Mainstream and sidestream cigarette smoke exposure increases Ca2+-dependent phospholipid binding proteins in guinea pig alveolar type II cells

We have earlier identified the presence of a 36 kDa Ca²⁺-dependent phospholipid-binding protein (PLBP) in guinea pig alveolar type II cells. PLBP has been suggested to act as a mediator in facilitating and regulating intracellular surfactant assembly and delivery to the plasma membrane of type II cells for secretion into alveolar space. It has been reported that cigarette smoke exposure (CSE) causes a decrease in the surfactant activity in bronchial washings. We have also reported earlier that mainstream (MS) and sidestream (SS) CSE causes desensitization of -adrenoreceptors in guinea pig alveolar type II cells. Since both Ca²⁺ and -adrenoreceptors are involved in surfactant secretion and PLBP is involved in surfactant delivery, it is important to know whether CSE causes any change in the PLBP level in alveolar type II cells. In the present study, we have demonstrated that MS and SS CSE causes a significant increase in the levels of PLBP in alveolar type II cells (107 and 150%, respectively) and in lung lavage (42 and 125%, respectively) in comparison to that in sham control (430 ng/mg protein in alveolar type II cells and 780 ng/mg protein in lung lavage). The mechanism by which smoke exposure causes an elevation in the levels of PLBP in alveolar type II cells and lung lavage remains to be investigated.     

Protein nitration in rat lungs during hyperoxia exposure: a possible role of myeloperoxidase

Several studies have suggested that exposure to hyperoxia causes lung injury through increased generation of reactive oxygen and nitrogen species. The present study was aimed to investigate the effects of hyperoxia exposure on protein nitration in lungs. Rats were exposed to hyperoxia (>95%) for 48, 60, and 72 h. Histopathological analysis showed a dramatic change in the severity of lung injury in terms of edema and hemorrhage between 48- and 60-h exposure times. Western blot for nitrotyrosine showed that several proteins with molecular masses of 29-66 kDa were nitrated in hyperoxic lung tissues. Immunohistochemical analyses indicate nitrotyrosine staining of alveolar epithelial and interstitial regions. Furthermore, immunoprecipitation followed by Western blot revealed the nitration of surfactant protein A and t1alpha, proteins specific for alveolar epithelial type II and type I cells, respectively. The increased myeloperoxidase (MPO) activity and total nitrite levels in bronchoalveolar lavage and lung tissue homogenates were observed in hyperoxic lungs. Neutrophils and macrophages isolated from the hyperoxia-exposed rats, when cocultured with a rat lung epithelial L2 cell line, caused a significant protein nitration in L2 cells. Inclusion of nitrite further increased the protein nitration. These studies suggest that protein nitration during hyperoxia may be mediated in part by MPO generated from activated phagocytic cells, and such protein modifications may contribute to hyperoxia-mediated lung injury.     

Acute lung injury edema fluid decreases net fluid transport across human alveolar epithelial type II cells

Most patients with acute lung injury (ALI) have reduced alveolar fluid clearance that has been associated with higher mortality. Several mechanisms may contribute to the decrease in alveolar fluid clearance. In this study, we tested the hypothesis that pulmonary edema fluid from patients with ALI might reduce the expression of ion transport genes responsible for vectorial fluid transport in primary cultures of human alveolar epithelial type II cells. Following exposure to ALI pulmonary edema fluid, the gene copy number for the major sodium and chloride transport genes decreased. By Western blot analyses, protein levels of alphaENaC, alpha1Na,K-ATPase, and cystic fibrosis transmembrane conductance regulator decreased as well. In contrast, the gene copy number for several inflammatory cytokines increased markedly. Functional studies demonstrated that net vectorial fluid transport was reduced for human alveolar type II cells exposed to ALI pulmonary edema fluid compared with plasma (0.02 +/- 0.05 versus 1.31 +/- 0.56 microl/cm2/h, p < 0.02). An inhibitor of p38 MAPK phosphorylation (SB202190) partially reversed the effects of the edema fluid on net fluid transport as well as gene and protein expression of the main ion transporters. In summary, alveolar edema fluid from patients with ALI induced a significant reduction in sodium and chloride transport genes and proteins in human alveolar epithelial type II cells, effects that were associated with a decrease in net vectorial fluid transport across human alveolar type II cell monolayers.     

