William M. Hull

Ontogeny of surfactant proteins A and B in human amniotic fluid as indices of fetal lung maturity.

ABSTRACT: Surfactant proteins A and B (SP-A and SP-B) were measured in human amniotic fluid by ELISA and correlated with lecithin to sphingomyelin ratio (L/S), phosphatidylglycerol (PG), and perinatal outcome. Amniotic fluid SP-A, SP-B, and L/S increased with advancing gestation. SP-A was detected at 19 wk gestation and increased dramatically in the 3rd trimester of pregnancy. SP-B was first detectable at 31 wk gestation and increased significantly to term. SP-A was a more specific predictor of non-respiratory distress syndrome (RDS) than L/S or SP-B; however, the sensitivity of SP-A in predicting RDS was less than L/S < 2.0 (26.3 versus 82.3%, respectively). In 209 pregnancies assessed within 48 h of delivery, the sensitivity of SP-B in predicting RDS (nondetectable SP-B) was comparable to the L/S, however, SP-B = 0 was frequently observed in mature infants, limiting its specificity for prediction of RDS. The greatest sensitivity and specificity were achieved with the measurement of L/S < 2.0 and negative PG, which correctly predicted 100% of the infants with RDS and 94% of those who did not develop the disorder. Measurement of SP-A or SP-B did not improve the prediction of RDS. SP-A, SP-B, and L/S were not affected by infant sex, Apgar score, rupture of membranes, size for gestational age, maternal diabetes, hypertension, or exposure to medications. SP-A, SP-B, and L/S were significantly elevated in amniotic fluid from black mothers. SP-A was significantly elevated in amniotic fluid from mothers who smoked during pregnancy. Although amniotic fluid SP-A and SP-B concentrations increased with advancing gestation, the predictive value of the L/S and PG for RDS was not enhanced by the additional measurement of SP-A and/or SP-B.

Stat-3 is required for pulmonary homeostasis during hyperoxia

Acute lung injury syndromes remain common causes of morbidity and mortality in adults and children. Cellular and physiologic mechanisms maintaining pulmonary homeostasis during lung injury remain poorly understood. In the present study, the Stat-3 gene was selectively deleted in respiratory epithelial cells by conditional expression of Cre-recombinase under control of the surfactant protein C gene promoter. Cell-selective deletion of Stat-3 in respiratory epithelial cells did not alter prenatal lung morphogenesis or postnatal lung function. However, exposure of adult Stat-3-deleted mice to 95% oxygen caused a more rapidly progressive lung injury associated with alveolar capillary leak and acute respiratory distress. Epithelial cell injury and inflammatory responses were increased in the Stat-3-deleted mice. Surfactant proteins and lipids were decreased or absent in alveolar lavage material. Intratracheal treatment with exogenous surfactant protein B improved survival and lung histology in Stat-3-deleted mice during hyperoxia. Expression of Stat-3 in respiratory epithelial cells is not required for lung formation, but plays a critical role in maintenance of surfactant homeostasis and lung function during oxygen injury.

Immunologic identification of a pulmonary surfactant-associated protein of molecular weight = 6000 daltons.

