Endocytosed SP-A and surfactant lipids are sorted to different organelles in rat type II pneumocytes

Heide Wissel1, Andrea Lehfeldt1, Petra Klein2, Torsten Müller2, and Paul A. Stevens1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Intracellular transport of endocytosed surfactant protein A (SP-A) and lipid was investigated in isolated rat type II cells. After internalization, SP-A and lipid are taken up via the coated-pit pathway and reside in a common compartment, positive for the early endosomal marker EEA1 but negative for the lamellar body marker 3C9. SP-A then recycles rapidly to the cell surface via Rab4-associated recycling vesicles. Internalized lipid is transported toward a Rab7-, CD63-, 3C9-positive compartment, i.e., lamellar bodies. Inhibition of calmodulin led to inhibition of uptake and transport out of the EEA1-positive endosome and thus of resecretion of both components. Inhibition of intravesicular acidification (bafilomycin A1) led to decreased uptake of both surfactant components. It inhibited transport out of early endosomes for lipid only, not for SP-A. We conclude that in type II cells, endocytosed SP-A and lipid are transported toward a common early endosomal compartment. Thereafter, both components dissociate. SP-A is rapidly recycled to the cell surface and does not enter classic lamellar bodies. Lipid is transported toward lamellar bodies.

surfactant protein A; calmodulin; intravesicular acidification; early endosome; lamellar body; dielectrophoretic field cage; confocal laser scanning microscopy


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AFTER THEIR SECRETION into the alveolar space, surfactant components are recycled by the type II pneumocytes. This mechanism presumably enables the lung with its limited surfactant pools to efficiently cope with the large and variable demand for functional surfactant under various conditions (43). Very little, however, is known about the mechanisms involved in this recycling process.

In previous work, Stevens et al. (29) have shown that surfactant protein (SP) A and surfactant lipid, after internalization by type II cells via the coated-pit pathway, are transported toward early endosomal compartments (29). SP-A and at least part of the lipid can then be rapidly resecreted (42). In this study, we characterized the pathways and molecular mechanisms involved in endocytosis, intracellular sorting, and resecretion of surfactant components using both morphological assays and pharmacological approaches. To follow intracellular transport along the endocytic compartments, in confocal laser scanning microscopy (CLSM) experiments, we used different markers for the various endosomal compartments as well as fluorescently labeled SP-A and lipids.

In most cells, sorting of internalized components occurs in so-called early or sorting endosomes, which are located in the peripheral cytoplasm. As done in this study, these organelles can be identified in confocal laser scanning assays by labeling with antibodies against the early endosomal antigen EEA1 as well as against Rab5, a small GTPase required for the import of endocytic material from the cell surface, and Rab4, another GTPase that regulates rapid recycling back to the plasma membrane via small vesicles (2, 20, 27, 34). In addition to this fast recycling pathway, some receptor systems such as the transferrin receptor will, under certain conditions, use a second, more slowly recycling compartment. With this so-called receptor-recycling compartment (RRC), another GTPase, Rab11, is associated (33).

Early endosomes will transport their contents destined for the degradative pathway to late endosomes and then to lysosomes. Late endosomes can be identified with antibodies against Rab7 (18). Interestingly, lamellar bodies also have Rab7 on their limiting membranes (40) as well as other late endosomal or lysosomal markers such as CD63 (37). These findings, in addition to a previous report (10a) on their enzymatic content, suggested that lamellar bodies may have additional properties of late endosomes in addition to characteristics of the de novo synthetic pathway.

Another property that lamellar bodies have in common with late endosomes and lysosomes is their low intravesicular pH. As done in this study, this property can be exploited to visualize such compartments with the use of LysoTracker dyes (3, 26). With the use of a recently developed antibody against an integral lamellar body-limiting membrane protein (3C9) (47), lamellar bodies can now be distinguished from other acidified organelles.

Additionally, pharmacological drugs were used to block intracellular transport pathways at various stages in biochemical and morphological assays. Specifically, we investigated the effects of the calmodulin inhibitor W-7 and of bafilomycin A1, a potent inhibitor of intravesicular acidification, on endocytic processes such as sorting and endosomal transport between organelles.

