Keratinocyte growth factor and glucocorticoid induction of human peroxiredoxin 6 gene expression occur by independent mechanisms that are synergistic.

AIMS: Peroxiredoxin 6 (Prdx6), a 1-cys Prdx has both peroxidase and phospholipase A2 activities, protecting against oxidative stress and regulating pulmonary surfactant phospholipid metabolism. This study determined the mechanism by which keratinocyte growth factor (KGF) and the glucocorticoid analogue, dexamethasone (Dex), induce increased Prdx6 expression. RESULTS: Transcriptional activation by KGF in both A549 lung adenocarcinoma cells and rat lung alveolar epithelial type II (ATII) cells utilizes an antioxidant response element (ARE), located between 357 and 349 nucleotides before the PRDX6 translational start, that is also necessary for upregulation of the human PRDX6 promoter in response to oxidative stress. Activation is mediated by binding of the transcription factor, Nrf2, to the ARE as shown by experiments using siRNA against Nrf2 and by transfecting ATII cells isolated from lungs of Nrf2 null mice. KGF triggers the migration of Nrf2 from cytoplasm to nucleus where it binds to the PRDX6 promoter as shown by chromatin immunoprecipitation assays. Activation of transcription by Dex occurs through a glucocorticoid response element located about 750 nucleotides upstream of the PRDX6 translational start. INNOVATION: This study demonstrates that KGF can activate an ARE in a promoter without reactive oxygen species involvement and that KGF and Dex can synergistically activate the PRDX6 promoter and protect cells from oxidative stress. CONCLUSION: These two different activators work through different DNA elements. Their combined effect on transcription of the reporter gene is synergistic; however, at the protein level, the combined effect is additive and protects cells from oxidative damage.

Pulmonary abnormalities in animal models due to Niemann-Pick type C1 (NPC1) or C2 (NPC2) disease.

Niemann-Pick C (NPC) disease is due to loss of NPC1 or NPC2 protein function that is required for unesterified cholesterol transport from the endosomal/lysosomal compartment. Though lung involvement is a recognized characteristic of Niemann-Pick type C disease, the pathological features are not well understood. We investigated components of the surfactant system in both NPC1 mutant mice and felines and in NPC2 mutant mice near the end of their expected life span. Histological analysis of the NPC mutant mice demonstrated thickened septae and foamy macrophages/leukocytes. At the level of electron microscopy, NPC1-mutant type II cells had uncharacteristically larger lamellar bodies (LB, mean area 2-fold larger), while NPC2-mutant cells had predominantly smaller lamellar bodies (mean area 50% of normal) than wild type. Bronchoalveolar lavage from NPC1 and NPC2 mutant mice had an approx. 4-fold and 2.5-fold enrichment in phospholipid, respectively, and an approx. 9-fold and 35-fold enrichment in cholesterol, consistent with alveolar lipidosis. Phospholipid and cholesterol also were elevated in type II cell LBs and lung tissue while phospholipid degradation was reduced. Enrichment of surfactant protein-A in the lung and surfactant of the mutant mice was found. Immunocytochemical results showed that cholesterol accumulated in the LBs of the type II cells isolated from the affected mice. Alveolar macrophages from the NPC1 and NPC2 mutant mice were enlarged compared to those from wild type mice and were enriched in phospholipid and cholesterol. Pulmonary features of NPC1 mutant felines reflected the disease described in NPC1 mutant mice. Thus, with the exception of lamellar body size, the lung phenotype seen in the NPC1 and NPC2 mutant mice were similar. The lack of NPC1 and NPC2 proteins resulted in a disruption of the type II cell surfactant system contributing to pulmonary abnormalities.

Stereoscopic digital mammography: improved specificity and reduced rate of recall in a prospective clinical trial.

