help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on November 25, 2003, doi:10.1165/rcmb.2003-0127OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2003-0127OCv1
30/5/751    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Poelma, D. L.
Right arrow Articles by van Iwaarden, J. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Poelma, D. L.
Right arrow Articles by van Iwaarden, J. F.
American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 751-758, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0127OC

A Common Pathway for the Uptake of Surfactant Lipids by Alveolar Cells

Davey L. Poelma, Michel R. Ju, Sjoerd C. Bakker, Luc J. Zimmermann, Burkhard F. Lachmann and J. Freek van Iwaarden

Department of Anesthesiology, and Laboratory of Pediatrics, Erasmus Medical Center—Faculty, University Medical Center Rotterdam, Rotterdam, The Netherlands

Address correspondence to: J. Freek van Iwaarden, Ph.D., Laboratory of Pediatrics, Erasmus MC—Faculty, University Medical Center Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail: fviw{at}paed.azm.nl


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 RESULTS
 Discussion
 References
 
The uptake of different surfactant lipids—dipalmitoylphosphatidylcholine (DPPC), phosphatidylglycerol (PG), or phosphatidylinositol (PI)—and liposomes with a surfactant-like composition by alveolar type II cells (alveolar type II cells) and macrophages (alveolar macrophages) was studied in vitro. Fluorescent-labeled liposomes containing either 86% of the studied lipid, i.e., DPPC, PG, PI, and 6% labeled phosphatidylethanolamine (PE) and 8% cholesterol or a lipid mixture similar to surfactant (DPPC, PG, PI, phosphatidylcholine, PE, and cholesterol in a weight ratio of 55:8:2:21:8:6) were incubated with alveolar macrophages and alveolar type II cells. The cell-associated fluorescence assessed by flow cytometry demonstrated a higher uptake of PG and PI by both alveolar macrophages and alveolar type II cells, and a lower uptake of DPPC by alveolar macrophages. In addition, fewer alveolar type II cells take up DPPC, whereas there are no differences for the alveolar macrophages in the number of cells involved in the uptake. Competition experiments with Texas Red–labeled liposomes and either DPPC liposomes or PI liposomes labeled with Bodipy indicated that all these liposomes are internalized via the same pathway by alveolar cells. Thus, lipid composition directly influences the (re)uptake of surfactant.

Abbreviations: dipalmitoylphosphatidylcholine, DPPC • phosphate-buffered saline, PBS • phosphatidylcholine, PC • phosphatidylethanolamine, PE • phosphatidylglycerol, PG • phosphatidylinositol, PI • surfactant proteins, SP


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 RESULTS
 Discussion
 References
 
The alveolus is lined with a thin layer of lipids and proteins, called surfactant, and this surface-active agent has an essential role in maintaining normal lung function. Pulmonary surfactant is produced by alveolar type II cells. By weight, ~ 90% of surfactant consists of lipids. Although the lipid composition varies in different species, its major component is phosphatidylcholine (PC; 70–80%), of which nearly 50% is saturated dipalmitoylphosphatidylcholine (DPPC). DPPC is the major surface tension–reducing component of surfactant. In addition, surfactant contains variable amounts of phosphatidylglycerol (PG; 7–18%), phosphatitylinositol (PI; 2–4%), and phosphatidylethanolamine (PE; 2–3%) (1). The remaining percentage consists of other phospholipids and cholesterol (1).

The presence of surfactant within the alveolus is the result of a complex system of production, secretion, uptake, and recycling. The production of newly synthesized surfactant has been suggested to be relatively slow, especially in newborn animals (2, 3). Therefore, inactivated surfactant is reused, i.e., recycled. It has been demonstrated that 50–90% of the PC is recycled depending on age and species, the contribution of recycling decreasing with increasing age (4). Inactivated surfactant is cleared from the alveolar space mainly by alveolar type II cells (5). This uptake of surfactant by alveolar type II cells is essential in enabling surfactant recycling. Therefore, uptake or reuptake of surfactant plays an important role in the homeostasis of the surfactant metabolism.

It has been shown that there are essential differences in the composition of surfactant between neonates and adults with regard to phospholipids, which, combined with the fact that neonates rely more on recycling, suggests an effect of the composition on the uptake of surfactant. Previous studies have demonstrated an effect of surfactant protein (SP), especially SP-A, on the uptake by alveolar cells (6–9). However, little is known about the effect of lipid composition on the clearance of surfactant from the alveolar space by alveolar cells, because most studies have focused on internalization of DPPC by both alveolar type II cells and alveolar macrophages. Quintero and colleagues have shown a more rapid clearance of PG by alveolar macrophages in contrast to DPPC (10), indicating an effect of lipid composition on uptake. It remains unclear, however, whether the uptake of surfactant by alveolar type II cells is also affected by lipid composition. Therefore, we studied the uptake of the major surfactant lipids by alveolar type II cells as well as alveolar macrophages using fluorescence-labeled liposomes (11). In addition, we examined how the different lipids influence each other's uptake by alveolar cells.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
RESULTS
 Discussion
 References
 
Ethical Guidelines
This study was approved by the Institutional Animal Committee at the Erasmus University Rotterdam and complied with National Institutes of Health guidelines. A total of 30 male Sprague Dawley rats (IFFA Credo, Someren, The Netherlands) with an average weight of 280 ± 41 g were used.