Calmodulin, a multifunctional calcium-binding protein, plays a role in the regulation of endocytosis, exocytosis, transcytosis, and receptor recycling (6, 39). Surfactant lipid secretion by type II pneumocytes is calmodulin dependent (38). Vacuolar H+-ATPases, which regulate intravesicular pH, are present in different cells in the plasma membrane as well as in intracellular membranes of various organelles such as endosomes (10, 30) and in lamellar bodies (4, 14). Disruption of their function by bafilomycin A1 in some systems inhibits ligand-receptor dissociation and intracellular transport of endosomal vesicles as well as recycling of receptors (24, 35).

The results presented here demonstrate that in freshly isolated type II cells, internalized SP-A and lipid are transported together to early endosomes but then take different intracellular pathways. SP-A is recycled rapidly toward the cell surface, whereas lipid is transported to the lamellar bodies.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

Monoclonal antibodies against Rab5 and Rab11 and the mouse anti-human monoclonal antibody reacting with the lysosomal glycoprotein CD63 were purchased from PharMingen (Heidelberg, Germany). Affinity-purified goat polyclonal antibodies against Rab4 and Rab7 were from Santa Cruz Biotechnology (Heidelberg, Germany). The anti-EEA1 monoclonal antibody as well as Cy3- and Cy2-labeled anti-mouse IgG antibodies and monovalent Fab-fluorochrome conjugates were obtained from Dianova (Hamburg, Germany). The monoclonal antibody 3C9 against the 180-kDa lamellar body-limiting membrane protein was from Berkeley Antibody (Richmond, CA). L-alpha -Phosphatidylethanolamine (PE)-N-(lissamine rhodamine B sulfonyl) (rhodamine-PE) was from Avanti Polar Lipids. ProLong antifade kit, LysoTracker, and secondary antibodies conjugated with Alexa 499 and Alexa 594 were from Molecular Probes Europe.

W-7 (Sigma, Deisenhofen, Germany) solutions were made up in Dulbecco's modified Eagle's medium (DMEM) and used at 10-200 µM. pH was adjusted to 7.4. Phorbol 12-myristate 13-acetate (PMA) and bafilomycin A1 (Sigma, Deisenhofen, Germany) were dissolved in DMSO, and stock solutions were kept at -20°C. For the assays, they were diluted with DMEM to 10-8 M and 1 µM, respectively.

All other materials and compounds were from major suppliers.

Isolation of SP-A

SP-A was purified from lavages of fresh lamb lungs obtained from the local slaughterhouse. All preparations were tested for purity and functional activity as described by Wissel et al. (41) and Young et al. (45).

Biotinylation of SP-A

Sheep SP-A was biotinylated with different biotin derivatives as previously described (28). Briefly, it was dialyzed overnight against 50 mM phosphate buffer, pH 6.3, at 4°C. It was then incubated with biotin derivatives for 3 h at room temperature at a molar ratio (biotin to SP-A) of 5:1 and a volume ratio of 1:50. Thereafter, it was dialyzed against the appropriate buffers for 24 h at 4°C. All biotinylated SP-A batches were tested for functional activity as previously described (29, 42). Activity was not different from that of unlabeled SP-A.

Labeling of SP-A With FITC

SP-A (4.6 mg/ml in 5 mM Tris · HCl, pH 7.4) was incubated with 400 µl of NaHCO3 (400 mM, pH 8.5) and 40 µl of FITC (5 mg/ml in DMSO) for 1 h at room temperature in the dark on a rotating wheel. The solution was then dialyzed exhaustively against double-distilled water. The labeled SP-A was then divided into aliquots and frozen at -80°C. Before use, it was tested for inhibition of surfactant secretion and for functional activity in lipid uptake assays as described above. Activity was not different from that of unlabeled SP-A.

Type II Pneumocyte Preparation

Type II cells were isolated from the lungs of adult male rats (body weight 120-140 g) according to Dobbs et al. (7). The cell pellets contained 90 ± 0.6% type II cells (n = 30 experiments) as determined by modified Papanicolaou stain. Cell viability after isolation as assayed by trypan blue exclusion was 98 ± 0.6% (n = 30 experiments). In all experiments, freshly isolated type II cells in suspension were used.