Macrophage and type II cell catabolism of SP-A and saturated phosphatidylcholine in mouse lungs

Type II cells and macrophages are the major cells involved in the alveolar clearance and catabolism of surfactant. We measured type II cell and macrophage contributions to the catabolism of saturated phosphatidylcholine and surfactant protein A (SP-A) in mice. We used intratracheally administered SP-A labeled with residualizing (125)I-dilactitol-tyramine, radiolabeled dipalmitoylphosphatidylcholine ([(3)H]DPPC), and its degradation-resistant analog [(14)C]DPPC-ether. At 15 min and 7, 19, 29, and 48 h after intratracheal injection, the mice were killed; alveolar lavage was then performed to recover macrophages and surfactant. Type II cells and macrophages not recovered by the lavage were subsequently isolated by enzymatic digestion of the lung. Radioactivity was measured in total lung, lavage fluid macrophages, alveolar washes, type II cells, and lung digest macrophages. Approximately equal amounts of (125)I-dilactitol-tyramine-SP-A and [(14)C]DPPC-ether associated with the macrophages (lavage fluid plus lung digest) and type II cells when corrected for the efficiency of type II cell isolation. Eighty percent of the macrophage-associated radiolabel was recovered from lung digest macrophages. We conclude that macrophages and type II cells contribute equally to saturated phosphatidylcholine and SP-A catabolism in mice.     

Compartmentalization of vascular endothelial growth factor to the epithelial surface of the human lung

BACKGROUND: Based on assessment of mRNA expression, the lung is a major site of expression of the vascular endothelial growth factor (VEGF) gene, largely from type II alveolar epithelial cells. With the knowledge that VEGF can function to induce vascular leak, we hypothesized that to protect the lung from pulmonary edema, the VEGF produced in the lung must be compartmentalized from the pulmonary endothelium, and thus must be compartmentalized to the surface of the respiratory epithelium. MATERIAL AND METHODS: To assess this hypothesis, we quantified the levels of VEGF in human respiratory epithelial lining fluid recovered by bronchoalveolar lavage from normal individuals. RESULTS: Strikingly, human respiratory epithelial lining fluid contains 11 +/- 5 ng/mL as quantified by ELISA, a 500-fold greater concentration than plasma (22 +/- 10 pg/mL, p < 0.0005). Western analysis of BAL fluid proteins showed the major VEGF isoform in respiratory epithelial lining fluid is VEGF165. CONCLUSIONS: With the knowledge that proteins of molecular mass like VEGF (34 to 46 kDa) slowly diffuse across the alveolar epithelium, it is likely that this high level "reservoir" of VEGF protein on the respiratory epithelial surface plays a role in normal lung endothelial biology. However, this compartmentalized VEGF reservoir may also be a "Damocles sword" poised to induce lung endothelial permeability in conditions of acute lung injury when the integrity of the alveolar epithelial barrier is breached.     

Vascular and epithelial damage in the lung of the mouse after X rays or neutrons

The response of the lung was studied in CFLP mice after exposure of the whole thorax to X rays (250 kVp) or cyclotron neutrons (16 MeV deuterons on Be, mean energy 7.5 MeV). To measure blood volume and leakage of plasma proteins, 51Cr-labeled red blood cells and 125I-albumin were injected intravenously and 24 h later lungs were lavaged via the trachea. Radioactivities in lung tissue and lavage fluid were determined to estimate the accumulation of albumin in the interstitial and alveolar spaces indicating damage to blood vessels and alveolar epithelium respectively. Function of type II pneumonocytes was assessed by the amounts of surfactant (assayed as lipid phosphorous) released into the lavage fluid. During the first 6 weeks, lavage protein and surfactant were increased, the neutron relative biological effectiveness (RBE) being unity. During pneumonitis at 12-24 weeks, surfactant levels were normal, blood volume was decreased, and both interstitial and alveolar albumin were increased. Albumin levels then decreased. At late times after exposure (42-64 weeks) alveolar albumin returned to normal but interstitial albumin was still slightly elevated. Values of RBE for changes in blood volume and interstitial and alveolar albumin at 15 weeks and for changes in blood volume and interstitial albumin at 46 weeks were 1.4, comparable with that for animal survival at 180 days. The results indicate that surfactant production is not critical for animal survival. They suggest that changes in blood vessels and alveolar epithelium occur during acute pneumonitis; epithelial repair follows but some vascular damage may persist. The time course of the changes in albumin levels did not correlate with increases in collagen biosynthesis which have been observed as early as 1 month after exposure and persist for up to 1 year. Furthermore, a dose which had no effect on leakage caused a marked increase in collagen biosynthesis. Thus the present results do not support a causal relationship between exudation of vascular protein during pneumonitis and the later development of fibrosis.     