ABSTRACT. Hydrophobic, small molecular weight, surfactant- associated protein of Mr = 6000 (SAP-6) was isolated from bovine, canine, and human alveolar lavage and identified by silver staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis gels. Lesser amounts of protein of Mr = 14,000, 20,000, and 26,000 daltons also copurified with SAP-6, likely representing oligomers of the Mr = 6,000 dalton protein. In the absence of sulfhydryl-reducing agents, increased amounts of the larger forms of the protein were observed. Antisera generated against bovine SAP-6 were used to further characterize the protein and distinguish it from the more abundant surfactant-associated glycoprotein of Mr = 35,000 (SAP- 35) present in mammalian surfactants. Rabbit antisera generated against the bovine hydrophobic protein recognized SAP-6 and lesser amounts of the proteins of Mr = 14,000, 20,000, and 26,000 daltons. The SAP-6 antisera were reactive against the hydrophobic proteins from human, bovine, and canine surfactants as assessed by immunoblot analysis after sodium dodecyl sulfate polyacrylamide gel electrophoresis. SAP-6 antisera did not detect bovine SAP-35 the abundant surfactant-associated glycoprotein, by immunoblot analysis; however, some reactivity of the anti-SAP-6 was detected against purified bovine SAP-35 by a sensitive enzyme-linked immune-adsorbant assay. Anti-SAP-6-did not react with bovine serum components either by immunoblot or by enzyme-linked immune- adsorbant assay. Monospecific antisera generated against bovine SAP-35 did not detect SAP-6 by immunoblot analysis. Immunoblot analysis of the protein in exogenous replacement surfactant preparations, Surfactant-TA and calf lung surfactant extract, utilizing anti-SAP-6 and anti-SAP-35 demonstrated that both preparations were enriched in SAP-6 and lacking in SAP-35. Surfactant-associated proteins, present in bovine lung-based replacement surfactants, are immunologically identified as SAP-6 and its oligomers.

Synthesis of surfactant-associated glycoprotein A by rat type II epithelial cells. Primary translation products and post-translational modification

Surfactant-associated glycoproteins A, 38 (A3), 32 (A2) and 26 (A1) kDa, pI (4.2-4.8), were identified as related proteins present in surfactant isolated from rat lung lavage fluid. Differences in size and charge among surfactant-associated glycoproteins A were related to differences in glycosylation as determined by reduction of the larger forms (38 and 32 kDa) to 26 kDa by endoglycosidase F and by increased isoelectric points of the glycosylated forms after treatment with neuraminidase. Synthesis and secretion of surfactant-associated glycoproteins A and precursors were demonstrated in purified rat Type II epithelial cells by immunoprecipitation of [35S]methionine-labelled proteins with anti-surfactant-associated glycoprotein A antisera. In pulse-chase experiments, labelled proteins 26-34 kDa, appeared within 10 min and smaller forms co-migrated with surfactant-associated glycoprotein A from alveolar lavage. The relative abundance of the larger molecular mass forms (30-34 kDa, pI 4.8) increased at later times up to 3 h. More acidic mature forms, which co-migrated with surfactant-associated glycoproteins A2 and A3 in surfactant (38 and 32 kDa), were readily detectable in the media, but were not abundant forms in lysates of labelled Type II cells after 1-3 h of incubation. Primary translation products of surfactant-associated glycoprotein A were immunoprecipitated with monospecific anti-surfactant-associated glycoprotein A antiserum after in vitro translation of poly(A)+ mRNA isolated from adult rat lung. The immunoprecipitated translation product migrated at 26 kDa, pI 4.8, and migrated slightly faster than surfactant-associated glycoprotein A1 from surfactant. Treatment of surfactant-associated glycoprotein A with bacterial collagenase resulted in proteolytic fragments 23-20 kDa, pI 4.2-4.8, which no longer underwent sulfhydryl-dependent cross-linking, suggesting that the collagen-like domain was required for the sulfhydryl-dependent oligomerization. Surfactant-associated glycoproteins A are synthesized by rat Type II epithelial cells as pre-proteins, 26-34 kDa. Larger forms result primarily from N-linked glycosylation of the 26 kDa primary translation product. Mature, more acidic forms result from further addition of sialic acid.

