Macrophages stimulate DNA synthesis in rat alveolar type II cells

Proliferation of alveolar type II cells after lung injury is crucial for repair of the epithelium. Because an influx of macrophages occurs as part of the inflammatory response associated with acute lung injury and macrophages produce mitogenic factors for a variety of cell types, experiments were conducted to determine if macrophages stimulated DNA synthesis in type II cells. Dialyzed medium conditioned by macrophages consistently stimulated type II cell DNA synthesis, whereas medium conditioned by a variety of other cell types did not. A SV40-transformed macrophage cell line, produced in our laboratory, also secreted substance(s) that enhanced 3H-thymidine incorporation into type II cells. In addition, coculturing rat alveolar macrophages with type II cells stimulated DNA synthesis in the epithelial cells. As determined by autoradiography, the addition of macrophages to type II cells cultured on plastic or on an endothelial cell extracellular matrix increased the labeling index of the epithelial cells from 1 to 15% and from 19 to 51%, respectively. The culture conditions that promoted the greatest increase in DNA synthesis, as well as an increase in cell number, occurred with type II cells plated on an extracellular matrix in medium containing macrophage-conditioned medium, cholera toxin, insulin, and epidermal growth factor. The results suggest that substances secreted by macrophages play a role in regulating alveolar type II cell proliferation in vivo.     

Stimulation of DNA synthesis in cultured rat alveolar type II cells

Restoration of the alveolar epithelium after injury is thought to be dependent on the proliferation of alveolar type II cells. To understand the factors that may be involved in promoting type II cell proliferation in vivo, we determined the effect of potential mitogens and culture substrata on DNA synthesis in rat alveolar type II cells in primary culture. Type II cells cultured in basal medium containing 10% fetal bovine serum (FBS) exhibited essentially no DNA synthesis. Factors that stimulated 3H-thymidine incorporation included cholera toxin, epidermal growth factor, and rat serum. The greatest degree of stimulation was achieved by plating type II cells on an extracellular matrix prepared from bovine corneal endothelial cells and then by culturing the pneumocytes in medium containing rat serum, cholera toxin, insulin, and epidermal growth factor. Under conditions of stimulation of 3H-thymidine incorporation there was an increased DNA content per culture dish but no increase in cell number. The ability of various culture conditions to promote DNA synthesis in type II cells was verified by autoradiography. Type II cells were identified by the presence of cytoplasmic inclusions, which were visualized by tannic acid staining before autoradiography. These results demonstrate the importance of soluble factors and culture substratum in stimulating DNA synthesis in rat alveolar type II cells in primary culture.     

Effect of ambroxol on surfactant secretion and synthesis in isolated type II alveolar cells

The effect of ambroxol is attributed in part to an effect on surfactant synthesis and secretion. Evidence supporting this hypothesis is largely indirect; a direct effect of ambroxol on surfactant synthesis and secretion remains to be demonstrated. In this study a direct effect of ambroxol was evaluated using isolated alveolar type II cells. Secretion of labelled phosphatidylcholine was measured following the addition of increasing concentrations (10(-8) M to 10(-4) M) of ambroxol to the culture medium for increasing time intervals. There was no significant increase in surfactant secretion with increased ambroxol concentration or prolonged exposure time. Uptake of 3H-choline and synthesis into 3H-phosphatidylcholine was analyzed as an indicator of surfactant synthesis. Again, increasing concentrations of ambroxol (10(-7) M to 10(-5) M) were followed for 1, 2, 4, 8 and 24 h. There was no significant effect on synthesis at any time point. Concentrations higher than those mentioned here resulted in LDH release from cultured cells. Ambroxol which also has anti-oxidative and anti-inflammatory effects does not exhibit direct stimulatory effects on surfactant synthesis and secretion in isolated (rat) alveolar type II cells as has been demonstrated e.g. for beta 2-adrenergic stimulation. A specific pharmacologic way to stimulate the surfactant system remains to be developed.     

Activated polymorphonuclear leukocytes inhibit phosphatidylcholine synthesis in cultured type II alveolar cells.