Proportional analysis of respiratory cells obtained by bronchoalveolar lavage.

Bronchoalveolar lavage was performed during fibreoptic bronchoscopy in 17 patients with biopsy-proven interstitial lung disease and in 12 control subjects who had focal lesions in the lung. The volume of fluid recovered was unrelated to disease activity or diagnosis. In the control subjects alveolar macrophages represented over 95% of the lavaged cells. The proportion of lymphocytes in the lavaged cells enabled a natural division of the diffuse interstitial lung diseases into two categories: active sarcoidosis, indicated by a large proportion of lymphocytes but a normal proportion of polymorphonuclear leukocytes; and idiopathic pulmonary fibrosis and asbestosis, indicated by a normal proportion of lymphocytes but a variable proportion of polymorphonuclear leukocytes. Bronchoalveolar lavage is a safe and well tolerated method for evaluating the role of alveolitis in diffuse interstitial lung disease through the sampling of respiratory alveolar cells.     

Expression of the major surfactant-associated protein, SP-A, in type II cells of human lung before 20 weeks of gestation

In a previous paper (Otto-Verberne et al., Anat. Embryol. 178, 29-39 (1988) we reported that the type II alveolar epithelial cell can be identified in fetal human lung on the basis of morphological and immunological characteristics from 10 to 12 weeks after conception (a.c.) onward. For immunological recognition we used a lung-specific antibody, called SALS-Hu (specific anti-lavage serum, rabbit antihuman). The present immunoblotting experiments, after one-and two-dimensional electrophoresis, showed that SALS-Hu-reactive proteins in lavage fractions obtained from alveolar proteinosis patients exhibited molecular masses of mainly 29, 31 to 36, and 62 to 66 kDa. All SALS-Hu-reactive proteins migrated in the same acidic isoelectric point range (pI 4.4-5.1) and were almost undetectable when we used SALS-Hu preabsorbed with recombinant surfactant-associated protein A. We concluded that SALS-Hu recognizes exclusively isoforms of the major surfactant-associated protein, SP-A. In vitro translation assays in which we used mRNA isolated from adult human lung confirmed that SALS-Hu recognized the 29 to 31 kDa SP-A precursor proteins. These SALS-Hu-immunoreactive precursors for SP-A were already detectable (though in much lower amounts) in human fetuses aged 17 to 18 weeks, indicating that mRNA coding for SP-A is present at that time. We concluded that the cytoplasmic staining of fetal (from 10-12 weeks a.c. onward) and adult human type II cells by SALS-Hu is due to the presence of SP-A.     

Identification of vitamin K-dependent carboxylase activity in lung type II cells but not in lung macrophages.

Fluorography of 14C-labelled glutamic acid residues in vitamin K-dependent protein precursors in lung microsomes (microsomal fractions) shows that the lung has several substrates that are not found in the liver. These precursor proteins unique to the lung have apparent molecular masses of 65, 53, 50, 36, 31 and 13 kDa. Type II epithelial cells appear to synthesize most of the vitamin K-dependent proteins in the lung. The 36 and the 31 kDa precursors also found in Type-II-cell microsomes have a similar molecular mass to those of surfactant-associated proteins, and we have previously shown [Rannels, Gallaher, Wallin & Rannels (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5952-5956] that the 36 kDa protein is one of the precursors for these proteins. Immunoblotting of membrane fragments of Type-II-cell microsomes with plasma prothrombin antibodies identified two prothrombin-like antigens of apparent molecular masses 68 and 65 kDa. This raises the question as to whether Type II cells are also a potential site for synthesis of prothrombin and possibly other vitamin K-dependent clotting factors. Pulmonary macrophages appear to be devoid of vitamin K-dependent carboxylase activity. However, Type II epithelial cells have significant activity, and this activity was unaltered when these cells were maintained in primary culture for 3 days, suggesting that carboxylase activity is expressed in lung alveolar epithelium independently of culture-induced changes in cellular differentiation. Carboxylase activity in Type II cells was enhanced 2-fold when cells were cultured for 24 h in the presence of 50 microM-warfarin. Type II cells, therefore, resemble hepatocytes with regard to their response to coumarin anticoagulant drugs.     