Materials
DPPC, egg PC, PG, PI, PE, and cholesterol were purchased from Sigma (Zwijndrecht, The Netherlands). PE labeled with Bodipy and that labeled with Texas Red were obtained from Molecular Probes (Leiden, The Netherlands). Both labeled PE lipids are labeled in the head-group.

Liposome Preparation
To prepare liposomes, the indicated concentrations of the different lipids were mixed. The mixture of lipids was dried under a stream of nitrogen gas. Liposomes were suspended in phosphate-buffered saline (PBS) at a concentration of 0.25 mg lipids/ml using glass pearls and vortexing. Immediately prior to use, the liposome suspension was sonicated for 2 min on ice using an ultrasonic disintegrator (Branson Sonifier 250, Danbury, CT) to prepare small, unilamellar liposomes (11). The size of the liposomes was determined by dynamic light scattering at 25°C with a Malvern 4700 system using a 25 mW Helium-Neon laser (NEC, Tokyo, Japan) and Automeasure version 3.2 software (Malvern Ltd, Malvern, UK). As a measure of particle size distribution of the dispersion, the system reports a polydispersity index. This index ranges from 0.0 for a monodisperse and up to 1.0 for an entirely polydisperse dispersion. After ultrasonification, liposome size ranged from 140–165 nm and the polydispersity index ranged from 0.20–0.35.

Isolation of Alveolar Cells
Before isolation of the cells from Sprague Dawley rats, the thorax was opened and the blood cells were removed from the lungs by perfusing the pulmonary artery with saline (37°C) supplemented with 20 IE heparin (Leo Pharma, Weesp, The Netherlands). The lungs were removed from the thoracic cavity en bloc and lavaged with 10 ml of Solution 1 (140 mM NaCl, 5 mM KCl, 2.5 mM Na2HPO4.2H2O, 10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethaesulfonic acid], 6 mM glucose, 0.2 mM EGTA (ethylene glycol-bis-[ß-amino ethyl ether] N,N'-tetraacetic acid, pH 7.40) at 22°C. This procedure was repeated four times. The lung lavages were pooled per animal and centrifuged (100 x g; 10 min; 4°C). The cellular pellet, i.e., alveolar macrophages, was suspended in Solution 2 (140 mM NaCl, 5 mM KCl, 2.5 mM phosphate buffer, 10 mM HEPES, 6 mM glucose, 2.0 mM CaCl2, and 1.3 mM MgSO4) to a concentration of 2 x 106 cells/ml and stored on ice until further use. The alveolar type II cells were isolated according to Dobbs and colleagues (12), suspended in Solution 2 at a concentration of 2 x 106 cells/ml, and stored on ice until further use. Alveolar macrophages were identified using monoclonal antibodies specific for rat macrophages (ED9) and alveolar type II cells were identified using an alkaline phosphatase assay as described by Edelson and collegues (13). The average yield per rat of alveolar type II cells was 16 x 106 and 5 x 106 alveolar macrophages. The purity of the alveolar type II cells was determined and 80 ± 5% of the cells in the type II cell isolate were identified as alveolar type II cells. The alveolar macrophage isolate had a purity of 92 ± 2% as determined by ED9 antibodies.

Uptake of Liposomes by Alveolar Cells
Alveolar type II cells and alveolar macrophages were isolated as described above and were suspended in Solution 2 to a concentration of 2 x 106 cells/ml. A total of 3 x 105 cells were incubated with various concentrations of the different liposomes at 37°C (unless stated otherwise) (final volume, 500 µl) in a shaking water bath. After 1 h, the incubation was terminated by addition of 2 ml of ice-cold PBS (11). The cell suspension was centrifuged at 100 x g for 10 min at 4°C. The supernatant was removed and the cells were suspended in 2 ml of ice-cold PBS and centrifuged again. This wash procedure was repeated twice. Finally, the pellet was resuspended in 200 µl cold PBS and cell-associated fluorescence was determined as described below.

Flow Cytometry
As a measure for the amount of internalized liposomes, cell-associated fluorescence of a total of 15,000 alveolar type II cells or alveolar macrophages was determined using flow cytometry (FACSCalibur; Becton Dickinson, Franklin Lakes, NY). The Bodipy fluorescence was determined in the FL 1 channel, whereas the Texas Red fluorescence was determined in the FL 4 channel. The spillover of Bodipy in the FL 4 channel as well as Texas Red in the FL 1 channel was less than 1% and was not corrected for in the double-labeling experiments. Control alveolar macrophages and alveolar type II cells were used in each experiment to determine the autofluorescence of the cells. Subsequently, the mean cell-associated fluorescence was determined only for those cells that had a higher fluorescence than that caused by autofluorescence (gated cells) (11). Cells with a higher fluorescence than that caused by autofluorescence were collected and identified using the methods described above, showing similar results for all types of liposomes; 81 ± 6% of the cells within the type II cell isolate were type II cells and 93 ± 5% in the alveolar macrophage isolate were alveolar macrophages.

Localization of Cell-Associated Fluorescence
Confocal laser microscopy was performed using a Zeiss LSM 410, with standard objectives and photomultiplier tubes dedicated to the appropriate excitation and emission spectra of the fluorescent label used (Bodipy: excitation, 503 nm; emission, 512 nm. Texas Red: excitation, 589 nm; emission, 615 nm). Images were serial-sectioned at a depth of 0.5 µm to distinguish cell-membrane associated fluorescence from true intracellular fluorescence.