PURPOSE: To compare stereoscopic digital mammography (DM) with standard DM for the rate of patient recall and the detection of cancer in a screening population at elevated risk for breast cancer. MATERIALS AND METHODS: Starting in September 2004 and ending in December 2007, this prospective HIPAA-compliant, institutional review board-approved screening trial, with written informed consent, recruited female patients at elevated risk for breast cancer (eg, personal history of breast cancer or breast cancer in a close relative). A total of 1298 examinations from 779 patients (mean age, 58.6 years; range, 32-91 years) comprised the analyzable data set. A paired study design was used, with each enrolled patient serving as her own control. Patients underwent both DM and stereoscopic DM examinations in a single visit, findings of which were interpreted independently by two experienced radiologists, each using a Breast Imaging Reporting and Data System (BI-RADS) assessment (BI-RADS category 0, 1, or 2). All patients determined to have one or more findings with either or both modalities were recalled for standard diagnostic evaluation. The results of 1-year follow-up or biopsy were used to determine case truth. RESULTS: Compared with DM, stereoscopic DM showed significantly higher specificity (91.2% [1167 of 1279] vs 87.8% [1123 of 1279]; P = .0024) and accuracy (90.9% [1180 of 1298] vs 87.4% [1135 of 1298]; P = .0023) for detection of cancer. Sensitivity for detection of cancer was not significantly different for stereoscopic DM (68.4% [13 of 19]) compared with DM (63.2% [12 of 19], P .99). The recall rate for stereoscopic DM was 9.6% (125 of 1298) and that for DM was 12.9% (168 of 1298) (P = .0018). CONCLUSION: Compared with DM, stereoscopic DM significantly improved specificity for detection of cancer, while maintaining comparable sensitivity. The recall rate was significantly reduced with stereoscopic DM compared with DM. SUPPLEMENTAL MATERIAL: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.12120382/-/DC1.

Characterization of the Niemann-Pick C pathway in alveolar type II cells and lamellar bodies of the lung.

The Niemann-Pick C (NPC) pathway plays an essential role in the intracellular trafficking of cholesterol by facilitating the release of lipoprotein-derived sterol from the lumen of lysosomes. Regulation of cellular cholesterol homeostasis is of particular importance to lung alveolar type II cells because of the need for production of surfactant with an appropriate lipid composition. We performed microscopic and biochemical analysis of NPC proteins in isolated rat type II pneumocytes. NPC1 and NPC2 proteins were present in the lung, isolated type II cells in culture, and alveolar macrophages. The glycosylated and nonglycosylated forms of NPC1 were prominent in the lung and the lamellar body organelles. Immunocytochemical analysis of isolated type II pneumocytes showed localization of NPC1 to the limiting membrane of lamellar bodies. NPC2 and lysosomal acid lipase were found within these organelles, as confirmed by z-stack analysis of confocal images. All three proteins also were identified in small, lysosome-like vesicles. In the presence of serum, pharmacological inhibition of the NPC pathway with compound U18666A resulted in doubling of the cholesterol content of the type II cells. Filipin staining revealed a striking accumulation of cholesterol within lamellar bodies. Thus the NPC pathway functions to control cholesterol accumulation in lamellar bodies of type II pneumocytes and, thereby, may play a role in the regulation of surfactant cholesterol content.

Cytoskeleton disruption in J774 macrophages: consequences for lipid droplet formation and cholesterol flux.

Macrophages store excess unesterified cholesterol (free, FC) in the form of cholesteryl ester (CE) in cytoplasmic lipid droplets. The hydrolysis of droplet-CE in peripheral foam cells is critical to HDL-promoted reverse cholesterol transport because it represents the first step in cellular cholesterol clearance, as only FC is effluxed from cells to HDL. Cytoplasmic lipid droplets move within the cell utilizing the cytoskeletal network, but, little is known about the influence of the cytoskeleton on lipid droplet formation. To understand this role we employed cytochalasin D (cyt.D) to promote actin depolymerization in J774 macrophages. Incubating J774 with acetylated LDL creates foam cells having a 4-fold increase in cellular cholesterol content (30-40% cholesterol present as cholesteryl ester (CE)) in cytoplasmic droplets. Lipid droplets formed in the presence of cyt.D are smaller in diameter. CE-deposition and -hydrolysis are decreased when cells are cholesterol-enriched in the presence of cyt.D or latrunculin A, another cytoskeleton disrupting agent. However, when lipid droplets formed in the presence of cyt.D are isolated and incubated with an exogenous CE hydrolase, the CE is more rapidly metabolized compared to droplets from control cells. This is apparently due to the smaller size and altered lipid composition of the droplets formed in the presence of cyt.D. Cytoskeletal proteins found on CE droplets influence droplet lipid composition and maturation in model foam cells. In J774 macrophages, cytoskeletal proteins are apparently involved in facilitating the interaction of lipid droplets and a cytosolic neutral CE hydrolase and may play a role in foam cell formation. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