Liposome Preparation

Small unilamellar liposomes were made by sonication as previously described (41). They contained 55% (by weight) dipalmitoylphosphatidylcholine (DPPC), 25% egg phosphatidylcholine, 10% dipalmitoylphosphatidylglycerol, and 10% cholesterol. A radioactive lipid marker, 1,2-dipalmitoyl-L-3-phosphatidyl-N-[methyl-3H]choline ([3H]DPPC; 1.46 nCi/µg lipid) was added.

For experiments with fluorescently labeled lipids, liposomes of the same lipid mixture were made, except that the DPPC was reduced to 53% (by weight) and 1% rhodamine-PE and 1% unlabeled PE were added.

The liposomes were kept at 3°C and used within 1 wk. Before use, they were brought to room temperature and centrifuged for 5 min at 1,000 g to remove large aggregates and multilamellar vesicles.

Liposome Internalization Assay

For each data point, 2.5 × 106 freshly isolated type II cells were taken up in 1 ml of cold DMEM-0.1% BSA with and without drugs. After the addition of 15 µl of [3H]DPPC-labeled liposomes (150 µg lipid)/2.5 × 106 cells, 10 µg SP-A/2.5 × 106 cells were added to the cells with and without drugs. The cells were quickly brought to 37°C for various times 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 and 10 mM EGTA. Thereafter, the cells were washed twice for 5 min each with DMEM-0.1% BSA. After the first wash, the samples were transferred to new tubes. The cells were washed twice more with DMEM and then pelleted. The final pellets were resuspended in distilled water and sonicated twice for 10 s with 40% power with a Bandelin sonicator (Bandelin, Berlin, Germany). An aliquot was taken for determination of protein content (Bio-Rad protein assay kit, Bio-Rad, Richmond, CA). For radioactivity measurements, 800 µl of methanol and 8 ml of BCS scintillation fluid (Amersham, Braunschweig, Germany) were added. Radioactivity was measured in a Wallac 1410 liquid scintillation counter equipped with an automatic quench correction (Pharmacia, Freiburg, Germany).

Surfactant Lipid Resecretion Assay

Type II cells were incubated with [3H]DPPC-labeled liposomes and SP-A in DMEM-0.1% BSA without drugs at 37°C and washed as described in Liposome Internalization Assay. Thereafter, half of the samples were pelleted to determine protein content and radioactivity. The other half were incubated for various times at 37°C in 1 ml of DMEM-0.1% BSA with and without drugs in liposome- and SP-A-free medium. Thereafter, the medium was collected, and the cells were washed as described by Wissel et al. (42). The final pellets were resuspended in distilled water and sonicated twice for 10 s at 40% power with a Bandelin sonicator (Bandelin). Subsequently, aliquots were taken for determination of protein content and radioactivity as described above.

Disulfide-Biotinylated SP-A Internalization Assay

The freshly isolated cells were washed once in DMEM to which 0.1% BSA was added. The cells (5 × 106 cells/sample) were taken up in 1 ml of cold DMEM-0.1% BSA and then incubated for 10 min with 3 µg/106 cells of SP-A labeled with NHS-SS-biotinylated SP-A in the absence and presence of drugs (W-7 or bafilomycin A1) at 37°C. In some experiments, the cells were pretreated with the drugs at 37°C for 10 min before incubation with SP-A and lipid in the continued presence of drugs.

During the incubation period, the cells were continuously and gently shaken. Thereafter, the medium was removed, and the cells were washed once with ice-cold PBS-0.1% BSA-10 mM EGTA (pH 7.4) for 5 min on ice. Subsequent removal of extracellular label was performed by the glutathione or iodoacetamide method described by Volz et al. (36) as modified by Wissel et al. (42). The final pellet was taken up in 200 µl of H2O and sonicated twice on ice for 10 s each at 40% maximal power with a Bandelin Sonopuls sonicator (Bandelin).

To visualize SP-A, equal amounts of total protein were added under nonreducing conditions to the wells of 10% SDS-PAGE gels and were subjected to electrophoresis. The proteins were then blotted onto nitrocellulose and visualized by enhanced chemiluminescence on film with the neutravidin-peroxidase system (Boehringer Mannheim, Mannheim, Germany). The results were quantitated by scanning densitometry.