Statistical Analysis
Differences in uptake of different components were determined by analysis of variance followed by a Bonferroni post hoc test; differences were considered statistically significant at a P < 0.05. Values are expressed as mean ± SEM.


   RESULTS
Top
 Abstract
 Introduction
 Material and Methods
 RESULTS
Discussion
 References
 
Concentration-Dependent Uptake of Liposomes
To study the uptake of the phospholipid components of surfactant, DPPC, PC, PG, and PI, isolated alveolar cells were incubated for 1 h with liposomes of different compositions. The mean cell-associated fluorescence, as a measure of liposome uptake, of the alveolar type II cells and alveolar macrophages demonstrates a concentration-dependent increase. With exception of the uptake of PI liposomes by alveolar macrophages and the uptake of PG liposomes by alveolar type II cells, the uptake of all individual liposomes reaches an apparent maximum at 25 µg/ml (Figures 1A and 1B). However, the maximum cell-associated fluorescence (e.g., maximal uptake) at the plateau differs between the various liposome compositions.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 1. Concentration-dependent uptake of liposomes. Alveolar type II cells (A) and macrophages (B) were isolated and incubated for 1 h at 37°C with the indicated concentrations of either 86% PG (PG:Chol:PE-Bodipy; weight ratio: 86:8:6), 86% PI (PI:Chol:PE-Bodipy; 86:8:6), 86% DPPC (DPPC:Chol.:PE-Bodipy; 86:8:6), 86% PC (PC: Chol:PE-Bodipy; 86:8:6), or normal (DPPC:PC: PG:PI:Chol:PE-Bodipy; 55:21:8:2:8:6) liposomes. The mean cell-associated fluorescence was determined for those cells with a higher fluorescence than autofluorescence (n = 4 incubations of 3 x 105 cells each at every concentration; values expressed as mean ± SEM). #Indicates a significant difference compared with the normal liposomes.

 
The 86% PG and 86% PI liposomes are internalized significantly more by alveolar type II cells than normal liposomes (PC, DPPC, PG, PI, PE, and cholesterol in a weight ratio of 21:55:8:2:6:8). The uptake of 86% PC liposomes and 86% DPPC liposomes does not differ from the normal liposomes, except at a higher lipid concentration of 50 µg/ml, more 86% PC liposomes are internalized (Figure 1A).

There is a significant difference in the uptake of 86% PG, 86% PI, 86% PC, and normal liposomes by alveolar macrophages at a concentration of 50 µg/ml. The internalization of 86% PG liposomes is significantly higher than that of normal liposomes, whereas the uptake of 86% DPPC liposomes by alveolar macrophages is significantly lower than normal liposomes. These results may indicate that uptake of liposomes by alveolar macrophages and alveolar type II is affected by the charge of the liposomes (i.e., 86% PG and 86% PI liposomes are more negatively charged).

Percentage Alveolar Type II Cells and Alveolar Macrophages Involved in Liposome Uptake
The percentage of gated alveolar type II cells and alveolar macrophages involved in the uptake of liposomes also demonstrate a concentration-dependent increase, reaching a plateau for all groups of liposomes (Figures 2A and 2B). The percentage of alveolar macrophages involved in the uptake of liposomes is not significantly affected by the charge of the liposomes (Figure 2B). In contrast, the number of alveolar type II cells internalizing liposomes is affected by the charge of the liposomes, although only the 86% PI liposomes are capable of significantly increasing the number of alveolar type II cells taking up liposomes. More interesting, however, is the significantly lower percentage of alveolar type II cells taking part in the uptake of 86% DPPC liposomes: 29 ± 4.5% versus 72.7 ± 2.3% involved in the internalization of normal liposomes at a concentration of 50 µg/ml (Figure 2A).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 2. Concentration-dependent uptake of liposomes. Alveolar type II cells (A) and macrophages (B) were isolated and incubated for 1 h at 37°C with the same fluorescent liposomes as described in the legend for Figure 1. The percentage of cells with a cell-associated fluorescence higher than autofluorescence was determined (= gated cells) (n = 4 incubations of 3 x 105 cells each at every concentration; values expressed as mean ± SEM). #Indicates a significant difference compared with the normal liposomes.

 
Localization of Cell-Associated Fluorescence
To ascertain that the cell-associated fluorescence was located within the cell rather than a specific binding to the outer membrane of the cell, confocal laser microscopy was used. The confocal scans through the middle of the cell show a punctuate fluorescence throughout the cell limited to its circumference with the exception of the nucleus. Both labels, the Texas Red–labeled PG liposomes (Figures 3A and 3C) and Bodipy-labeled DPPC liposomes (Figures 3B and 3D), were internalized by the alveolar type II cells and alveolar macrophages; untreated cells did not show any fluorescence at the same microscopic settings.



View larger version (152K):
[in this window]
[in a new window]
  		Figure 3. Localization of cell-associated fluorescence. Both alveolar type II cells (A, B) and alveolar macrophages (C, D) were isolated and incubated with 86% Bodipy-labeled DPPC liposomes and 86% Texas Red–labeled PG liposomes. One h after incubation, cells were washed 3 times with cold PBS and the cells were mounted on glass coverslips. Confocal laser microscopy demonstrates a punctuate distribution of Texas Red–labeled PG liposomes (A, C) and Bodipy-labeled DPPC liposomes (B, D) that is not limited to the cell surface, and is not localized in the nucleus of the cell. This distribution of fluorescence throughout the cell indicates internalization of liposomes, strengthened by the fact that untreated cells did not show any fluorescence at the same microscopic settings.

