1 Kinderpoliklinik and 2 Department of Anatomy, Ludwig-Maximilians-University, D-80336 Munich, Germany
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ABSTRACT |
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Animal experiments suggest developmental changes in surfactant homeostasis. The uptake and metabolism of [3H]dipalmitoylphosphatidylcholine-labeled liposomes with a surfactant-like composition were evaluated in type II cells isolated from rats of different postnatal ages. The early part of the uptake process (0-60 min) was more rapid and reached higher levels in cells from 2-day-old rats than in those from 7-day-old, 14-day-old, or adult rats. Temperature independence of this initial phase, differences in response to trypsin-EDTA or neuraminidase treatment, and the dependency of increased neonatal uptake on the presence of phosphatidylglycerol in liposomes suggested binding as a major mechanism of cell-lipid interaction. Although a two to three times larger amount of lipid was associated with neonatal cells, the metabolism of phosphatidylcholine, indicated by a decrease in label in phosphatidylcholine and an accompanying increase in sphingomyelin, was significantly smaller in 2-day-old than in adult cells. These studies support the hypothesis that neonatal and adult cells may have differences in the interaction with alveolar phospholipids and in the metabolism of phosphatidylcholine.
development; endocytosis; liposomes
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INTRODUCTION |
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PULMONARY SURFACTANT is a complex mixture of lipids and surfactant-specific proteins that exists in a variety of functional and morphological forms (28). Although the exact sequence of events and the mechanisms by which surfactant phospholipids are removed from the alveolar compartment and the subsequent intracellular metabolism are largely unknown, the overall homeostasis seems to be tightly controlled (18, 28). Under steady-state conditions, the rate of secretion must balance the rate of uptake.
Experiments with whole animals suggest developmental changes in the rate and efficiency of the uptake process and in the subsequent intracellular processing (18). Newborn, 3-day-old rabbits recycled surfactant at a much higher rate than adult rabbits. However, the turnover and degradation processes were substantially slower than those for adult animals (11, 17). Type II pneumocytes synthesize, secrete, take up and catabolize, or recycle surfactant. They are the major cellular components in the surfactant life cycle as shown by cellular fractionation studies in a variety of animal experiments (5, 24) and by autoradiographic studies on whole lungs (4, 31).
In experiments with isolated adult type II cells, which were cultured
on plastic, the uptake of surfactant lipids appeared not to be very
closely linked to
L--phosphatidylcholine (PC) secretion because stimulation by agonists affected secretion but not
uptake (12). Similarly, the lectins concanavalin A and
Maclura pomifera agglutinin inhibited
secretion, but only the latter had an effect on uptake (12). Other
investigators (1, 5) found small increases in the rate of uptake in
response to some, but not all, secretagogues when the cells were
cultured on certain microporous membranes. Type II cells, which were
isolated from rats of different postnatal ages and cultured on plastic,
had a strong developmentally changing pattern of surfactant lipid secretion in response to
-adrenergic as well as to
P1- and
P2-purinergic agonists (13).
Because the activity of endocytotic and other uptake processes may also be developmentally regulated, we hypothesized that the differences in surfactant lipid uptake rates, metabolism, and changes in stimulation by agonists could be observed during postnatal development. We therefore used exactly the same methods of cell isolation and culture that had been previously employed in secretion experiments (13) to study the ontogenic development and regulation of lipid uptake in these cells. Some of the results have been presented in abstract form (15).
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MATERIALS AND METHODS |
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Chemicals and drugs. Dulbecco's
modified Eagle's medium (DMEM), Earle's balanced salt solution, and
fetal bovine serum were purchased from GIBCO BRL (Karlsruhe, Germany).
Porcine pancreatic elastase was from Elastin Products
(Owensville, MO).
[2-Palmitoyl-9,10-3H]dipalmitoyl-L--phosphatidylcholine
([ palmitoyl-3H]DPPC)
and
dipalmitoyl-L-
-[methyl-3H]phosphatidylcholine
([methyl-3H]DPPC)
were from NEN-DuPont (Frankfurt, Germany). Terbutaline sulfate was from
Geigy (Basel, Switzerland). The following chemicals were purchased from
Sigma (Deisenhofen, Germany): ATP,
12-O-tetradecanylphorbol 13-acetate,
rat immunoglobulin G, DPPC, PC (type XI-E) from egg yolk (egg PC),
L-
-phosphatidyl-DL-glycerol
ammonium salt from egg (PG), and
L-
-phosphatidylinositol
sodium salt from soybean (PI), authentic standards of choline,
CDP-choline, phosphorylcholine, glycerophosphorylcholine, betaine,
lysophosphatidylcholine, sphingomyelin, phosphatidylserine,
phosphatidylethanolamine, phosphatidic acid, cardiolipin, and
neuraminidase.
