Pulmonary Surfactant Apoprotein Research Articles

The “wisdom” of lung surfactant: balancing host defense and surface tension-reducing functions

the mixture of lipids and proteins that comprises pulmonary surfactant is both complex and multifunctional. Although surfactant was originally identified as a lipoprotein complex that reduces surface tension at the air-liquid interface of the lung ([4][1], [20][2]), both in vitro and in vivo studies

Novel approach to quantitative reverse transcription PCR assay of mRNA component in autopsy material using the TaqMan fluorogenic detection system: dynamics of pulmonary surfactant apoprotein A

A novel approach to quantitative reverse transcription (RT)-PCR assay of mRNA component using fluorescent TaqMan methodology and a new instrument (ABI Prism 7700 sequence detection system) was developed for autopsy materials. Pulmonary surfactant apoprotein A (SP-A) mRNA from a cadaveric lung was quantitated in real-time. The target SP-A gene and the endogenous reference of glyceraldehyde-3-phosphate (GAPDH) were amplified in the same tube, and an amount of the target was normalized to the reference. This assay had a high reproducibility and discrimination even in forensic autopsy materials up to 96 h postmortem. Elevated SP-A expressions were determined in some cases. This system without post-PCR sample handling would be a very useful tool in pathological diagnosis and DNA analysis.

Immunohistochemistry of pulmonary surfactant apoprotein A in forensic autopsy: reassessment in relation to the causes of death

To reassess the immunohistochemical distribution of pulmonary surfactant apoprotein A (SP-A) in relation to the causes of death, 282 forensic autopsy cases were reviewed. The most intense and dense granular immunostaining of intra-alveolar SP-A was observed in the hyaline membrane syndrome from various traumas, protracted death from drowning, and perinatal aspiration of amniotic fluid. Similar granular staining pattern was found in fatal poisoning by a muscle relaxant and organophosphate pesticides. An evident increase of intra-alveolar granular staining was noted in most fatalities from mechanical asphyxia and drowning, and some cases of fire death. SP-A staining was usually very weak or sparse in alcohol intoxication, poisoning by hypnotics and also carbon monoxide poisoning. These findings suggest that the amount of intra-alveolar granular SP-A staining may be a possible indicator of severity and duration of respiratory distress (agony) from peripheral (non-central nervous system) origin and alveolar damage.

Ovine surfactant protein cDNAs: use in studies on fetal lung growth and maturation after prolonged hypoxemia.

cDNAs for ovine surfactant-associated protein (SP) A, SP-B, and SP-C have been cloned and shown to possess strong similarity to cDNAs for surfactant apoproteins in other species. These reagents were employed to examine the effect of fetal hypoxia on the induction of surfactant apoprotein expression in the fetal lamb. Postnatal lung function is dependent on adequate growth and maturation during fetal development. Insulin-like growth factor (IGF) I and IGF-II, which are present in all fetal tissues studied, possess potent mitogenic and proliferative actions, and their effects can be modulated by IGF-specific binding proteins (IGFBPs). Hypoxia can lead to increases in circulating cortisol and catecholamines that can influence lung maturation. Therefore, the effects of mild hypoxia in chronically catheterized fetal lambs at gestational days 126-130 and 134-136 (term 145 days) on the expression of pulmonary surfactant apoproteins and IGFBPs were examined. Mild hypoxia for 48 h resulted in an increase in plasma cortisol that was more pronounced at later gestation, and in these animals, there was a twofold increase in SP-A mRNA. SP-B mRNA levels also increased twofold, but this was not significant. SP-C mRNA was not altered. No significant changes in apoprotein mRNA were observed with the younger fetuses. However, these younger animals selectively exhibited reduced IGFBP-5 mRNA levels. IGF-I mRNA was also reduced at 126-130 days, although this conclusion is tentative due to low abundance. IGF-II levels were not affected at either gestational age. We conclude that these data suggest that mild prolonged fetal hypoxia produces alterations that could affect fetal cellular differentiation early in gestation and can induce changes consistent with lung maturation closer to term.

