Identification of specific proteins synthesized by type II pneumocytes in primary culture

Proteins from primary cultures of type II granular pneumocytes have been examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to identify type II cell-specific proteins. The distribution of Coomassie Blue-stained bands in preparations of cellular proteins, culture medium, lavage and lamellar bodies have been compared. The most prominent stained band in the serum-free medium from type II cell cultures (HS1; Mr 39900) corresponds to a major protein in acellular sedimentable (20000 g for 30 min) crude surfactant obtained from rat lungs by saline (0.9% NaCl) lavage. A second protein (HS2; Mr 12000) is also found both in type II cell-conditioned medium and in lavage. Neither rat serum nor donor calf serum (used in the isolation of the type II cells) contains a protein co-migrating with HS1 or HS2 proteins. HS1 is also found in Coomassie Blue-stained gels of cellular proteins and of lamellar bodies isolated from whole lungs. Cultures of type II cells incorporate [14C]phenylalanine into HS1 and HS2 as shown by autoradiography of sodium dodecyl sulphate/polyacrylamide gels of culture medium. Rat lungs perfused in situ incorporate [35S]methionine into HS1 in the lamellar body fraction. A third protein (HS3; Mr 47000) is observed only in autoradiographs of cell culture medium; no corresponding Coomassie Blue-stained band can be identified in medium, in cells or in lung lavage. No protein bands corresponding to HS1, HS2 or HS3 are found in conditioned media from pulmonary alveolar macrophages, rat fibroblasts or bovine aorta endothelial cells. Two-dimensional gel electrophoresis of HS1 shows a single polypeptide with an isoelectric point of 6.3; HS3 appears as a chain of spots with a range of isoelectric points from 6.3 to 6.6. HS2 has not been identified on two-dimensional gels. The amino acid composition of HS1 does not differ significantly from that of surfactant apoproteins studied previously; however, HS1 is not detected by glycoprotein stains, nor does it appear to be a subunit of a thiol-linked multimer.     

Stimulation of pulmonary surfactant secretion by activating neutrophils in rat type II pneumocytes culture

The influence of activating neutrophils on the secretion of phosphatidylcholine (PC), the predominant component of pulmonary surfactant, was examined using primary culture of rat type II pneumocytes. Simultaneous addition of neutrophils and opsonized zymosan, but not neutrophils or opsonized zymosan alone, to type II pneumocytes caused a significant increase in PC secretion without affecting the release of lactate dehydrogenase, a marker of cytotoxicity. The increase in PC secretion was dependent on the number of activating neutrophils. In addition, pretreatment of culture with the combination of superoxide dismutase and catalase inhibited the increase in PC secretion. These findings indicate that activating neutrophils stimulate the secretion of pulmonary surfactant and that the stimulation is mediated by oxygen radicals.     

An albumin-associated PLA2-like activity inactivates surfactant phosphatidylcholine secreted from fetal type II pneumocytes

Type II pneumocytes are responsible for the synthesis and secretion of pulmonary surfactant, which reduces surface tension in lung alveoli, thus decreasing their tendency to collapse during expiration. For this effect to be sustained, the integrity of the surface-active components of surfactant must be maintained. This study has shown that, when cultured type II pneumocytes are exposed to lipoprotein-free serum (LFS), the level of lyso-phosphatidylcholine (lyso-PC) in the secreted surfactant phospholipids is markedly elevated with a concomitant decline in the level of phosphatidylcholine (PC). This effect is the result of hydrolysis of surfactant PC by a phospholipase A(2) (PLA(2))-like activity present within serum. Anion-exchange chromatography, gel filtration chromatography and preparative electrophoresis of human LFS have shown that this PLA(2)-like activity coelutes with albumin and is biochemically distinct from the secretory form of PLA(2). Furthermore, specific inhibitors of PLA(2) such as p-bromophenacyl bromide, aristolochic acid, and palmitoyl trifluoromethyl ketone do not inhibit this activity of serum. Commercially purified human serum albumin fraction V and recombinant human serum albumin (rHSA) are almost as effective as LFS in enhancing the level of lyso-PC in the media. The latter finding implies that rHSA directly generates lyso-PC from secreted PC and suggests that this PLA(2)-like activity may be an intrinsic attribute of albumin.     