Standard amounts of biotinylated SP-A as well as control samples (cells carried through the experiments without added SP-A) were run on each gel to allow identification of the SP-A-specific bands. Under the conditions used, SP-A runs at molecular masses of 28-35 and 60-64 kDa. Both bands were included for quantification.

SP-A Resecretion Assay

After the incubation period with biotinylated SP-A (without drugs; pulse) as described in Disulfide-Biotinylated SP-A Internalization Assay, the cells were washed as described by Wissel et al. (42). An aliquot of the cells was taken and immediately processed as described in Disulfide-Biotinylated SP-A Internalization Assay. The rest of the cells was resuspended in DMEM-0.1% BSA with and without drugs at 37°C without SP-A for various amounts of time as indicated in RESULTS and Figs. 1-9. After the resecretion period, the cell pellets were washed and treated with the glutathione or iodoacetamide procedure as described in Disulfide-Biotinylated SP-A Internalization Assay. The final pellets were resuspended in distilled water and sonicated for 2 × 10 s with a Bandelin sonicator at 40% power (Bandelin). An aliquot was removed for determination of protein content. Visualization of SP-A was performed as described above.


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Fig. 1.   Identification of early intracellular uptake compartments for surfactant protein (SP) A and lipid with confocal laser scanning microscopy (CLSM). A: colocalization of EEA1 (green) and Rab5 (red). The cells were processed for double-labeled fluorescence with primary mouse monoclonal anti-EEA1 antibody or Cy3-conjugated Fab fragment goat anti-mouse IgG followed by mouse monoclonal anti-Rab5 antibody or Cy2-conjugated goat anti-mouse IgG. B: colocalization of internalized SP-A labeled with FITC-labeled SP-A (green) and lipids labeled with L-alpha -phosphatidylethanolamine (PE)-N-(lissamine rhodamine B sulfonyl) (rhodamine-PE)-labeled liposomes (red) at early time points (5 min at 37°C). C: internalized lipid labeled with rhodamine-PE liposomes (red) did not colocalize with the lamellar body marker 3C9 labeled with 3C9 anti-lamellar body antibody or Alexa 488-labeled anti-mouse IgG (green) at early time points (5 min after start of internalization at 37°C). D: colocalization of SP-A labeled with FITC-SP-A (green) with Rab5 labeled with anti-Rab5 antibody or Alexa 594-labeled anti-mouse IgG (red) and unlabeled liposomes at early time points (5 min after start of internalization). E: partial colocalization of SP-A labeled with FITC-SP-A (green) with Rab4 labeled with anti-Rab4 antibody or Alexa 594-labeled anti-mouse IgG antibody (red) and unlabeled liposomes at early time points (5-7 min after start of internalization). F: SP-A labeled with FITC-SP-A (green) was not associated with Rab11-positive compartments labeled with anti-Rab11 antibody or Alexa 594-labeled anti-mouse IgG antibody (red) and unlabeled lipid after 30-min internalization. FITC-SP-A (green) label was localized at the cell periphery, whereas the Rab11- or Alexa-labeled compartment (red) was found in perinuclear structures. Sections were made through the center of the cell. A representative sample of 10 cells is shown. In all pictures, yellow indicates areas of overlap between red and green markers.