 
Effect of 86% PG Texas Red–Labeled Liposomes on the Uptake of 86% PI Bodipy-Labeled Liposomes
The increased uptake of negatively charged PI and PG liposomes, combined with an increased percentage alveolar type II cells involved in this uptake, raised the question whether these two types of liposomes are taken up via the same "charge" dependent pathway. Therefore, the effect of 86% PG–Texas Red–labeled liposomes on the uptake of 86% PI-Bodipy–labeled liposomes by alveolar type II cells and alveolar macrophages was studied by keeping the Bodipy-labeled PI liposomes constant and increasing the concentration of added Texas Red–labeled PG liposomes. In both cell types, alveolar type II cells and alveolar macrophages, Bodipy-associated mean fluorescence (i.e., uptake of 86% PI liposomes) significantly decreases with an increasing concentration of 86% PG liposomes. On the other hand, the cell-associated Texas Red mean fluorescence (i.e., uptake of 86% PG liposomes) increases significantly with an increasing concentration of 86% PG liposomes (Figures 4A and 4B).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 4. Competition between PI liposomes and PG liposomes (mean fluorescence). Alveolar cells were isolated and incubated for 1 h at 37°C with 12.5 µg Bodipy-labeled PI liposomes (PI:Chol:PE-Bodipy; 86:8:6) and the indicated concentrations of Texas Red–labeled PG liposomes (PG:Chol:PE Texas Red; 86:8:6). The mean cell-associated Bodipy fluorescence (PI; squares) as well as the mean cell-associated Texas Red fluorescence (PG; triangles) was determined in alveolar type II cells (A) and alveolar macrophages (B). Subsequently, the cell-associated Bodipy fluorescence in the presence of PG Texas Red was expressed as a percentage of the cell-associated Bodipy fluorescence of the incubation with 12.5 µg PI-Bodipy alone (cell-associated PI [%]). The cell-associated Texas Red fluorescence in the presence of PI Bodipy was expressed as a percentage of the cell-associated Texas Red fluorescence of the incubation with 50 µg PG Texas Red alone (cell-associated PG [%]). Values are expressed as mean ± SEM (n = 4). #Indicates a significant difference compared with 0 µg/ml Texas Red–labeled PG liposomes.

 
In addition, when the concentration of 86% PG liposomes is increased, significantly fewer cells take up 86% PI liposomes preferentially; there is a significant increase in the number of alveolar cells, either alveolar type II cells or alveolar macrophages, that take up both 86% PI liposomes and 86% PG liposomes (Figures 5A and 5B). There are hardly any alveolar cells that internalize only 86% PG liposomes, except at the highest PG concentration, 50 µg/ml, at which there is a significant percentage (3%) of the alveolar type II cells that internalize only 86% PG liposomes. The total amount of alveolar cells involved in the uptake does not change significantly (83% for alveolar macrophages and 79% for alveolar type II cells) (Figures 5A and 5B). These results may indicate that both types of liposomes are taken up via the same pathway. Next, the question arose whether this pathway was the same for DPPC.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 5. Competition between PI liposomes and PG liposomes (gated cells). Alveolar cells type II cells (A) and macrophages (B) were isolated and incubated for 1 h at 37°C with the same fluorescent liposomes as described in the legend for Figure 3. Squares (%PI+, PG– cells) are the percentage cells whose Bodipy cell–associated fluorescence is higher than the autofluorescence and whose fluorescence caused by Texas Red is within the range of autofluorescence. Triangles (%PG+, PI– cells) are the percentage cells that have a Texas Red cell–associated fluorescence higher than autofluorescence and a Bodipy fluorescence within the range of autofluorescence. Circles (%PG+, PI+) are the percentage of cells that have a Texas Red cell–associated fluorescence higher than the autofluorescence, as well as a Bodipy cell–associated fluorescence higher than the autofluorescence. The dotted line and asterisks are the total percentage of cells with a fluorescence higher than the autofluorescence, either by Bodipy or Texas Red. Values are expressed as mean ± SEM (n = 4). #Indicates a significant difference compared with 0 µg/ml Texas Red–labeled PG liposomes.

 
Effect of 86% PG Texas Red–Labeled Liposomes on the Uptake of 86% DPPC Bodipy–Labeled Liposomes
To determine if 86% DPPC liposomes are taken up via the same pathway as 86% PG, alveolar cells were incubated with 86% DPPC Bodipy–labeled liposomes and increasing concentrations of 86% PG Texas Red–labeled liposomes. Increasing the concentration of 86% PG Texas Red–labeled liposomes results in a significant increase of Texas Red–associated mean fluorescence, together with a significant decrease in Bodipy-associated mean fluorescence (Figures 6A and 6B).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 6. Uptake of DPPC liposomes in presence of Texas Red–labeled PG liposomes (mean fluorescence). Alveolar cells were isolated and incubated for 1 h at 37°C with 12.5 µg Bodipy labeled DPPC liposomes (DPPC:Chol:PE-Bodipy; 86:8:6) and the indicated concentrations of Texas Red–labeled PG liposomes (PG:Chol:PE-Texas Red; 86:8:6). The mean cell-associated Bodipy fluorescence (DPPC; squares) as well as the mean cell-associated Texas Red fluorescence (PG; triangles) was determined in alveolar type II cells (A) and alveolar macrophages (B). Subsequently, the cell-associated Bodipy fluorescence in the presence of PG Texas Red was expressed as a percentage of the cell-associated Bodipy fluorescence of the incubation with 12.5 µg DPPC Bodipy alone (%cell-associated DPPC). The cell-associated Texas Red fluorescence in the presence of DPPC Bodipy was expressed as a percentage of the cell-associated Texas Red fluorescence of the incubation with 50 µg PG Texas Red alone (%cell-associated PG). Values are expressed as mean ± SEM (n = 4). #Indicates a significant difference compared with 0 µg/ml Texas Red–labeled PG liposomes.

