Internalized SP-A and lipid are differentially resecreted by type II pneumocytes

Heide Wissel1, Stefan Zastrow1, Ekkehard Richter2, and Paul A. Stevens1

1 Clinic of Neonatology, University Children's Hospital Charité, Humboldt-University Berlin, 10098 Berlin; and 2 Institute of Biology, Department of Membrane Physiology, Humboldt-University Berlin, 10115 Berlin, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Biochemical and morphological assays were developed to study surfactant protein A (SP-A) and lipid resecretion kinetics by isolated type II cells in vitro. After a 10-min uptake period with SP-A (3 µg/106 cells) in combination with liposomes (60 µg/106 cells), the cells were allowed to resecrete. After 5 min of resecretion, only 21.7 ± 4.6% of the internalized SP-A remained intracellularly compared with 54 ± 2.9% of the lipids. Extracellular SP-A present during the resecretion period partially inhibited resecretion (SP-A, 36% at 5 min; lipid, ~16% at 5 min). Lipid resecretion was also dependent on the SP-A concentration present during the uptake period. Although, as shown by confocal laser scanning microscopy, after a 10-min uptake period at 37°C, most of the fluorescein isothiocyanate-labeled SP-A and rhodamine-phosphatidylethanolamine-labeled lipids colocalized within the cells, after an additional 10 min of resecretion, both the strength of the fluorescence signals and the extent of colocalization had markedly decreased. These data indicate that internalized lipid and SP-A can be resecreted rapidly by type II cells, likely via different pathways.

surfactant protein A; liposomes; endocytosis; internalization


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AFTER ITS SYNTHESIS by alveolar type II cells, surfactant is secreted into the alveolar space via lamellar bodies (8, 26, 27). It then converts from lamellar bodies to tubular myelin, which then gives rise to the monolayer. Surfactant is also cleared from the alveolus, partly by alveolar macrophages and partly by reinternalization by type II cells. It has been shown that >60% of alveolar surfactant clearance takes place via type II cells (29).

Time-dependent reuptake of surfactant by type II cells was first shown by Hallman et al. (14). Analysis of the specific activity time curves in this study also suggested that at least some of the lipids that had been taken up by the cells were resecreted into the alveoli. In recent years, repeated cycling of surfactant components between intra- and extracellular compartments was shown by several authors (1, 14, 21, 22, 40). Although rapid recycling could be of biological and therapeutic significance as a way to cope with limited surfactant pools under conditions of stress, very little is known about the time course and mechanisms of lipid resecretion by type II cells. It is unclear whether all internalized surfactant enters the lamellar body before resecretion or whether there exist other resecretion pathways. Virtually nothing is known about its regulatory mechanisms. The aim of our study was to establish assays to study resecretion in vitro and to clarify its time course and possible mechanisms.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals

Lipids, drugs, glutathione, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC)-labeled Maclura pomifera lectin, and FITC-labeled concanavalin A were obtained from Sigma (Deisenhofen, Germany). Radioactive isotopes as well as biodegradable scintillation counting fluid were from Amersham (Braunschweig, Germany). L-alpha -phosphatidylethanolamine (PE)-N-(lissamine rhodamine B sulfonyl) was obtained from Avanti Polar Lipids. Elastase, BM chemiluminescence kits, and fatty acid-free serum albumin (BSA fraction V) were purchased from Boehringer Mannheim (Mannheim, Germany). Biotin compounds and NeutrAvidin-horseradish peroxidase conjugate were obtained from Pierce (Rockford, IL). Bio-Rad protein assay kit was from Bio-Rad (Richmond, CA). ProLong Antifade Kit was obtained from Molecular Probes Europe. Cell culture media and supplements were obtained from Life Technologies (Gaithersburg, MD).

Type II Cell Preparation

Type II cells were prepared from pathogen-free male Wistar rats with a modification of the method of Dobbs et al. (7). The time of preparation was approx 2 h. In all experiments, freshly isolated cells in suspension were used. Purity was 92 ± 0.6% type II cells as determined by Papanicolaou stain (n = 50 experiments). Viability of the cells as determined by trypan blue exclusion was 96 ± 0.7% (n = 50 experiments).

Preparation of Liposomes

Small unilamellar radiolabeled liposomes were prepared as described by Wissel et al. (35). The phospholipid composition of the liposomes was 55% (wt) dipalmitoylphosphatidylcholine (DPPC), 25% egg phosphatidylcholine, 10% dipalmitoylphosphatidylglycerol, and 10% cholesterol. As radioactive lipid markers, either 1,2-dipalmitoyl-L-3-phosphatidyl-N-[methyl-3H]choline (1.46 nCi/µg lipid) or 1-palmitoyl-2-[1-14C]linoleoyl-L-3-phosphatidylethanolamine (0.25 nCi/µg lipid) was added. Unlabeled PE (1% ) was added in exchange for an equal amount of DPPC. In some experiments, liposomes were labeled with the fluorescent lipid L-alpha -PE-N-(lissamine rhodamine B sulfonyl). These samples consisted of the same lipid mixture except that DPPC was reduced to 53% by weight and unlabeled PE and L-alpha -PE-N-(lissamine rhodamine B sulfonyl) were each added to 1% by weight.

Surfactant Protein A Isolation

Sheep surfactant protein A (SP-A) from lamb lungs obtained fresh from a local slaughterhouse was isolated according to Hawgood et al. (16). All preparations were tested for purity with SDS-PAGE and subsequent silver staining as well as with Western blot. Contamination with IgG was tested with ELISA and Western blot against anti-sheep IgG. Lipid aggregation was tested with the method of Hawgood et al. (15). The preparations were also tested for functional activity in surfactant secretion assays according to Dobbs et al. (9) and in lipid uptake assays as described by Wissel et al. (35).