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Fig. 2.   Late endosomal compartments for SP-A and lipid as visualized by CLSM. A: SP-A did not reach acidified compartments. Type II cells were preincubated with LysoTracker Red (red) for 10 min at 37°C and then incubated with FITC-SP-A (green) and unlabeled lipid for 5 min at 37°C followed by a 10-min chase in label-free DMEM. B: endocytosed lipid was colocalized with acidified compartments at later (>15-min) time points. Type II cells were preincubated with LysoTracker Green for 10 min at 37°C and then incubated with rhodamine-PE-labeled liposomes (red) and unlabeled SP-A for 5 min at 37°C followed by a 10-min chase period in DMEM. C-F: cells were incubated with SP-A and lipid for 5 min at 37°C and then chased in medium for 10 min. They were then washed, fixed, and processed as described in METHODS. C: SP-A labeled with FITC-SP-A (green) and unlabeled lipid did not colocalize with Rab7 labeled with polyclonal rabbit anti-Rab7 antibody or anti-rabbit IgG secondary Alexa 594-labeled antibody (red). D: endocytosed lipid labeled with rhodamine-PE-labeled liposomes (red) and unlabeled SP-A colocalized with Rab7 labeled with polyclonal rabbit anti-Rab7 antibody or anti-rabbit IgG secondary Alexa 488-labeled antibody (green) at late time points. E: SP-A labeled with FITC-SP-A (green) and unlabeled lipid did not colocalize with the lamellar body marker 3C9 labeled with 3C9 monoclonal antibody or anti-mouse IgG secondary Alexa 594-labeled antibody (red) at late time points. F: endocytosed lipid labeled with rhodamine-PE-labeled liposomes (red) and unlabeled SP-A was in lamellar bodies labeled with unlabeled SP-A, 3C9 monoclonal antibody, or anti-mouse IgG secondary Alexa 488-labeled antibody (green) at late time points. Sections were made through the center of the cell. A representative sample of 10 cells is shown. In all pictures, yellow indicates areas of overlap between red and green markers.



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Fig. 3.   In situ localization of FITC-SP-A and rhodamine-PE lipid. FITC-labeled SP-A and rhodamine-PE-labeled lipid were instilled in vivo into rat lungs 7 min before death. Thereafter, the lungs were processed for CLSM as described in METHODS. A: SP-A (green) did not colocalize with 3C9-positive compartments (red) 7 min after internalization (zoom 4). B: in contrast, instilled lipid (red) partially colocalized with 3C9-positive compartments (green; zoom 4).



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Fig. 4.   Uptake of biotinylated SP-A in type II cells is affected by calmodulin and proton pump inhibitors. Type II cells were incubated with 3 µg disulfide-biotinylated SP-A/1 million cells in DMEM-0.1% BSA in the absence and presence of W-7 (200 µM) and bafilomycin A1 (Baf A1; 1 µM) for 10 min at 37°C. Detection of intracellular SP-A was by neutravidin-peroxidase and enhanced chemiluminescence. Results were quantified by densitometric analysis and are means ± SE expressed as relative optical density (OD); n = 3 experiments. * P < 0.0001 vs. control. dagger  P = 0.0371 vs. control.



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Fig. 5.   SP-A-mediated lipid uptake in the presence and absence of calmodulin and proton pump inhibitors. Type II cells were incubated with [3H]dipalmitoylphosphatidylcholine (DPPC)-labeled liposomes and 4 µg SP-A/1 million cells at 3°C (time = 0 min) and then warmed to 37°C for 10 min in the absence and presence of W-7 (200 µM) or bafilomycin A1 (1 µM). Results are means ± SE; n = 3 experiments. * P < 0.0001 vs. control. dagger  P = 0.0011 vs. control.



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Fig. 6.   Influence of calmodulin and proton pump inhibitors on SP-A resecretion. Type II cells were incubated with disulfide-biotinylated SP-A for 10 min at 37°C (time = 0 min). Then the cells were washed by the glutathione or iodoacetamide procedure as described in METHODS and then allowed to resecrete into fresh DMEM-0.1% BSA in the presence and absence of W-7 (200 µM) or bafilomycin A1 (1 µM) for 30 min at 37°C. Intracellular SP-A was detected by enhanced chemiluminescence. Results are means ± SE; n = 3 experiments. * P = 0.034 vs. 0 min.



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Fig. 7.   Influence of W-7 and bafilomycin A1 on lipid resecretion after SP-A-mediated lipid uptake. Type II cells were incubated with [3H]DPPC-labeled liposomes and SP-A in DMEM at 37°C for 10 min (time = 0 min). Thereafter, the cells were then allowed to resecrete into DMEM-0.1% BSA in the absence and presence of W-7 (200 µM) or bafilomycin A1 (1 µM) for 30 min at 37°C. Results are means ± SE; n = 4 experiments. * P = 0.0245 vs. 0 min. dagger  P = 0.0130 vs. 0 min.



