Yoshinori Ogasawara

Immunocytochemical localization of surfactant protein D(SP-D) in type II cells, Clara cells, and alveolar macrophages of rat lung

We investigated the cellular and subcellular distribution of surfactant protein D (SP-D) by immunogold labeling in lungs of adult rats that had been given bovine serum albumin coupled to 5-nm gold (BSAG) for 2 hr to visualize the endocytotic pathway. Specific gold labeling for SP-D was found in alveolar Type II cells, Clara cells, and alveolar macrophages. In Type II cells abundant labeling was observed in the endoplasmic reticulum, whereas the Golgi complex and multivesicular bodies were labeled to a limited extent only. Lamellar bodies did not seem to contain SP-D. Gold labeling in alveolar macrophages was restricted to structures containing endocytosed BSAG. In Clara cells labeling was found in the endoplasmic reticulum, the Golgi complex, and was most prominent in granules present in the apical domain of the cell. Double labeling experiments with anti-surfactant protein A (SP-A) showed that both SP-A and SP-D were present in the same granules. However, SP-A was distributed throughout the granule contents, whereas SP-D was confined to the periphery of the granule. The Clara cell granules are considered secretory granules and not lysosomes, because they were not labeled for the lysosomal markers cathepsin D and LGP120, and they did not contain endocytosed BSAG.

Binding specificity of lung surfactant protein SP-D for glucosylceramide

The specificities of the binding of lung surfactant protein SP-D to glycolipids were examined using 125I-labeled SP-D as a probe. When the binding study was performed on TLC plates, SP-D bound exclusively to GlcCer, whereas it failed to bind to GalCer, GM1, GM2, asialo-GM1, asialo-GM2, sulfatide, Forssman antigen, ceramide dihexoside, ceramide trihexoside, globoside, paragloboside or ceramide. Excess native SP-D competed with 125I-SP-D for the binding to GlcCer. Antibody to rat SP-D inhibited 125I-SP-D binding to GlcCer. Ca2+ was absolutely required for the binding of SP-D to GlcCer; Mg2+ failed to substitute for Ca2+. SP-D bound to ceramide monohexoside in glycolipids isolated from rat lung and bronchoalveolar lavage fluids of rats.

Enzyme-linked immunosorbent assay for human pulmonary surfactant protein D

We developed a simple and sensitive enzyme-linked immunosorbent assay (ELISA) for human pulmonary surfactant protein D (SP-D). Human SP-D was purified from bronchoalveolar lavage fluids of patients with pulmonary alveolar proteinosis. Nine monoclonal antibodies (MAbs) were established from BALB/c mice immunized with the purified human SP-D. All MAbs were directed to either 43 kDa SP-D contained in lung lavage fluids of patients with pulmonary alveolar proteinosis or in amniotic fluids from healthy normal pregnancies. The ELISA is based on a sandwich method using two MAbs, 6B2 and 7C6. Cross-reactivity to human SP-A or rat SP-D was evaluated as below 0.6%. The recovery of different concentrations of SP-D ranged from 94.4% to 111.2%, and serial dilutions of amniotic fluids showed good linearity. SP-D concentrations in 21 amniotic fluids from normal pregnancies were measured by the ELISA. The mean concentration in amniotic fluids from pregnancies in the third trimester was significantly higher than that from earlier stages of gestation (p < 0.001), indicating that this ELISA may be applicable for prediction of fetal lung maturity.

Ontogeny of surfactant apoprotein D, SP-D, in the rat lung

Surfactant protein D (SP-D) is a collagenous surfactant-associated glycoprotein synthesized by alveolar type II cells. Antiserum against rat SP-D was raised in rabbits and an enzyme-linked immunosorbant assay (ELISA) has been developed using anti-rat SP-D IgG. In the present study we examined the developmental profile of SP-D in the rat lung compared with that of surfactant protein A (SP-A). SP-A content in the lungs increased during late gestation and reached its maximum on day 1 of neonate, and then gradually decreased until at least day 5. SP-D content during early gestation was less than 10 ng/mg protein until day 18, but on day 19 there was a 4-fold increase in SP-D (compared to that on day 18). It increased twice between day 21 and the day of birth, when it reached the adult level of 250 ng/mg protein, which is about one fourth that of the adult level of SP-A. Unlike SP-A there seemed to be no decrease in SP-D content after birth. These results demonstrate that SP-D is regulated developmentally as are the other components of surfactant, but the inconsistency in the developmental profiles of SP-A and SP-D suggests that these proteins may play different roles in lung maturation.

Characterization of pulmonary surfactant protein D: its copurification with lipids

Surfactant protein D (SP-D) is a collagenous surfactant associated protein synthesized by alveolar type II cells. SP-D was purified from the supernatant of rat bronchoalveolar lavage fluids obtained by centrifugation at 33,000 x gav for 16 h. The contents of SP-D and SP-A in fractions obtained by the centrifugation of rat bronchoalveolar lavage were determined by enzyme-linked immunoassay. The total content of SP-D was approximately 12% of that of SP-A in these lavage fluids. 99.1% of SP-A was present in the 33,000g pellet, whereas 71.1% of SP-D was in the 33,000g supernatant. Analysis by high performance liquid chromatography reveals that lipids are copurified with isolated SP-D. Phosphatidylcholine accounted for 84.8% of the phospholipids copurified with SP-D. Unlike SP-A, SP-D in the purified and delipidated form failed to compete with 125I-labeled SP-A for phosphatidylcholine binding, and to aggregate phospholipid liposomes. The present study demonstrates that lipids are copurified with SP-D, that SP-D and SP-A distribute differently in rat bronchoalveolar lavage fluids, and that SP-D in the purified and delipidated form does not exhibit interaction with lipids in the same fashion as SP-A.