Activated human neutrophils (PMNs) were demonstrated to inhibit total de novo phosphatidylcholine (PC) synthesis in monolayered rat alveolar type II cells (T2C). Non-activated PMNs had no effect on PC synthesis in this system. The magnitude of inhibition T2C PC synthesis by phorbol myristate acetate-activated PMNs in six experiments averaged 59.0 +/- 13%. Exogenous chelated iron (ferric pyrophosphate) did not appear to augment the PMN-mediated inhibition of T2C PC production in this model. Alpha-1-antiprotease usually provided no protection relative to the PMN insult towards the T2C. However, superoxide dismutase and catalase alone or in combination generally provided a significant, protective effect. Although activated PMNs consistently decreased T2C PC synthesis, this effect did not appear to involve generalized T2C cytotoxicity, as assessed by lack of release of cytosolic lactate dehydrogenase. These results indicate that PMNs can inhibit T2C PC synthesis in vitro, probably via oxyradical injury. This type of pulmonary host autoinjury may be operative in a variety of acute lung injury syndromes involving pulmonary sequestration of activated PMNs.     

Pulmonary surfactant transport in alveolar type II cells

Abstract:   Pulmonary surfactant (PS) is a mixture of several lipids (mainly phosphatidylcholine; PC) and four apoproteins (A, B, C and D). The classical hypothesis of PS transport suggests that PS is synthesized in the endoplasmic reticulum and transported to the lamellar body (LB) via the Golgi apparatus. However, recent studies have raised questions regarding this single route. This study examined, independently, the intracellular trafficking route of three different components of PS, that is, PC, SP-A and SP-B. Alveolar type II cells were isolated from Sprague–Dawley rats or Japanese white rabbits. The cells were cultured with either [³H]choline or [³⁵S]methionine/cysteine with or without brefeldin A, which disassembles the Golgi apparatus. LB was purified from disintegrated cells with sucrose density gradient centrifugation. [³H]PC was extracted from radiolabeled media, cells, and the LB fraction with Bligh–Dyer's method. [³⁵S]SP-A or [³⁵S]SP-B was immunoprecipitated from each sample with a specific antibody. [³H]PC was transported and stored to the LB via a Golgi-independent pathway. [³⁵S]SP-A was transported to the Golgi apparatus, underwent glycosylation, and was then constitutively secreted. The secreted [³⁵S]SP-A was re-uptaken into the LB. [³⁵S]SP-B was transported and stored to the LB via the Golgi-dependent pathway. These results indicate that, rather than a single route, surfactant components take different pathways to reside in the LB. These different pathways may reflect the different nature and role of each surfactant component such as surface tension-lowering activity and innate host defense.     

Endocytosis in alveolar type II cells: effect of charge and size of tracers.

The type II cell of the alveolar epithelium of adult rats has been studied by electron microscopy to determine its ability to endocytose electron-dense tracers of differing molecular charge and size. The tracers studied were native (anionic) ferritin, cationic ferritin, 70-kilodalton dextran, and colloidal carbon. Alveolar macrophages ingested all tracers in large amounts, while type II cells took up significant amounts of cationic ferritin only. This tracer was observed, in sequence, within small pinocytic vesicles, large electron-lucent multivesicular bodies, small electron-dense multivesicular bodies, and, by 30 min after instillation, within the nonlamellar matrix of lamellar bodies. By 2 hr all lamellar bodies in any labeled cell contained cationic ferritin. Cationic ferritin also appeared to be transported from alveoli to interstitium by vesicles of type II, but not type I, cells. Native ferritin and dextran were observed in the same organelles as cationic ferritin but in much smaller amounts; colloidal carbon was not taken up by type II cells. These tracers were not observed in the alveolar interstitium. Type II cells therefore appear to internalize preferentially the tracer that binds to the cell membrane. Once within the cell, the tracer may enter a pathway that terminates in a lamellar body or, in the case of cationic ferritin, may be ferried across the cell.     

Effects of ozone on phospholipid synthesis by alveolar type II cells isolated from adult rat lung

