Surfactant apoprotein in nonmalignant pulmonary disorders.

Formalin-fixed, paraffin-embedded lungs exhibiting a variety of nonmalignant disorders were studied by immunoperoxidase staining using antibodies specific for surfactant apoprotein, IgG, IgM, IgA, albumin, fibrinogen, and lysozyme. Normal Type II pneumocytes showed staining for surfactant apoprotein in the perinuclear region only. The extent and intensity of staining for apoprotein was markedly increased in reactive Type II pneumocytes. This increase appeared to be a nonspecific reaction to lung injury. The intra-alveolar material in pulmonary alveolar proteinosis stained intensely for surfactant apoprotein, indicating that the accumulated proteinaceous material contained pulmonary surfactant. Type II pneumocytes in pulmonary alveolar proteinosis exhibited hyperplasia as well as hypertrophy. The few macrophages in lung affected by pulmonary alveolar proteinosis stained intensely for lysozyme. The excessive intraalveolar accumulation of proteinaceous material in pulmonary alveolar proteinosis may be the result of both an over-production as well as a deficient removal of pulmonary surfactant.     

Presence of apocrine epithelial antigen (AEA) in type II pneumocytes and in hyaline membranes of neonatal RDS

We have investigated the distribution of an apocrine membrane antigen (AEA) in pulmonary tissue using a rabbit antiserum raised against fat globule glycoproteins isolated from human milk. In indirect immunostaining (PAP, IF) of sections from normal lung tissue, the membranes facing the alveolar lumen of cells corresponding to the type II pneumocytes in the alveolar walls were decorated. The selective distribution of AEA to the membranes of type II pneumocytes was confirmed in double immunostaining by identification of these cells with rat antibodies against surfactant apoprotein. In fetal lung tissue, the AEA antigen was detected by the 9th week of gestation. In lung samples from newborns which had died of respiratory distress syndrome (RDS) the intra-alveolar hyaline membranes stained for the AEA antigen. SDS-PAGE of the immunoprecipitate obtained with anti-AEA serum from radiolabelled glycoprotein fraction of normal lung tissue revealed a single band of 79,000 dalton apparent molecular weight. These findings indicate that the AEA constitutes a membrane marker of the type II pneumocytes and might be involved in the secretory process of surfactant. Immunohistological evidence for the presence of AEA in the hyaline membranes of neonatal RDS is also presented.     

Identification of a cell membrane protein that binds alveolar surfactant.

Alveolar surfactants are complex mixtures of proteins and phospholipids produced by type II alveolar cells and responsible for lowering pulmonary surface tension. The process by which surfactant is produced and exported and by which its production by pulmonary cells is regulated are not well understood. This study was designed to identify a cellular receptor for surfactant constituents. To do so, monoclonal anti-idiotypic antibodies directed against antibodies to porcine and rabbit surfactant proteins were prepared. These monoclonal anti-idiotypic antibodies bind both alveolar lining and bronchial epithelial cells in rabbit, porcine, and human lungs. Macrophages and other nonepithelial cells do not react with these antibodies. Western blot analysis indicates that both A2R and A2C recognize the same proteins in both pig and rabbit lungs: a 30-kd protein and additional proteins at 52 and 60 kd. Preincubating lung wash cells with A2C or A2R prevents binding of porcine or rabbit surfactant preparations, respectively, by these cells. Preincubating frozen sections of lung tissue with surfactant inhibits binding of A2R and A2C to the lung. Antibody directed to a cell membrane protein that recognizes alveolar surfactant may be useful in elucidating the structure and function of this receptor and in understanding the cellular physiology and pathophysiology of the surfactant system.     