Binding of pulmonary surfactant protein A to galactosylceramide and asialo-GM2

The binding of pulmonary surfactant protein A (SP-A) to glycolipids was examined in the present study. The direct binding of SP-A on a thin-layer chromatogram was visualized using 125I-SP-A as a probe. 125I-SP-A bound to galactosylceramide and asialo-GM2, but failed to exhibit significant binding to GM1, GM2, asialo-GM1, sulfatide, and Forssman antigen. The study of 125I-SP-A binding to glycolipids coated onto microtiter wells also revealed that SP-A bound to galactosylceramide and asialo-GM2. SP-A bound to galactosylceramides with non-hydroxy or hydroxy fatty acids, but showed no binding to either glucosylceramide or galactosylsphingosine. Excess native SP-A competed with 125I-SP-A for the binding to asialo-GM2 and galactosylceramide. Specific antibody to rat SP-A inhibited 125I-SP-A binding to glycolipids. In spite of chelation of Ca2+ with EDTA or EGTA, SP-A retained a significant binding to glycolipids. Inclusion of excess monosaccharides in the binding buffer reduced the glycolipid binding of SP-A, but failed to achieve complete abolishment. The oligosaccharide isolated from asialo-GM2 is also effective at reducing 125I-SP-A binding to the solid-phase asialo-GM2. From these data, we conclude that SP-A binds to galactosylceramide and asialo-GM2, and that both saccharide and ceramide moieties in the glycolipid molecule are important for the binding of SP-A to glycolipids.

A novel type of binding specificity to phospholipids for rat mannose-binding proteins isolated from serum and liver.

Mannose‐binding protein (MBP) belongs to the collectin subgroup of C‐type lectins with specificity for mannose and N‐acetylglucosamine sugars. We investigated whether rat MBPs isolated from serum (S‐MBP) and liver (L‐MBP) interact with phospholipids using antibody against each MBP. Both S‐ and L‐MBPs bound to phosphatidylinositol coated onto microtiter wells in a concentration‐ and a Ca2+‐dependent manner. L‐MBP also bound to phosphatidylglycerol and weakly to phosphatidylserine. MBPs interacted with liposomes composed of these lipids. S‐ and L‐MBPs bound to phosphatidylinositol 4‐monophosphate. L‐MBP also bound to cardiolipin. These results provide evidence for a novel type of ligand binding specificity for MBPs, and raise the possibility that phospholipids are ligands for collectins.

Pre- and postnatal stimulation of pulmonary surfactant protein D by in vivo dexamethasone treatment of rats

Fetal (days 18 and 20 of gestation), neonatal (days 0, 2 and 4 of neonate) and adult rats were injected with dexamethasone (1 mg/kg) in vivo and 24 hours later the effect on the contents of surfactant protein D (SP-D) in the rat lungs were examined in comparison with surfactant protein A, disaturated phosphatidylcholine and phosphatidylglycerol. In vivo dexamethasone treatment resulted in significant increases of SP-D content as the other 3 components of surfactant in both fetuses and neonates, but not in adults. Responsiveness to glucocorticoid treatment on SP-D content was maximum on day 1 of neonate (2.7 times control value). The contents of surfactant components examined tend to respond better to steroid in postnatal rats. These data demonstrated that glucocorticoid treatment in vivo for short durations exhibits the stimulatory effect on the contents of SP-D in the fetal and neonatal rat lungs.

Interaction of phospholipid liposomes with plasma membrane isolated from alveolar type II cells: effect of pulmonary surfactant protein A.

Pulmonary surfactant protein A (SP-A) augments the uptake of phospholipid liposomes containing dipalmitoylphosphatidylcholine (DPPC) by alveolar type II cells. The SP-A-mediated uptake process of lipids by type II cells have not been well understood. In the present study we investigated the SP-A-mediated interaction of phospholipids with plasma membrane isolated from alveolar type II cells. SP-A increased the amount of liposomes containing radiolabeled DPPC associated with type II cell plasma membrane by 4-fold compared to the control without SP-A when analyzed by sucrose density gradient centrifugation. This effect is dependent upon the SP-A concentration. The enhancement was inhibited by anti-SP-A antibody and EGTA. When type II cell plasma membrane and liposomes containing [14C]DPPC and [3H]triolein were coincubated with or without SP-A, analysis on sucrose density gradients revealed that the profiles of [14C]DPPC and [3H]triolein in each fraction were almost identical with or without SP-A, indicating that SP-A mediates the binding of liposomes to plasma membrane but not transfer of DPPC. SP-A increased the association of liposomes containing DPPC with the membrane by 2-fold more than that containing 1-palmitoyl-2-linoleoyl-phosphatidylcholine (PLPC). SP-A induced aggregation of phospholipid liposomes containing PLPC as well as those containing DPPC, but the final turbidity of DPPC liposomes aggregated by SP-A was only by 15% greater than that of PLPC liposomes. The amount of DPPC liposomes associated with the plasma membrane derived from type II cells was 2-fold greater than that from liver. We speculate that the SP-A-mediated interaction of lipids with type II cell plasma membrane may contribute, in part, to the lipid uptake process by type II cells.