Distinct effects of SP-B and SP-C on the uptake of surfactant-like liposomes by alveolar cells in vivo and in vitro (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1998
Surfactant protein A (SP-A) binds to dipalmitoylphosphatidylcholine (DPPC) and induces phospholipid vesicle aggregation. It also regulates the uptake and secretion of surfactant lipids by alveolar type II cells. We introduced the single mutations Glu195CGln (rE195Q), Lys201CAla (rK201A) and Lys203CAla (rK203A) for rat SP-A, Arg199CAla (hR199A) and Lys201CAla (hK201A) for human SP-A, and the triple mutations Arg197, Lys201 and Lys203CAla (rR197A/K201A/K203A) for rat SP-A, into cDNAs for SP-A, and expressed the recombinant proteins using baculovirus vectors. All recombinant proteins avidly bound to DPPC liposomes. rE195Q, rK201A, rK203A, hR199A and hK201A function with activity comparable to wild type SP-A. Although rR197A/K201A/K203A was a potent inducer of phospholipid vesicle aggregation, it failed to stimulate lipid uptake. rR197A/K201A/K203A was a weak inhibitor for lipid secretion and did not competed with rat [ 125 I]SP-A for receptor occupancy. From these results, we conclude that Lys201 and Lys203 of rat SP-A, and Arg199 and Lys201 of human SP-A are not individually critical for the interaction with lipids and type II cells, and that Glu195 of rat SP-A can be replaced with Gln without loss of SP-A functions. This study also demonstrates that the SP-A-mediated lipid uptake is not directly correlated with phospholipid vesicle aggregation, and that specific interactions of SP-A with type II cells are involved in the lipid uptake process. ß 1998 Elsevier Science B.V. All rights reserved.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1991
A wide range of liposome compositions have previously been examined in vivo for their ability te affnct the uptake of liposomes into cells of the reticuloendothelial (RE, nmnonnclear phagocyte) system (Allen, T,M. and Chonn, A. (1987) FEBS Lett. 223, 42-46; Allen et al. (1989) Biochim. Biophys. Acta 981, 27-35). in this study we have examined the ability of cultured routine bone marrow macrophages to endocytose liposomes of various compositions and have looked for correlations between the in vivo and the in vitro observations. Compounds which substantially decreased RE uptake of liposomes in vivo, such as monosialogangiioside (GMt) and a novel synthetic lipid derivative of polyethylenegiyeol (PEG-PE), also greatly decreased liposome uptake by bone marrow ma(xopheges in a eoncentrafion.depoodent manner. Lipids which increase bilayer rigidity, such as splfingomyelin (SM) and cholesterol (CHOL), decreased both in vivo and in vitro uptake of liposomes. Likewise, positive correlation~ were observed between the in vivo behavior of liposom~ containing phosplmfidylserine (PS) or various gangliosides and the ability of these liposomes to be taken up by bone marrow macropheges. Total liposome uptake by macrophages increased with incubation time at 37°C while very little liposome association with the macrophages was observed at 4oc. Liposome uptake increased with liposome concentration and for liposomes composed of egg phosphatidylcholine (PC) uptake plateaued at 40 nmol lipid I~r mg cell protein.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1990
The interaction of liposomes with macrophage cells was monitored by a new fluorescence method (Hong, K., Straubinger, R.M. and Papahadjopoulos, D., J. Cell Biol. 103 (1986) 56a) that allows for the simultaneous monitoring of binding, endocytosis, acidification and leakage. Profound differences in uptake, cell surface-induced leakage and leakage subsequent to endocytosis were measured in liposomes of varying composition. Pyranine (1-hydroxypyrene-3,6,8-trisulfonic acid, I-IllS), a highly fluorescent, water-soluble, pH sensitive dye, was encapsulated at high concentration into the lumen of large unilamellar vesicles. HPTS exhibits two major fluorescence excitation maxima (403 and 450 nm) which have a complementary pH dependence in the range 5-9: the peak at 403 nm is maximal at low pH values while the peak at 450 nm is maximal at high pH values. The intra-and extracellular distribution of liposomes and their approximate pH was observed by fluorescence microscopy using appropriate excitation and barrier filters. The uptake of liposomal contents by cells and their subsequent exposure to acidified endosomes or secondary lysosomes was monitored by spectrofluorometry via alterations in the fluorescence excitation maxima. The concentration of dye associated with cells was determined by measuring fluorescence at a pH independent point (413 nm). The average pH of ceil-associated dye was determined by normalizing peak fluorescene intensities (403 nm and 450 nm) to fluorescence at 413 nm and comparing these ratios to a standard curve. HPTS-containing liposomes bound to and were acidified by a cultured murine macrophage cell line (J774) with att/ 2 of 15-20 min. The acidification of liposomes exhibited biphasic kinetics and 50-80% of the liposomes reached an average pH lower than 6 within 2 h. A liposomal lipid marker exhibited a rate of uptake similar to HPTS, however the lipid component selectively accumulated in the cell; after an initial rapid release of liposome contents, 2.5-fold more lipid marker than liposomal contents remained associated with the cells after 5 h. Coating haptenated iiposomes with antibody protected liposomes from the initial release. The leakage of iiposomal contents was monitored by co-encapsulating I-IPTS and p-xylene-bis-pyridinium bromide, a fluorescence quencher, into liposomes. The time course of dilution of liposome contents, detected as an increase in HPTS fluorescence, was coincident with the acidification of HPTS. The rate and extent of uptake of neutral and negatively charged liposomes was similar; however, liposomes opsonized with antibody were incorporated at a higher rate (2.9-fold) and to a greater extent (3.4-fold). In addition, the rate and extent of incorporation of liposome encapsulated HPTS was dependent on temperature and the metabolic state of the cell, consistent with uptake of liposomes by endocytosis. The use of HlYFS allowed accurate and simultaneous quantitation of iiposome uptake, acidification, cell-induced leakage of liposomes, and regurgitation of liposome contents. In addition to cell-surface induced leakage, liposomes leaked extensively during endocytosis coincident with acidification; half of the cell-induced dilution of liposome contents was accounted for by leakage at the cell surface, while the remainder occurred coincident with acidification. Liposome contents labeled the aqueous space of endosomes and lysosomes and were regurgitated rapidly as liposomal lipid accumulated selectively. Opsonization of iiposomes, to induce Fc-mediated endocytosis, afforded protection to the initial dilution of liposome contents, but not the rate of leakage, after endocytosis. Implications of these studies for the use of liposomes as drug delivery vehicles are discussed.