Four monoclonal antibodies,AMH-1, -2, -3, and -4, give varied reactivities with monocytes,alveolar macrophages,and epithelioid-cell granulomas

Four monoclonal antibodies, termed AMH-1, AMH-2, AMH-3, and AMH-4, raised against human lung macrophages in bronchoalveolar lavaged fluid, alveolar spaces, and interstitia of lung tissue are described. The antibodies were produced according to hybridoma technique by immunizing mice with bronchoalveolar lavaged cells. All four monoclonal antibodies reacted with macrophages in bronchoalveolar lavaged fluid and alveolar spaces by immunohistochemical staining and flow cytometric analysis, but they gave different reactivity patterns with the monocyte-macrophage lineage. AMH-1 did not react with peripheral blood monocytes, peritoneal macrophages, or pulmonary interstitial macrophages. Although AMH-2 reacted weakly with blood monocytes and with some of the pulmonary interstitial macrophages, it did not react with peritoneal macrophages. AMH-3 did not show reactivities with either blood monocytes or peritoneal macrophages but was positive for most of the pulmonary interstitial macrophages. AMH-4 was reactive with cells from the monocyte-macrophage lineage. There was a correlation between the reactivity patterns of all four antibodies to macrophages in bronchoalveolar lavaged fluid and the patients' smoking habits. Most significantly, epithelioid cells of lung granulomas obtained from patients with sarcoidosis and hypersensitivity pneumonitis were negative for AMH-1 but were strongly stained by AMH-2, AMH-3, and AMH-4. Differences among the four antibodies in their reactivities with macrophages and granulomas in lungs indicate that lung macrophages contain heterogeneous populations which are in various states of differentiation and maturation and that the epithelioid cells and lung macrophages share the same membrane antigens. Therefore, these antibodies would be useful reagents for investigating the subpopulations and functions of macrophages in lungs and for clarifying the pathogenesis of granulomatous lung diseases.     

Morphogenetic and functional activity of type II cells in early fetal rhesus monkey lungs. A comparison between primates and rodents.

To evaluate further the role of type II alveolar epithelial cells in primate lung development, lungs of fetal (46 to 155 days gestational age [DGA]), postnatal, and adult rhesus monkeys were investigated with antibodies against surfactant protein A (SP-A), Alcian blue (AB) staining, and periodic acid-Schiff (PAS) staining with/without alpha-amylase pre-treatment. In adult and postnatal lungs, type II cells (cuboid shape; large, roundish nucleus) displayed a unique cytoplasmic staining for SP-A. In prenatal lungs, a low-columnar to cuboid type of cell with a large, roundish nucleus was first detectable by 62 DGA. It was the only cell type to line the distalmost tubules or buds of the prospective respiratory tract. It exhibited (initially partial) cytoplasmic staining for SP-A. AB and PAS stainings showed the presence of acid glycoconjugates and large apical and/or basal glycogen fields. After 95 DGA, the lining of the distal respiratory tract additionally displayed flatter cells with immunoreactivity for SP-A and non-reactive zones. Columnar epithelium (pseudostratified or simple) never stained for SP-A. We conclude that morphologically identifiable type II cells first appear in fetal rhesus monkey lungs by 62 DGA (pseudoglandular period). The cells may already synthesize surfactant and extracellular matrix components. They generate type I cells, and thus the entire pulmonary acinus lining. These conclusions for the rhesus monkey fully agree with our earlier conclusions for another primate, the human, and for rodents. However, as presently shown, primates differ greatly from rodents with respect to the timing of type II cell differentiation (at 29-38% versus 73-75% of gestation or at 22-25% versus 48-49% of prenatal lung development).     

Classification of pulmonary alveolar proteinosis in newborns, infants, and children

Pulmonary alveolar proteinoses are rare pulmonary diseases characterised by an intraalveolar accumulation of surfactant protein A. Subtyping of alveolar proteinoses: Type I alveolar proteinoses: severe respiratory insufficiency in newborns, which will take a lethal course without lung transplant; hereditary SP-B deficiency and an intraalveolar accumulation of N-terminal incompletely processed SP-C. Type II alveolar proteinoses: occur in newborns and infants; often take a lethal course; show intraalveolar accumulation of precursors of SP-B and mature SP-B as well as an accompanying interstitial lung disease of variable severity. Type III alveolar proteinoses: in infants and children; do not generally take a lethal course; they are characterised by an intraalveolar accumulation of precursors of SP-B and mature SP-B without accompanying interstitial lung disease. "Cryptogenic" congenital, acquired (idiopathic), and secondary type III alveolar proteinoses can be distinguished.In newborns, infants, and children with pulmonary alveolar proteinosis, a detailed pathological-anatomical examination including immunohistochemical and molecular genetic analyses, should be performed in order to optimise the therapeutical management.     