Early alveolar epithelial dysfunction promotes lung inflammation in a mouse model of Hermansky-Pudlak syndrome.

RATIONALE: The pulmonary phenotype of Hermansky-Pudlak syndrome (HPS) in adults includes foamy alveolar type 2 cells, inflammation, and lung remodeling, but there is no information about ontogeny or early disease mediators. OBJECTIVES: To establish the ontogeny of HPS lung disease in an animal model, examine disease mediators, and relate them to patients with HPS1. METHODS: Mice with mutations in both HPS1/pale ear and HPS2/AP3B1/pearl (EPPE mice) were studied longitudinally. Total lung homogenate, lung tissue sections, and bronchoalveolar lavage (BAL) were examined for phospholipid, collagen, histology, cell counts, chemokines, surfactant protein D (SP-D), and S-nitrosylated SP-D. Isolated alveolar epithelial cells were examined for expression of inflammatory mediators, and chemotaxis assays were used to assess their importance. Pulmonary function test results and BAL from patients with HPS1 and normal volunteers were examined for clinical correlation. MEASUREMENTS AND MAIN RESULTS: EPPE mice develop increased total lung phospholipid, followed by a macrophage-predominant pulmonary inflammation, and lung remodeling including fibrosis. BAL fluid from EPPE animals exhibited early accumulation of both SP-D and S-nitrosylated SP-D. BAL fluid from patients with HPS1 exhibited similar changes in SP-D that correlated inversely with pulmonary function. Alveolar epithelial cells demonstrated expression of both monocyte chemotactic protein (MCP)-1 and inducible nitric oxide synthase in juvenile EPPE mice. Last, BAL from EPPE mice and patients with HPS1 enhanced migration of RAW267.4 cells, which was attenuated by immunodepletion of SP-D and MCP-1. CONCLUSIONS: Inflammation is initiated from the abnormal alveolar epithelial cells in HPS, and S-nitrosylated SP-D plays a significant role in amplifying pulmonary inflammation.

Role of the PI3-kinase signaling pathway in trafficking of the surfactant protein A receptor P63 (CKAP4) on type II pneumocytes.

Surfactant protein A (SP-A) plays an important role in the maintenance of lung lipid homeostasis. Previously, an SP-A receptor, P63 (CKAP4), on type II pneumocyte plasma membranes (PM) was identified by chemical cross-linking techniques. An antibody to P63 blocked the specific binding of SP-A to pneumocytes and the ability of SP-A to regulate surfactant secretion. The current report shows that another biological activity of SP-A, the stimulation of surfactant uptake by pneumocytes, is inhibited by P63 antibody. cAMP exposure resulted in enrichment of P63 on the cell surface as shown by stimulation of SP-A binding, enhanced association of labeled P63 antibody with type II cells, and promotion of SP-A-mediated liposome uptake, all of which were inhibited by competing P63 antibody. Incubation of A549 and type II cells with SP-A also increased P63 localization on the PM. The phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway was explored as a mechanism for the transport of this endoplasmic reticulum (ER)-resident protein to the PM. Treatment with LY-294002, an inhibitor of the PI3-kinase pathway, prevented the SP-A-induced PM enrichment of P63. Exposure of pneumocytes to SP-A or cAMP activated Akt (PKB). Blocking either PI3-kinase or Akt altered SP-A-mediated lipid turnover. The data demonstrate an important role for the PI3-kinase-Akt pathway in intracellular transport of P63. The results add to the growing body of evidence that P63 is critical for SP-A receptor-mediated interactions with type II pneumocytes and the resultant regulation of surfactant turnover.