Biotin Labeling of SP-A

Sheep SP-A was biotinylated as previously described with NHS-LC-biotin or NHS-SS-biotin at pH 6.3 (31). SP-A was stored at -20°C until needed and used within 4 wk after biotinylation. Biotinylated SP-A batches were tested for functional activity in lipid uptake assays. Their biological activity was 98 ± 3% of that of unlabeled SP-A (difference not significant; n = 4 experiments).

FITC Labeling of SP-A

FITC was diluted in DMSO to prepare a 5 mg/ml solution. One milligram of sheep SP-A in 5 mM HEPES was incubated with 100 µg of FITC in 200 µl of 400 mM NaHCO3 (pH 8.5, final volume ~500 µl) for 1 h in the dark at 22°C with gentle agitation. Labeled SP-A was dialyzed with distilled water three times, at 2, 5, and 23 h, at 3°C to remove unconjugated fluorophore and was stored in aliquots at -20°C. FITC-labeled SP-A batches were tested for functional activity in lipid uptake assays. Their activity was not different from that of unlabeled SP-A (105 ± 7.5%; difference not significant; n = 3 experiments).

SP-A Resecretion Assay

Freshly isolated type II cells were washed once in Dulbecco's modified Eagle's medium to which 0.1% bovine serum albumin (BSA) was added. The cells (5 × 106/sample) were taken up in 1 ml of cold DMEM-0.1% BSA and incubated with 3 µg/106 cells of NHS-SS-biotin-SP-A. The cells were incubated for 10 min at 37°C with gentle continuous shaking. At the end of the incubation period, the cells were centrifuged and the medium was removed. The samples were placed on ice, and 1 ml/sample of cold PBS-0.1% BSA-10 mM EGTA, pH 7.4, was added to the cells for 5 min. Then the cells were resuspended in 1 ml of glutathione (GSH) solution (75 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 190 mM GSH, and 1% BSA, pH 8.2) and incubated for 30 min at 3°C. The cells were then washed again for 5 min in PBS-0.1% BSA-10 mM EGTA followed by another 20-min incubation period with GSH solution. The cells were washed in PBS-0.1% BSA. One milliliter per sample of cold iodoacetamide solution (50 mM iodoacetamide, 75 mM Tris acetate, 1 mM CaCl2, and 1 mM MgCl2) was added to the cells followed by incubation for another 30 min on ice. After the cells were washed with PBS for 5 min, the samples were separated in 1.5-ml microtubes. For resecretion, the cells were warmed to 37°C for various periods of time in DMEM-0.1% BSA without SP-A as indicated in RESULTS and in Figs. 1-7. Thereafter, the cell pellets were washed as described before: PBS-BSA-EGTA, GSH solution, followed by PBS-BSA-EGTA and a second round of GSH solution, washed in PBS-BSA, and finally incubated in iodoacetamide-PBS. The final pellets were resuspended in distilled water and sonicated two times for 10 s each with a Bandelin sonicator at 40% power (Bandelin, Berlin, Germany). An aliquot was removed for determination of the protein content.

To visualize SP-A, equal amounts of total protein were added under nonreducing conditions to the wells of SDS-PAGE gels and subjected to electrophoresis. The gel was then blotted onto nitrocellulose, and the proteins were visualized by enhanced chemiluminescence (BM chemiluminescence, Boehringer Mannheim) on film with the NeutrAvidin-horseradish peroxidase system. The results were quantitated by scanning densitometry (imaging densitometer model GS-670, Bio-Rad). Standard amounts of biotinylated SP-A and control samples (cells carried through the experiments without added SP-A) were also run on each gel. Under the conditions used, SP-A runs at molecular masses of 28-35 and 60-64 kDa. Both bands were included for quantitation.

Surfactant Lipid Resecretion Assay

Type II cells (2.5 × 106/sample) were taken up in 1 ml of DMEM-0.1% BSA. The cells were suspended for 5 min at 37°C. After the addition of 15 µl liposomes (150 µg lipid)/2.5 × 106 cells, 2, 4, or 10 µg SP-A/106 cells were added to half of the cells. Subsequently, these cells were incubated for different periods of time at 37°C and then centrifuged. The medium was removed, and the cells were washed three times for 5 min each with DMEM containing 5% fatty acid-free BSA (Boehringer Mannheim) and 10 mM EGTA. Then the cells were washed two times for 5 min each with DMEM-0.1% BSA. After the first wash, the samples were transferred to new tubes. Some of the cells (either with or without SP-A) were washed once with pure DMEM and then pelleted to determine the radioactivity after uptake of the lipids.

After removal of material adhering to the cell membrane, some of the samples were incubated for different times at 37°C in 1 ml of DMEM-0.1% BSA (liposome- and SP-A-free medium) to allow resecretion. After the resecretion period, medium was collected, and the cells were washed three times with DMEM containing 5% fatty acid-free BSA and 10 mM EGTA, once with medium containing 0.1% BSA, and once with pure DMEM. The final pellets were resuspended in distilled water and sonicated two times for 10 s each at 40% power (Bandelin sonicator). Subsequently, aliquots were taken for the determination of protein content. For analysis of radioactivity, 8 ml of biodegradable scintillation counting fluid were added to the samples (cells and medium). Radioactivity was measured in a Wallac 1410 liquid scintillation counter (Pharmacia, Freiburg, Germany).

The secretion rate is expressed as a ratio of the disintegrations per minute in the medium to the sum of the disintegrations per minute in the medium and cells.

Determination of Protein and Lipid Phosphorus Contents

Protein content was determined by the assay described by Bradford (4) with the Bio-Rad protein assay kit (Bio-Rad).

After lipid extraction, lipid phosphorus content was determined as described by Bartlett (2).

Determination of Phospholipid Species

After lipid extraction (3), the phospholipids were fractionated into different classes by two-dimensional TLC on silica gel H60 TLC plates (Merck) with unlabeled compounds as tracers. First, neutral fats were separated by development with hexane-diethyl ether (40:60 vol/vol) as the solvent. Subsequently, the phospholipids were separated by chloroform-methanol-ammonia (130:50:10) as the solvent in the first dimension and chloroform-acetone-methanol-acetic acid-water (60:80:20:20:10) in the second dimension. The spots were visualized after a short exposure to iodine vapor and then scraped into 8 ml of scintillation fluid for measurement of radioactivity in a scintillation counter.