Klassifikation der Alveolarproteinosen im Neugeborenen-, Säuglings- und Kindesalter

Pulmonary alveolar proteinoses are rare pulmonary diseases characterised by an intraalveolar accumulation of surfactant protein A. Subtyping of alveolar proteinoses: Type I alveolar proteinoses: severe respiratory insufficiency in newborns, which will take a lethal course without lung transplant; hereditary SP-B deficiency and an intraalveolar accumulation of N-terminal incompletely processed SP-C. Type II alveolar proteinoses: occur in newborns and infants; often take a lethal course; show intraalveolar accumulation of precursors of SP-B and mature SP-B as well as an accompanying interstitial lung disease of variable severity. Type III alveolar proteinoses: in infants and children; do not generally take a lethal course; they are characterised by an intraalveolar accumulation of precursors of SP-B and mature SP-B without accompanying interstitial lung disease. “Cryptogenic” congenital, acquired (idiopathic), and secondary type III alveolar proteinoses can be distinguished. In newborns, infants, and children with pulmonary alveolar proteinosis, a detailed pathological-anatomical examination including immunohistochemical and molecular genetic analyses, should be performed in order to optimise the therapeutical management.     

Morphogenetic and functional activity of type II cells in early fetal rhesus monkey lungs

To evaluate further the role of type II alveolar epithelial cells in primate lung development, lungs of fetal (46 to 155 days gestational age [DGA]), postnatal, and adult rhesus monkeys were investigated with antibodies against surfactant protein A (SP-A), Alcian blue (AB) staining, and periodic acid-Schiff (PAS) staining with/without alpha-amylase pre-treatment. In adult and postnatal lungs, type II cells (cuboid shape; large, roundish nucleus) displayed a unique cytoplasmic staining for SP-A. In prenatal lungs, a low-columnar to cuboid type of cell with a large, roundish nucleus was first detectable by 62 DGA. It was the only cell type to line the distalmost tubules or buds of the prospective respiratory tract. It exhibited (initially partial) cytoplasmic staining for SP-A. AB and PAS stainings showed the presence of acid glycoconjugates and large apical and/or basal glycogen fields. After 95 DGA, the lining of the distal respiratory tract additionally displayed flatter cells with immunoreactivity for SP-A and non-reactive zones. Columnar epithelium (pseudostratified or simple) never stained for SP-A. We conclude that morphologically identifiable type II cells first appear in fetal rhesus monkey lungs by 62 DGA (pseudoglandular period). The cells may already synthesize surfactant and extracellular matrix components. They generate type I cells, and thus the entire pulmonary acinus lining. These conclusions for the rhesus monkey fully agree with our earlier conclusions for another primate, the human, and for rodents. However, as presently shown, primates differ greatly from rodents with respect to the timing of type II cell differentiation (at 29–38% versus 73–75% of gestation or at 22–25% versus 48–49% of prenatal lung development). © 1992 Wiley-Liss, Inc.     

Expression of the 35kDa and low molecular weight surfactant-associated proteins in the lungs of infants dying with respiratory distress syndrome.