 
Raising the concentration of 86% PG liposomes results in a significant increase in double-labeled alveolar macrophages, together with a significant decrease in Bodipy-labeled alveolar macrophages.

At a concentration of 12.5 µg/ml 86% PG liposomes, the amount of double-labeled alveolar macrophages decreases again and the amount of alveolar macrophages that take up only 86% PG liposomes increases (Figure 7B). The alveolar type II cells demonstrate a similar change in cells involved in the uptake; first the amount of double-labeled cells increase, and at a concentration of 5 µg/ml, which is lower than for alveolar macrophages, the amount of double-alveolar type II cells decreases, together with an increase in alveolar type II cells that take up only 86% PG liposomes (Figure 7A).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 7. Uptake of DPPC liposomes in presence of Texas Red–labeled PG liposomes (gated cells). Alveolar type II cells (A) and macrophages (B) were isolated and incubated for 1 h at 37°C with the same fluorescent liposomes as described in the legend of Figure 5. The squares (%DPPC+, PG– cells) are the percentage of cells that have a Bodipy cell–associated fluorescence higher than the autofluorescence and a Texas Red fluorescence not higher than the autofluorescence. The triangles (%PG+, DPPC– cells) are the percentage of cells that have a Texas Red cell–associated fluorescence higher than the autofluorescence and a Bodipy fluorescence not higher than the autofluorescence. The circles (%PG+, DPPC+) are the percentage of cells that have a Texas Red cell–associated fluorescence higher than the autofluorescence as well as Bodipy cell–associated fluorescence higher than the autofluorescence. The dotted line and asterisks are the total percentage of cells with a fluorescence higher than autofluorescence, either by Bodipy or Texas Red. Values are expressed as mean ± SEM (n = 4). #indicates a significant difference compared with 0 µg/ml Texas Red–labeled PG liposomes.

 
Effect of 86% DPPC Texas Red–Labeled Liposomes on the Uptake of 86% PG Bodipy–Labeled Liposomes
To determine whether 86% DPPC liposomes can inhibit the uptake of 86% PG liposomes, suggesting a similar pathway for uptake of liposomes, alveolar cells were incubated with 86% PG Bodipy–labeled liposomes and increasing concentrations of 86% DPPC Texas Red–labeled liposomes. Increasing the concentration of 86% DPPC Texas Red–labeled liposomes results in a significant decrease of Bodipy-associated mean fluorescence in alveolar type II cell, but no changes were seen in cell-associated fluorescence in alveolar macrophages (Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Uptake of phosphatidylglycerol liposomes in the presence of increasing concentrations of dipalmitoylphosphatidylcholine liposomes

 
Increasing the concentration of 86% DPPC liposomes results in a significant decrease in single-labeled, Bodipy-associated alveolar cells, and a concomitant increase in double-labeled, Bodipy- and Texas Red–associated alveolar cells. There is a slight, nonsignificant increase in alveolar cells that are only Texas Red–labeled and thus takes up only 86% DPPC liposomes.


   Discussion
Top
 Abstract
 Introduction
 Material and Methods
 RESULTS
 Discussion
References
 
In the current study, we investigated the uptake of the main phospholipid components of surfactant in vitro by alveolar type II cells and alveolar macrophages. We used fluorescence-labeled liposomes containing 86% of the studied component, 8% cholesterol, and 6% labeled PE. Confocal laser microscopy demonstrated the intracellular presence of cell-associated fluorescence, indicating that the fluorescence-labeled liposomes are indeed taken up by the alveolar cells (Figure 3). The uptake of the liposomes shows a concentration-dependent increase in cell-associated fluorescence (Figure 1), reaching a plateau at 25 µg/ml, similar to the results of our previous study (11). The uptake of the more negatively charged liposomes (i.e., 86% PG and 86% PI liposomes) by alveolar type II cells and alveolar macrophages is significantly higher than that of the more neutrally charged liposomes (i.e., 86% PC, 86% DPPC, and normal liposomes). These results indicate that the alveolar cells have a higher affinity for negatively charged liposomes.

The mechanism of internalization of liposomes by the alveolar cells is not clear at the moment. For macrophages as well as alveolar type II cells, at least part of the uptake of liposomes seems to be mediated by the coated-pit pathway (14–16), suggesting a receptor-mediated process, but for which the receptor and the specificity of the receptor are unknown. For argument's sake, we assume that the uptake of liposomes by alveolar cells is receptor-mediated. This is a receptor with a high affinity for negatively charged liposomes per se and which does not discriminate between PG and PI liposomes, as was shown by competition experiments with 86% PI- and 86% PG-containing liposomes.