Confocal Laser Scanning Microscopy

Type II cells (5 × 106) were suspended in 1 ml of DMEM-0.1% BSA. The cells were incubated for 10 min at 37°C with 100 µg of liposomal phospholipid containing the fluorescent lipid rhodamine-PE and FITC-labeled SP-A (2 µg/106 cells). The cells were separated from the medium by centrifugation and washed three times with cold DMEM-10 mM EGTA-5% fatty acid-free BSA and then three times with cold DMEM. Thereafter, the cells were transferred to new tubes. At this time point, some of the cells were reserved for fixation. The other cells were treated for 10 min at 37°C in DMEM without SP-A and lipid as described in Surfactant Lipid Resecretion Assay. The final pellets were treated for microscopy as described below. To visualize the cell boundaries, after the uptake period, the cells were washed at 3°C as described in SP-A Resecretion Assay and then incubated for 30 min at 3°C with TRITC-labeled Maclura pomifera agglutinin (MPA; 5 µg/ml) or FITC-labeled concanavalin A (1 µg/ml).

For microscopy, the cells were fixed in 1% paraformaldehyde-500 mM HEPES (pH 7.4) for 15 min on ice and then transferred to a fresh tube and washed two more times with PBS. The cells were placed on glass slides and covered with ProLong Antifade Kit and a glass coverslip. They were then examined with a confocal laser scanning microscope (CLSM; Leica CLSM, Leica Lasertechnic, Heidelberg, Germany) equipped with an argon-krypton laser. Images of the cells were created with standard objectives and photomultiplier tubes dedicated to fluorescent excitation and emission spectra for rhodamine (excitation = 541 nm and emission = 572 nm) and FITC (excitation = 490 nm and emission = 520 nm). With the dual-channel system of the confocal microscope, dual-emission (535/590 nm) images were recorded simultaneously with a scanning speed of 16 s/frame (512 lines).

Serial sectioning of the cells at 1-µm depth was performed to distinguish label adherent to the cell membrane from internalized materials and to assess colocalization of labeled SP-A and lipid.

Quantitative and statistical functions were performed with the Quantify Tool-Window Leica (TCS, NT version 1.5.451, Leica Lasertechnik). With the stacks function, intensity profiles of vertical optical sections through the cell volume were recorded, and the ratio of the two fluorescence intensities (rhodamine and FITC) was calculated.

Data Analysis

All results are means ± SE. Statistical analysis was done by paired and unpaired t-tests or, for multiple comparisons, by analysis of variance with subsequent Fisher's protected least significant difference test where appropriate. The level of significance was set at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Validation of Internalization

To ensure that our conclusions regarding resecretion were valid, it was important to demonstrate true effective internalization of surfactant components as the starting condition for resecretion. Using a rigorous protocol to remove adherent surfactant from the cell membrane, Wissel et al. (35) have previously reported internalization of lipids by type II cells in the presence of SP-A. In the present study, we confirmed the effective intracellular localization of lipids after 10 min of incubation with the use of fluorescence CLSM on freshly isolated type II cells. The cells were incubated in suspension for 10 min at 37°C with rhodamine-PE-labeled liposomes and FITC-labeled SP-A as described in MATERIALS AND METHODS.

As shown by CLSM (Fig. 1A), after 10 min, most of the internalized SP-A and lipid label colocalized within intracellular vesicles. To validate these findings, we investigated by fluorescence microscopy the presence and absence of the labels. We found both labels to be present in 76 ± 0.7% of the cells counted (n = 3 separate experiments; in each experiment, 300 cells were counted); 16 ± 0.9% of the cells contained only one label, and 7.3 ± 0.3% of the cells were without label. Very little label could be seen at the cell membrane and in the extracellular space.


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Fig. 1.   Uptake and resecretion of FITC-labeled surfactant protein A (SP-A) and rhodamine-phosphatidylethanolamine (PE)-labeled liposomes with time by type II pneumocytes. Alveolar type II cells (5 × 106) were incubated with 100 µg of liposomal phospholipid containing fluorescent lipid rhodamine-PE and with FITC-SP-A (2 µg/106 cells) for 10 min (A-C). Some of the cells were allowed to resecrete in DMEM for 10 min (D-F). Thereafter, cells were washed and fixed as described in MATERIALS AND METHODS. Cells were examined with an epifluorescence microscope interfaced with a confocal laser scanning microscope. Dual-emission (535/590 nm) images were simultaneously recorded. Equatorial sections are shown. A: uptake of FITC-labeled SP-A (green) and rhodamine-PE-labeled lipid (red) by type II cells. In most intracellular vesicles, both markers colocalized (yellow). To demonstrate true intracellular localization of internalized surfactant components, type II cells were incubated with unlabeled liposomes and FITC-labeled SP-A for 10 min at 37°C, then incubated at 3°C for 30 min with tetramethylrhodamine isothiocyanate (TRITC)-labeled Maclura pomifera agglutinin to visualize cell boundaries (B). Alternatively, cells were incubated with rhodamine-PE-labeled liposomes and unlabeled SP-A and then incubated with FITC-labeled concanavalin A for 30 min at 3°C (C). D: resecretion of SP-A and lipid. After 10-min uptake of rhodamine-PE-labeled liposomes and FITC-labeled SP-A, cells were incubated for 10 min in DMEM at 37°C without added SP-A and lipid. In addition to vesicles containing both labels, vesicles containing only 1 label appear. E and F: corresponding fluorescence images to D showing FITC-labeled SP-A only and rhodamine-PE label only, respectively.