2-(12-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-PC12) was from Molecular
Probes (Eugene, OR). Surfactant protein (SP) A was prepared from human
alveolar proteinosis patients and was a gift from Henk Haagsman
(Laboratory of Veterinary Biochemistry, Utrecht University, Utrecht,
The Netherlands) (16). Other biochemicals were purchased
at the highest grade available from Merck (Darmstadt, Germany) or Fluka
(Neu-Ulm, Germany).
Isolation and culture of type II cells. Type II cells were isolated from the lungs of Sprague-Dawley rats (200-300 g; Charles River, Hannover, Germany) by the elastase digestion and immunoglobulin G panning method (7) as previously described (12, 13). The freshly isolated cells were plated at a density of 3 × 106 cells/dish on 35-mm plastic dishes and cultured in 1.5 ml of DMEM containing 10% fetal bovine serum, 10 mg/ml of streptomycin, and 100 U/ml of penicillin for 18-20 h at 37°C in a humidified atmosphere of 95% air-5% CO2. Of the cells from 2-day-old rats 89 ± 4% (n = 32 experiments), 85 ± 5% from 7-day-old rats (n = 12 experiments), 90 ± 3% from 14-day-old rats (n = 9 experiments), and 94 ± 2% from adult rats (n = 32 experiments) could be identified as type II cells after this period of culture by phase-contrast microscopy and staining with phosphine 3R. Viability was assessed by exclusion of erythrosin B and was >95%. The amount of protein recovered from the dishes after overnight culture did not differ between adult and neonatal cell preparations [81 ± 9 (n = 32 experiments), 87 ± 15 (n = 12 experiments), 87 ± 24 (n = 9 experiments), and 82 ± 10 (n = 32 experiments) µg protein/dish in cells from 2-day-old, 7-day-old, 14-day-old, and adult rats, respectively]. The preparation of cells from different ages was done in random order.
Electron microscopy. The morphological characteristics and integrity of the cultured cells isolated from 2-day-old and adult rats (three preparations each) were analyzed in more detail. After 20 h of culture on plastic, the cells were detached as in the uptake experiments, fixed for 30 min with 2% glutaraldehyde in 10 mM phosphate buffer containing 150 mM NaCl [phosphate-buffered saline (PBS)], pH 7.4, and collected by centrifugation at 180 g. The pellet was rinsed repeatedly in PBS, embedded in 4% agarose, and thereafter cut in pieces 2-3 mm in length. Before being embedded in araldite, the samples were postfixed in 2% OsO4 in PBS for 1 h. Semithin sections were stained with toluidin blue for general orientation. Thin sections were stained with uranyl acetate (saturated solution in 70% methanol) and lead citrate and studied in a Philips CM 10 electron microscope.
Liposome preparation. Liposomes were
freshly prepared for each experimental day from a mixture of DPPC-egg
PC-PG-cholesterol (10:5:2:3 mol/mol) as previously described (12). In
some experiments, PG was replaced by PI. Briefly, aliquots of the lipid
mixture were evaporated to dryness under nitrogen and stored at
70°C. When liposomes were to be prepared, the lipids were
dissolved in chloroform, and
[ palmitoyl-3H]DPPC
for uptake experiments or
[methyl-3H]DPPC
or
[ palmitoyl-3H]DPPC
for metabolism experiments was added to a final specific activity of
1.5 Ci/mol. The mixture was again dried under nitrogen, suspended in
PBS, and sonicated (5 × 1.5-min bursts) at 50°C with a probe
sonifier HD800 equipped with a MS73D tip (Bandelin, Berlin, Germany) at
20% maximum output. When assessed by thin-layer chromatography, the
result was that <0.5% of the DPPC was degraded under these conditions, even when the sonication was continued for 20 min. After
overnight storage at 4°C, the liposome suspension was centrifuged at 1,200 g for 20 min to remove
aggregates. The supernatant contained unilamellar liposomes with a mean
diameter of 110 nm (width 70 nm) as determined by laser light
scattering (Lo-Sizer, Malvern, Seefeld, Germany) and negative staining
(12).