The receptor is not specific for negatively charged liposomes. The competition experiments of PG and DPPC liposomes demonstrate that the uptake of DPPC by the alveolar cells can be fully blocked by PG, leading, instead, to uptake of PG, whereas in the reverse, competing PG with DPPC leads to only a partial reduction in the PG uptake by alveolar cells (Table 1). Therefore, the receptor affinities for DPPC and PG and/or PI liposomes seem to differ; the highest affinity being for PG, followed by PI and DPPC. There seem to be very few differences in the uptake of phospholipids by alveolar macrophages and alveolar type II cells, but from our experiments, it cannot be concluded that the "phospholipid receptor" of alveolar macrophages and alveolar type II cells is the same. The only striking difference is the number of cells involved in the uptake of DPPC liposomes. The number of alveolar type II cells that internalize DPPC liposomes is significantly lower than the number of alveolar macrophages (29 versus 72%), whereas the number of cells involved in the uptake of PG liposomes is approximately the same for alveolar macrophages and type II cells (75–80%). A possible explanation is that the distribution of receptors on alveolar macrophages and type II cells differs. Most of the alveolar macrophages may have a high density of receptors, whereas the alveolar type II cell population consists of several subpopulations of cells with different receptor densities. Hence, adding DPPC liposomes to alveolar cells will lead to association of DPPC with practically all macrophages and, in the case of type II cells, association will occur only with the subpopulation of type II cells with the highest receptor density. PG or PI liposomes will also associate with the subpopulations of alveolar type II cells with lower receptor densities due to the high affinity of the receptor for these two phospholipids. This hypothesis may explain the competition experiments of DPPC with PG and vice versa using alveolar type II cells (Figures 6 and 7 and Table 1). In experiments in which DPPC is placed in competition with PG, a rapid drop in the cell-associated DPPC is observed, whereas PG competing with DPPC demonstrates only a slight reduction in the cell-associated PG, which is indicative of a higher affinity of the alveolar cells for PG than for DPPC. The maximal number of alveolar type II cells that can internalize DPPC is ~ 30%, adding PG to the incubation results in a rapid decline in the number of cells that contain only DPPC with a concomitant increase in PG+ cells. The double-positive cells increase initially, but at higher PG concentrations the number decreases. Apparently, at relatively low PG concentrations, PG competes with DPPC for the cells with the highest receptor density, leading to cells that internalize both lipids as well as small numbers of PG+ and DPPC+ cells. At higher PG concentrations, the PG uptake by cells with high receptor density is saturated, leading to the uptake of PG by the subpopulation of cells with low receptor density: those that cannot internalize DPPC. Therefore, there are more alveolar type II cells that can take up PG, which consist of the subpopulations of cells with high as well as with low receptor densities, and fewer cells that can internalize DPPC, namely the subpopulation with high receptor density. The results of the competition between PG and DPPC can be explained in a similar way. In the absence of DPPC, the number of PG+ cells is ~ 80%, which consists of the subpopulations with high as well as low receptor densities. Including DPPC in the incubations leads to a reduction in the number of the PG+ cells that remain at 30–40% PG+ cells at a 10-fold higher concentration of DPPC over PG. This cell population reflects the low receptor density cells. Hence, ~ 40–50% of the alveolar type II cells have a low receptor density and cannot internalize DPPC, and ~ 30–40% of the alveolar type II cells have a high receptor density and are able to internalize both DPPC and PG.

Up to now, the focus has been on the role of SPs in the uptake of DPPC by alveolar cells in vitro. At least three of the four SPs (SP-A, SP-B, and SP-C) were implicated in the internalization of surfactant lipids by alveolar cells. SP-A and SP-C were shown to stimulate the uptake of liposomes by alveolar cells (6–9, 16, 17), whereas the influence of SP-B on the uptake of liposomes is more complex. SP-B was reported to stimulate the uptake by alveolar cells (18–20), whereas others have shown that SP-B can inhibit the SP-C–mediated uptake by alveolar type II cells (7, 8). In vivo the role of the surfactant proteins becomes more complex and unclear. In SP-A knockout mice, the uptake of surfactant by alveolar cells appears to be normal (21, 22), whereas one would expect that, based on the vitro results, the uptake would be disturbed in these animals; only under inflammatory conditions are differences in lipid uptake between SP-A (+/+) and SP-A (–/–) mice observed (23). In contrast, for SP-D no influence on lipid uptake could be demonstrated in vitro (17), whereas the surfactant lipid metabolism in SP-D (–/–) mice was disturbed (24, 25). Therefore, in spite of numerous studies, the role of the SPs in the surfactant lipid metabolism in vivo still needs to be further clarified.