Characteristics of Human Surfactant-Associated Glyoproteins A

ABSTRACT: Surfactant-associated glycoprotein A [molecular weight (Mr) = 34,000, isoelectric point (pi) 4.6-5.0] and its sulfhydryl dependent oligomers were purified and partially characterized from surfactant obtained from human alveolar lavage. Two major forms of the protein were identified by silver stain and immunoblot analysis of surfactant using human surfactant-associated glycoprotein A antisera: glycoprotein A2, Mr = 34,000 and glycoprotein Ai, Mr = 28,000. The larger form was reduced to Mr = 28,000 by treatment with endogylcosidase F, indicating the presence of complex N-linked oligosaccharide on the molecule. Charge heterogeneity was decreased and the isoelectric point increased by treatment with neuroaminidase, supporting the presence of sialic acid. Homology between the proteins Mr = 34,000 and 28,000 was confirmed by analysis of two-dimensional tryptic and chymotryptic peptides of 125I-iodo-glycoproteins Ai and A2 which were identical. The protein was very rich in glycine and its amino acid composition was similar to that of glycoprotein A previously reported for the dog and rat. Treatment of glycoproteins A with bacterial collagenase resulted in the generation of highly glycosylated peptides Mr = 20,000- 22,000, pi 4.6-5.0, which no longer formed sulfhydryldependent oligomers, supporting the presence of significant collagen-like region in the molecule. In the absence of reducing agents, glycoprotein A from surfactant was present as sulfhydryl-dependent dimers and larger oligomers. Higher molecular weight aggregates of glycoproteins A were also present in lavage material even after sulfhydryl reduction. Glycoproteins A were identified in surfactant from amniotic fluid, normal adult lung lavage, human cadaver lung lavage, and material obtained from lung lavage from a patient with alveolar proteinosis. Alveolar proteinosis proteins contained larger amounts of the higher molecular weight aggregates and smaller molecular weight proteolytic fragments of glycoproteins A than material obtained from other sources. Peptide mapping of the I25Iiodinated aggregates, approximately Mr = 50,000, 70,000, 100,000, and greater were identical to glycoproteins A (Mr = 34,000 and 28,000) from normal human lung lavage. A smaller immunoreactive form (Mr = 20,000) shared several peptides but lacked others, supporting its origin as a proteolytic fragment of glycoproteins Ai or A2. Human glycoprotein A2 is a complex N-linked glycoprotein likely representing the glycosylated form of a polypeptide precursor Mr = 28,000.

Compound SFTPB 1549C→GAA (121ins2) and 457delC heterozygosity in severe congenital lung disease and surfactant protein B (SP-B) deficiency

Several human respiratory disorders have been linked to an abnormality of pulmonary surfactant synthesis or turnover. Among those conditions, hereditary deficiency in the hydrophobic surfactant protein B (SP‐B) has been recognized as a rare cause of respiratory failure in term newborn infants. Homozygosity for a common mutation (1549C→GAA, or 121ins2) of the SP‐B‐encoding gene (SFTPB) results in rapidly fatal respiratory failure, with complete absence of the mRNA and protein observed in lung fluid or biopsy specimens. Hereditary SP‐B deficiency is also associated with aberrant processing of proSP‐C and deficiency of the active SP‐C peptide. In the present study, we characterized the SFTPB gene in an infant with severe unexplained respiratory distress and identified a paternally derived 1549C→GAA lesion, as well as a hitherto unreported mutation (457delC) inherited from the mother. Analysis of bronchoalveolar lavage fluid demonstrated the complete absence of SP‐B. However, unlike previous infants with hereditary SP‐B deficiency, proSP‐C was processed to the active SP‐C peptide, suggesting that the defect in SP‐B, rather than SP‐C, caused the respiratory distress in this infant. The present findings demonstrate the importance of SFTPB in pulmonary function and support the need for further genotype–phenotype correlations in patients with SP‐B deficiency. Hum Mutat 14:502–509, 1999.