Liposome uptake. As previously described in detail (12), after overnight culture, the cells were rinsed three times with fresh Earle's balanced salt solution containing 25 mM HEPES buffer (pH 7.4) and incubated for 30 min at 37°C. Liposomes and test agents were then added, and the incubation was continued for varying periods of time. The reaction was stopped by rapidly removing the medium, the cells were washed five times with cold PBS, and PBS containing 0.25% trypsin and 0.54 mM EDTA was added. After 5 min at room temperature, the dish was gently scraped with a cell lifter (Costar, Bodenheim, Germany), and the cells and medium were separated by rapid filtration through GF/A glass-fiber filters (Whatman, Maidstone, UK). The dish and filter were washed six times with ice-cold PBS. The filter was transferred to a scintillation vial and incubated in 0.1 N NaOH (0.6 ml) for 30 min at 50°C to solubilize the protein. After the filters were cooled to room temperature, Optifluor (10 ml) was added, and radioactivity was measured in a liquid scintillation counter. In some experiments, the cells were scraped (cell lifter) by mechanical detachment only, omitting treatment with trypsin-EDTA. Uptake of liposomes is expressed in nanomoles of DPPC per milligram of type II cell protein. In all experiments, blank culture dishes with DMEM containing serum and antibiotics but without cells were treated in parallel exactly as described above. The radioactivity that was recovered from these dishes was not dependent on the method used to remove the cells (mechanical detachment or trypsin-EDTA) and was routinely subtracted from the cell-associated radioactivity as described previously (12). The general term uptake was used to indicate cell-associated label under various experimental conditions without differentiating binding, adhesion, internalization, or endocytosis.
Fluorescence microscopy. Liposomes containing 10% of the PC replaced with NBD-PC12, a nonexchangeable, fluorescently labeled analog of PC, were prepared as described in Liposome preparation. The molar ratio of the NBD-PC12-DPPC-egg PC-PG-cholesterol mixture was 1.5:9.1:4.4:2:3 mol/mol. After incubation and washing, the cells were observed with an inverted IMT 2 microscope (Olympus, Hamburg, Germany) equipped with a silicon-intensified tube camera (Hamamatsu, Herrsching, Germany) and recorded on videotape. An XF-19 filter (Omega Optical, Brattleboro, VT) equipped with a dichroic barrier at 505 nm was used for excitation at 470 nm and emission at 515 nm.
Analysis of lipids and water-soluble choline-containing metabolites. The cells were incubated with [methyl-3H]DPPC- or [ palmitoyl-3H]DPPC-labeled liposomes, washed, treated with trypsin-EDTA as described in Liposome uptake, and collected by centrifugation (200 g for 10 min at 4°C). To facilitate recovery and subsequent detection of minor components, a mixture of unlabeled phospholipids and water-soluble choline-containing compounds was added as a cold carrier, and the suspension was extracted (12). Phospholipids in the "lower" phase were fractionated into individual components by thin-layer chromatography on 60C silica-gel plates (Merck) in 0.25% chloroform-methanol-2-propanol-potassium chloride-triethylamine (30:9:25:6:18 by volume) (27). Lipids were made visible on the thin-layer plates by exposure to iodine vapor, identified by comparison with standards, and quantified by liquid scintillation counting. Similarly, water-soluble choline-containing metabolites were isolated from the "upper" layer and then separated by thin-layer chromatography on 60C silica-gel plates with methanol-0.55% NaCl-NH4OH (50:50:1 by volume) (30). CDP-choline was made visible with ultraviolet light and the other choline-containing compounds by a brief exposure to iodine vapors.
Other assays. Protein was measured with a Coomassie blue binding method with an assay kit from Bio-Rad (Munich, Germany). Phospholipids were quantitated after lipid extraction with a phosphorus assay (12).
Data analysis. Type II cells were isolated from the pooled lungs of 3-4 adult, 18-22 two-day-old, 8-14 seven-day-old, and 5-8 fourteen-day-old rats and distributed among the various treatment groups. Two dishes with cells and two blank dishes without cells per group were used in each uptake experiment. Each dish was processed separately, and the values were averaged to yield a single data point per group per experiment. All data are means ± SE from the number (n) of individual experiments indicated. The data were analyzed statistically as indicated in RESULTS with GraphPad Software (San Diego, CA) (23).
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RESULTS |
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The ultrastructural analysis of adult type II cells and cells from
2-day-old rats isolated after 20 h in culture showed morphologically intact and metabolically active type II pneumocytes (Fig.
1). The majority of cells contained several
lamellar bodies (Fig. 1, A,
C, and
D). The cytoplasm was rich in rough
endoplasmatic reticulum, free ribosomes, and mitochondria. The amount
of these organelles was somewhat higher in neonatal cells, whereas the amount of lamellar bodies appeared to be lower. The neonatal cells regularly had a large Golgi apparatus, frequently multivesicular bodies, and an euchromatin-rich nucleus with a large prominent nucleolus (Fig. 1B). Only
occasionally, single ciliated cells, macrophages, or leukocytes were
seen, supporting the high purity of the primary cultures as assessed by
light microscopy (see MATERIALS AND
METHODS).