Survival signaling in type II pneumocytes activated by surfactant protein-A

Surfactant-associated protein-A (SP-A) is a component of pulmonary surfactant that acts as a cytokine through interaction with a cell-surface receptor (SPAR) on lung epithelial cells. SP-A regulates important physiological processes including surfactant secretion, gene expression, and protection against apoptosis. Tyrosine kinase and PI3K inhibitors block effects of SP-A, suggesting that SPAR may be a receptor tyrosine kinase and activate the PI3K-PKB/Akt pathway. Here we report that SP-A treatment leads to rapid tyrosine-specific phosphorylation of several important proteins in lung epithelial cells including insulin receptor substrate-1 (IRS-1), an upstream activator of PI3K. Analysis of anti-apoptotic signaling species downstream of IRS-1 showed activation of PKB/Akt but not of MAPK. Phosphorylation of IkappaB was minimally affected by SP-A as was NFkappaB gel shift activity. However, FKHR was rapidly phosphorylated in response to SP-A and its DNA-binding activity was significantly reduced. Since FKHR is pro-apoptotic, this may play an important role in signaling the anti-apoptotic effects of SP-A. Therefore, we have characterized survival-enhancing signaling activated by SP-A leading from SPAR through IRS-1, PI3K, PKB/Akt, and FKHR. The activity of this pathway may explain, in part, the resilience of type II cells to lung injury and their survival to repopulate alveolar epithelium after peripheral lung damage.     

Palmitate and glucose oxidation by fetal type II pneumocytes

Fetal type II pneumocytes in organotypic culture can oxidize both palmitate and glucose, with glucose being converted to CO2 at a rate substantially greater than that of palmitate. Glucose can be oxidized by both the pentose shunt pathway and the tricarboxylic acid cycle. Palmitate oxidation to CO2 is increased by carnitine and reduced by glucose and unsaturated fatty acids. These data suggest that glucose may be an important oxidative substrate during late fetal life and that fatty acids may play a relatively minor role in type II cell oxidative metabolism.     

Repopulation of a human alveolar matrix by adult rat type II pneumocytes in vitro : A novel system for type II pneumocyte culture

This paper describes the preparation of lung acellular alveolar matrix fragments and culture of rat type II pneumocytes directly on the alveolar epithelial basement membrane, thereby permitting study of the effect of lung basement membrane on the morphology and function of type II cells. Collagen types I, III, IV and V, laminin and fibronectin were located by immunofluorescence in the lung matrix with the same patterns as those described for the normal human lung. Transmission electron microscopy (TEM) of the fragments revealed intact epithelial and endothelial basement membranes. The matrix maintained the normal three-dimensional alveolar architecture. Glycosaminoglycans were still present by Alcian Blue staining. Isolated adult rat type II pneumocytes cultured on 150 micron thick fragments of acellular human alveolar extracellular matrix undergo gradual cytoplasmic flattening, with loss of lamellar bodies, mitochondria, and surface microvilli. These changes are similar to the in vivo differentiation of type II pneumocytes into type I pneumocytes. The type II pneumocyte behaviour on the lung epithelial basement membrane contrasted sharply with that of the same cell type cultured on a human amnionic basement membrane. On the latter surface the cells retained their cuboidal shape, lamellar bodies and surface microvilli for up to 8 days. These observations suggest that the basement membranes from different organ systems exert differing influences on the morphology and function of type II pneumocytes and that the alveolar and amnionic basement membranes may have differing three-dimensional organizations. The technique of direct culture of type II cells on the lung basement membrane provides a useful tool for studying the modulating effect of the basement membrane on alveolar epithelial cells.     

Developmental regulation of phospholipid secretion by fetal type II pneumocytes

Surfactant sufficiency is dependent upon adequate synthesis and secretion of surfactant by the type II alveolar epithelium. Our laboratory has previously shown that basal secretion of surfactant phospholipid by differentiated fetal type II cells is lower than the basal secretion by adult cells. The purposes of this study were to determine if undifferentiated fetal type II cells can secrete phosphatidylcholine, to determine if terbutaline, a β-adrenergic agonist, stimulates secretion of surfactant phospholipids by undifferentiated fetal cells and to examine the effects of differentiation on secretion of surfactant phospholipids by fetal cells. Constitutive (basal) secretion of phosphatidylcholine increased linearly as a function of time in both undifferentiated and differentiated cells, but the rate of secretion was greater in differentiated cells than the rate of secretion in undifferentiated cells. Terbutaline caused a concentration-dependent increase in secretion in both undifferentiated and differentiated cells. Maximal effective concentration and EC₅₀ were similar for undifferentiated (10⁻⁶ M, 0.2 μM) and differentiated (10⁻⁵ M, 0.3 μM) cells. The relative stimulation of secretion above control values was greater for undifferentiated cells. The kinetics of terbutaline stimulation varied significantly with cellular differentiation. Terbutaline resulted in 230% stimulation of secretion in undifferentiated cells at 30 min followed by a decline in the response to terbutaline at 60 to 120 min. In contrast, terbutaline stimulated secretion by differentiated cells showed a sustained linear increase from 0 to 120 min. This regulation of stimulated secretion is not present in undifferentiated cells. We conclude that undifferentiated type II cells are capable of the secretion of phosphatidylcholine and that terbutaline stimulates secretion by undifferentiated cells. Furthermore, basal secretion increases as a function of differentiation of type II cells and the regulation of stimulated secretion seen in differentiated cells is not developed in undifferentiated cells. The developmental regulation of the secretion of surfactant is complex and probably involves both excitatory as well as inhibitory mechanisms which develop at different stages of differentiation of the type II cell.     

