‐I/LC3‐II and P62 also developed in these mice. Furthermore, AT2 cell lysates from 32 wk mice showed accompanying increases in the levels of marker antigens for both mitochondria (Tom20) and lysosomes (Lamp1). We conclude that expression of the ABCA3 E292V mutation produces changes in AT2 organellar homeostasis which result in time‐dependent disruption of macroautophagy‐dependent cellular quality control pathways. We speculate that these changes contribute to the phenotype of a vulnerable epithelium susceptible to second hit injuries that promote the pathogenesis of ILD.

Effect of Cigarette Smoke Extract on Insulin Transport in Alveolar Epithelial Cell Line A549

The main purpose of this study was to evaluate the effect of cigarette smoke extract (CSE) on insulin transport in alveolar epithelial cells. We first examined the effect of CSE pretreatment on cell viability, mRNA expression, and lamellar body structures in A549 cells. Then the effect of CSE pretreatment on FITC-insulin transport was examined. When A549 cells were treated with 30 μg/ml of CSE for 48 h, the expression of some mRNAs abundantly expressed in type II alveolar epithelial cells such as surfactant protein B was significantly increased. Lamellar bodylike structures became more evident with CSE treatment. FITC-insulin uptake from the apical side and subsequent efflux to the basal side was enhanced by CSE treatment in A549 cells. The enhancing effect of CSE on FITC-insulin uptake was concentration-dependent and reversible. A concentration-dependent enhancing effect of CSE on FITC-insulin uptake was also observed in normal, primary cultured alveolar type II epithelial cells isolated from rats. Treatment of A549 cells by CSE may direct the cells to a more type II-like phenotype. In accordance with this observation, FITC-insulin uptake was enhanced by CSE treatment. These results may partly explain the higher insulin absorption from the lung in smokers than in nonsmokers.

Type II pneumocyte lamellar bodies contain components of the Niemann‐Pick type C (NPC) pathway

Type II cell lamellar bodies, the primary storage organelle for lung surfactant, contain about 10% cholesterol. Cellular cholesterol trafficking has been shown to be regulated by the NPC pathway. A form of alveolar proteinosis characterized by high cholesterol to phospholipid ratio has been reported in an NPC2 patient, which suggested the NPC pathway played a role in regulating surfactant cholesterol in type II cells. Our objective was to define the localization and function of components of the NPC pathway in alveolar type II cells. Immunocytochemical analysis for NPC1, NPC2, and lysosomal acid lipase (LAL) was performed in isolated primary rat type II cells. The ATP‐binding cassette transporter (ABCA3) was used to identify the limiting membrane of lamellar bodies. 3D projections of a single lamellar body acquired by confocal microscopy confirmed NPC1 expression along the entirety of the limiting membrane. NPC1 was localized in alternating adjacent domains with ABCA3. NPC2 and LAL were located within the lumen of the organelle. Upon pharmacological inhibition of the NPC pathway with U18666A compound, cholesterol was found to accumulate within lamellar bodies. These results suggest that the NPC pathway is important for regulating lamellar body cholesterol. [HL‐19737 and NRSA T32‐HL‐07748‐17]

Bacteremia Does Not Affect Cellular Uptake of Ultrafine Particles in the Lungs of Rats

To assess the effects of intra-abdominal bacteremia on lung cellular function in vivo, we used electron microscopy to quantify the uptake of 6 nm diameter, albumin-coated colloidal gold particles (overall diam. 20.8 nm) by cells in the lungs of rats made septic by the introduction of live bacteria (E.coli and B. fragilis) into their abdomens. Gold particles were instilled into the trachea 24 hr after bacteremia induction, and lungs were harvested and prepared for electron microscopy 24 hr later. Because bacteremia produces an increase in metabolism, we hypothesized that this might be associated with increased cellular uptake of these particles and also with increased permeability of the alveolar epithelial barrier to them, as bacteremia is also associated with lung injury. We quantified particle uptake by counting particle densities (particles/μm²) within type I and type II epithelial cells, capillary endothelial cells, erythrocytes and neutrophils in the lungs of five septic rats and five sham-sepsis controls. We also counted particle densities within organelles of these cells (nuclei, mitochondria, type II cell lamellar bodies) and within the alveolar interstitium. We found particles to be present within all of these compartments, although we found no differences in particle densities between bacteremic rats and sham-sepsis controls. Our results suggest that these 6 nm particles were able to freely cross cell and organelle membranes, and further suggest that this ability was not altered by bacteremia.

Aberrant lung structure, composition, and function in a murine model of Hermansky-Pudlak syndrome.