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Fig. 8.   Effect of drugs on intracellular surfactant transport processes as visualized with CLSM. A: calmodulin inhibitors inhibit SP-A uptake. Type II cells were preincubated for 10 min with W-7, and then FITC-SP-A (green) was added in the presence of the drug for 10 min. The cells were then washed, fixed, and incubated with anti-EEA1 antibody or Alexa 594-labeled anti-mouse IgG antibody (red). B: decreased colocalization of SP-A and lipid after resecretion. Type II cells were allowed to internalize FITC-SP-A (green) and rhodamine-PE liposomes (red) for 10 min followed by a 30-min chase period in DMEM without SP-A and lipid. C: internalized SP-A did not colocalize with CD63 after resecretion. Type II cells were allowed to internalize FITC-SP-A (green) and unlabeled liposomes (red) for 10 min followed by a 30-min chase period in DMEM without SP-A and lipid. The cells were fixed and incubated with anti-CD63 antibody or Alexa 594-labeled anti-mouse IgG antibody (red) as described in A. D: internalized lipid and SP-A were trapped in early endosomes in W-7-treated cells. Type II cells were allowed to internalize FITC-SP-A (green) and rhodamine-PE-labeled liposomes (red) for 10 min followed by a 30-min chase period in DMEM without SP-A and lipid in the presence of 200 µM W-7. Both surfactant components accumulated in common large structures (yellow), which labeled for EEA1 (data not shown). E: internalized lipid, but not SP-A, was trapped in bafilomycin A1-treated cells. Type II cells were allowed to internalize FITC-SP-A and rhodamine-PE-labeled liposomes for 10 min followed by a 30-min chase period in DMEM without SP-A and lipid in the presence of 1 µM bafilomycin A1. Although lipid accumulated in large irregular structures (red), SP-A (green) was found separately in peripheral small vesicles. F: internalized lipid in bafilomycin A1-treated cells colocalized with early endosomal markers. Type II cells were allowed to internalize unlabeled SP-A and rhodamine-PE-labeled liposomes (red) for 10 min followed by a 30-min chase period in DMEM without SP-A and lipid in the presence of 1 µM bafilomycin A1. The cells were then washed, fixed, and incubated with anti-EEA1 antibodies or anti-mouse IgG Alexa 488-labeled antibody (green). Sections were made through the center of the cell. A representative sample of 10 cells is shown. In all pictures, yellow indicates areas of overlap between red and green markers.



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Fig. 9.   Effect of drugs on intracellular surfactant transport processes in single living type II cells assayed by CLSM combined with a dielectrophoretic field cage (DFC). A: image of a single type II cell trapped within the electrodes of the DFC. Distance between the opposite electrodes was 40 µm. Single type II cell with lipid (red) was internalized via the SP-A-mediated pathway. B: colocalization of internalized SP-A and lipid at early stages. Type II cells were incubated with FITC-SP-A and rhodamine-PE liposomes for 30 min at 3°C as described in METHODS and then introduced into the DFC and allowed to warm up. After 13 min at 26°C, the majority of internalized SP-A (green) and lipid (red) colocalized (yellow). C: intracellular SP-A and lipid were in vesicles. One minute later, i.e., 14 min after the start of incubation at 26°C, FITC-SP-A and rhodamine-PE-labeled lipid began to separate. SP-A and lipid collected in vesicles in close proximity to the cell membrane. D: rapid resecretion of SP-A and some of the internalized lipid. Two minutes later, i.e., after 15 min at 26°C, the amount of intracellular FITC-SP-A had decreased. Some extracellular lipid label can be seen in the top right corner. E: loss of lipid label as vesicles from the cell. Three minutes later at 26°C, the amount of FITC-SP-A still present intracellularly had decreased even more. It was partially dissociated from the internalized lipid (red). An exocytosis figure (lipid; red) can be recognized in the bottom right corner. F: in bafilomycin A1-treated type II cells, lipid was trapped in an intracellular compartment separate from SP-A. After 3 min of uptake of FITC-SP-A and rhodamine-PE-labeled lipid at 37°C in normal medium, cells were washed, incubated in bafilomycin A1 (1 µM)-containing medium, and trapped in the DFC. Resecretion was allowed for 40 min at 26°C. At the end of the resecretion period, small amounts of SP-A (green) are seen in small vesicles distinct from the lipid (red), which is mainly in larger, irregular intracellular vesicular structures. Images were created with the standard ×40 oil-immersion, 1.3-numerical aperture objective (A, zoom 2.7; B-E, zoom 3.4; F, zoom 4). For all pictures, sections were made through the center of the cell. Representative samples of 6 cells are shown.