Identification of Genes (SPON2 and C20orf2) Differentially Expressed between Cancerous and Noncancerous Lung Cells by mRNA Differential Display

mRNA differential display was applied to three small cell lung carcinoma (SCLC) cell lines, six non-small cell lung carcinoma (NSCLC) cell lines, and three normal lung tissues to identify genes differentially expressed between lung carcinoma cells and normal lung tissues and between SCLC cells and NSCLC cells. We isolated five differentially expressed genes, two that were novel and three that were already known. DIL-1 (differentially expressed in cancerous and noncancerous lung cells; HGMW-approved symbol SPON2) and pulmonary surfactant apoprotein A were expressed in normal lung tissues but not in lung carcinoma cell lines, whereas DIL-2 (HGMW-approved symbol C20orf2) and nm23-H1 were expressed in lung carcinoma cell lines but not in normal lung tissues. The remaining gene, Annexin II, was expressed at a lower level in SCLC than in NSCLC and normal lung tissues. These genes were also differentially expressed in primary lung cancers. One of the two novel genes, DIL-1, encodes a secreted protein homologous to the Mindin/F-spondin family. The other, DIL-2, encodes a protein with a putative ATP/GTP binding site motif. These data provide basic information necessary to understand the differences in gene expression profiles between lung carcinoma and normal lung and between SCLC and NSCLC. Further characterization of these genes will help to clarify the molecular mechanisms of human lung carcinogenesis.

Immunohistochemical localization of surfactant apoproteins in usual interstitial pneumonia associated with pulmonary carcinoma.

Surfactant apoproteins A and B (SP-A and SP-B) are antigenic determinants of pulmonary surfactant complexes. The role and functional significance of these proteins are largely unknown and the pattern of expression is probably related to the functional maturation of type II pneumocytes. Differential expression of SP-A and SP-B was reported in the developing human lung but little is known of their expression in the chronic injury. We studied 5 surgical cases of usual interstitial pneumonia (UIP) associated with carcinoma to evaluate the expression of pulmonary surfactant apoproteins. These cases were immunohistochemically examined by the streptavidin-biotin complex method using monoclonal antibodies HS-1 and HS-2 against pulmonary surfactant apoprotein A (SP-A) and B (SP-B), respectively. In UIP, SP-B was expressed strongly in type II pneumocytes and Clara cells but bronchiolar epithelium and metaplastic squamous cell lines in the honeycomb lesion were non-reactive. SP-A showed a similar pattern but much weaker reactivity when compared to that of SP-B. Type II pneumocytes in normal lung tissue exhibited weak immunoreactivity and no difference in the intensity of staining between SP-A and SP-B. Neither carcinomatous area nor metaplastic lining cells at honeycomb lesion show immunoreactivity to SP-A and SP-B. These results suggest that type II pneumocytes in the UIP are functionally immature in their expression of the apoprotein types and the metaplastic squamous cells or neoplastic transformed cells do not have molecular characteristics of type II pneumocytes.

Protein 1 and Clara cell 10-kDa protein distribution in normal and neoplastic tissues with emphasis on the respiratory system.

Thirty-six different normal tissues and 13 different malignant epithelial tumours, were examined immunohistochemically for the presence of protein 1 (P1) and Clara cell 10-kDa protein (CC10). Adenocarcinomas of the lung were also examined for the expression of pulmonary surfactant apoprotein using a monoclonal antibody (PE-10). The staining results of P1 and CC10 were almost identical both in normal tissues and in malignant tumours. In normal lung, Clara cells were strongly positive for both P1 and CC10. In addition, some goblet cells and non-ciliated non-mucus cells in the upper airways were moderately positive for both proteins. In the malignant tumours, some lung cancers were positive for P1 and CC10, both of which were positive in the same tumour cells on sequential sections. In 117 lung cancers, P1 and CC10 were positive in 10.2% of adenocarcinomas, 20.5% of squamous cell carcinomas, and 12.5% of large cell carcinomas. PE-10 stained positively in 65.3% of adenocarcinomas, a frequency significantly higher than that of P1 and CC10 (P < 0.01). These results suggest that P1 and CC10 are nearly identical proteins, that both are useful markers of Clara cells, and that many pulmonary adenocarcinomas express surfactant apoprotein rather than Clara cell proteins.

An expression profile of active genes in human lung.