Isolated alveolar type II cells were exposed to ozone by gas diffusion through the thin Teflon bottom of culture dishes. After exposure, type II cells were further incubated in the presence of labeled substrates to assess the capacity to synthesize surfactant lipids. The incorporation of [Me-14C]choline into both total and disaturated phosphatidylcholines in inhibited to 50% of the control values under conditions that result in a diffusion of 0.4 microgram O3/18 cm2-dish per 2.5 h. The incorporation rates of [1-14C]palmitate, [1-14C]acetate, D[U-14C]glucose, and [1,3-3H]glycerol into phosphatidylcholines are also lower after ozone exposure. Moreover, the synthesis of phosphatidylglycerols and phosphatidylethanolamines from these substrates is also inhibited by exposure of type II cells to ozone. These incorporation studies indicate that the effect of ozone is early in the biosynthetic pathway, probably at the step catalyzed by the enzyme glycerolphosphate acyltransferase. Determination of the activity of this enzyme after the ozone exposure shows that it is decreased, whereas the activity of lysophosphatidylcholine acyltransferase is increased. The activity of choline phosphotransferase also appears to be decreased after exposure of type II cells to ozone, although this enzyme was less susceptible than glycerolphosphate acyltransferase. Studies with the sulfhydryl reagent 5,5'-dithiobis (2-nitrobenzoic acid) indicate a positive correlation between the effect of this compound on enzyme activities in sonicated type II cells and the sensitivity of these enzymes in intact cells to ozone. This suggests that the effect of ozone on the synthesis of surfactant lipids is at least partially exerted via oxidation of the sulfhydryl groups of glycerolphosphate acyltransferase.     

Bronchoalveolar lavage fluid from normal rats stimulates DNA synthesis in rat alveolar type II cells

Proliferation of alveolar type II cells after lung injury is important for the restoration of the alveolar epithelium. Bronchoalveolar lavage fluid (BALF) may represent an important source of growth factors for alveolar type II cells. To test this possibility, BALF fluid was collected from normal rats, concentrated 10-fold by Amicon filtration, and tested for its ability to stimulate DNA synthesis in rat alveolar type II cells in primary culture. BALF induced a dose-dependent increase in type II cell DNA synthesis resulting in a 6-fold increase in [3H]thymidine incorporation. Similar doses also stimulated [3H]thymidine incorporation into rat lung fibroblasts by 6- to 8-fold. Removal of pulmonary surface active material by centrifugation did not significantly reduce the stimulatory activity of BALF for type II cells. The stimulation of type II cell DNA synthesis by BALF was reduced by 100% after heating at 100 degrees C for 10 min, and by approximately 80% after reduction with dithiothreitol, and after trypsin treatment. Dialysis of BALF against 1 N acetic acid resulted in a 27% reduction in stimulatory activity. The effect of BALF in promoting type II cell DNA synthesis was more pronounced when tested in the presence of serum, although serum itself has very little effect on type II cell DNA synthesis. When BALF was tested in combination with other substances that stimulate type II cell DNA synthesis (cholera toxin, insulin, epidermal growth factor, and acidic fibroblast growth factor), additive effects or greater were observed. When BALF was chromatographed over Sephadex G150, the activity eluted with an apparent molecular weight of 100 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and comparison of the role of beta-agonists on in vivo and in vitro surfactant-related phospholipid synthesis and secretion by fetal rabbit lung and isolated type II alveolar cells

The role of beta-adrenergic stimulation in surfactant synthesis and secretion was investigated in the fetal lung. Fetuses were treated with isoxsuprine or saline on gestational day 24 by ip injection. Three days later the fetal lungs were lavaged and intracellular surfactant was isolated on a sucrose gradient. Concurrently undifferentiated type II alveolar cells were isolated from 24-day fetal rabbit lung and grown in vitro. In the in vivo portion of the study, examination of surfactant pool sizes revealed that only saline treatment produced a significant elevation in tissue-stored or secreted surfactant compared to untreated controls. Isoxsuprine appeared to inhibit the saline-induced increase. In the case of the intracellular surfactant, the phosphatidylcholine content per gram of lung was significantly increased after saline treatment. In vitro response of isolated type II alveolar cells to isoxsuprine was dependent on prior incubation of the cells for 24 h with conditioned medium. Isoxsuprine stimulated a dose-dependent decrease in the intracellular stores of radioactively labeled DSPC after 24 h of exposure to the drug. A corresponding increase in labeled DSPC in the culture medium was observed. Forth-eight hours after exposure to the drug, those cells that had secreted the highest level of DSPC displayed the highest levels of renewed synthesis of DSPC. This study indicates that the immature fetal lung can be induced to synthesize surfactant-related phospholipid by the stress of laparotomy and/or drug administration. Short-term exposure to beta-agonists is insufficient to stimulate secretion of surfactant stores. In contrast, isolated type II alveolar cells exposed to isoxsuprine respond by secreting DSPC.     