Transfer of phospholipids by a protein fraction obtained from canine pulmonary lavage

Surfactant phospholipid exists in multicompartment pools within the subphase of the lung. Movement among these pools and back into type II alveolar cells may be catalyzed by a phospholipid transfer protein resident in the subphase. We demonstrate here that a protein fraction obtained from canine lung lavage catalyzes the intermembrane transfer of all the major surfactant phospholipids. The protein is probably not derived from serum and is unrelated to surfactant proteins that have already been described.     

Role of CD13/aminopeptidase N in rat lymphocytic alveolitis caused by thoracic irradiation

CD13/aminopeptidase N is a cell surface glycoprotein that is widely distributed in a variety of mammalian cells. It was recently shown to have chemotactic activity for T lymphocytes. This study examined the role of CD13/aminopeptidase N in lymphocytic alveolitis in radiation-induced lung injury caused by a single-dose thoracic irradiation (15 Gy) in rats. Significantly increased aminopeptidase activity was detected in bronchoalveolar lavage fluid obtained from irradiated rats at 4 weeks after irradiation compared to the activity in unirradiated rats. Significantly higher aminopeptidase activity was detected on alveolar macrophages from irradiated rats at 2 and 4 weeks than on those from unirradiated rats. Western blot analysis showed an increased expression of CD13/aminopeptidase N protein in alveolar macrophages from irradiated rats at 4 weeks. Chemotactic activity for normal rat lymphocytes was detected in bronchoalveolar lavage fluid from irradiated rats at 4 weeks, and approximately 60% of the activity was inhibited by pretreatment of bronchoalveolar lavage fluid with bestatin, a specific aminopeptidase inhibitor. This study suggests that CD13/aminopeptidase N may play an important role as a lymphocyte chemoattractant in lymphocyte-mediated alveolitis in experimental radiation-induced lung injury.     

Cytoplasmic enzyme patterns in isolated hamster pulmonary alveolar type II cells

Three cytoplasmic enzyme patterns were studied in pulmonary alveolar type II cells isolated from normal adult hamster lung: lactate dehydrogenase (total and isoenzymes), peroxidase, and beta-N-acetylglucosaminidase. Enzyme patterns of freshly-isolated type II cells were found to be different from those of freshly-isolated pulmonary hamster fibroblasts. After both types of cells had been cultured for seven days, no difference in cytoplasmic enzyme patterns remained. Lactate dehydrogenase isoenzyme patterns for type II cells were different from those obtained from polymorphonuclear leukocytes and alveolar macrophages. These data may be useful in detecting sources of lung injury by assessment of enzyme patterns in bronchoalveolar lavage fluid.     

Inhibition of phospholipase A2 activity of guinea-pig alveolar macrophages by lipocortin-like proteins purified from mice lung

Guinea-pig alveolar macrophages were harvested by bronchoalveolar lavage and purified by differential adhesion. They were labeled with 14C-Arachidonic acid and then exposed to platelet-activating factor or to the calcium ionophore A23187. The activity of cellular phospholipase A2 was considered as the release of free 14C-Arachidonic acid in the cell supernatant. The pretreatment of guinea-pig alveolar macrophages with two lipocortin-like proteins (36 kDa and 40 kDa) purified from mice lung induced a significant inhibition of their phospholipase A2 activity upon platelet-activating factor and calcium ionophore stimulation. These results indicate that lipocortin-like proteins can modulate the phospholipase A2 activity of isolated cells in vitro.     

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.     