Newborn respiratory distress syndrome (RDS) results from a deficiency of pulmonary surfactant. Surfactant has three ultrastructural forms: lamellar bodies, which, when secreted from Type II pneumocytes, transform into tubular myelin; tubular myelin in turn gives rise to the phospholipid monolayer at the air-fluid interface in the alveolus that constitutes functional surfactant. It has been shown previously that the lungs of infants dying from RDS lacked tubular myelin despite the presence of abundant lamellar bodies, whereas the lungs of control infants dying from other causes had both tubular myelin and lamellar bodies. An abnormality in the conversion of lamellar bodies to tubular myelin in RDS was proposed as a possible explanation for this finding. To evaluate the role of surfactant proteins (SPs) in this conversion, the authors re-examined the lungs of 11 RDS infants and 10 control infants for reactivity with antisera to high and low molecular weight SPs. In control infants, abundant intense staining with antisera to both types of SPs was found, but in the RDS lungs, staining was weaker than that in controls and less intense for high molecular weight compared to low molecular weight SPs. In lungs from patients with RDS, although staining increased with increasing gestational and post-natal ages, the intensity was less than control levels at all ages. The correlation of deficiency of SPs in RDS with lack of tubular myelin suggests that SPs may be involved in the conversion of normal lamellar bodies to tubular myelin and that the deficiency of SPs could explain the persistent respiratory distress in the presence of surfactant phospholipid synthesis.     

An immunohistological comparison of primary lung carcinosarcoma and sarcoma.

We wished to assess the antigenic expression of primary lung tumors diagnosed as either carcinosarcoma or sarcoma in order to determine whether this information would be useful in distinguishing the two. We therefore immunohistochemically analyzed six pulmonary carcinosarcomas and five primary lung sarcomas for the presence of carcinoembryonic antigen (CEA), S100 protein, cytokeratin and vimentin using commercially available monoclonal and polyclonal antibodies on formalin fixed tissues. Six of six carcinosarcomas stained positively for cytokeratin while none of the sarcomas stained. In three carcinosarcomas both the carcinomatous and sarcomatous areas were positive while in three only the carcinomatous areas were positive. CEA staining was present in five carcinosarcomas and absent in all the sarcomas. CEA positivity was strong and not confined to those tumors with obvious gland formation. Staining for S100 protein was positive in two carcinosarcomas but only in those areas showing chondroid differentiation. Immunohistochemical staining for vimentin using two different monoclonal antibodies gave inconsistent results. We conclude that in differentiating between a carcinosarcoma and a sarcoma of the lung, immunohistochemical staining for both cytokeratin and CEA are useful with cytokeratin marginally preferable. The data indicate that carcinosarcoma of the lung, like that of the upper aerodigestive tract, expresses antigens suggesting both epithelial and mesenchymal differentiation.     

Immunohistological detection of the β subunit of prolyl 4-hydroxylase in rat and mini pig lungs with radiation-induced pulmonary fibrosis

Polyclonal and monoclonal antibodies to the subunit of prolyl 4-hydroxylase, the protein disulphide isomerase, were used to compare the pulmonary cells in 13 normal and in 20 fibrotic rat and mini-pig lungs made fibrotic by X-ray irradiation, using the ABC immunoperoxidase technique. In normal lungs, prominent staining of Clara cells and type II pneumocytes and weaker reactivity with alveolar macrophages, fibroblasts, endothelial and smooth muscle cells were detectable. In pulmonary disease, in which interstitial fibrosis was the characteristic feature, the immunoreactivity was increased in both the epithelial and interstitial cells. Type I pneumocytes remained negative. In the early stages of disease (3 to 4 weeks after irradiation) when little morphological alteration was seen, capillary endothelial cells had already become immunoreactive. These results underline the complex involvement and interaction of different lung cell populations in the process of pulmonary fibrogenesis.     

Immunolocalization of cathepsin D in pneumocytes of normal human lung and in pulmonary fibrosis

Cathepsin D expression has been assessed by immunohistochemistry and immunoelectron microscopy in fetal, normal adult and injured lungs of human beings. In addition to the well known positivity of alveolar macrophages and the bronchial epithelial cells, normal type I and to a lesser extent type II pneumocytes showed a granular, cytoplasmic staining pattern. Using immunogold labelling of lowicryl embedded human lung, cathepsin D was present in lysosomes of epithelial cells. Double immunofluorescence labelling employing type I and type II specific antibodies or lectins confirmed the epithelial staining for cathepsin D. At the terminal sac period during lung development cathepsin D appears in the alveolar epithelium. In fibrotic specimens, enhanced immunoreactivity was found in epithelial and non-epithelial cells. Proliferative epithelial formations were strongly stained with cathepsin D antibodies, whereas detached, desquamated epithelial cells were weakly positive or negative. We suggest that cathepsin D plays a role in the remodelling process during fibrogenesis.     