Hermansky-Pudlak syndrome (HPS) is a genetically heterogeneous inherited disease causing hypopigmentation and prolonged bleeding times. An additional serious clinical problem of HPS is the development of lung pathology, which may lead to severe lung disease and premature death. No cure for the disease exists, and previously, no animal model for the HPS lung abnormalities has been reported. A mouse model of HPS, which is homozygously recessive for both the Hps1 (pale ear) and Hps2 (pearl) genes, exhibits striking abnormalities of lung type II cells. Type II cells and lamellar bodies of this mutant are greatly enlarged, and the lamellar bodies are engorged with surfactant. Mutant lungs accumulate excessive autofluorescent pigment. The air spaces of mutant lungs contain age-related elevations of inflammatory cells and foamy macrophages. In vivo measurement of lung hysteresivity demonstrated aberrant lung function in mutant mice. All these features are similar to the lung pathology described in HPS patients. Morphometry of mutant lungs indicates a significant emphysema. These mutant mice provide a model to further investigate the lung pathology and therapy of HPS. We hypothesize that abnormal type II cell lamellar body structure/function may predict future lung pathology in HPS.

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

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.

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

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 betaHC9 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.

Geometrical exact solutions for confocal lamellar textures yielding confinement and faceting phenomena

We study the equilibrium textures of lamellar bodies in cells limited by two parallel plates interacting only with the orientations of the layers but not with their positions. For such ‘loose’ anchorings, confocal textures are expected, i.e. textures of equidistant and parallel curved layers. When all defects lie outside the smectic body, with the aid of a fortunate change of variables, we find an exact solution of the nonlinear equilibrium equation describing the textures. Instead of being governed by a differential equation, the equilibrium textures are governed by a finite equation that gives the common evolute f of the layers. The equilibrium texture can then be deduced from f by a geometrical construction. This approach is reminiscent of that introduced by Wulff (1901) in order to determine the equilibrium shape of solid crystals. Our main result is the existence, for certain boundary conditions and according to the shape of the surface energy function, of confined distortions and faceted textures. Faceted textures characterize the presence of an angular point in the surface energy, a feature currently debated.

Glutathione metabolism in newborns: evidence for glutathione deficiency in plasma, bronchoalveolar lavage fluid, and lymphocytes in prematures.

Respiratory distress in premature newborns is associated with deficiency of surfactant in the bronchoalveolar lining fluid; this may be influenced by a local deficiency of antioxidants. Severe L-buthionine-S,R-sulfoximine-induced depletion of glutathione (GSH, a major antioxidant) in rodents is associated with lung type 2 cell lamellar body damage and decreased concentrations in lung and bronchoalveolar lavage fluid (BALF) of phosphatidyl choline (a major component of surfactant). At birth, prematurely born newborns (30-34 weeks) had lower peripheral venous plasma GSH concentrations than term (> 36 weeks) babies; these levels decreased further with increasing prematurity (< 27 weeks, with respiratory distress). On day 2, the peripheral venous plasma GSH concentrations reached a nadir, and the lowest levels were found in the most premature newborns. Lymphocyte GSH concentrations were lowest on day 2 and day 7, and in prematures (< 27 weeks, with respiratory distress) remained below adult lymphocyte GSH levels for at least 4 weeks. At birth, prematures (< 27 weeks, with respiratory distress) had a central plasma arterio-venous (A-V) GSH gradient across the lung (an estimate of lung uptake of GSH) of 0.72 +/- 0.15 (mean +/- SD) mumol/L; on day 2, the A-V gradient did not change significantly (0.49 +/- 0.09 mumol/L). At birth, these prematures had markedly decreased BALF GSH concentrations (compared with adult levels), and they were not significantly changed during the first 4 weeks of life. These results suggest that GSH deficiency is present in prematures and that it increases with the degree of prematurity. At birth, GSH deficiency will compromise the lungs' defense against oxidative stress injury. Oxidative stress is likely to increase if hyperoxic treatment is given for respiratory distress in these infants.

Synthesis of type II cell lamellar body lysozyme-15 kD protein (lbl-15) by perfused rat lung.