The influence of the surfactant phospholipid composition, in particular the charge of the phospholipids, on the uptake of surfactant liposomes by alveolar cells is also not clear. The first data are derived from in vivo experiments by Jacobs and colleagues (26, 27), demonstrating the disappearance of radiolabeled PG in the lungs of neonatal and adult rabbits after intratracheal injection. Bates and Chandler have shown that radiolabeled PG–containing vesicles are internalized more rapidly by alveolar type II cells in vitro (28, 29). Recently, a study by Quintero and colleagues indicated that alveolar macrophages internalize PG more readily than DPPC in vitro (10). The present study indicates that, besides alveolar macrophages, alveolar type II cells also have a higher affinity for PG- or PI-containing liposomes than for the more neutrally charged liposomes. In addition, the pathway of uptake of PG, PI, and DPPC by alveolar cells appears to be the same. Uptake of surfactant lipids by alveolar cells is an important step in the recycling of surfactant. Surfactant recycling itself is crucial in maintaining normal lung functions. Therefore, changes in the surfactant lipid composition, for instance, more or less PG or other negatively charged lipids, may not only alter the uptake of these lipids but also of DPPC, the most important surface tension–lowering component of surfactant. In various diseases, such as respiratory distress syndrome, extrinsic allergic alveolitis, and respiratory infections, changes in the surfactant lipid composition have been observed (30, 31). Whether an altered uptake of surfactant lipids by alveolar cells plays a role in the pathogenesis of respiratory diseases is not yet known. Further studies on the role of the surfactant lipid composition on the uptake of surfactant by alveolar cells are therefore warranted, particularly with regard to the influence of lipid composition on the in vivo uptake of surfactant and the effects on lung mechanics. Not only will this information expand our knowledge of the surfactant metabolism, but it might also provide new tools for therapeutic interventions for various respiratory diseases.


   Acknowledgments
 
This study was supported by LEO Pharmaceutical Products, Ballerup, Denmark. The authors thank A. Kersbergen for her technical assistance and Laraine Visser-Isles for English-language editing.

Received in original form April 4, 2003

Received in final form November 25, 2003


   References
Top
 Abstract
 Introduction
 Material and Methods
 RESULTS
 Discussion
 References
 