Pathway to lamellar bodies for surfactant protein A.

Alveolar surfactant protein A (SP-A) is endocytosed by type II epithelial cells through clathrin-dependent uptake and targeted to lamellar bodies for resecretion. However, the mechanism for secretion of newly synthesized SP-A, whether regulated exocytosis of lamellar bodies or constitutive secretion, is unresolved. If it is the latter, lamellar body SP-A would represent endocytosed protein. Amantadine, an inhibitor of clathrin-coated vesicle budding, was used to evaluate the role of endocytosis in accumulation of SP-A in lamellar bodies. In isolated rat lungs, amantadine (10 mM) inhibited uptake of endotracheally instilled (35)S-labeled biosynthesized surfactant proteins by >80%. To study trafficking of newly synthesized SP-A, lungs were perfused for up to 6 h with [(35)S]methionine, and surfactant was isolated from lung lavage fluid and lamellar bodies were isolated from lung homogenate. With control lungs, the mean specific activity of [(35)S]SP-A (disintegrations per minute per microgram of SP-A) increased linearly with time of perfusion: it was significantly higher in isolated lamellar bodies than in surfactant and was increased in both compartments by 50-60% in the presence of 0.1 mM 8-bromo-cAMP. These results suggest a precursor-product relationship between lamellar body and extracellular [(35)S]SP-A. Specific activities in both compartments were unaffected by addition of amantadine (10 mM) to the lung perfusate, indicating that uptake from the alveolar space was not responsible for the increase in lamellar body [(35)S]SP-A. Thus the pathway for secretion of newly synthesized SP-A is by transfer from the site of synthesis to the storage/secretory organelle prior to lamellar body exocytosis.

P63 (CKAP4) as an SP-A receptor: implications for surfactant turnover.

Surfactant protein-A (SP-A) plays an important role in the clearance of surfactant from the lung alveolar space and in the regulation of surfactant secretion and uptake by type II pneumocytes in culture. Two pathways are important for the endocytosis of surfactant by type II cells and the intact lung, a receptor-mediated clathrin-dependent pathway and a non-clathrin, actin-mediated pathway. The critical role of the clathrin/receptor-mediated pathway in normal mice is supported by the finding that SP-A gene-targeted mice use the actin-dependent pathway to maintain normal clearance of surfactant. Addition of SP-A to the surfactant of the SP-A null mice "rescued" the phenotype, further emphasizing the essential role of the SP-A/receptor-mediated process in surfactant turnover. This review presents an overview of the structure of SP-A and its function in surfactant turnover. The evidence that the interaction of SP-A with type II cells is a receptor-mediated process is presented. A newly identified receptor for SP-A, P63/CKAP4, is described in detail, with elucidation of the specific structural features of this 63 kDa, nonglycosylated, highly coiled, transmembrane protein. The compelling evidence that P63 functions as a receptor for SP-A on type II cells is summarized. Regulation of P63 receptor density on the surface of pneumocytes may be a novel approach for the regulation of surfactant homeostasis by the lung.

Reed-Sternberg cells in breast FNA of a patient with left breast mass.

Hodgkin's lymphoma is a potentially curable malignancy of the lymphatic system characterized by a variable number of scattered and large mononucleated and multinucleated tumor cells, the Hodgkin and Reed-Sternberg cells residing in an abundant heterogeneous admixture of non-neoplastic inflammatory cells. It represents approximately 30% of all lymphomas according to the World Health Organization (WHO). Patients with Hodgkin's lymphoma typically present with painless peripheral adenopathy, fever, night sweats, and weight loss. We report a rare case of Hodgkin's lymphoma presented as a breast mass in a 23-year-old woman diagnosed on fine needle aspiration (FNA). At presentation, she had no B symptoms, or palpable lymphadenopathy.