SP-A Binding Assay

Specific binding of NHS-LC-biotinylated SP-A in the presence and absence of drugs was done with the slope peeling method as previously described (29).

Agonist-Mediated Secretion of Internalized Lipids

Type II cells in DMEM were incubated with [3H]DPPC-labeled liposomes (150 µg lipid/2.5 × 106 cells and /ml) in the presence of SP-A (10 µg/ml) for 10 min at 37°C (see Liposome Internalization Assay). Thereafter, the cells were washed as described in Liposome Internalization Assay. An aliquot (1 ml of cell suspension containing 2.5 million cells) was then taken to determine protein and radioactivity content (time = 0 min; 100% internalization). The cells were then incubated in DMEM without lipid and SP-A for up to 30 min without drugs at 37°C and subsequently incubated in the presence and absence of drugs (10-8 M PMA, 10-4 M ATP, and 2 µM A-23187) for an additional 30 min (time = 60 min). Aliquots were taken at 30 and 60 min to determine protein and radioactivity contents.

CLSM

To visualize intracellular SP-A or lipid, for each time point, 5 × 106 type II cells suspended in 1 ml of DMEM-0.1% BSA were incubated with 20 µg of FITC-SP-A or 100 µg of rhodamine-PE-labeled liposomes in the absence and presence of W-7 (200 µM) or bafilomycin A1 (1 µM) at 37°C. Thereafter, the cells were separated from the medium by centrifugation and washed three times with cold DMEM-5% fatty acid-free BSA-10 mM EGTA and then three times with cold PBS. The cells were then fixed in 1% paraformaldehyde-250 mM HEPES, pH 7.4, for 10 min at 3°C.

In SP-A and lipid resecretion assays, for each time point, four aliquots of 5 million freshly isolated cells suspended in 1 ml of DMEM-0.1% BSA were incubated with 100 µg of liposomal phospholipid containing rhodamine-PE and 5 µg of FITC-SP-A at 37°C. Thereafter, the cells were separated from the medium by centrifugation and washed three times with cold DMEM-5% fatty acid-free BSA-10 mM EGTA and then three times with cold PBS. One portion of the cells was fixed directly after the uptake period as described above. The other portions were incubated in 1 ml of DMEM-0.1% BSA in the presence and absence of drugs at 37°C for resecretion. The cells were then washed and fixed as described above.

For immunocytochemistry, after incubation with FITC-SP-A or rhodamine-PE liposomes, the cells were washed, incubated in PBS-1% BSA, fixed as described above, and permeabilized in 0.02% saponin-PBS-5% BSA on ice for 5 min before incubation with antibodies labeling different subcellular organelles (specificities indicated in RESULTS). The cells were incubated with the antibodies for 1 h at 3°C and washed three times with cold PBS-0.1% BSA. Detection was carried out with a Cy3-, Cy2-, Alexa 499-, or Alexa 594-labeled anti-mouse IgG or anti-goat IgG antibody. The cells were then transferred to fresh tubes and washed twice with cold PBS-0.1% BSA and once with PBS. They were then placed on glass slides and covered with ProLong antifade kit under a glass coverslip.

For double labeling, the cells were fixed in 1% paraformaldehyde-250 mM HEPES, pH 7.4, for 10 min at 3°C followed by a 5-min incubation with 0.02% saponin in PBS-5% BSA on ice before incubation for 1 h on ice with mouse monoclonal EEA1 antibodies diluted 1:100 followed by antibody Cy3-conjugated Fab fragment goat anti-mouse IgG (heavy plus light; diluted 1:200) for 3 h on ice. The next monoclonal antibody, directed against Rab5, was applied (1:250) for 1 h on ice followed by incubation with Cy2-conjugated goat anti-mouse IgG (1:1,000) for 1 h on ice. The cells were then washed, fixed, and mounted as described above.