An expression profile of genes active in the human lung was obtained by collecting 797 partial sequences from a 3'-directed cDNA library. Three genes were found to produce mRNA each of which comprised more than 1% of total mRNA. These three have been identified as genes for pulmonary surfactant apoprotein (PSP-A), Clara cells 10-kDa secretory protein, and HLA-E heavy chain. In the remaining 745 clones, 221 were composed of 89 species that occurred recurrently, and 524 clones appeared only once. Because the 3'-directed cDNA library faithfully represents the mRNA population in the source tissue, these numbers represent the relative activities of the gene expression. Altogether 437 gene species were novel, and 179 gene species were identified in GenBank. A significant portion of these genes encode proteins found in secretory proteins, cell surface proteins, and components in the protein synthesis machinery, representing the function of the lung.

Laryngeal Metastasis from a Pulmonary Papillary Adenocarcinoma: A Case Report

Metastases to the larynx from distant primaries are very rare. The present article reports a case of metastatic papillary adenocarcinoma of the larynx of lung origin. The patient was a 59-year-old female non-smoker, who had a history of adenocarcinoma of the right lung. For the laryngeal tumor, we performed a partial laryngectomy following biopsy. The tumor of the larynx was a papillary adenocarcinoma resembling the lung tumor, both demonstrating positive immunohistochemical staining for pulmonary surfactant apoprotein. The findings emphatically indicated the laryngeal tumor to be metastasis from the primary papillary adenocarcinoma of the lung. The present case report presents the clinical findings, course of disease and histopathological findings with brief reviews of the literature.

Collectins: pattern recognition molecules involved in first line host defense

A group of chimeric molecules comprising globular heads, which contain the carbohydrate recognition domain, and collagen tails are defined as Collectins. The mannose-binding proteins, pulmonary surfactant apoproteins A and D and conglutinin all qualify as members of this family, whose function appears to be as pattern recognition molecules involved in the first line of defense in the pre-immune host.

Amphipathic α-helical peptides based on surfactant apoprotein SP-A

Three peptides based on the putative amphipathic helical region of the major pulmonary surfactant apoprotein (SP-A) were synthesized by solid-phase techniques, mixed with DPPC and tested for efficacy as lung surfactants in an in vitro adult rat lavaged lung model. The peptides correspond to residues 81–102 (SP-A 81–102) and 78–101 (SP-A 78–101) of the native human sequence and an analog with increased hydrophobicity, Leu 84,90SP-A 78–101. Neither native sequence was effective in simple mixtures with DPPC. However, substitution of leucine residues for Asp 84 and Thr 90 of SP-A 81–102 yielded a peptide which was active in mixtures with DPPC, restoring quasi-static lung compliance to 90% of the unlavaged value. In the absence of peptide, DPPC had no effect on the P-V curve of the lavaged lung. The activity of the Leu 84,90 analog correlated with an increased amphipathic α-helical potential and an improvement in several predictive parameters for lipid-binding. The similarities between this active peptide and other active amphipathic α-helical peptides lend support to the hypothesis that amphipathic α-helical potential and the size of the hydrophobic face are critical for functional synthetic surfactant peptides in simple mixtures with dipalmitoylphosphatidylcholine.

Overexpression of pulmonary surfactant apoprotein A mRNA in alveolar type II cells and nonciliated bronchiolar (Clara) epithelial cells in streptozotocin-induced diabetic rats demonstrated by in situ hybridization.

Pulmonary surfactant is critical for gas exchange and is composed of both phospholipids and specific surfactant-associated proteins. The most abundant surfactant protein is termed surfactant apoprotein A (SP-A). This protein is thought to be important in the formation of tubular myelin, in absorption of surfactant to the air-liquid interface, in recycling of surfactant in alveolar type II cells, and in the regulation of secretion. We have examined the expression and localization of SP-A mRNA in streptozotocin-induced diabetic rats by in situ hybridization using a specific rat cDNA probe. Diabetes was induced by intraperitoneal injection of 60 mg/kg streptozotocin. After 10 wk, lungs were excised and examined by in situ hybridization and by light and electron microscopy. The ultrastructural examination demonstrated the marked changes of endoplasmic reticulum of alveolar type II cells, as reported previously. Immunohistostaining of SP-A in diabetic lungs was weak in alveolar type II cells. However, by autoradiographs of in situ hybridization, compared with the control lungs, a larger number of silver grains for the SP-A mRNA were shown in alveolar type II cells and also in some bronchiolar epithelial (Clara) cells from the diabetic lungs. Alveolar type II cells having high contents of silver grains were also increased in number. These results were confirmed by measurement of the SP-A content and by Northern blot analysis. The present study demonstrates an overexpression of SP-A mRNA despite the ultrastructural changes in the endoplasmic reticulum of alveolar type II cells in the diabetic lungs, which will provide new information on the regulatory mechanism of SP-A gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acid sequences of the two subunits of a phospholipase A2 inhibitor from the blood plasma of Trimeresurus flavoviridis. Sequence homologies with pulmonary surfactant apoprotein and animal lectins.