Characterization of rat alveolar type II cells in vitro by immunological, biochemical, and morphological criteria

Using an anti-rat surfactant apoprotein antiserum which specifically reacts with cytoplasmic structures in alveolar type II cells on histopathology sections of rat lung, we have examined the immunoreactivity of pulmonary type II cells in vitro. Single cell suspensions of lung tissue were prepared from male Fischer 344 rats by intratracheal elastase digestion according to standard published methods. Cytocentrifuged preparations of the resulting cell suspensions revealed that approximately 40% of the cells stained positive for surfactant apoprotein using an immunoperoxidase staining technique. Without further cell fractionation steps, the cell suspensions were plated at colonial densities in growth medium. The cells that attached after 24 hours of incubation and at daily intervals were analyzed for surfactant apoprotein immunoreactivity as well as for proliferation, morphology, and phospholipid biosynthesis. The percentage of immunopositive cells increased with time from 75% at day 1 to 94% at 4 days after plating. This increase was paralleled by a linear increase in the number of immunopositive cells, which expanded into cell colonies. During the initial 5 days in vitro, the immunopositive cells retained their epithelial morphology and contained cytoplasmic osmiophilic bodies. Phospholipid biosynthesis by the isolated lung cells was analyzed and the data revealed that the rate of incorporation of 14C-choline into phosphatidylcholine increased with time in culture. These studies indicated that the anti-rat surfactant apoprotein antisera can be used to identify and quantitate functional alveolar type II cells in vitro. Thus the specific antisera may facilitate studies of type II cells undergoing various environmental alterations both in vivo and in vitro.     

Enzymes of phospholipid biosynthesis in rat alveolar type II cells

Some enzymes responsible for de novo synthesis of phosphatidylcholine and the reacylation of phosphatidylcholine to disaturated phosphatidylcholine have been examined in isolated rat type II alveolar cells. Compared to the enzyme levels found in whole lung homogenate, levels of choline kinase, choline phosphotransferase and lysolecithin acyltransferase are enriched 10 fold in isolated type II alveolar cells. No enrichment of cholinephosphate cytidyltransferase was observed suggesting that this enzyme may be rate limiting in phosphatidylcholine biosynthesis.     

The effect of dietary intervention on serum lipid levels in type 2 diabetes mellitus

Dietary therapy is the cornerstone of lipid management in patients with type 2 diabetes mellitus. The key strategies are the reduction of intake of saturated fat, trans unsaturated fat and cholesterol, and the reduction of energy intake to promote weight loss. This approach will produce significant improvements in the serum levels of low-density lipoprotein (LDL) cholesterol, triglycerides, and high-density lipoprotein (HDL) cholesterol. According to both the American Diabetes Association and the National Cholesterol Education Program (NCEP), the primary target of therapy is the serum LDL cholesterol level, with the secondary targets being non-HDL cholesterol, triglycerides, and HDL cholesterol. The recently updated guidelines of the NCEP place new emphasis on increasing soluble fiber intake to 10 to 25 g/d and adding foods fortified with plant stanols/sterols (2 g/d) as options to enhance the LDL cholesterol-lowering effect of diet.     

The effect of glycemic control on salivary lipid peroxidation in type II diabetic patients

Background and objectiveHyperglycemia and some disturbance in antioxidant system lead to free radicals production and oxidative stress. Assessment of some products of oxidative stress could be effective in evaluation of diabetic control. This study aimed at evaluation of glycemic control on salivary lipid peroxidation in diabetic patients.MethodsThis case control study has been done on 44 diabetic (type II) and 44 healthy subjects. Un-stimulated saliva was collected and correlation between malondialdehid (MDA) as an end -product of lipid peroxidation and HbA1c was assessed.ResultsMDA and HbA1c of diabetic patients were significantly higher than control group. There was a indirect correlation between MDA and glycemic control level.ConclusionEvaluation of salivary MDA levels could be useful in prediction of glycemic control.     

Identification and characterization of a lysophosphatidylcholine acyltransferase in alveolar type II cells