Isolated alveolar type II pneumocytes of the rat have been shown to secrete a 14 to 15 kD protein that has some sequence homology and immunoreactivity with lysozyme. Using immunochemical analyses of rat lung subcellular fractions and 35S metabolic labeling of isolated perfused lung preparations, we studied the subcellular distribution and synthetic pathway for this protein. SDS-PAGE and Western blotting of lamellar bodies (LB) using a polyclonal anti-human lysozyme (anti-HLZ) demonstrated a single band at 15 kD that was significantly enriched over rat lung homogenates, isolated lysosomes, and type II cell lysates. This 15 kD protein isolated from LB by immunoprecipitation with anti-HLZ also demonstrated functional lysozyme activity and was termed lamellar body lysozyme (lbl-15). Analysis of LB and surfactant (SF) isolated from 12 separate perfused lung preparations labeled for 6 h with 35S-labeled amino acids demonstrated that lbl-15 represented a significant portion of the radiolabeled LB proteins (5.9% of total LB radioactivity). Lamellar bodies and an extracellular fraction (surfactant) obtained from rat lungs perfused with 35S-methionine-cysteine for varying times both showed time-dependent appearance of lbl-15. At all time points, the specific activity of lbl-15 was greater in LB than in SF. The kinetics for appearance of lbl-15 in LB and SF was similar to that for surfactant protein A. These results indicate that in the rat lung, type II cells synthesize a 15 kD protein (lbl-15) that is secreted into the alveolar space via an organellar pathway involving LB.(ABSTRACT TRUNCATED AT 250 WORDS)

Ascorbic acid prevents oxidative stress in glutathione-deficient mice: effects on lung type 2 cell lamellar bodies, lung surfactant, and skeletal muscle.

Glutathione deficiency in adult mice leads to lung type 2 cell lamellar body and mitochondrial damage; as reported here, these effects are associated with marked decrease of the levels of phosphatidylcholine (the main component of lung surfactant) in the lung and the bronchoalveolar lining fluid. Severe mitochondrial damage was also found in skeletal muscle. Treatment with ascorbate (1-2 mmol per kg of body weight per day), which led to greatly increased (approximately 2-fold) levels of lung and muscle mitochondrial glutathione, prevented damage to lamellar bodies and mitochondria as well as the decline of phosphatidylcholine levels in lung and alveolar lining fluid. The findings indicate that glutathione deficiency leads to depletion of lung surfactant and that this can be prevented with ascorbate. Administration of ascorbate spares glutathione and prevents cellular damage. Lamellar body degeneration in glutathione deficiency appears to be associated with oxidative damage to the perilamellar membrane, which contains the enzymes required for phosphatidylcholine synthesis. It is notable that although severe glutathione deficiency is lethal to newborn rats, which apparently do not synthesize ascorbate, adult mice are better able to survive such a deficiency because they can synthesize ascorbate. The present studies, which suggest that high doses of ascorbate may be of therapeutic value, emphasize that ascorbate and glutathione have actions in common and that they function together in a physiologically significant antioxidant system.

Ontogeny of rat lung type II cells correlated with surfactant lipid and surfactant apoprotein expression

During the last stages of intrauterine growth, remarkable changes occur in the alveolar epithelium that include cellular differentiation and increased production of surfactant lipid and apoprotein. We made morphometric measurements of type II cell characteristics from rats aged gestational day 20 to 14 days postnatal. We also measured the amounts of disaturated phosphatidylcholine (DSPC) and surfactant apoprotein (SP-A) in lung tissue, bronchoalveolar lavage, and a lamellar body-rich fraction, and we estimated the lung content of mRNAs for SP-A, SP-B, and SP-C. Lavage and lamellar body surfactant lipid and apoprotein content per lung showed a pattern of a sharp rise in the early postnatal period, then a substantial decline, and a second increase by day 14. When normalized for dry lung weight, the highest DSPC values were found on postnatal day 1 in all compartments. The fraction of whole lung DSPC found in lamellar body or lavage was greatest in the 48-h period surrounding birth. Lamellar body SP-A was greater than lavage SP-A on gestational day 22, but a day later the lavage SP-A was 16 times greater than the lamellar body SP-A. The lung tissue content of all three apoprotein mRNAs increased sharply before birth, fell during the 1st postnatal wk, and then rose again to adult levels. Type II cell number and lamellar body number per milligram of dry lung tissue was highest on post-natal day 1 and fell by one-half during the 1st postnatal wk. The amount of DSPC per unit of lamellar body volume rose to its greatest value on postnatal day 1 and then decreased more than threefold. These findings indicate a pattern of expansion of surfactant cellular and biochemical pools at the time of birth in the rat.