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To evaluate development of postnatal surfactant lipid uptake, type II
cells that had been isolated from rats at different postnatal ages were
exposed to
[ palmitoyl-3H]DPPC-labeled
liposomes of surfactant-typical composition. Cellular uptake,
determined as trypsin-EDTA-resistant cell-associated radioactivity, increased with time in adult type II cells as previously shown (3, 12)
(Fig.
2A). In
contrast, the uptake by cells from 2-day-old rats was much more rapid
during the first hour, resulting in significantly higher initial
cell-associated
[ palmitoyl-3H]DPPC
(Fig. 2A). After 2-4 h, uptake
did not further increase, and the final level reached was not much
higher than that in adult cells. In separate experiments with various
concentrations of liposomes, the substantially higher initial uptake
rate of cells from 2-day-old animals was confirmed (Fig.
2B). The curve of surfactant lipid
uptake in type II cells from 7- and 14-day-old rats was early (4, 30, and 60 min), somewhat, although not significantly, higher than that in
adult cells, but converged at later time points (90, 120, 180, and 240 min; n = 5-7; data not shown).
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Developmental regulation of surfactant secretion by various agonists
has already been shown in type II cells during the first 28 days of
life (13). Therefore, a possible influence on uptake by these agents
was investigated. Although optimal concentrations for surfactant
secretion and the induction of various second messengers were used, no
effects on uptake in all age groups investigated were noted (Table
1). The uptake rate after 2 h was
significantly reduced to ~25% of the control value when the cells
were incubated at 4°C to block energy-dependent processes or when a
short treatment with glutaraldehyde to fix cellular membrane proteins
was carried out. Although the processes involved in the lipid uptake
observed may be related to classic endocytosis, they were neither
influenced by the activation of various cellular second messenger
systems nor did they show a developmental regulation beyond 2 days of life.
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For a more detailed study of the nature of the differences during the
initial phase of uptake between cells from 2-day-old and adult rats,
the 30-min time point was chosen. Surprisingly, incubation at 4°C
did not significantly inhibit uptake in both age groups at this early
time point (Table 2). To determine the total amount of cell-associated DPPC, the cells were carefully lifted
from their plates at the end of incubation by mechanical means only.
Under these conditions, similar amounts of cell-associated DPPC were
detected for neonatal and adult cells (Table 2). Trypsin-EDTA treatment
was only able to remove substantial amounts of cell-associated radioactivity in adult cells (Table 2). These data indicate that initially mainly temperature-independent mechanisms are responsible for
the uptake of liposomal DPPC and that the substantially higher uptake
rate was, to a great part, due to differences in binding characteristics (e.g., differences in response to trypsin-EDTA treatment) between neonatal and adult type II cells.
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Phospholipid head groups determine the surface charge and, in the case
of neutral substituents, the coverage of the negative phosphate group
of the liposomes and thus may determine the initial interaction with
the cell surface. Therefore, a lipid mixture of an identical negative
charge but with a more bulky head group was studied, e.g., PG was
replaced with PI, thus replacing the glycerol moiety with an inositol
moiety. Although a tendency toward higher values in cells from
day 2 was observed and although a large number of experiments were therefore performed, no significant differences in initial cellular uptake between cells from 2-day-old and
adult lungs were found with these liposomes, even when their concentrations varied over a broad range (Table
3).
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Negatively charged sialic acid can be removed from the surface of adult
type II cells in the whole lung by neuraminidase treatment (8). In
separate experiments, we preincubated type II cells from adult and
day 2 rats with and without
neuraminidase (2 U/ml) for 30 min, washed the cells two times with PBS
containing 5% bovine serum albumin, and then incubated them with the
PG-containing liposomes at 100 µM DPPC. Uptake was significantly
enhanced by neuraminidase treatment at 30 min in cells from adult rats
only (Table 4). This indicated differences
in sialization and associated surface charges between neonatal and
adult cells that might be involved in the differences in liposome
uptake between these cells.
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Fluorescence microscopy with NBD-PC12-labeled liposomes localized the majority of the label after 30 min of incubation to the outside of the cells from 2-day-old rats. These cells appeared to have more label at the cell margin than adult cells (data not shown). After 2 h of incubation, a significant amount of label was found within the cells. Due to a relatively low level of signal, no quantitative analysis was performed.