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.     

Fetal mouse alveolar type II cells in culture express several type II cell characteristics found in vivo, together with major histocompatibility antigens.

Alveolar type II cells were isolated from fetal mouse lung by differential adherence and obtained in monolayer culture. Cultures display a high degree of purity as shown by histochemical and immunocytochemical staining procedures. Seventy-five percent of cells stained positive with specific anti-lavage serum mouse (SALS-M), an antiserum specific for (pre)alveolar type II cells of the mouse, and osmiophilic bodies were present in 82% of cells. These and other characteristics of type II cells in culture correspond to those of alveolar type II cells in fetal mouse lung. The pattern of reactivity of these cells with various anti-cytokeratin antibodies is described, and we show that, in contrast to rat type II cells, they do not exhibit alkaline phosphatase activity. Identity of the type II cell cultures was shown by their specific phospholipid composition and surfactant protein A (SP-A) content. The fetal alveolar type II cells in culture were found to synthesize and express class I but not class II major histocompatibility complex (MHC) antigens. The possibility to culture fetal alveolar type II cells of the mouse and the availability of genetically well-defined inbred and transgenic mouse strains opens ways to study the genetics of type II cell differentiation and function. Also, the in vitro availability of alveolar type II cells, the progenitor cells of mouse lung tumors, will enable us to study in vitro several of the processes involved in lung tumorigenesis in the mouse.     

Specific localization of lysosomal aminopeptidases in type II alveolar epithelial cells of the rat lung

We previously demonstrated that lysosomal cysteine proteinases, cathepsins B, H, and L were localized in lysosomes of alveolar macrophages and bronchial epithelial cells in the rat lung, while cathepsin H, a typical aminopeptidase, was additionally distributed in lamellar bodies containing surfactant in type II alveolar epithelial cells (ISHII et al., 1991). The present immunohistochemical study further examined the localization of lysosomal aminopeptidases, cathepsin C, and tripeptidyl peptidase I (TPP-I) in the rat lung. Western blotting confirmed the presence of cathepsin C and TPP-I as active forms in the pulmonary tissue, showing 25 kD and 47 kD, respectively. Immunohisto/cytochemical observations demonstrated that positive staining for cathepsin C and TPP-I was more intensely localized in alveolar epithelial regions than in bronchial or bronchiolar epithelial cells. By double immunostaining using confocal laser microscopy, immunoreactivity for cathepsin H was found to be co-localized with that for cathepsin C or TPP-I in both type II cells and macrophages. Moreover, when doubly stained with anti-cathepsin C and ED2, single-positive type II cells could be clearly distinguished from double-positive macrophages in the alveolar region. Immunoelectron microscopy revealed the gold labeling of cathepsin C or TPP-I in multivesicular and composite bodies, and lamellar bodies of Type II cells. These results showing that lysosomal aminopeptidases such as cathepsin H, cathepsin C and TPP-I are localized in lamellar bodies of type II alveolar epithelial cells strongly argue for the participation of lysosomal aminopeptidases in the formation process of surfactant containing specific proteins.     

Bronchogenic squamous cell carcinomas with invasion along alveolar walls

In bronchogenic squamous cell carcinoma, a growth pattern along the alveolar walls of the peripheral lung parenchyma is unusual. In order better to understand the way tumour cells invade the peripheral lung parenchyma, we studied two cases of squamous cell carcinoma with invasion along the alveolar walls (in 30% to 40% of the area surrounding the tumour). We used immunohistochemical staining with antibodies against pulmonary surfactant, apoproteins (PE-10) and collagen type IV, and electron microscopy. Tumour cells invading the peripheral lung tissue were located between one layer of type II alveolar epithelial cells and the basement membrane of the alveolar walls. These results suggest that the cells of a squamous carcinoma (unlike an adenocarcinoma) have the ability to spread along the basement membrane of the alveolar walls without destroying pre-existing normal peripheral lung parenchyma.     