Phospholipase A2 inhibitor (PLI), purified from the blood plasma of the Habu snake (Trimeresurus flavoviridis), was separated into two distinct subunits, PLI-A and PLI-B. These subunits were shown to be glycoproteins with molecular weights of around 21,000-22,000. When they were deglycosylated chemically with trifluoromethanesulfonic acid, the molecular weights were found to be 17,000. Their amino acid sequences were determined by alignment of peptides obtained by lysyl endopeptidase digestion and Staphylococcus aureus V8 protease digestion. PLI-A and PLI-B were each composed of 147 amino acid residues with one residue, Asn103, being for N-linked glycosylation, and the molecular weights of their protein portions were calculated to be 16,368 and 16,408, respectively. Each subunit contained four cysteine residues, all of which exist in disulfide linkages (Cys64-Cys141 and Cys119-Cys133). The sequences of PLI-A and PLI-B showed 89.9% homology to each other. When the sequences were compared with those of lipocortins, no significant homologies were detected. But the sequences were significantly homologous to those of COOH-terminal carbohydrate recognition portions of pulmonary surfactant apoprotein and animal lectins.

Surfactant Apoprotein A (SP-A) Is Synthesized in Airway Cells

The pulmonary surfactant apoproteins A, B, and C (SP-A, SP-B, and SP-C, respectively) function in concert with surfactant phospholipids to reduce surface pressure in the alveolus. Surfactant apoproteins also regulate surfactant synthesis, secretion, adsorption, and recycling. SP-A and B have been localized by immunocytochemistry to alveolar epithelial (type II) cells, alveolar macrophages, and nonciliated bronchiolar epithelial (Clara) cells. In contrast, in situ hybridization to SP-A and B mRNA in human lung has shown SP-A and B transcripts in type II cells, but only SP-B message in Clara cells, implying that synthesis of SP-A occurs exclusively in type II cells. In this report, in situ hybridization to SP-A mRNA was performed on adult and developing rabbit lung and on human lung. SP-A transcripts were found in type II cells and bronchiolar epithelium of both species. The distribution of SP-A message-containing cells in the bronchiolar epithelium of rabbits and humans was similar to the distribution of Clara cells in these two species. These data indicate that SP-A is not only synthesized in type II cells but also in Clara cells.

Pulmonary Corpora Amylacea Contain Surfactant Apoprotein

The case of a 53-year-old female with interstitial pneumonitis is described with special regard to biochemical characterization of pulmonary corpora amylacea which were found in the lung specimen obtained by bronchial biopsy from the patient. The main protein component in bronchoalveolar lavage (BAL) fluid of the patient was albumin, but proteins in the precipitate fraction of BAL fluid, where the corpora amylacea were recovered, predominantly consisted of 36 kD protein which was stained with the monoclonal antibody PE 10 to human pulmonary surfactant apoprotein by immunoblot. Histologically the pulmonary corpora amylacea were stained with eosin and PAS. The particles were stained immunohistochemically by immunoperoxidase reaction using PE 10, but not by antibodies to human albumin. The pulmonary surfactant apoprotein seems, therefore, to be not simply adsorbed in the particles, but to be contained in them. Thus, the surfactant apoprotein may, at least in this case, be involved in the formation of pulmonary corpora amylacea.

Primary Lung Cancer in an 18-year-old Boy: Case Report

An autopsy case of an 18-year-old boy with adenocarcinoma of the lung is reported. He experienced dyspnea and hemosputum in July 1988. Chest radiographs showed a diffuse bilateral streaky shadow, bilateral pleural effusion and cardiac enlargement. The diagnosis of adenocarcinoma was made by transbronchial biopsy at another hospital. He visited the National Cancer Center Hospital on October 7, 1988. The diagnosis of lung cancer was strongly suggested by positive immunohistochemical staining for pulmonary surfactant apoprotein in biopsy specimens from supraclavicular lymph nodes. Intensive systemic survey demonstrated no other primary site than the lung. The patient was treated with cisplatin, adriamycin and etoposide and his subjective symptoms such as cough and dyspnea significantly improved over the next three months. Tumor shadows in the lung increased steadily, however after February, 1989. A significant lymphangitic spread of the carcinoma and marked obsteoblastic bone metastases were revealed at autopsy.