The metabolism of liposomal DPPC that is taken up by type II cells was
investigated in cells from 2-day-old and adult rat lungs with
[methyl-3H]DPPC,
a DPPC labeled in the choline moiety. Similar to the above, we also
found that with this label substantially more radioactivity was taken
up after 30 min by cells from 2-day-old rats (Table 5). In neonatal cells, most of this label
was confined to PC, without much redistribution over the 2-h
observation period. In contrast, there was a significant linear trend
for the percentage of PC with time in adult but not in neonatal cells
(P < 0.05 by ANOVA). When adult type
II cells were compared directly with cells from 2-day rats, the
percentage of label in PC was significantly different at the 30-min but
not at the 2-h time point because there was also some loss of label
from the neonatal cells (Table 5). The sphingomyelin fraction increased
at the same time. Only insignificant amounts of radioactivity were
detected in the other phospholipid fractions (Table 5). In control
experiments performed at 4°C, no significant metabolism of PC after
2 h of incubation was seen in either adult type II cells (99.7 ± 0.4% PC; 0.3 ± 0.3% sphingomyelin;
n = 2) or cells from 2-day-old rats
(99.6 ± 0.5% PC; 0.7 ± 1.2% sphingomyelin;
n = 2). Only a minor portion (<0.1%
of total radioactivity) was found in the water-soluble fraction in
which the label migrates mainly with free choline and CDP-choline.
However, no differences for the two age groups investigated and no
changes with time were noted.
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The same experiments were performed with [ palmitoyl-3H]DPPC as the label. Again, at 30 min, the total amount of liposomes taken up was smaller in adult cells [1.2 ± 0.2 × 105 vs. 2.4 ± 0.4 × 105 (neonatal) dpm/mg protein; P < 0.05], and the degradation of PC was significantly larger in adult cells (66.8 ± 2.7 vs. 73.4 ± 4.3% of total radioactivity in neonatal cells; P < 0.05). Under these conditions, PC was also metabolized to some extent to sphingomyelin and lyso-PC.
To further characterize the modulation of liposome uptake by the cells,
the effect of SP-A was assessed. SP-A has been shown to specifically
increase phospholipid uptake and to alter the metabolic degradation of
internalized phospholipids. In both adult and day
2 neonatal cells, phospholipid uptake was increased
two- to threefold over control values (Table
6). Consistently, in the
presence of SP-A, the amounts of label in lyso-PC, PG, and phosphatidylethanolamine were reduced without systematic changes in the
other phospholipids, supporting the concept of reduced phospholipid
metabolism by SP-A (Table 6). Such a response pattern was expected for
SP-A; however, no differences between adult and neonatal cells were
demonstrated.
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DISCUSSION |
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The cumulative evidence now supports the concept that type II pneumocytes represent the major cellular compartment for surfactant phospholipid clearance from the alveolar space under normal conditions (5, 18). However, not much is known on a cellular basis about the mechanisms, the regulation, or a possible developmental dependency of the lipid uptake process.
In a previous study, Griese et al. (13) demonstrated developmental changes in agonist-stimulated surfactant secretion from type II cells that were isolated from rats of different ages. Because activation of the secretatory process may be closely linked to the activity of endocytotic or other uptake processes, we hypothesized that under our culture conditions, the developmental changes of both basal and agonist-stimulated uptake may be detectable among the various age groups.
For the early part of the uptake process, e.g., from 0 to 60 min, we found substantial differences between adult and day 2 neonatal type II cells. The rate of uptake was significantly faster in the cells from newborn animals. However, this very rapid rate and the lack of dependency on temperature together with the inaccessibility to degrading metabolism characterized this uptake mainly as cell association by binding or fusion processes. This notion was further supported by our fluorescence microscopy observations that the liposomes localized initially mainly to the outer surface of type II cells from 2-day-old rats. The fact that treatment with trypsin-EDTA did not remove labeled lipids suggests either binding to inaccessible glycoproteins (of the cell surface glycocalix) or fusion with lipids in the cell membrane. No significant fusion of the liposomes with adult type II cells was detected in quenching experiments, which used octadecyl rhodamine B chloride (R18)-labeled liposomes (Griese and Beck, unpublished data).
During development of the lung, an increased addition of terminal sialic acid to the cell surface glycoproteins of type II cells in the postnatal period is observed (2, 8). This leads to less negative charge on adult cells. Treatment of adult cells with neuraminidase may increase the negative cell surface charge (8). Thus the higher binding capacity of neonatal cells may be related to less sialic acid on their cell surface. This is in agreement with our data on adult type II cells. There, an increased lipid uptake was observed after treatment with neuraminidase. However, differences in surface charge alone cannot account for all differences between neonatal and adult cells.
The enhanced uptake of neonatal cells was reduced when liposomes were used in which PG was replaced with PI, e.g., the charge of liposomes was kept constant. Type II cells were able to distinguish small differences in the lipid head group structure on the bilayer surface of the liposome. A similar observation has been made in CV1 cells, a green monkey kidney cell line (22). Developmental differences in phospholipid composition of the surfactant with respect to these phospholipids are unlikely to play a role for surfactant homeostasis during neonatal development because in the rat, the switch from PI to PG as the predominant acidic phospholipid of surfactant precedes birth (6).