Pulmonary surfactant is a complex of lipids and proteins produced and secreted by alveolar type II cells that provides the low surface tension at the air-liquid interface. The phospholipid most responsible for providing the low surface tension in the lung is dipalmitoylphosphatidylcholine. Dipalmitoylphosphatidylcholine is synthesized in large part by phosphatidylcholine (PC) remodeling, and a lysophosphatidylcholine (lysoPC) acyltransferase is thought to play a critical role in its synthesis. However, this acyltransferase has not yet been identified. We have cloned full-length rat and mouse cDNAs coding for a lysoPC acyltransferase (LPCAT). LPCAT encodes a 535-aa protein of approximately 59 kDa that contains a transmembrane domain and a putative acyltransferase domain. When transfected into COS-7 cells and HEK293 cells, LPCAT significantly increased lysoPC acyltransferase activity. LPCAT preferred lysoPC as a substrate over lysoPA, lysoPI, lysoPS, lysoPE, or lysoPG and prefers palmitoyl-CoA to oleoyl-CoA as the acyl donor. This LPCAT was preferentially expressed in the lung, specifically within alveolar type II cells. Expression in the fetal lung and in rat type II cells correlated with the expression of the surfactant proteins. LPCAT expression in fetal lung explants was sensitive to dexamethasone and FGFs. KGF was a potent stimulator of LPCAT expression in cultured adult type II cells. We hypothesize that LPCAT plays a critical role in regulating surfactant phospholipid biosynthesis and suggest that understanding the regulation of LPCAT will offer important insight into surfactant phospholipid biosynthesis.     

Detection of antibodies in serum of patients with idiopathic pulmonary fibrosis against isolated rat alveolar type II cells

The serum of 2 patients suffering from idiopathic pulmonary fibrosis (IPF) repeatedly demonstrated a positive antibody reaction against rat alveolar type II cells isolated by the use of a combination of elastase and trypsin for disaggregation and a Percoll density gradient for purification. Neither of the other 8 IPF patients examined nor any of the controls showed a positive reaction. Because the 2 patients with serum antibodies against alveolar type II cells did not show any antinuclear antibodies, it is concluded that the antibodies against the surface of rat alveolar type II cells found in the serum of IPF patients may be autoantibodies involved in the pathogenesis of IPF.     

In vitro effects of hyperoxia on alveolar type II pneumocytes: inhibition of glutathione synthesis increases hyperoxic cell injury.

An in vitro model of alveolar epithelial oxidant injury was developed based on exposure to hyperoxia of cultured guinea pig type II pneumocytes using a biphasic cell culture system in aerobiosis. The present study investigates the roles of intracellular antioxidant enzymes and of glutathione in providing protection against hyperoxia. A 2-day type II cell culture in normoxia was associated with a significant decrease in protein, catalase, and Cu-Zn SOD cell content, whereas ATP cell content, Mn-SOD, and glutathione peroxidase (GPx) activities did not change and glutathione cell content significantly increased. Exposure of type II cells to hyperoxia did not induce significant changes in cell content in protein, SOD, catalase, GPx, or glutathione cell content when compared to control cells (exposed to normoxia). With ATP cell content expressed as a cell injury index (CII), type II cell injury was found to increase with increasing O2 concentrations. Indeed, a 2-day 50% O2 and 95% O2 exposure resulted in a CII of -7.5 +/- 6.2% and 17.9 +/- 5.9%, respectively, LDH release by type II cells was not significantly increased after hypoxic exposure. Cell injury effects of hyperoxia did not correlate with the endogenous antioxidant enzyme activities (SOD, Mn-SOD, catalase). In marked contrast, there was a significant correlation between the CII and total glutathione content of type II cells (p < .01). This correlation was largely due to the close relationship between CII and reduced glutathione. Hyperoxic induced cell injury (as demonstrated by CII > 0) was clearly associated with significantly lower intracellular glutathione level when compared to experiments without hyperoxia induced cell injury (CII < 0). In addition, in the presence of buthionine sulfoximine (BSO), the ability of type II cells to synthetize new glutathione was severely impaired, whereas ATP cell content and cell antioxidant enzyme activities did not change. As a consequence, the reduction of intracellular glutathione significantly increased the susceptibility of cells to hyperoxia injury (p < .05). The results strongly support the hypothesis that the regulation of glutathione levels is an important mechanism in protecting hyperoxia-induced type II cell injury.     

The effect of O-acetylsalicylic acid on lipid synthesis by guinea pig gastric mucosa in vitro

The aim of this work was to investigate the involvement of lipids as possible components of the gastric mucosal barrier by studying the synthesis and secretion of lipids by the epithelial cell lining of gastric mucosa and the effect of salicylate on these processes. O-Acetylsalicylic acid reversibly reduced in vitro incorporation of [U-14C]acetate and of DL-[2-14C]mevalonic acid into lipids by isolated epithelial cells and by intact mucosa of guinea pig stomach, indicating reversible inhibition of lipid synthesis by the tissue in the presence of the drug. Inhibition of incorporation of both precursors into total lipids, into their fatty acid components, and into cholesterol is demonstrated.