Biophysical and biological activity of a synthetic 8.7-kDa hydrophobic pulmonary surfactant protein SP-B.

We have synthesized pulmonary surfactant apoprotein SP-B peptides by solid-phase chemistry and demonstrated their ability to enhance the surface-active properties of synthetic lipid mixtures. The synthetic peptides were reactive with antiserum generated against the native bovine surfactant peptide. Both peptides conferred surfactant-like properties to synthetic lipid mixtures as assessed by a Wilhelmy balance and pulsating bubble surfactometer. Likewise, mixtures of synthetic SP-B peptides and lipid restored compliance of isolated surfactant-deficient rat lungs. This work demonstrates the utility of SP-B as a functional component of pulmonary surfactant mixtures for treatment of respiratory distress syndrome or other disorders characterized by surfactant deficiency.

Changes in gene expression in hyperoxia-induced neonatal lung injury

Exposure to high concentrations of oxygen (hyperoxia) can result in lung injury. The biochemical basis of this injury is poorly understood, but it is likely to include alterations in gene expression. Hyperoxia-induced (H-I) cDNAs have been molecularly cloned (Horowitz et al. J. Biol. Chem. 264: 7092-7095, 1989) from the lungs of an adult rabbit exposed to toxic levels of oxygen. One of them (H-I 1) was identified as encoding the tissue inhibitor of metalloproteinases (TIMP), a key regulatory protein of extracellular matrix turnover. Here we identify another clone (H-I 3) as encoding pulmonary surfactant apoprotein A (SP-A). We also show that in neonatal rabbits exposed to 100% oxygen for 96 h, the mRNAs corresponding to TIMP, SP-A, and another H-I gene are increased. These studies have begun to explore specific changes in gene expression associated with neonatal hyperoxic lung injury.

Lamellar body formation precedes pulmonary surfactant apoprotein expression during embryonic mouse lung development in vivo and in vitro

Abstract The purpose of this investigation was to determine whether lamellar inclusion body (LB) formation and surfactant apoprotein (SP-35) production are directly coordinated by temporal and positional information during development. In the present study we report a comparison between embryonic B10.A mouse lung morphogenesis and cytodifferentiation in vivo with that observed during organ culture in serumless medium. Precursor LB were first detected at embryonic day 12 (E12d), and progressively larger numbers and forms were produced during subsequent differentiation of respiratory alveolar duct epithelium. SP-35 was first detected during the canalicular period (E16.5d). Lung cultures (E12 d) showed pseudoglandular and canalicular periods of morphogenesis, and both ciliated epithelial and type II cell differentiation. Nonciliated cells produced increasing numbers of lamellar inclusion bodies throughout the culture period. SP-35 was detected at 9 days in vitro (d.i.v.). These observations indicate (i) precursor LB formation precedes SP-35 expression and is not dependent on apoprotein synthesis; (ii) E12d lung development in vitro using serumless medium proceeds at a rate equivalent to 0.5 days in vivo through 11 d.i.v.; and (iii) morphogenesis and differentiation occur in the absence of exogenous hormones and growth factors. The cell-cell interactions that play a role in morphogenesis and cell differentiation appear to be intrinsic to the developmental program for embryonic lung development and are likely to be mediated by autocrine and/or paracrine factors.

Pulmonary surfactant apoproteins: a review of protein and genomic structure.

In recent years, as the complexity of the surfactant system has become more apparent, investigators with an increasingly diverse set of skills have been attracted to the study of this secretory product of the alveolar epithelium. In addition to advancing our knowledge of the mechanisms underlying the mechanical stability of the lung, recent studies of the surfactant system have also contributed information to less organ-specific biological phenomena such as exocytosis, endocytosis, cell differentiation and lipid-protein interactions in biomembranes. Pulmonary surfactant is not composed of a single class of molecules but, rather, is a collection of interrelated macromolecular lipoprotein complexes that differ in composition, structure, and function. The purpose of this review is to describe the structure of the lung-specific proteins that are associated with the phospholipids of surfactant in the alveolar space. The organization of the genes for the surfactant proteins is outlined and the affects of these proteins on the properties of phospholipid membranes are discussed.