Reactive type II pneumocytes in bronchoalveolar lavage fluid

OBJECTIVE: To evaluate the prevalence of reactive type II pneumocytes (RPII) in bronchoalveolar lavage (BAL) fluid samples obtained from patients with various pulmonary disorders. STUDY DESIGN: Consecutive BAL fluid samples were screened for the presence of RPII on May-Grünwald-Giemsa-stained cytocentrifuge preparations. BAL fluid samples with and without RPII were compared with regard to prevalence, associated clinical diagnoses and cytologic findings. RESULTS: RPII were generally large cells with a high nuclear:cytoplasmic ratio and deeply blue-stained, vacuolated cytoplasm. Most RPII occurred in cohesive cell groups, and the vacuoles tended to be confluent. Cytologic findings associated with RPII were foamy alveolar macrophages, activated lymphocytes and plasma cells. RPII were present in 94 (21.7%) of 433 included BAL fluid samples. The highest prevalences were noted in patients with systemic inflammatory response syndrome and alveolar hemorrhage. In addition, RPII tended to occur more frequently in ventilator-associated pneumonia, Pneumocystis carinii pneumonia, extrinsic allergic alveolitis and drug-induced pulmonary disorders. In contrast, RPII were not observed in BAL fluid samples obtained from patients with sarcoidosis. CONCLUSION: RPII were prevalent in about 20% of BAL fluid specimens. They were associated mainly with conditions of acute lung injury and not observed in sarcoidosis.     

Monoclonal antibody isolation of type II pneumocytes

A monoclonal antibody that identifies a membrane molecule unique in rat lung for type II alveolar epithelial cells was used to isolate these cells from enzymatically dispersed lung cells by fluorescence-activated cell sorting. Although multistep physical separation techniques have permitted the isolation of large quantities of these cells and flow cytometry has been used by others to isolate lamellar body-containing cells, the application of this antibody-directed sorting has distinct advantages. Because the marker molecule is expressed on immature type II cells prior to the development of lamellar bodies, the antibody will also permit their isolation and study.     

Natural protection from apoptosis by surfactant protein A in type II pneumocytes

Surfactant-associated protein A (SP-A) is a component of pulmonary surfactant that binds to a specific receptor (SPAR) on the surface of type II alveolar cells of the lung and regulates gene expression and surfactant secretion. Previously we have shown that activation of SPAR by SP-A binding initiates a signal through pathways that involve tyrosine phosphorylation, include IRS-1, and entail activation of phosphatidylinositol 3-kinase (PI3K). In other cell types, cytokines that activate the PI3K signaling pathway promote cell survival. Therefore we investigated whether there was an effect of SP-A on apoptosis as measured by DNA laddering, FACS analysis, TUNEL assay, and annexin V binding. SP-A protected primary cultures of rat type II alveolar cells against the apoptotic effects of etoposide and UV light and also protected the H441 human Clara lung tumor cell line against staurosporine-induced apoptosis. The protective effects of SP-A were abrogated by inhibition of either tyrosine-specific protein kinase activity or PI3K. SP-A/SPAR interaction thus initiates a signaling pathway that regulates apoptosis in type II cells. These findings may be important in understanding the pathogenesis of acute lung injury and pulmonary tumorigenesis and may suggest new therapeutic options.     