15(S)-Lipoxygenase-1 associates with neutral lipid droplets in macrophage foam cells: evidence of lipid droplet metabolism.

15(S)-lipoxygenase-1 (15-LO-1) was present in the whole-cell homogenate of an acute human monocytic leukemia cell line (THP-1). Additionally, 15-LO-1 was detected on neutral lipid droplets isolated from THP-1 foam cells. To investigate if 15-LO-1 is active on lipid droplets, we used the mouse leukemic monocytic macrophage cell line (RAW 264.7), which are stably transfected with human 15-LO-1. The RAW 15-LO-1 cells were incubated with acetylated low density lipoprotein to generate foam cells. 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], the major 15-LO-1 metabolite of arachidonic acid, was produced in the 15-LO-1 RAW but not in the mock transfected cells when incubated with arachidonic acid. Lipid droplets were isolated from the cells and incubated with arachidonic acid, and production of 15(S)-HETE was measured over 2 h. 15(S)-HETE was produced in the incubations with the lipid droplets, and this production was attenuated when the lipid droplet fraction was subjected to enzyme inactivation through heating. Efflux of 15(S)-HETE from cholesteryl ester-enriched 15-LO RAW cells, when lipid droplets are present, was significantly reduced compared with that from cells enriched with free cholesterol (lipid droplets are absent). We propose that 15-LO-1 is present and functional on cytoplasmic neutral lipid droplets in macrophage foam cells, and these droplets may act to accumulate the anti-inflammatory lipid mediator 15(S)-HETE.

Role of P63 (CKAP4) in binding of surfactant protein-A to type II pneumocytes.

We have recently described a putative receptor for lung surfactant protein-A (SP-A) on rat type II pneumocytes. The receptor, P63, is a 63-kDa type II transmembrane protein. Coincubation of type II cells with P63 antibody (Ab) reversed the inhibitory effect of SP-A on secretagogue-stimulated surfactant secretion from type II cells. To further characterize SP-A interactions with P63, we expressed recombinant P63 protein in Escherichia coli and generated antibodies to P63. Immunogold electron microscopy confirmed endoplasmic reticulum and plasma membrane localization of P63 in type II cells with prominent labeling of microvilli. Binding characteristics of iodinated SP-A to type II cells in the presence of P63 Ab were determined. Binding (4 degrees C, 1 h) of (125)I-SP-A to type II cells demonstrated both specific (calcium-dependent) and nonspecific (calcium-independent) components. Ab to P63 protein blocked the specific binding of (125)I-SP-A to type II cells and did not change the nonspecific SP-A association. A549 cells, a pneumocyte model cell line, expressed substantial levels of P63 and demonstrated specific binding of (125)I-SP-A that was inhibited by the P63 Ab. The secretagogue (cAMP)-stimulated increase in calcium-dependent binding of SP-A to type II cells was blocked by the presence of P63 Ab. Transfection of type II cells with small interfering RNA to P63 reduced P63 protein expression, attenuated P63-specific SP-A binding, and reversed the ability of SP-A to prevent surfactant secretion from the cells. Our results further substantiate the role of P63 as an SP-A receptor protein localized on the surface of lung type II cells.

Surfactant protein-A plays an important role in lung surfactant clearance: evidence using the surfactant protein-A gene-targeted mouse.