To determine cell boundaries in the CLSM experiments, we labeled the cell surface with TRITC-labeled MPA, which is a well-known marker for the apical surface of type II pneumocytes (25). Alternatively, FITC-labeled concanavalin A was used to visualize the cell border. The combination of these markers with either of the surfactant labels was used to confirm the intracellular location of the surfactant labels. Also, as shown in Fig. 1B, MPA bound in a polar fashion to the cell surface, indicating that the cells used in these experiments at this point in time had not yet lost all polarity. Concanavalin A labeled the complete cell boundaries (Fig. 1C).

Lipid Resecretion Assay

To demonstrate resecretion, in biochemical assays type II cells in suspension were incubated with radioactively labeled liposomes with and without 2 µg/106 cells of SP-A for 10 min with the same procedure as described before (35). Thereafter, the cells were carefully washed to remove adhering lipid and SP-A from the cell membranes. Then the cell aliquots were incubated in lipid- and SP-A-free DMEM for different periods of time. Protein content of the cell pellet and radioactivity in both the pellet and supernatant were assayed.

With time, the intracellular amount of lipid that had been taken up with SP-A decreased significantly (P < 0.05 for 0- vs. 10-, 15-, 20-, and 25-min uptake; Fig. 2A). At the same time, the extracellular amount of label increased significantly (P < 0.05 for 5 vs. 15, 20 and 25 min of resecretion; Fig. 2B). After 20 min of resecretion, the intracellular lipid content had reached a minimum (P = 0.0072 vs. 0 min). At the same time, the extracellular amount of label was maximal (P = 0.0112 vs. 5 min). The time course of lipid secretion after SP-A-mediated uptake therefore shows a fast phase within the first 10 min and a slow phase thereafter. After 30 min of resecretion, the intracellular amount of label increased again. At the same time, the extracellular amount of label decreased.


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Fig. 2.   Resecretion of internalized lipids with time after SP-A-mediated uptake. Alveolar type II cells (2.5 × 106) were incubated with 150 µg of [3H]dipalmitoylphosphatidylcholine (DPPC)-labeled liposomes for 10 min at 37°C in absence (control) and presence of SP-A (2 µg/106 cells). Thereafter, cells were washed and then allowed to resecrete for indicated times after 10 min of uptake time as described in MATERIALS AND METHODS. Values are means ± SE; n = 3 experiments. A: intracellular label. open circle , Resecretion in presence of SP-A; , changes within cells incubated with lipids without SP-A were not significant. P < 0.02 for 0 min vs. all time points up to 25 min and 5 vs. 20 and 25 min. B: extracellular label. open circle , Resecretion after uptake in presence of SP-A; , changes within cells incubated with lipids without SP-A were not significant. P < 0.02 for 5 vs. 15, 20, and 25 min.

In those assays run without SP-A present in the uptake phase, far less lipid was internalized. Changes in the amount of label, either intracellular or extracellular, were not significant.

This resecretion effect was dependent on the concentration of SP-A used in the uptake phase. Increasing the concentration of SP-A while holding the amount of lipid presented to the cells constant changed the extent of secretion significantly (Fig. 3). At the lowest concentration of SP-A used (0.5 µg /106 cells), after a 5-min resecretion time, 94 ± 4.6% of the internalized lipids remained within the cells and 82 ± 7% remained after 20 min. In comparison, in cells that had taken up lipid in the absence of SP-A, 96 ± 6% remained after 5 min of resecretion and 92 ± 7.7% remained after 20 min intracellularly. With 2 µg SP-A /106 cells, 85 ± 5.7% of the internalized lipid remained in the cells after 5 min. After 20 min, 50% of the internalized lipid had left the cell. With 4 µg SP-A/106 cells, the fast phase of resecretion was more prominent. After 5 min, 46% of the lipid had been resecreted. This fraction then did not change much anymore (50% at 20 min). With 10 and 50 µg SP-A/106 cells, the fraction resecreted with time was similar to the situation with 4 µg/106 cells (50% at 5 and 20 min).


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Fig. 3.   Concentration dependency of SP-A-mediated lipid resecretion. Cells (2.5 × 106) in suspension were incubated for 10 min at 37°C with [3H]DPPC-labeled liposomes (60 µg/106 cells) and different concentrations of SP-A. Material adherent to cell surface was then removed as described in MATERIALS AND METHODS. Thereafter, some of the samples were incubated in DMEM-0.1% BSA for 5 and 20 min of resecretion. At end of this period, cells were again washed extensively. Values are means ± SE; n = 5 experiments. 0 Min, start of resecretion period after 10 min of internalization; 5 and 20 min, times of resecretion subsequent to 10-min internalization period. open circle , No extracellular SP-A present during resecretion period; , 0.5 µg SP-A/106 cells present during resecretion period; triangle , 2 µg SP-A/106 cells (P = 0.0164 for 0 vs. 20 min); black-triangle, 4 µg SP-A/106 cells (P = 0.0002 for 0 vs. 5 min; P = 0.0004 for 0 vs. 20 min); , 10 µg SP-A/106 cells (P = 0.0121 for 0 vs. 5 min; P = 0.0163 for 0 vs. 20 min); , 50 µg SP-A/106 cells (P = 0.00038 for 0 vs. 5 min; P = 0.0032 for 0 vs. 20 min). Inset: SP-A-mediated lipid uptake is dependent on concentration (0, 0.5, 2, 4, 10, and 50 µg SP-A/106 cells) of SP-A. Time of incubation was 10 min. Increasing amounts of SP-A led to increasing amounts of lipid internalized. A plateau is reached at ~10 µg SP-A/106 cells. P = 0.0005 for 0 vs. 4 µg SP-A. P < 0.0001 for 0 vs. 10 µg of SP-A and 0 vs. 50 µg of SP-A.

Validation of Lipid Resecretion

To exclude that resecretion was due to removal of material simply adhering to the cell membrane, cells that had internalized [3H]DPPC-labeled liposomes in the presence of SP-A were washed with the same methods as described in SP-A Resecretion Assay. Then the cells were put in new medium at 3 or 37°C.