Intracellular and oligomeric forms of surfactant-associated apolipoproteins(s) A in the rat

Sulfhydryl-dependent oligomeric forms of the surfactant-associated apolipoprotein(s) A, obtained from particulate preparations of adult rat lung lavage, were characterized by immunoblot analysis and by silver staining of proteins separated by one- and two-dimensional SDS-polyacrylamide gel electrophoresis. Under non-reducing conditions, these proteins migrated as oligomers, Mr approx. 50-70, 115, 160 kDa and greater. The large oligomers were reduced to the apolipoprotein(s) A subunits by treatment with beta-mercaptoethanol; Mr 38 (A3), 32 (A2) and 26 kDa (A1), pI 4.2-4.8. Mr 50 kDa protein was composed of sulfhydryl-dependent homo-dimers of protein(s) A1 (Mr 26 kDa). 55 kDa protein was a hetero-dimer composed primarily of A1 and A2 (Mr 26 and 32 kDa). 62 kDa protein was composed of hetero-dimers of A3 and apolipoprotein A2 (Mr 38 and 32 kDa). 70 kDa protein was a homodimer composed of apolipoprotein A3 A3 (38 kDa). Larger molecular forms were composed primarily of 38 and 32 kDa and lesser amounts of 26 kDa. Treatment with endoglycosidase F reduced A2 and A3 to 26 kDa. Apolipoprotein A1 co-migrated with a protein of Mr 26 kDa immunoprecipitated from [35S]methionine-labelled Type II epithelial cells. Chymotryptic-tryptic peptide maps of apolipoproteins A1, A2 and A3 were identical, suggesting that apolipoproteins A3 and A2 arise through extensive glycosylation of apolipoprotein A1.

Human Surfactant Protein-A Contains Blood Group A Antigenic Determinants

ABSTRACT: A major blood group antigenic epitope was identified on human pulmonary surfactant protein A (SPA). MAb and polyclonal antibodies generated against purified human SP-A aggregated blood group A human erythrocytes and immunostaine epithelial cells in a variety of human tissues, consistent with the tissue distribution of major blood group antigens. SP-A MAb (MAb-8) agglutinated red cells and immunostained tissues from A or AB blood groups, but did not react with cells or tissues from O or B individuals. MAb-8 immunostaining of tissue from blood group A individuals was ablated by incubation with blood group A red cells. MAb and polyclonal antibodies directed against A blood group antigens reacted strongly with purified SP-A obtained from a blood group A individual with alveolar proteinosis. MAb and polyclonal antibodies specific for B blood group antigen failed to react with SP-A from this patient or from patients who were in blood group B. Reactivity of anti-blood group MAb was lost after treatment of SP-A with endoglycosidase-F, demonstrating its reactivity with an epitope dependent on the asparagine-linked oligosaccharide at asparagine 187. Reactivity of MAb-8 with SP-A persisted after endoglycosidase-F treatment, but was lost after digestion with collegenase as assessed by Western blot after SDS-PAGE. Reactivity of MAb to SP-A was sensitive to β-elimination, supporting the presence of another blood group antigenic site distinct from the epitope dependent on the asparagine-linked carbohydrate. The finding that the SP-A molecule contains a major blood group epitope has implication for the clinical use of surfactant replacement preparations and diagnostic reagents based on this protein.