Lysozyme is an ozone-sensitive component of alveolar type II cell lamellar bodies

Exposure of rats to 3 ppm ozone for up to 8 h results in significant changes in lamellar bodies, the surfactant storing organelles of type II cells. We have previously shown that a 14 kDa lamellar body protein is decreased as early as 4 h after the onset of ozone exposure. We have isolated this ozone-sensitive protein from rat lung lamellar bodies and identified it as lysozyme by immunochemical methods, as well as by its amino acid composition, N-terminal amino acid sequence and bacteriolytic activity. Reduced lysozyme activity in isolated lamellar bodies is detected as early as 4 h after the start of ozone exposure. Following an 8 h ozone exposure, the activity does not return to control levels for at least 48 h. Lamellar body lysozyme is expected to be secreted with surfactant phospholipids, thereby contributing to the antimicrobial defense of the alveolar lining layer. The acute lysozyme deficiency seen in ozone-induced oxidant injury may reduce the resistance of the lung to infection.

Evidence that fatty acid chain length is a type II cell lipid-sorting signal

This study was undertaken to determine those structural features of phospholipid molecules which influence their enrichment in type II cell lamellar body material. Cultured fetal rabbit lung tissue was labeled with [1-14C]acetate, type II cells were isolated, and extracellular lamellar body and microsomal fractions were prepared. Radiolabeled molecular species of phosphatidylcholine (PC) and phosphatidylethanolamine were analyzed by high-performance liquid chromatography (HPLC), followed by silver nitrate thin-layer chromatography of HPLC peak fractions that overlapped. Compared with microsomes, lamellar body PC was selectively enriched with molecular species containing 14- and 16-carbon fatty acids and depleted of species containing 18-carbon fatty acids. Palmitoleic acid and an ether linkage positively influenced the enrichment of PC molecular species in the lamellar body material, but these structural features were secondary to the predominant influence of fatty acid chain length. In vivo, lung tissue normally contains low levels of palmitoleic acid; hence most unsaturated fatty acids are 18-carbons or longer. A cellular lipid-sorting mechanism that selects PCs by recognition of 14- and 16-carbon fatty acid chains (and not by recognition of fatty acid saturation) should serve to enrich the resulting pulmonary surfactant with disaturated molecular species of PC.

Glutathione metabolism in the lung: inhibition of its synthesis leads to lamellar body and mitochondrial defects.

Mice treated with buthionine sulfoximine, an inhibitor of glutathione synthesis, showed striking alterations of morphology of lung type 2 cell lamellar bodies (swelling and disintegration) and mitochondria (degeneration) and of lung capillary endothelial cells (mitochondrial swelling). These effects probably may be ascribed to glutathione deficiency; administration of glutathione monoester protects against them. Measurements of arteriovenous plasma glutathione levels across the lung indicate that the net uptake of glutathione by this organ is substantial. Thus, glutathione exported from the liver to the blood plasma is utilized by the lung which, like the liver, kidney, and lymphocytes (and unlike skeletal muscle), exhibits a high overall rate of glutathione turnover. Intraperitoneal injection of glutathione into buthionine sulfoximine-treated mice leads to very high levels of plasma glutathione without significant increase in the glutathione levels of liver, lung, and lymphocytes; on the other hand, administration of glutathione monoester leads to markedly increased tissue and mitochondrial levels of glutathione. Administration of glutathione monoester (in contrast to glutathione) to control mice also increases mitochondrial glutathione levels. The findings indicate that glutathione is required for mitochondrial integrity and that it probably also functions in the processing and storage of surfactant in lamellar bodies. The morphological changes observed after treatment with buthionine sulfoximine and their prevention by glutathione monoester as well as findings on glutathione metabolism indicate that this tripeptide plays an important role in the lung. The previously observed failure of buthionine sulfoximine-treated mice to gain weight is mainly due to glutathione deficiency in the intestinal mucosa.

Alteration of pulmonary structure by Pseudomonas aeruginosa exoenzyme S.

Intratracheal administration of purified Pseudomonas aeruginosa exoenzyme S elicited extensive, grossly observable damage in the rat lung within 2 h. Light and electronmicroscopy revealed injury and necrosis of bronchial epithelium, type I pneumocytes and capillary endothelial cells after 1 h; associated haemorrhage, fibrinous exudation and released type II cell lamellar bodies in alveolar lumina after 1-12 h; progressively increasing accumulations of polymorphonuclear leucocytes in the bronchi and alveoli and in alveolar septae (interstitial pneumonia) after 1-12 h; collapse of alveolar septal connective tissue and damage to pulmonary arterioles and venules. Treatment of monolayer cultures of bronchial fibroblasts with purified exoenzyme S elicited vacuolation of the cells with apparent membrane damage as revealed by light and electronmicroscopy. In-vivo production and activity of P. aeruginosa exoenzyme S may be an important pathogenicity determinant in the necrotising lung injury characteristic of P. aeruginosa pneumonia.