Previous studies with the isolated perfused rat lung showed that both clathrin- and actin-mediated pathways are responsible for endocytosis of dipalmitoylphosphatidylcholine (DPPC)-labeled liposomes by granular pneumocytes in the intact lung. Using surfactant protein-A (SP-A) gene-targeted mice, we examined the uptake of [(3)H]DPPC liposomes by isolated mouse lungs under basal and secretagogue-stimulated conditions. Unilamellar liposomes composed of [(3)H]DPPC: phosphatidylcholine:cholesterol:egg phosphatidylglycerol (10:5:3:2 mol fraction) were instilled into the trachea of anesthetized mice, and the lungs were perfused (2 h). Uptake was calculated as percentage of instilled disintegrations per minute in the postlavaged lung. Amantadine, an inhibitor of clathrin and, thus, receptor-mediated endocytosis via clathrin-coated pits, decreased basal [(3)H]DPPC uptake by 70% in SP-A +/+ but only by 20% in SP-A -/- lung, data compatible with an SP-A/receptor-regulated lipid clearance pathway in the SP-A +/+ mice. The nonclathrin, actin-dependent process was low in the SP-A +/+ lung but accounted for 55% of liposome endocytosis in the SP-A -/- mouse. With secretagogue (8-bromoadenosine 3',5'-cyclic monophosphate) treatment, both clathrin- and actin-dependent lipid clearance were elevated in the SP-A +/+ lungs while neither pathway responded in the SP-A -/- lungs. Binding of iodinated SP-A to type II cells isolated from both genotypes of mice was similar indicating a normal SP-A receptor status in the SP-A -/- lung. Inclusion of SP-A with instilled liposomes served to "rescue" the SP-A -/- lungs by reestablishing secretagogue-dependent enhancement of liposome uptake. These data are compatible with a major role for receptor-mediated endocytosis of DPPC by granular pneumocytes, a process critically dependent on SP-A.

Expression and biological activity of ABCA1 in alveolar epithelial cells.

The mechanisms used by alveolar type I pneumocytes for maintenance of the lipid homeostasis necessary to sustain these large squamous cells are unknown. The processes may involve the ATP-binding cassette transporter A1 (ABCA1), a transport protein shown to be crucial in apolipoprotein A-I (apoA-I)-mediated mobilization of cellular cholesterol and phospholipid. Immunohistochemical data demonstrated the presence of ABCA1 in lung type I and type II cells and in cultured pneumocytes. Type II cells isolated from rat lungs and cultured for 5 days in 10% serum trans-differentiated toward cells with a type I-like phenotype which reacted with the type I cell-specific monoclonal antibody VIIIB2. Upon incubation of the type I-like pneumocytes with agents that up-regulate the ABCA1 gene (9-cis-retinoic acid [9cRA] and 22-hydroxycholesterol [22-OH, 9cRA/22-OH]), ABCA1 protein levels were enhanced to maximum levels after 8 to 16 hours and remained elevated for 24 hours. In the presence of apoA-I and 9cRA/22-OH, efflux of radioactive phospholipid and cholesterol from pneumocytes was stimulated 3- to 20-fold, respectively, over controls. Lipid efflux was inhibited by Probucol. Sucrose density gradient analysis of the media from stimulated cells incubated with apoA-I identified heterogeneous lipid particles that isolated at a density between 1.063 and 1.210 g/ml, with low or high apoA-I content. Thus, pneumocytes with markers for the type I phenotype contained functional ABCA1 protein, released lipid to apoA-I protein, and were capable of producing particles resembling nascent high-density lipoprotein, indicating an important role for ABCA1 in the maintenance of lung lipid homeostasis.

Identification and characterization of p63 (CKAP4/ERGIC-63/CLIMP-63), a surfactant protein A binding protein, on type II pneumocytes.

Surfactant protein A (SP-A) binds to alveolar type II cells through a specific high-affinity cell membrane receptor, although the molecular nature of this receptor is unclear. In the present study, we have identified and characterized an SP-A cell surface binding protein by utilizing two chemical cross-linkers: profound sulfo-SBED protein-protein interaction reagent and dithiobis(succinimidylpropionate) (DSP). Sulfo-SBED-biotinylated SP-A was cross-linked to the plasma membranes isolated from rat type II cells, and the biotin label was transferred from SP-A to its receptor by reduction. The biotinylated SP-A-binding protein was identified on blots by using streptavidin-labeled horseradish peroxidase. By using DSP, we cross-linked SP-A to intact mouse type II cells and immunoprecipitated the SP-A-receptor complex using anti-SP-A antibody. Both of the cross-linking approaches showed a major band of 63 kDa under reduced conditions that was identified as the rat homolog of the human type II transmembrane protein p63 (CKAP4/ERGIC-63/CLIMP-63) by matrix-assisted laser desorption ionization and nanoelectrospray tandem mass spectrometry of tryptic fragments. Thereafter, we confirmed the presence of p63 protein in the cross-linked SP-A-receptor complex by immunoprobing with p63 antibody. Coimmunoprecipitation experiments and functional assays confirmed specific interaction between SP-A and p63. Antibody to p63 could block SP-A-mediated inhibition of ATP-stimulated phospholipid secretion. Both intracellular and membrane localized pools of p63 were detected on type II cells by immunofluorescence and immunobloting. p63 colocalized with SP-A in early endosomes. Thus p63 closely interacts with SP-A and may play a role in the trafficking or the biological function of the surfactant protein.