A back-exchange procedure with unlabeled liposomes, as previously described (11), was used to try to remove any material still adhering to the cell membrane. Thereafter, the intracellular label was quantified as described in Lipid Resecretion Assay. The results were compared with those of resecretion experiments done at 3°C without lipid in the back-exchange buffer and with those of resecretion experiments done at 37°C (Fig. 4). The back-exchange procedure at 3°C led to a small nonsignificant decrease in the intracellular amount of label. Differences between the two methods were not significant. In comparison, when a conventional resecretion assay was done at 37°C with an aliquot from the same preparation, 35% of label had been resecreted after 5 min.


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Fig. 4.   Resecretion vs. adherence to cell membrane by back-exchange procedure. After a 10-min uptake period (60 µg/106 cells of [3H]DPPC-labeled liposomes and 4 µg/106 cells of SP-A), type II cells were washed with 5% fatty acid-free BSA-10 mM EGTA as described in MATERIALS AND METHODS. Thereafter, half of the cells were incubated in DMEM-1% BSA with unlabeled liposomes (100 µg/106 cells) for 5 or 20 min at 3°C (). The other half was incubated in DMEM at 3 () or 37°C () for 5 or 20 min. Radioactivity and protein content were determined. Times indicated were times allowed for resecretion, i.e., after 10 min of uptake time. Values are means ± SE; n = 3 experiments. * P < 0.0001 for 0 vs. 5 and 20 min.

In additional experiments, we checked the efficiency of the washing procedures used by allowing liposomes to adhere to type II cells in the presence of SP-A at 3°C. Then the cells were washed three times with 5% fatty acid-free BSA-10 mM EGTA as described in MATERIALS AND METHODS. Thereafter, an aliquot was taken, and the amount of label associated with the cells was measured. Some of the washed samples were then warmed to 37°C for different time periods, and the amount of label associated with the cells was measured. After 10 and 30 min of binding time, 7,350 ± 2,420 and 10,330 ± 1,190 dpm/106 cells, respectively, were associated with the cells. After being washed, ~3% of the label remained associated with the cells (222 ± 11 and 221.3 ± 9 dpm/106, respectively). Longer incubation of the cells in lipid- and SP-A-free medium at 37°C did not change the amount of cell-associated label.

Cell viability after the repeated washing as assayed by trypan blue exclusion was 94 ± 0.7% (n = 30 experiments). Lactate dehydrogenase (LDH) release was 2% of the total amount (n = 10 experiments).

These results suggest that the washing procedure itself was effective and that the resecretion phenomenon could not easily be explained by the shedding of cell membrane components.

SP-A Resecretion

After incubation of the type II cells with 3 µg of biotin-labeled SP-A/106 cells for 10 min, extracellular biotin was removed from the SP-A still adhering to the cell membrane with the GSH-iodoacetamide procedure as described in MATERIALS AND METHODS. Then the cells were allowed to resecrete into DMEM. Internalized biotinylated SP-A was visualized by SDS-PAGE and Western blotting as described in MATERIALS AND METHODS. A representative blot is shown in Fig. 5A. After 5 min, 21.7 ± 4.9% of SP-A remained in the cells (P = 0.0010 vs. 0 min), i.e., 70-80% of the internalized SP-A had been resecreted (Fig. 5B). This fraction did not change with time. Values were similar after 10 (P = 0.0014 vs. 0 min) and 20 (P = 0.0022 vs. 0 min) min.



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Fig. 5.   SP-A resecretion. Freshly isolated type II cells (5 × 106) were incubated with biotin-labeled SP-A (3 µg/106 cells) for 10 min at 37°C. At end of uptake period, cells were washed by GSH-iodoacetamide procedure as described in MATERIALS AND METHODS and then allowed to resecrete into fresh DMEM-0.1% BSA for 5, 10, and 20 min at 37°C. Thereafter, cells were washed again. Intracellular SP-A was detected by NeutrAvidin-horseradish peroxidase and enhanced chemiluminescence. A: representative Western blot of biotinylated SP-A resecretion by type II cells. Lane 1, biotinylated SP-A (10 ng); lane 2, cells without added SP-A; lane 3, type II cells incubated for 10 min at 37°C with SP-A; lanes 4-6, SP-A in type II cells after 5, 10, and 20 min of resecretion, respectively. On each gel, control samples (cells without added SP-A) were also run. Nos. on left, molecular mass in kDa. B: scanning densitometric quantitation of SP-A resecretion with time. Values are means ± SE of percentage of relative optical density (OD) at 0 min; n = 3 experiments.

To assay whether type II cells remain functional after our washing procedures, type II cells were first washed with the GSH-iodoacetamide procedure and then incubated with 4 µg SP-A/106 cells and liposomes (60 µg lipid/106 cells) for 10 min at 37°C and then centrifuged and washed as described in Surfactant Lipid Resecretion Assay. Thus treated cells internalized 1,261 ± 19 vs. 1,291 ± 18 pmol DPPC · 106 cells-1 · 10 min-1 in untreated cells (n = 3 experiments).

CLSM

With CLSM, we demonstrated that after 10 min of internalization time (Fig. 1, A-C), SP-A and lipid labels colocalized intracellularly in vesicles of different size (Fig. 1A). After 10 min of internalization followed by 10 min of resecretion, the extent of colocalization of SP-A and lipid had decreased. SP-A and lipid label colocalized in a markedly smaller fraction of the cells (14.1 ± 1%; Fig. 1, D-F). Compared with cells that had internalized label for 20 min in the presence of extracellular labeled SP-A and lipids (data not shown), the fluorescence intensity had decreased considerably and the size of the label-containing vesicles was smaller; the extent of colocalization, however, was similar.

As far as could be determined from measurements on nine cells from three different experiments, 20% of the lipid label was lost during resecretion vs. 61% of the SP-A label, consistent with the results from our biochemical assays. For technical reasons, it was not possible to evaluate >3 cells/run.