An electrophoretic and immunochemical characterization of human surfactant-associated proteins

We have prepared an antiserum against a serum-free extract of alveolar proteinosis lavage that recognizes the same proteins as an antiserum to human surfactant. Using one and two-dimensional gel electrophoresis, protein blotting and immunostaining we have found proteins with Mr of approx. 35 and 60 kDa to be present in every source of human surfactant we have examined. These proteins are immunologically related to those found in the lavage from alveolar proteinosis patients, have the same electrophoretic characteristics and are not found in serum. The 35 kDa protein is a group of at least eight isoforms ranging in relative molecular mass Mr from 32 to 36 kDa with isoelectric points between 4.8 and 5.5. Neuraminidase digestion studies have shown that at least part of this charge heterogeneity may be due to sialic acid residues. The less abundant form, with a Mr of about 60 kDa is also a sialoglycoprotein with similar isoelectric points.     

Analysis of the native murine bone morphogenetic protein serine threonine kinase type I receptor (ALK-3)

The bone morphogenetic proteins, members of the transforming growth factor-β cytokine family, induce the osteoblast phenotype and promote osteogenesis in the bone marrow stroma. Simultaneously, these cytokines inhibit other mesodermal differentiation pathways, such as adipogenesis and myogenesis. The receptors for the bone morphogenetic proteins belong to a family of transmembrane serine/threonine kinase TGFβ type I and type II receptor proteins. In man, these include the activin receptor like kinase-3 (ALK-3), a type I receptor protein. We have used a polyclonal antibody to examine the expression of the native murine ALK-3 protein in murine tissues and bone morphogenetic protein-responsive cell lines. On Western blot analyses, we found that the native 85 kDa native ALK-3 protein was expressed in a number of murine tissues; protein and mRNA levels did not necessarily correlate. Two bone morphogenetic protein-responsive cell lines, BMS2 bone marrow stromal cells and C2C12 myoblasts, expressed the ALK-3 protein constitutively. Cell differentiation was accompanied by modest changes in ALK-3 protein levels. Immunoprecipitation of the ALK-3 protein cross linked to [¹²⁵I] BMP-4 revealed two major receptor complexes of approximately 90 kDa and 170 kDa in size. Biotin surface-labeling experiments revealed that the 85 kDa ALK-3 protein was constitutively associated with a novel 140 kDa surface glycoprotein. Deglycosylation reduced the protein's size to 116 kDa, comparable in size to that of the recently described BMP type II receptor. These findings support the current model that BMP interacts with a pre-existing complex consisting of a type I and type II receptor protein. © 1996 Wiley-Liss, Inc.     

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase: immunochemical characterization of the lung enzyme from pregnant rabbits

A polyclonal antibody was produced in guinea pig against the lung NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) purified from pregnant rabbits. Western blot analysis demonstrated that the protein identified by this antibody in the 105,000g supernatant fraction of lung tissue from pregnant rabbits had a molecular mass of 30 kDa and comigrated with the purified PGDH. The specific activity of the lung PGDH in pregnant rabbits (25- to 28-day gestations) was 36.7 nmol NADH formed/min/mg protein compared to 0.3 nmol NADH formed/min/mg protein in nonpregnant rabbits. Although the PGDH activity in the lung cytosol of nonpregnant rabbits was inhibited by the anti-lung PGDH antibody, the 30-kDa protein was not detected by Western blot analysis. An examination of this 30-kDa protein during the gestational period indicated that the protein was present after 10 days and the amount of the protein increased from Day 10 to Day 28. This increase in the immunochemically reactive protein correlated with the marked increase in PGDH specific activity between 10 and 28 days. An immunochemically reactive protein also was observed in the ovary of 25- to 28-day pregnant rabbits and the specific activity of the ovary PGDH was 19.3 nmol NADH formed/min/mg protein. Only trace levels of the PGDH activity were detected in the ovaries of nonpregnant rabbits. A 30-kDa protein was not detected by the anti-rabbit lung PGDH in brain, kidney, bladder, uterus, liver, and heart tissue of pregnant or nonpregnant rabbits. When rabbit or human placental cytosol was examined with the anti-rabbit lung PGDH only faint 30-kDa bands were observed by Western blot analysis. A monoclonal antibody prepared against human placental PGDH did not recognize the 30-kDa band in the pregnant rabbit lung. Localization studies indicated a marked increase in immunochemical staining in pulmonary epithelial cells of pregnant rabbits as compared to nonpregnant rabbits. Lung epithelial cells but not endothelial cells were identified as containing the PGDH.