Pulmonary abnormalities due to ABCA1 deficiency in mice.

Mice gene targeted for ATP-binding cassette transporter A1 (ABCA1; Abca1(-/-)) have been shown to have low-serum high-density lipoprotein and abnormal lung morphology. We examined alterations in the structure and function of lungs from -/- mice (DBA1/J). Electron microscopy of the diseased mouse lung revealed areas of focal disease confirming previous results (47). Lipid analysis of the lung tissue of -/- mice showed a 1.2- and 1.4-fold elevation in total phospholipid (PL) and saturated phosphatidylcholine, respectively, and a marked 50% enrichment in total cholesterol content predominantly due to a 17.5-fold increase in cholesteryl ester compared with wild type (WT). Lung surfactant in the -/- mice was characterized by alveolar proteinosis (161%), a slight increase in total PL (124%), and a marked increase in free cholesterol (155%) compared with WT. Alveolar macrophages were enriched in cholesterol (4.8-fold) due to elevations in free cholesterol (2.4-fold) and in cholesteryl ester (14.8-fold) compared with WT macrophages. More PL mass was cleared from the alveolar space of -/- mice lungs, measured using intratracheal installation of (3)H-PL liposomes. Compared with WT mice, the Abca1(-/-) mice demonstrated respiratory distress with rapid, shallow breathing. Thus the lungs of mice lacking ABCA1 protein demonstrated abnormal morphology and physiology, with alveolar proteinosis and cholesterol enrichment of tissue, surfactant, and macrophages. The results indicate that the activity of ABCA1 is important for the maintenance of normal lung lipid composition, structure, and function.

Pathways for clearance of surfactant protein A from the lung.

Uptake and degradation of (125)I-surfactant protein A (SP-A) over a 1-h period was studied in alveolar cells in culture and in isolated perfused lungs to elucidate the mechanism for clearance of the protein from the alveolar space. Specific inhibitors of clathrin- and actin-dependent endocytosis were utilized. In type II cells, uptake of SP-A, compared with controls, was decreased by 60% on incubation with clathrin inhibitors (amantadine and phenylarsine oxide) or with the actin inhibitor cytochalasin D. All agents reduced SP-A metabolism by alveolar macrophages. Untreated rat isolated perfused lungs internalized 36% of instilled SP-A, and 56% of the incorporated SP-A was degraded. Inhibitors of clathrin and actin significantly reduced SP-A uptake by approximately 54%, whereas cytochalasin D inhibited SP-A degradation. Coincubation of agents did not produce an additive effect on uptake of SP-A by cultured pneumocytes or isolated perfused lungs, indicating that all agents affected the same pathway. Thus SP-A clears the lung via a clathrin-mediated pathway that requires the polymerization of actin.

Paracrine stimulation of surfactant secretion by extracellular ATP in response to mechanical deformation.