  1. Veldhuizen, R., K. Nag, S. Orgeig, and F. Possmayer. 1998. The role of lipids in pulmonary surfactant. Biochim. Biophys. Acta 1408:90–108.[Medline]
  2. Jacobs, H., A. Jobe, M. Ikegami, and S. Jones. 1982. Surfactant phosphatidylcholine source, fluxes, and turnover times in 3-day-old, 10-day-old, and adult rabbits. J. Biol. Chem. 257:1805–1810.[Abstract/Free Full Text]
  3. Jobe, A., M. Ikegami, I. Sarton-Miller, and L. Barajas. 1980. Surfactant metabolism of newborn lamb lungs studied in vivo. J. Appl. Physiol. 49:1091–1098.[Abstract/Free Full Text]
  4. Martini, W. Z., D. L. Chinkes, R. E. Barrow, E. D. Murphey, and R. R. Wolfe. 1999. Lung surfactant kinetics in conscious pigs. Am. J. Physiol. 277:E187–E195.
  5. Rider, E. D., M. Ikegami, and A. H. Jobe. 1992. Localization of alveolar surfactant clearance in rabbit lung cells. Am. J. Physiol. 263:L201–L209.
  6. Bates, S. R., C. Dodia, and A. B. Fisher. 1994. Surfactant protein A regulates uptake of pulmonary surfactant by lung type II cells on microporous membranes. Am. J. Physiol. 267:L753–L760.
  7. Horowitz, A. D., B. Moussavian, E. D. Han, J. E. Baatz, and J. A. Whitsett. 1997. Distinct effects of SP-A and SP-B on endocytosis of SP-C by pulmonary epithelial cells. Am. J. Physiol. 273:L159–L171.
  8. Horowitz, A. D., B. Moussavian, and J. A. Whitsett. 1996. Roles of SP-A, SP-B, and SP-C in modulation of lipid uptake by pulmonary epithelial cells in vitro. Am. J. Physiol. 270:L69–L79.
  9. Tsuzuki, A., Y. Kuroki, and T. Akino. 1993. Pulmonary surfactant protein A-mediated uptake of phosphatidylcholine by alveolar type II cells. Am. J. Physiol. 265:L193–L199.
  10. Quintero, O. A., and J. R. Wright. 2000. Metabolism of phosphatidylglycerol by alveolar macrophages in vitro. Am. J. Physiol. Lung Cell. Mol. Physiol. 279:L399–L407.[Abstract/Free Full Text]
  11. Poelma, D. L., L. J. Zimmermann, H. H. Scholten, B. Lachmann, and J. F. van Iwaarden. 2002. In vivo and in vitro uptake of surfactant lipids by alveolar type II cells and macrophages. Am. J. Physiol. Lung Cell. Mol. Physiol. 283:L648–L654.[Abstract/Free Full Text]
  12. Dobbs, L. G., R. Gonzalez, and M. C. Williams. 1986. An improved method for isolating type II cells in high yield and purity. Am. Rev. Respir. Dis. 134:141–145.[Medline]
  13. Edelson, J. D., J. M. Shannon, and R. J. Mason. 1988. Alkaline phosphatase: a marker of alveolar type II cell differentiation. Am. Rev. Respir. Dis. 138:1268–1275.[Medline]
  14. Straubinger, R. M., K. Hong, D. S. Friend, and D. Papahadjopoulos. 1983. Endocytosis of liposomes and intracellular fate of encapsulated molecules: encounter with a low pH compartment after internalization in coated vesicles. Cell 32:1069–1079.[CrossRef][Medline]
  15. Muller, W. J., K. Zen, A. B. Fisher, and H. Shuman. 1995. Pathways for uptake of fluorescently labeled liposomes by alveolar type II cells in culture. Am. J. Physiol. 269:L11–L19.
  16. Stevens, P. A., H. Wissel, S. Zastrow, D. Sieger, and K. P. Zimmer. 2001. Surfactant protein A and lipid are internalized via the coated-pit pathway by type II pneumocytes. Am. J. Physiol. Lung Cell. Mol. Physiol. 280:L141–L151.[Abstract/Free Full Text]
  17. Wright, J. R., and D. C. Youmans. 1995. Degradation of surfactant lipids and surfactant protein A by alveolar macrophages in vitro. Am. J. Physiol. 268:L772–L780.
  18. Bates, S. R., M. F. Beers, and A. B. Fisher. 1992. Binding and uptake of surfactant protein B by alveolar type II cells. Am. J. Physiol. 263:L333–L341.
  19. Bates, S. R., and A. B. Fisher. 1993. Degradation of surfactant protein B by alveolar type II cells. Am. J. Physiol. 265:L448–L455.
  20. Sane, A. C., and S. L. Young. 1994. The stimulation of cellular phospholipid uptake by surfactant apoproteins. Biochim. Biophys. Acta 1213:107–112.[Medline]
  21. Ikegami, M., T. R. Korfhagen, M. D. Bruno, J. A. Whitsett, and A. H. Jobe. 1997. Surfactant metabolism in surfactant protein A–deficient mice. Am. J. Physiol. 272:L479–L485.
  22. Korfhagen, T. R., A. M. LeVine, and J. A. Whitsett. 1998. Surfactant protein A (SP-A) gene targeted mice. Biochim. Biophys. Acta 1408:296–302.[Medline]
  23. Quintero, O. A., T. R. Korfhagen, and J. R. Wright. 2002. Surfactant protein A regulates surfactant phospholipid clearance after LPS-induced injury in vivo. Am. J. Physiol. Lung Cell. Mol. Physiol. 283:L76–L85.[Abstract/Free Full Text]
  24. Botas, C., F. Poulain, J. Akiyama, C. Brown, L. Allen, J. Goerke, J. Clements, E. Carlson, A. M. Gillespie, C. Epstein, and S. Hawgood. 1998. Altered surfactant homeostasis and alveolar type II cell morphology in mice lacking surfactant protein D. Proc. Natl. Acad. Sci. USA 95:11869–11874.[Abstract/Free Full Text]
  25. Ikegami, M., J. A. Whitsett, A. Jobe, G. Ross, J. Fisher, and T. Korfhagen. 2000. Surfactant metabolism in SP-D gene-targeted mice. Am. J. Physiol. Lung Cell. Mol. Physiol. 279:L468–L476.[Abstract/Free Full Text]
  26. Jacobs, H. C., A. H. Jobe, M. Ikegami, and S. Jones. 1985. Reutilization of phosphatidylglycerol and phosphatidylethanolamine by the pulmonary surfactant system in 3-day-old rabbits. Biochim. Biophys. Acta 834:172–179.[Medline]
  27. Jacobs, H. C., D. M. Lima, J. M. Fiascone, and M. R. Mercurio. 1988. Reutilization of surfactant phosphatidylglycerol and lysophosphatidylcholine by adult rabbits. Biochim. Biophys. Acta 962:227–233.[Medline]
  28. Chander, A., W. D. Claypool, Jr., J. F. Strauss, III, and A. B. Fisher. 1983. Uptake of liposomal phosphatidylcholine by granular pneumocytes in primary culture. Am. J. Physiol. 245:C397–C404.
  29. Bates, S. R., P. B. Ibach, and A. B. Fisher. 1989. Phospholipids co-isolated with rat surfactant protein C account for the apparent protein-enhanced uptake of liposomes into lung granular pneumocytes. Exp. Lung Res. 15:695–708.[Medline]
  30. Jouanel, P., C. Motta, J. Brun, C. Molina, and B. Dastugue. 1981. Phospholipids and microviscosity study in broncho-alveolar lavage fluids from control subjects and from patients with extrinsic allergic alveolitis. Clin. Chim. Acta 115:211–221.[CrossRef][Medline]
  31. Mander, A., S. Langton-Hewer, W. Bernhard, J. O. Warner, and A. D. Postle. 2002. Altered phospholipid composition and aggregate structure of lung surfactant is associated with impaired lung function in young children with respiratory infections. Am. J. Respir. Cell Mol. Biol. 27:714–721.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Respir. Crit. Care Med.Home page
Huqun, S. Izumi, H. Miyazawa, K. Ishii, B. Uchiyama, T. Ishida, S. Tanaka, R. Tazawa, S. Fukuyama, T. Tanaka, et al.
Mutations in the SLC34A2 Gene Are Associated with Pulmonary Alveolar Microlithiasis
Am. J. Respir. Crit. Care Med., February 1, 2007; 175(3): 263 - 268.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
D. L. H. Poelma, B. Lachmann, J. J. Haitsma, L. J. Zimmermann, and J. F. van Iwaarden
Influence of phosphatidylglycerol on the uptake of liposomes by alveolar cells and on lung function
J Appl Physiol, May 1, 2005; 98(5): 1784 - 1791.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2003-0127OCv1
30/5/751    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Poelma, D. L.
Right arrow Articles by van Iwaarden, J. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Poelma, D. L.
Right arrow Articles by van Iwaarden, J. F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2004 American Thoracic Society.