The ratio of the fluorescence intensity of the lipid to that of the SP-A label after the uptake period versus after 10 min of resecretion was also compared with CLSM. After 10 min of uptake, the fluorescence ratio of lipid to SP-A label was 0.85 ± 0.02. After 10 min of subsequent resecretion, the ratio of lipid to protein had increased to 1.79 ± 0.1. Although loss of fluorescence intensity could be due to several factors, such as quenching of the FITC label by low pH in acidified vesicles, these results are consistent with the results of the biochemical assays (see Lipid Resecretion Assay) and suggest differential loss of lipid and SP-A label during resecretion.

Specificity of the Resecretion Processes

Resecretion process can be inhibited by SP-A. To investigate whether extracellular SP-A could inhibit the resecretion process, as had been shown for agonist-stimulated surfactant lipid secretion (9), unlabeled SP-A was added to the extracellular medium at the start of the resecretion period (Fig. 6A). With 3 µg SP-A/106 cells present during the resecretion phase, 57.5 ± 10.4% of the labeled SP-A remained intracellularly at 5 min (value not significantly different from that at 0 min). In comparison, without extracellular SP-A, only 21.7 ± 4.6% of the internalized SP-A remained within the cells (P = 0.001 vs. 0 min). Therefore, extracellular SP-A partially inhibits SP-A resecretion. Increasing the amount of SP-A did not further enhance inhibition (results not shown).


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Fig. 6.   Extracellular SP-A partially inhibits resecretion. A: effect on SP-A resecretion. Freshly isolated type II cells (5 × 106) were incubated with biotin-labeled SP-A (3 µg/106 cells) for 10 min at 37°C. At end of uptake period, cells were washed by GSH-iodoacetamide procedure as described in MATERIALS AND METHODS and then allowed to resecrete into fresh DMEM-0.1% BSA with and without 3 µg SP-A/106 cells for 5, 10, and 20 min at 37°C. Intracellular SP-A was detected by NeutrAvidin-horseradish peroxidase and enhanced chemiluminescence. open circle , Resecretion in presence of SP-A in extracellular medium; , resecretion in absence of SP-A. Values are means ± SE; n = 3 experiments. B: effect on lipid resecretion. [3H]DPPC-labeled liposomes (60 µg/106 cells) were incubated with type II cells in absence and presence of SP-A (4 µg/106 cells) for 10 min at 37°C. Thereafter, cells were carefully washed and then allowed to resecrete into fresh medium in absence (open circle ) and presence () of SP-A (4 µg/106 cells). At end of resecretion period, medium was harvested and cells were washed. Radioactivity associated with lipid and protein content were determined. open circle , Resecretion in presence of SP-A in extracellular medium; , resecretion in absence of SP-A. Values are means ± SE in percentage of intracellular label at 0 min; n = 3 experiments. * P = 0.0126 for 0 vs. 5 min and P = 0.0026 for 0 vs. 20 min.

To investigate the effect of extracellular SP-A on lipid resecretion, type II cells were incubated with labeled liposomes with and without 4 µg SP-A/106 cells for 10 min at 37°C. The cells were then carefully washed. Radioactivity and protein contents were measured in an aliquot (0 min). Some of the cells were then put into medium to which 4 µg SP-A/106 cells were added (Fig. 6B). SP-A inhibited the early phase of lipid resecretion of liposomes internalized in its presence. After 5 min, 75 ± 3.6% of the intracellular label remained in the cells and 69.8 ± 5.7% remained after 20 min when SP-A was present in the extracellular medium during the resecretion period (not significant vs. 0 min). Without extracellular SP-A present during resecretion, only 59.5 ± 5.8% of the internalized lipid remained within the cells at 5 min (P = 0.0127 vs. 0 min); at 20 min, this value had decreased even more, to 45.9 ± 5.7% of the value at the start of the resecretion period (P = 0.0026 vs. 0 min). In cells that had been incubated with liposomes in the absence of SP-A, no effect of SP-A on the loss of intracellular label could be detected (Fig. 2).

Adding unlabeled lipids to the medium of the cells resecreting at 37°C did not have any effect on lipid resecretion in the cells that had taken up lipid in the presence or absence of SP-A (results not shown).

Resecretion process is temperature sensitive. To demonstrate a temperature dependency of the resecretion process, resecretion was studied at 3°C (see Fig. 4) as well as at 16°C in comparison to 37°C (Fig. 7). After uptake of lipid plus SP-A at 37°C and subsequent resecretion at 37°C, the intracellular lipid content decreased significantly (P = 0.0057 for 0 vs. 5 min). After uptake of lipid in the presence of SP-A at 37°C and subsequent resecretion at 16°C, the intracellular lipid content did not significantly decrease.


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Fig. 7.   Temperature dependency of resecretion. Type II cells were incubated with [3H]DPPC-labeled liposomes (60 µg/106 cells) and SP-A (4 µg/106 cells) at 37 (10 min) and 16°C (30 min). Cells were washed as described in MATERIALS AND METHODS and then incubated in DMEM-0.1% BSA at 16 or 37°C for 5 or 20 min. Values are means ± SE; n = 3 experiments. , Uptake and resecretion at 37°C (P <=  0.02 for 5 and 20 vs. 0 min; * P = 0.0057 for 0 vs. 5 min and P = 0. 0075 for 0 vs. 20 min); open circle , uptake at 37°C and resecretion at 16°C; , uptake and resecretion at 16°C.

To prove that the cells could still internalize at 16°C, cells were incubated at 16°C with liposomes and SP-A. At this temperature, internalization did take place, albeit at a slower rate. Therefore, to obtain enough label intracellularly to enable measurements, the uptake period was extended. After a 30-min uptake period at 16°C, the cells were incubated in lipid- and SP-A-free DMEM at 16°C. At this temperature, no resecretion could be detected.