We developed a heterologous system to study the effect of mechanical deformation on alveolar epithelial cells. First, isolated primary rat alveolar type II (ATII) cells were plated onto silastic substrata coated with fibronectin and maintained in culture under conditions where they become alveolar type I-like (ATI) cells. This was followed by a second set of ATII cells labeled with the nontransferable, vital fluorescent stain 5-chloromethylfluorescein diacetate to distinguish them from ATI cells. By morphometric analysis, equibiaxial deformation (stretch) of the silastic substratum induced comparable changes in cell surface area for both ATII and ATI cells. Surfactant lipid secretion was measured using cells metabolically labeled with [(3)H]choline. In response to 21% tonic stretch for 15 min, ATII cells seeded with ATI cells secreted nearly threefold more surfactant lipid compared with ATII cells seeded alone. ATI cells did not secrete lipid in response to stretch. The enhanced lipid secretion by ATII plus ATI cocultures was inhibited by treatment with apyrase and adenosine deaminase, suggesting that ATP release by ATI cells enhanced surfactant lipid secretion at 21% stretch. This was confirmed using a luciferase assay where, in response to 21% stretch, ATI cells released fourfold more ATP than ATII cells. Because ATI cells release significantly more ATP at a lower level of stretch than ATII cells, this supports the hypothesis that ATI cells are mechanosensors in the lung and that paracrine stimulation of ATII cells by extracellular ATP released from ATI cells plays a role in regulating surfactant secretion.

Defective surfactant secretion in a mouse model of Hermansky-Pudlak syndrome.

Hermansky-Pudlak syndrome (HPS) in humans represents a family of disorders of lysosome-related organelle biogenesis associated with severe, progressive pulmonary disease. Human case reports and a mouse model of HPS, the pale ear/pearl mouse (ep/pe), exhibit giant lamellar bodies (GLB) in type II alveolar epithelial cells. We examined surfactant proteins and phospholipid from ep/pe mice to elucidate the process of GLB formation. The 2.8-fold enrichment of tissue phospholipids in ep/pe mice resulted from accumulation from birth through adulthood. Tissue surfactant protein (SP)-B and -C were increased in adult ep/pe mice compared with wild-type mice (WT), whereas SP-A and -D were not different. Large aggregate surfactant (LA) from adult ep/pe mice had decreased phospholipid, SP-B, and SP-C, with no differences in SP-A and -D compared with WT. Although LA from ep/pe animals exhibited an increased total protein-to-total phospholipid ratio compared with WT, surface tension was not compromised. Phospholipid secretion from isolated type II cells showed that basal and stimulated secretion from ep/pe cells were approximately 50% of WT cells. Together, our data indicate that GLB formation is not associated with abnormal trafficking or recycling of surfactant material. Instead, impaired secretion is an important component of GLB formation in ep/pe mice.

IL-4 induces production of the lung collectin surfactant protein-D.

BACKGROUND: Surfactant protein (SP)-D is an epithelial cell product of the distal air spaces that aids uptake and clearance of inhaled pathogens and allergens. Allergic airway inflammation significantly increases SP-D levels in the bronchoalveolar lavage fluid in asthmatic patients and mouse models, but the mechanisms involved remain unknown. OBJECTIVE: To investigate the effects of the TH2-type cytokine IL-4 on SP-D production by isolated pulmonary epithelial cells. METHODS: Rat type II alveolar epithelial cells were purified and cultured with dexamethasone, cAMP, and isobutyl-1-methylxanthine (DCI). The effects of IL-4 on SP-D expression were investigated at the protein and mRNA levels by means of Western and Northern blot analyses. RESULTS: In contrast to a lamellar body protein ABCA3 and surfactant protein-A, expression of SP-D significantly declined when cells were cultured in medium alone for 24 hours. The presence of DCI in the culture medium restored SP-D levels, which were enhanced by 2-fold after addition of recombinant IL-4. The enhancing effects of IL-4 were concentration-dependent, with maximum effects observed at 20 ng/mL (1.43 nmol/L). IL-4 did not rescue cycloheximide-induced decrease of intracellular SP-D levels and did not inhibit extracellular release of SP-D. However, IL-4 significantly augmented DCI-induced SP-D mRNA expression by approximately 2.5-fold over control levels. CONCLUSIONS: IL-4 selectively upregulates SP-D expression, and it may act at the level of mRNA in isolated pulmonary epithelial cells. Since SP-D has a potent anti-inflammatory function, this mechanism may be part of a negative feedback loop providing a regulatory link between adaptive and innate immunity during allergic inflammation.