Dilemmas about the antenatal use of corticosteroids for prevention of neonatal morbidity and mortality

Neonatal respiratory distress syndrome (RDS) is one of the biggest problems in modern obstetrics. The incidence of RDS is 1%-2%. RDS is a condition of insufficient surfactant production. Surfactant is a complex molecule which is responsible for maturation of fetal lungs. The most important factor for insufficient surfactant production and pulmonary immaturity is shortening of gestation, i.e. preterm delivery. Antenatal corticosteroids for maturation of fetal lungs are in use for over thirty years. Corticosteroids decrease the incidence and intensity of RDS, the severity of intracerebral hemorrhage, and overall neonatal morbidity and mortality. The mechanism of corticosteroid action is probably induction of fetal pulmonary enzyme complex that is responsible for adequate surfactant production and regulation of pulmonary interstitial fluids. In this literature review, we analyze long- and short-term benefits and risks of single and multiple antenatal corticosteroid administration.     

A MONOCLONAL ANTIBODY POOL FOR ROUTINE IMMUNOHISTOCHEMICAL DETECTION OF HUMAN RESPIRATORY SYNCYTIAL VIRUS ANTIGENS IN FORMALIN-FIXED,PARAFFIN-EMBEDDED TISSUE

Four monoclonal antibodies (MAbs) with specificities for epitopes on human respiratory syncytial virus (RSV) proteins preserved after formalin fixation and paraffin embedding were identified in fixed and embedded virus-infected HEp-2 cell pellets. The MAbs bound epitopes on the fusion protein, the nucleoprotein, the phosphoprotein, and the M2 protein of the virus. Following high-temperature antigen unmasking, immunohistochemical staining revealed RSV antigens in the lungs of five of seven children who died with confirmed RSV infection and in none of nine children who died for other reasons, with no evidence of RSV infection. Staining was cytoplasmic, granular, and confined to epithelial cells. Intense staining was seen at the apex of ciliated bronchial and bronchiolar epithelial cells in all five positive cases. In one case, of pneumonitis, infected pneumocytes were present in the alveoli and in several cases, CD68-positive, cytokeratin-negative alveolar macrophages stained for viral antigens. These antibodies may prove useful in studies of the pathogenesis of RSV infection. © 1997 John Wiley & Sons, Ltd.     

Arrested pulmonary alveolar cytodifferentiation and defective surfactant synthesis in mice missing the gene for parathyroid hormone-related protein.

Parathyroid hormone-related protein (PTHrP) and PTH/PTHrP receptor expression are developmentally regulated in lung epithelium and adepithelial mesenchyme, respectively. To test the hypothesis that PTHrP is a developmental regulator of terminal airway development, we investigated in vivo and in vitro models of alveolar cytodifferentiation using mice in which the gene encoding PTHrP was ablated by homologous recombination. We have determined that fetal and newborn PTHrP(-/-) lungs showed delayed mesenchymal-epithelial interactions, arrested type II cell differentiation, and reduced surfactant lamellar body formation and pulmonary surfactant production. Embryonic PTHrP(-/-) lung buds cultured in the absence of skeletal constriction or systemic compensating factors also exhibited delayed alveolar epithelial (type II cell) and mesenchymal cytodifferentiation, as well as a > 40% inhibition of surfactant phospholipid production (n = 3-5). Addition of exogenous PTHrP to embryonic PTHrP(-/-) lung cultures normalized interstitial cell morphology and surfactant phospholipid production. The importance of PTHrP as an endogenous regulatory molecule in mammalian lung development is supported by the findings that ablation of PTHrP expression in isolated developing lung is sufficient to disrupt normal development of the alveolar ducts and the centriacinar regions.