It is possible that small differences in purity between neonatal and adult type II cell cultures (see MATERIALS AND METHODS) or a differential effect of the culture conditions on cells from different postnatal ages might be responsible for the observed differences. Because contamination by alveolar macrophages may be responsible for increased degradation (see below), the somewhat less pure cultures from neonatal cells would have been expected to have a higher degree of degradation. However, the opposite was observed. The ultrastructural studies also demonstrated the morphological integrity of the type II cells used. There was no evidence that the isolation and culture conditions resulted in marked structural differences between the age extremes. In agreement with the data previously reported by others (32), we found that the number of lamellar bodies was lower in neonatal cells and that both multivesicular and composite bodies were found more frequently. The observed differences in binding and metabolic changes are very unlikely to result from differences in cell purity or cell culture-induced morphological differences, although this cannot be excluded definitely. From a functional point of view, SP-A has been shown to affect uptake and potentially metabolism of lipids by type II cells (12, 29). Therefore, ontogenic increases in the expression and secretion of SP-A by these cells (10) could also be responsible for the differences between neonatal and adult cells. Again, however, the opposite effects to those observed, e.g., increased uptake and less degradation in adult cells, would be expected. Further support that the differences between neonatal and adult type II cells were not related to SP-A-dependent processes and that the cells were functionally intact comes from the data that the addition of SP-A had the same specific effects in both age groups. This characteristic response of type II cells to SP-A has recently been shown to be acquired by day 19 fetal type II cells cultured for 2 days (19). Taken together, all these findings make it unlikely that differences from culture of the cells might be responsible for the observed changes.
During the later phase of the uptake process, e.g., from 90 to 180 min, ~75% of the liposome uptake was dependent on the temperature and integrity of cell membrane proteins. Therefore, we took care to reproduce the culture conditions exactly as previously described (13). Bates et al. (1) and Chinoy et al. (5) recently reported that cells cultured on a membranous support show small (10-50%) increases in uptake in response to some secretagogues but not to others. Nevertheless, we found no modulation of uptake by agonists even under conditions in which surfactant secretion was stimulated substantially in neonatal and adult cells (14).
Our data on the metabolism of PC represent distribution of the radiolabel and are therefore indirect. In this study, no direct measurement of enzyme activities or specific activities in defined cellular compartments was made. We observed that the loss of label from PC and the concomitant increase in lyso-PC and sphingomyelin were significantly lower in neonatal compared with adult cells. Although we observed some differences depending on the moiety in DPPC that was labeled, we did not observe such a large degradation of DPPC (50-60%) in neonatal cells as reported by Kresch and colleagues in one (20) but not in another study (19). Unfortunately, no comparisons to adult cells were made (19, 20). The difference from our results may be related to the completely different conditions of cell isolation, which included fetal explant culture. In the adult cells, we observed a loss of label from PC, e.g., by degrading or remodeling metabolism, which was similar to that shown previously by others (9, 29) and by Griese et al. (12). With DPPC labeled in the fatty acid moiety, 4-7% of the label was found in PG (12, 29) and 5% in sphingomyelin (12). In this study, substantial label was also localized in sphingomyelin. Sphingomyelin can be synthesized by transferring phosphocholine to ceramide. Phosphocholine may be derived from either PC or CDP-choline (25, 26). Similar to the data of Lecerf et al. (21) but in contrast to the data of Chander et al. (3), we did not find a significant accumulation of water-soluble choline products in our system. A high activity of sphingomyelin biosynthesis by transfer of phosphocholine from phosphatidylcholine has been shown to take place in rat lung lamellar bodies or the plasma membrane (21). Thus these data together with the very low content of CDP-choline strongly support the hypothesis that under our conditions exogenous PC may be directly utilized for the synthesis of sphingomyelin.
These data on isolated cells are in agreement with in vivo data and may help to explain some observations made in whole animals or in isolated perfused lungs. First, the initial and very rapid association of labeled surfactant or liposomes observed after exogenous delivery to the lungs may represent tight binding to cell surface structures that cannot be removed by extensive washing or depletion of divalent ions. This might be more pronounced in neonatal than in adult rat lungs. Second, because the uptake with ongoing time does not differ between neonatal and adult cells, differences in endocytosis between neonatal and adult animals may not play a primary role. Third, the smaller degradation of PC in type II cells from 2-day-old rats in comparison to adult type II cells may be one of the mechanisms leading to lower surfactant turnover rates observed in newborn animals.