AMNIOTIC FLUID CONCENTRATION OF SURFACTANT PROTEINS IN INTRA-AMNIOTIC INFECTION

Objective. Pulmonary surfactant is a complex molecule of lipids and proteins synthesized and secreted by type II alveolar cells into the alveolar epithelial lining. Both lipid and protein components are essential for lung function in postnatal life. Infection is a well-established cause of preterm delivery, and several inflammatory cytokines play a role in the mechanisms of preterm parturition. An increased concentration of inflammatory cytokines in amniotic fluid or fetal plasma has been linked to the onset of preterm parturition and fetal/neonatal injury, including cerebral palsy and chronic lung disease. Experimental evidence indicates that inflammatory mediators also regulate surfactant protein synthesis, and histologic chorioamnionitis is associated with a decreased incidence of hyaline membrane disease in neonates. This study was conducted to determine if amniotic fluid concentrations of surfactant protein (SP)-A, SP-B, and SP-D change in patients with and without intra-amniotic infection (IAI). Materials and methods. A case–control study was conducted to determine amniotic fluid concentrations of SP-A, SP-B, SP-D, and total protein in patients who had an amniocentesis performed between 18 and 34 weeks of gestation for the detection of IAI in patients with spontaneous preterm labor with intact membranes (n = 42) and cervical insufficiency prior to the application of cerclage (n = 6). Amniotic fluid samples were selected from a bank of biological specimens and included patients with (n = 16) and without (n = 32) IAI matched for gestational age at amniocentesis. Intra-amniotic infection was defined as a positive amniotic fluid culture for microorganisms. Each group was further subdivided according to a history of corticosteroid administration within 7 days prior to amniocentesis into the following subgroups: (1) patients without IAI who had received antenatal corticosteroids (n = 21), (2) patients with IAI who had received antenatal corticosteroids (n = 9), (3) patients without IAI who had not received antenatal corticosteroids (n = 11), and (4) patients with IAI who had not received antenatal corticosteroids (n = 7). Amniotic fluid was obtained by transabdominal amniocentesis. SP-A, SP-B, and SP-D concentrations in amniotic fluid were determined by enzyme-linked immunosorbent assay (ELISA). Non-parametric statistics were used for analysis. Results. Women with IAI had a higher median amniotic fluid concentration of SP-B and of SP-B/total protein, but not other SPs, than those without IAI (both p = 0.03). Among patients who had received antenatal corticosteroids, the median amniotic fluid concentration of SP-B and of SP-B/total protein was significantly higher in patients with IAI than in those without IAI (SP-B, IAI: median 148 ng/mL, range 37.3–809 ng/mL vs. without IAI: median 7.2 ng/mL, range 0–1035 ng/mL; p = 0.005 and SP-B/total protein, IAI: median 14.1 ng/mg, range 4.3–237.5 ng/mg vs. without IAI: median 1.45 ng/mg, range 0–79.5 ng/mg; p = 0.003). Among women who had not received antenatal corticosteroids, the median amniotic fluid concentrations of SP-B and of SP-B/total protein were not significantly different between patients with and without IAI (SP-B, IAI: median 4 ng/mL, range 0–31.4 ng/mL vs. without IAI: median 3.4 ng/mL, range 0–37 ng/mL; p = 0.8 and SP-B/total protein, IAI: median 0.55 ng/mg, range 0–6.96 ng/mg vs. without IAI: median 0.59 ng/mg, range 0–3.28 ng/mg; p = 0.9). The median amniotic fluid concentrations of SP-A, SP-A/total protein, SP-D, and SP-D/total protein were not significantly different between patients with and without IAI whether they received antenatal corticosteroids or not (all p > 0.05). Conclusions. IAI was associated with an increased amniotic fluid concentration of SP-B in patients who received antenatal corticosteroids within 7 days prior to amniocentesis.

In vitro acetylation of rat pulmonary surfactant-associated glycoprotein(s) A primary translation products

The primary translation products of pulmonary surfactant-associated glycoprotein(s) A, the major apolipoprotein in mammalian surfactants, exhibit extensive charge heterogeneity. After in vitro translation of poly(A)+ mRNA from rat lung, the primary translation products of glycoprotein(s) A were identified as a charge train of five proteins of 26 kDa (pI 4.6-5.0), the predominant forms being the more acidic members (pI less than 4.8). Inhibition of acetylation during in vitro translation of rat lung poly(A)+ mRNA resulted in a predominance of the more basic isoforms (pI greater than or equal to 4.8). Intracellular forms of glycoprotein(s) A were immunoprecipitated from rat Type II epithelial cells after treatment with tunicamycin or after deglycosylation with endoglycosidase H. Five intracellular precursors consisting primarily of acidic members of the charge train were identified, this being consistent with the intracellular acetylation of the protein. In contrast, canine glycoprotein(s) A translation products consisted of only three proteins of 26 kDa (pI 4.8-5.0), in which most of the radiolabel was concentrated in the more basic components. Acetylation may account for some, but not all, of the charge heterogeneity in the primary translation products and processed forms of surfactant-associated glycoprotein(s) A in the rat.