Lipids Resecreted After SP-A-Mediated Uptake Are Unmodified

Freshly isolated type II cells were incubated with PE-labeled liposomes in the presence and absence of SP-A (2 µg SP-A/106 cells) for 10 min at 37°C. Thereafter, the cells were washed as described in Lipid Resecretion Assay. Half of the cells were incubated for 15 min at 37°C in DMEM-0.1% BSA. The supernatant was separated from the cells. Intra- and extracellular lipid species were determined by two-dimensional TLC after lipid extraction as described in MATERIALS AND METHODS. Phosphorus was measured, and radioactivity associated with the different lipid species was assayed to calculate the fraction of label in PE as described by Wissel et al. (35).

The results are summarized in Table 1. PE taken up by the SP-A-mediated pathway and then resecreted is largely unmodified. Although after 10 min of uptake at 37°C, 93.5 ± 2.2% of the label remained associated with PE, after 15 min of resecretion, the fraction of intracellular label still in PE was 87.1 ± 5%. The fraction of label still in PE in the extracellular medium was 94.0 ± 0.7%. In contrast, the lipid taken up without SP-A was significantly degraded. After 10 min of uptake, only 79.4 ± 0.8% of the label was still associated with PE. After an additional 15 min of resecretion, the fraction of label still in PE had further decreased to 54.7 ± 1.5% for the intracellular compartment and 27.6 ± 5.9% for the extracellular compartment.

                              
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Table 1.   Fate of resecreted lipids


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

De novo surfactant synthesis is time and energy consuming, and surfactant pools are limited. Therefore, in stress situations, alternative mechanisms must exist that enable the lung to cope with the increased demand. One such possible mechanism is recycling of surfactant components. Experimental evidence gathered from in vivo animal studies indicates that surfactant components can indeed cycle back and forth several times between the alveolar space and lung tissue before being degraded (for a review, see Ref. 36).

This recycling process can be divided roughly into three steps: reuptake by type II cells, intracellular transport between the endocytic and secretory pathways, and resecretion by the pneumocyte. Several studies (5, 12, 23, 28, 30, 33, 35, 37, 39, 41) have demonstrated that both surfactant lipids and proteins can be reinternalized by type II cells, partly by receptor-mediated uptake and partly by other mechanisms. In contrast, relatively little is known about the mechanisms and kinetics of intracellular transport and resecretion of surfactant components after reuptake.

The aim of our study was to develop in vitro resecretion assays for both lipid components to begin to describe the time course and mechanisms of resecretion of surfactant components internalized by the type II cell in the presence of SP-A. The possible role of SP-A in lipid metabolism by type II cells is controversial. Although some in vitro studies (32, 38), including that by Wissel et al. (35), found a stimulatory effect of SP-A on lipid uptake, others (18) using different cell systems and methods found no effect on internalization and suggested that SP-A would simply enhance lipid adherence to the cell membrane. Therefore, it was necessary to confirm that surfactant components were indeed internalized by type II cells and did not simply adhere to the cell membrane.

With CLSM, we could demonstrate that 10 min after the start of the uptake, most of the SP-A and the lipid colocalized within the intracellular vesicles (Fig. 1, A-C). After the extensive washing procedure, very little material was left adhering to the cell membrane.

These results were further corroborated by back- exchange (Fig. 4) and washing experiments as described in RESULTS. Back exchange is a very effective procedure to remove lipid adhering to cell membrane. However, this procedure, when employed at 3°C on cells that had internalized labeled liposomes, did not release any more lipid than if the cells were simply left in normal medium. Incubation of the cells at 37°C in the presence of unlabeled lipids also did not release more label than if no lipids were present (results not shown). We therefore feel confident that in our assays SP-A and lipid were indeed internalized by the cells.

SP-A can be rapidly and effectively resecreted by the cells as shown in Fig. 5. After 5 min, 70-80% of the internalized SP-A was resecreted (Fig. 5B). This process is temperature dependent (Fig. 7) and can be partially inhibited by extracellular SP-A (Fig. 6A).

Lipid taken up by the SP-A-mediated pathway is also rapidly resecreted (Fig. 2). This was not due to a simple lipid gradient between cells and medium because extracellular lipid added at the start of the resecretion period did not affect lipid resecretion. The extent and kinetics of lipid resecretion were dependent on the concentration of SP-A used in the uptake phase. With 2 µg SP-A/106 cells, the rate of loss of label from the intracellular compartment was slower than with 4, 10, or 50 µg SP-A/106 cells (Fig. 3). As can be seen best with the results with the higher amounts of SP-A, resecretion was biphasic, with a rapid phase within 5 min depleting most of what could eventually be resecreted and a slower phase thereafter. Essentially, all resecretable material had left the cells 20 min after the start of the resecretion phase. After 30 min, the amount of intracellular lipid label again increases, paralleled by a decrease in the extracellular label. Most likely, this indicates another round of reinternalization, but this phenomenon has not yet been investigated in more detail.

We suggest that resecretion is an active process for the following reasons. 1) As far as could be determined from LDH release, trypan blue exclusion assays, and functional tests done before and after the washing procedures, the vitality, permeability, and functionality of the cells in suspension seemed normal. 2) We found no evidence for high levels of labeling of the cell membrane. Therefore, shedding or lipid leaking of the membrane is unlikely. Also, extracellular unlabeled lipids would likely exchange with the labeled lipids inserted in the cell membrane, thereby causing a false positive "resecretion." However, even at 37°C, the presence of extracellular lipids did not affect resecretion. 3) The resecretion assay involves numerous steps that could damage the cells. As far as could be determined from LDH release and trypan blue exclusion assays, at all times during uptake and resecretion, the vitality and permeability of the cells were good. Also, the cells were still able to internalize lipid via the SP-A-mediated pathway. Furthermore, leaking of labeled cell surface membrane lipids does not explain our results. As mentioned above, no evidence for high levels of labeling of the cell membrane could be found. The results of our back-exchange experiments also argue against leaking of the cell membrane. Extracellular unlabeled lipids would likely exchange with the labeled lipids still in the cell membrane, thereby causing a false positive resecretion. However, even at 37°C, the presence of extracellular lipid did not affect resecretion. 4) The process is temperature dependent. At 3°C, no resecretion occurs. At 16°C, endocytosis does take place, albeit at a slower pace, as described in other cell systems (11, 34). However, no resecretion occurs as shown in Fig. 7. Even after considerable time, no intracellular lipid enters the medium, suggesting the existence of a temperature-sensitive step in the resecretion process. Therefore, we suggest that resecretion is not due to simple shedding or leaking from the membrane of lipid but is likely an active, energy-dependent process.