Although the mechanisms of surfactant lipid uptake by type II cells are not yet completely defined, our data are in agreement with the view that adsorption reactions, which are relatively independent of regulatory processes, initially play a substantial role and that the internalization of the liposomes occurs via an endocytotic process. The modulatory influence of surfactant secretagogues on uptake appears to be relatively small, and SP-A enhances uptake in both neonatal and adult cells. PC, which has been taken up, is metabolically processed more actively in adult than in neonatal cells.
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ACKNOWLEDGEMENTS |
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The expert technical assistance of J. Gnielka is appreciated. We thank Hank Haagsman for the gift of surfactant protein A.
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FOOTNOTES |
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This research was supported by Deutsche Forschungsgemeinschaft Grant Gr 970/3-1.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Griese, The Lung Research Group, Kinderpoliklinik, Ludwig-Maximilians-Univ., Pettenkoferstr. 8a, D-80336 Munich, Germany (E-mail: griese{at}pk-i.med.uni-muenchen.de).
Received 30 December 1998; accepted in final form 22 June 1999.
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REFERENCES |
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1.
Bates, S. R.,
C. Dodia,
and
A. B. Fisher.
Surfactant protein A regulates uptake of pulmonary surfactant by lung type II cells on microporous membranes.
Am. J. Physiol.
267 (Lung Cell. Mol. Physiol. 11):
L753-L760,
1994
2.
Castells, M. T.,
J. Ballesta,
J. F. Madrid,
M. Aviles,
and
J. A. Martinez-Menarguez.
Characterization of glycoconjugates in developing rat respiratory system by means of conventional and lectin histochemistry.
Histochemistry
95:
419-426,
1991[Medline].
3.
Chander, A.,
W. D. Claypool,
J. F. Strauss,
and
A. B. Fisher.
Uptake of liposomal phosphatidylcholine by granular pneumocytes in primary culture.
Am. J. Physiol.
245 (Cell Physiol. 14):
C397-C404,
1983[Abstract].
4.
Chevalier, G.,
and
A. J. Collet.
In vivo incorporation of choline-3H, leucine-3H and galactose-3H in alveolar type II pneumocytes in relation to surfactant synthesis. A quantitative radioautographic study in mouse by electron microscopy.
Anat. Rec.
174:
289-310,
1972[Medline].
5.
Chinoy, M. R.,
A. B. Fisher,
and
H. Shuman.
Confocal imaging of time-dependent internalization and localization of NBD-PC in intact rat lungs.
Am. J. Physiol.
266 (Lung Cell. Mol. Physiol. 10):
L713-L721,
1994
6.
Cockshutt, A.,
and
F. Possmayer.
Metabolism of surfactant lipids and proteins in the developing lung.
In: Pulmonary Surfactant: From Molecular Biology to Clinical Practice, edited by B. Robertson,
L. van Golde,
and J. Batenburg. Amsterdam: Elsevier, 1992, p. 339-377.
7.
Dobbs, L. G.,
R. Gonzalez,
and
M. C. Williams.
An improved method for isolating type II cells in high yield and purity.
Am. Rev. Respir. Dis.
134:
141-145,
1986[Medline].
8.
Faraggiana, T.,
D. Villari,
J. Jagirdar,
and
J. Patil.
Expression of sialic acid on the alveolar surface of adult and fetal human lungs.
J. Histochem. Cytochem.
34:
811-816,
1986[Abstract].
9.
Fisher, A. B.,
A. Chander,
and
J. Reicherter.
Uptake and degradation of natural surfactant by isolated rat granular pneumocytes.
Am. J. Physiol.
253 (Cell Physiol. 22):
C792-C796,
1987
10.
Floros, J.,
D. S. Phelps,
H. P. Harding,
S. Church,
and
J. Ware.
Postnatal stimulation of rat surfactant protein A synthesis by dexamethasone.
Am. J. Physiol.
257 (Lung Cell. Mol. Physiol. 1):
L137-L143,
1989
11.
Glatz, T.,
M. Ikegami,
and
A. H. Jobe.
Metabolism of exogenously administered natural surfactant in the newborn lamb.
Pediatr. Res.
16:
711-715,
1982[Abstract].
12.
Griese, M.,
L. I. Gobran,
and
S. A. Rooney.
Surfactant lipid uptake and secretion in type II cells in response to lectins and secretagogues.
Am. J. Physiol.
261 (Lung Cell. Mol. Physiol. 5):
L434-L442,
1991
13.
Griese, M.,
L. I. Gobran,
and
S. A. Rooney.
Ontogeny of surfactant secretion in type II pneumocytes from fetal newborn and adult rats.
Am. J. Physiol.
262 (Lung Cell. Mol. Physiol. 6):
L337-L343,
1992
14.