Leukotrienes stimulate phosphatidylcholine secretion in cultured type II pneumocytes

We previously reported that arachidonic acid stimulates secretion of phosphatidylcholine in cultures of type II pneumocytes and, based on studies with cyclooxygenase and lipoxygenase inhibitors, suggested that this effect was mediated by lipoxygenase products of arachidonic acid metabolism (Gilfillan, A.M. and Rooney, S.A. (1985) Biochim. Biophys. Acta 833, 336-341). We have now examined the effect of leukotrienes on phosphatidylcholine secretion in type II cells as well as the effect of a leukotriene antagonist, FPL55712, on the stimulatory effect of arachidonic acid. Leukotrienes C4, D4 and E4 stimulated phosphatidylcholine secretion and this effect was dependent on concentration in the range 10(-12)-10(-6) M. Leukotriene E4 was the most stimulatory, followed by D4 and C4. Leukotriene B4 had no effect. Incubation of the cells with 10(-7) M leukotriene E4 for 90 min resulted in a 107% increase in the rate of phosphatidylcholine secretion. Incubation with 10(-6) M leukotrienes D4 and C4 for the same period resulted in 81% and 63% stimulation, respectively. The leukotrienes had no effect on cellular phosphatidylcholine synthesis or on lactate dehydrogenase release. The stimulatory effects of leukotrienes E4 and D4 were abolished by FPL55712. Similarly, the stimulatory effect of 6 X 10(-6) M arachidonic acid on phosphatidylcholine secretion was reduced from 74% to 25% by 10(-5) M FPL55712. Thus, the stimulatory effect of arachidonic acid on surfactant phospholipid secretion in type II cells is mediated at least in part by leukotrienes.     

Internalized SP-A and lipid are differentially resecreted by type II pneumocytes

Biochemical and morphological assays were developed to study surfactant protein A (SP-A) and lipid resecretion kinetics by isolated type II cells in vitro. After a 10-min uptake period with SP-A (3 microg/10(6) cells) in combination with liposomes (60 microg/10(6) cells), the cells were allowed to resecrete. After 5 min of resecretion, only 21.7 +/- 4.6% of the internalized SP-A remained intracellularly compared with 54 +/- 2.9% of the lipids. Extracellular SP-A present during the resecretion period partially inhibited resecretion (SP-A, 36% at 5 min; lipid, approximately 16% at 5 min). Lipid resecretion was also dependent on the SP-A concentration present during the uptake period. Although, as shown by confocal laser scanning microscopy, after a 10-min uptake period at 37 degrees C, most of the fluorescein isothiocyanate-labeled SP-A and rhodamine-phosphatidylethanolamine-labeled lipids colocalized within the cells, after an additional 10 min of resecretion, both the strength of the fluorescence signals and the extent of colocalization had markedly decreased. These data indicate that internalized lipid and SP-A can be resecreted rapidly by type II cells, likely via different pathways.     

Antifreeze proteins are secreted by winter rye cells in suspension culture

During cold-acclimation, winter rye ( Secale cereale L) leaves secrete antifreeze proteins (AFPs) into the apoplast. The AFPs bind to ice and modify its growth, which is easily observed in vitro . However, it is not yet known whether in planta AFPs interact with ice or whether they exert cryoprotective effects. These experiments are difficult to conduct with intact plants, so the aim of this work was to determine whether AFPs are produced in response to cold temperature in cell culture and to examine their function by using suspension cells. We showed that suspension cells secreted three of the six known winter rye AFPs into the culture medium during acclimation at 4°C. These AFPs were not present in washed suspension cells, thus indicating that they are not firmly bound to the cell walls. In order to examine the function of extracellular AFPs, non-acclimated (NA) winter rye suspension cells and protoplasts isolated from NA winter rye leaves were then frozen and thawed in the presence of AFPs extracted from cold-acclimated winter rye leaves. The AFPs had no effect on the survival of NA protoplasts after freezing; however, they lowered the lethal temperature at which 50% of the cells are killed by freezing (LT₅₀) of NA suspension cells by 2.5°C. We conclude that low above-zero temperatures induce winter rye suspension cells to secrete AFPs free in solution where they can protect intact suspension cells, but not protoplasts, from freezing injury, presumably by interacting with extracellular ice.     

Corticosteroid receptor in type II pneumocytes of the rat

Glucocorticoid receptor in rat type II pneumocytes has been characterized. The Scatchard plot analysis of 3H-dexamethasone binding to type II cells showed a single class of binding sites. The apparent Kd of 3H-dexamethasone binding by a whole cell assay was 9.1 nM and the maximal binding capacity was 78.0 f mol/10(6) cells (0.31 pmol/mg cytosol protein).