Biochemical Journal, 2011
Proteins SP-B and SP-C are essential to promote formation of surface-active films at the respiratory interface, but their mechanism of action is still under investigation. In the present study we have analysed the effect of the proteins on the accessibility of native, quasi-native and model surfactant membranes to incorporation of the fluorescent probes Nile Red (permeable) and FM 1-43 (impermeable) into membranes. We have also analysed the effect of single or combined proteins on membrane permeation using the soluble fluorescent dye calcein. The fluorescence of FM 1-43 was always higher in membranes containing SP-B and/or SP-C than in protein-depleted membranes, in contrast with Nile Red which was very similar in all of the materials tested. SP-B and SP-C promoted probe partition with markedly different kinetics. On the other hand, physiological proportions of SP-B and SP-C caused giant oligolamellar vesicles to incorporate FM 1-43 from the external medium into apparently most of t...
American journal of physiology. Lung cellular and molecular physiology, 2001
Infection of the respiratory tract is a frequent cause of lung pathologies, morbidity, and death. When bacterial endotoxin [lipopolysaccharide (LPS)] reaches the alveolar spaces, it encounters the lipid-rich surfactant that covers the epithelium. Although binding of hydrophilic surfactant protein (SP) A and SP-D with LPS has been established, nothing has been reported to date on possible cross talks between LPS and hydrophobic SP-B and SP-C. We designed a new binding technique based on the incorporation of surfactant components to lipid vesicles and the separation of unbound from vesicle-bound LPS on a density gradient. We found that among the different hydrophobic components of mouse surfactant separated by gel filtration or reverse-phase HPLC, only SP-C exhibited the capacity to bind to a tritium-labeled LPS. The binding of LPS to vesicles containing SP-C was saturable, temperature dependent, related to the concentrations of SP-C and LPS, and inhibitable by distinct unlabeled LPSs...
Monocytes incubated with surfactant: a model for human alveolar macrophages?
1997
Monocytes migrate to the lungs and enter the alveoli where they come into contact with surfac- tant and differentiate into alveolar macrophages. This study focused on the question of the extent to which monocytes and monocyte-derived macrophages (MDM) incubated with surfactant resemble alveolar macro- phages. Surfactant-incubated monocytes shared with alveolar macrophages the intracellular presence of sur- factant, efficient phagocytosis of
PLoS ONE, 2012
The peripheral lungs are a potential entrance portal for nanoparticles into the human body due to their large surface area. The fact that nanoparticles can be deposited in the alveolar region of the lungs is of interest for pulmonary drug delivery strategies and is of equal importance for toxicological considerations. Therefore, a detailed understanding of nanoparticle interaction with the structures of this largest and most sensitive part of the lungs is important for both nanomedicine and nanotoxicology. Astonishingly, there is still little known about the bio-nano interactions that occur after nanoparticle deposition in the alveoli. In this study, we compared the effects of surfactant-associated protein A (SP-A) and D (SP-D) on the clearance of magnetite nanoparticles (mNP) with either more hydrophilic (starch) or hydrophobic (phosphatidylcholine) surface modification by an alveolar macrophage (AM) cell line (MH-S) using flow cytometry and confocal microscopy. Both proteins enhanced the AM uptake of mNP compared with pristine nanoparticles; for the hydrophilic ST-mNP, this effect was strongest with SP-D, whereas for the hydrophobic PL-mNP it was most pronounced with SP-A. Using gel electrophoretic and dynamic light scattering methods, we were able to demonstrate that the observed cellular effects were related to protein adsorption and to protein-mediated interference with the colloidal stability. Next, we investigated the influence of various surfactant lipids on nanoparticle uptake by AM because lipids are the major surfactant component. Synthetic surfactant lipid and isolated native surfactant preparations significantly modulated the effects exerted by SP-A and SP-D, respectively, resulting in comparable levels of macrophage interaction for both hydrophilic and hydrophobic nanoparticles. Our findings suggest that because of the interplay of both surfactant lipids and proteins, the AM clearance of nanoparticles is essentially the same, regardless of different intrinsic surface properties.