Griese, M.,
L. I. Gobran,
and
S. A. Rooney.
Signal-transduction mechanisms of ATP-stimulated phosphatidylcholine secretion in rat type II pneumocytes: interactions between ATP and other surfactant secretagogues.
Biochim. Biophys. Acta
1167:
85-93,
1993[Medline].
15.
Griese, M.,
and
D. Reinhardt.
Uptake of surfactant like liposomes by neonatal and adult type II pneumocytes (Abstract).
Am. J. Respir. Crit. Care Med.
151:
A632,
1995.
16.
Haagsman, H. P.,
S. Hawgood,
T. Sargeant,
D. Buckley,
R. T. White,
K. Drickamer,
and
B. J. Benson.
The major lung surfactant protein, SP 28-38, is a calcium-dependent, carbohydrate-binding protein.
J. Biol. Chem.
262:
13877-13880,
1987
17.
Jacobs, H. C.,
A. Jobe,
M. Ikegami,
and
S. Jones.
Surfactant phosphatidylcholine, source, fluxes, and turnover times in 3-day-old, 10-day-old, and adult rabbits.
J. Biol. Chem.
257:
1805-1810,
1982
18.
Jobe, A. H.,
and
E. D. Rider.
Catabolism and recycling of surfactant.
In: Pulmonary Surfactant: From Molecular Biology to Clinical Practice, edited by B. Robertson,
L. G. van Golde,
and J. Batenburg. Amsterdam: Elsevier, 1992, p. 313-337.
19.
Kresch, M. J.,
and
C. Christian.
Developmental regulation of the effects of surfactant protein A on the phospholipid uptake by fetal rat type II pneumocytes.
Lung
176:
45-61,
1998[Medline].
20.
Kresch, M. J.,
L. A. Ciriani,
H. Lu,
and
C. Christian.
Developmental regulation of reuptake of phosphatidylcholine by type II alveolar epithelium.
Biochim. Biophys. Acta
1210:
167-173,
1994[Medline].
21.
Lecerf, J.,
L. Fouilland,
and
J. Gagniarre.
Evidence for a high activity of sphingomyelin biosynthesis by phosphocholine transfer from phosphatidylcholine to ceramides in lung lamellar bodies.
Biochim. Biophys. Acta
918:
48-59,
1987[Medline].
22.
Lee, K. D.,
K. Hong,
and
D. Papahadjopoulos.
Recognition of liposomes by cells: in vitro binding and endocytosis mediated by specific lipid headgroups and surface charge density.
Biochim. Biophys. Acta
1103:
185-197,
1992[Medline].
23.
Motulsky, H.
Intuitive Biostatistics. New York: Oxford University Press, 1995.
24.
Rider, E. D.,
I. Machiko,
and
A. H. Jobe.
Localization of alveolar surfactant clearance in adult rabbit cells.
Am. J. Physiol.
263 (Lung Cell. Mol. Physiol. 7):
L201-L209,
1992
25.
Rooney, S. A.
Phospholipid composition, biosynthesis, and secretion.
In: Comparative Biology of the Normal Lung, edited by R. Parent. Boca Raton, FL: CRC, 1992, p. 511-544.
26.
Scherphof, G. L.
Phospholipid metabolism in animal cells.
In: Phospholipids Handbook, edited by G. Cevc. New York: Dekker, 1993, p. 777-800.
27.
Touchstone, J. C.,
J. C. Chen,
and
K. M. Beaver.
Improved separation of phospholipids in thin layer chromatography.
Lipids
15:
61-62,
1980.
28.
Wright, J. R.,
and
J. A. Clements.
Metabolism and turnover of lung surfactant.
Am. Rev. Respir. Dis.
136:
426-444,
1987[Medline].
29.
Wright, J. R.,
R. E. Wager,
S. Hawgood,
L. G. Dobbs,
and
J. A. Clements.
Surfactant apoprotein Mr = 26,000-36,000 enhances uptake of liposomes by type II cells.
J. Biol. Chem.
262:
2888-2894,
1987
30.
Yavin, E.
Regulation of phospholipid metabolism in differentiating cells from rat brain cerebral hemispheres in culture.
Biochem. J.
251:
1392-1397,
1976.
31.
Young, S. L.,
E. K. Fram,
E. Larson,
and
J. R. Wright.
Recycling of surfactant lipid and apoprotein-A studied by electron microscopic autoradiography.
Am. J. Physiol.
265 (Lung Cell. Mol. Physiol. 9):
L19-L26,
1993
32.
Yung, S. L.,
E. K. Fram,
C. L. Spain,
and
E. W. Larson.
Development of type II pneumocytes in rat lung.
Am. J. Physiol.
260 (Lung Cell. Mol. Physiol. 4):
L113-L122,
1991