Although most of the SP-A was resecreted within a few minutes of uptake (Figs. 5 and 6A), resecretion of lipids was not as effective. Within the time frame of our experiments (up to 30 min), only 50% of the internalized lipid is resecreted (Figs. 2, 3, and 6B). Lipids reappearing in the medium after SP-A-mediated uptake remained largely intact. In contrast, lipids resecreted after uptake in the absence of SP-A were degraded (Table 1). It must be pointed out, however, that in addition to true SP-A-mediated lipid uptake, some lipid is also internalized by other nonspecific pathways such as diffusion and membrane exchange as described before (35). If recycled at all, these lipids most likely are recycled via different pathways with different kinetics. This would contribute to the incomplete recycling of lipids found at early time points. The finding that SP-A and lipid labels left within the cell after 10 min of resecretion were partially segregated in separate vesicles, as seen in Fig. 1, D-F, is compatible with the idea of several distinct sources contributing to SP-A and lipid resecretion. On resecretion, these vesicles decrease in size and fluorescence intensity, most likely because of loss of their contents to the extracellular space. Also, the change in the ratio of the fluorescence of the two labels is compatible with selective loss from different compartments.

A model could therefore be constructed in which, after uptake as a lipoprotein complex intracellularly, the lipid partially separates from SP-A. The part still associated with SP-A rapidly recycles and leaves the cells again. The part of the lipid released from the SP-A would recycle with different (slower) kinetics.

In this regard, it is interesting that SP-A present in the extracellular medium inhibits resecretion of both components to a different extent (SP-A, 36%; lipid, 16%; Fig. 6). Therefore, with the assumption that SP-A-mediated inhibition of secretion is a specific process, this would again suggest that there is more than one pathway for resecretion both for lipids and for SP-A. In fact, this had been suggested by several studies in the literature. For example, Froh et al. (13) found that most of the newly synthesized SP-A was not found in the classic secretory organelles of type II cells, which contain most of the de novo synthesized lipid. Also, in preterm lambs, newly synthesized SP-A is secreted independent of lamellar bodies (20).

The identity of the transport processes involved in resecretion is unknown. It could be that part of the lipid entering the cells with SP-A is rapidly returned to the surface, remaining associated with SP-A as this recycles. Rapidly recycling vesicles have been described in other cell systems, for example, vesicles recycling lipoprotein-derived cholesterol from the endosomes to the plasma membrane of hepatocytes (17). Also, in other systems, distinct populations of recycling vesicles exist, for example, in the transferrin/transferrin receptor pathway (6).

It is tempting to speculate that the part of the lipid that is not rapidly recycled with SP-A is transported to the classic lamellar bodies. In subfractionation experiments with sucrose gradients, we found that most of the lipid internalized is indeed found in those fractions containing lamellar bodies. However, a substantial amount of lipid was also detected in overlying lighter fractions, consistent with the hypothesis that there might be considerable overlap between "classic" lamellar bodies and other vesicular fractions containing endocytosed lipids, such as endosomes (P. A. Stevens, H. Wissel, S. Zastrow, D. Sieger, and K. P. Zimmer, unpublished results). More incisive studies need to be done to clarify these issues as well as the regulatory mechanisms involved.

These in vitro findings and those of other groups (32, 37, 38, 41) in both in vivo and in vitro situations are in contrast to some newer data on the role of SP-A in surfactant homeostasis obtained by targeted gene disruption. Mice in which the SP-A gene has been inactivated show no obvious disturbance of surfactant pool sizes, at least in later life (24). This, however, does not necessarily argue against a role for SP-A in surfactant pool regulation. In early life, there is a clear difference in surfactant pool sizes between knockout and wild-type mice, which disappears in later life (19). It is also possible that as yet unknown redundant mechanisms exist, which can compensate for the lack of SP-A. Third, SP-A might still play an important role in in vivo stress situations, which is not apparent under the conditions investigated until now. More detailed studies are clearly required to define the physiological role of SP-A in surfactant metabolism in these mice.

The results in this study also suggest that at least in the in vitro situation, the true extent of lipid uptake by type II cells might be considerably higher than previously assumed. If there is continuous resecretion occurring at the same time as uptake, just measuring the amount of intracellular label at different times would give a false low impression of the uptake rate. This should be taken into account when evaluating quantitative studies of uptake by type II pneumocytes.

In summary, our study demonstrates that SP-A and lipid in vitro can be rapidly resecreted after uptake by type II cells in vitro. Although SP-A seems to be resecreted by a fast process, resecretion of lipids could be by more than one pathway. Not much is known yet about the mechanisms involved in this recycling process. Reentry of SP-A and lipid in the extracellular medium was partially inhibitable by extracellular SP-A but not by extracellular lipid. Further dissection of the pathways involved and their regulation will be needed to better understand surfactant pool size regulation.


    ACKNOWLEDGEMENTS

This work was supported by grants from the Deutsche Forschungsgemeinschaft (to P. A. Stevens) and by German Ministry for Research and Technology Grant 01 ZZ 9511.


    FOOTNOTES

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: P. A. Stevens, Clinic of Neonatology, University Children's Hospital Charité, Humboldt-University Berlin, Schumannstrasse 20-21, 10098 Berlin, Germany (E-mail: paul_a.stevens{at}charite.de).

Received 11 January 1999; accepted in final form 13 October 1999.


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DISCUSSION
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