For in situ localization experiments, 100 µg of FITC-labeled SP-A with 375 µl of unlabeled liposomes or 100 µg of unlabeled SP-A with 375 µl of rhodamine-PE liposomes in 1 ml of PBS were instilled intratracheally in vivo into the lungs of anesthetized rats 7 min before death. The lungs were then perfused with an ice-cold solution containing 154 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1.3 mM MgSO4, 2.6 mM sodium phosphate, 10 mM HEPES, and 6 mM glucose, pH 7.4, until they were blood free. The unfixed freshly removed rat lung tissues were snap-frozen by liquid nitrogen before cryostat sectioning. The sections were postfixed in 5% paraformaldehyde for 10 min at 4°C and then incubated overnight at 4°C with the dilution of the monoclonal antibody 3C9 (diluted 1:50 in PBS-5% BSA), washed as described above, and then incubated for 2 h at room temperature with goat anti-mouse IgG Alexa 594-conjugated or Alexa 488-conjugated antibodies (diluted 1:100 in PBS-5% BSA). The sections were covered with ProLong antifade kit and a glass coverslip.

Subsequent preparation procedures for CLSM are described in Figs. 1, 2, and 7-9. For each time point or assay, four slides were prepared. At least 10 cells on each slide were scored. All experiments were repeated four times. Reproducibility of the internalization events was similar to the results described in previous work (29).

The cells were examined with an epifluorescence microscope interfaced with a confocal laser scanning microscope (Leica Lasertechnik, Heidelberg, Germany) equipped with an argon or krypton laser. Images of cells were created with a standard objective (×40 oil, numerical aperture 1.3) and photomultiplier tubes dedicated to fluorescent excitation and emission spectra for rhodamine, Cy3, and Alexa 594 (excitation 541 nm, emission 572 nm) and FITC, Cy2, and Alexa 488 (excitation 490 nm, 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 at 16 s/frame (512 lines). The FITC, Cy2, and Alexa 488 signals are displayed in Figs. 1-3, 8, and 9 in the pseudocolor green and the rhodamine, Cy3, and Alexa 594 signals in red.

Quantitative and statistical functions were performed with the Quantify Tool-Window, Leica TCS NT version 1.5.451 (Leica Lasertechnik, Heidelberg, Germany). With the Stacks function, xz intensity profiles of marked areas of representative cells were built and the ratio of the two fluorescence intensities (FITC and rhodamine labels) was calculated.

For each data point, cells from four different animals were investigated (10 cells/animal). Reproducibility was similar to previously described experiments (29).

Controls with the cell membrane markers concanavalin A and Maclura pomifera were regularly done to ascertain that the internalization labels were inside the cell boundaries as described in previous work (29, 42).

Dielectric Field Cage Combined With CLSM

Technical specifications of this system are described in detail elsewhere (12, 21, 25). The three-dimensional microelectrode structures were purchased from Evotec BioSystems (Hamburg, Germany). Briefly, the cells in this system were trapped by a dielectric field between the eight electrodes of a field cage. The distance between opposite electrodes was 40 µm. The dielectric field cage (DFC) was energized with a field with a frequency of 1.9 MHz and a root mean square voltage of 2.8. In the cage center, the negative dielectrophoresis held the cell in the middle plane of the microelectrode (see Fig. 8A).

Freshly isolated type II cells preincubated with FITC-SP-A and rhodamine-PE liposomes were introduced into the medium in the presence and absence of bafilomycin A1 (1 µM) at 26°C into the microsystem channel of the field cage with a low-volume injector (Evotec BioSystems) and a Hamilton needle syringe. With a syringe pump (SP 210iw; WPI), the cells were streamed into the channel at 2-10 µl/h (corresponding to a streaming velocity of 50-250 µm/s). When a cell entered the central range of the DFC, the electrical field was switched on to trap the cell. For the duration of the confocal-microscopic observation period, the pump was stopped. With the dual-channel system of the confocal microscope, dual-emission (535/590-nm) images in the equatorial plane were recorded simultaneously with a scanning speed of 4 s/frame at 60-s intervals.

Determination of Protein Content

Protein content was determined by the assay described by Bradford (1) with a Bio-Rad protein assay kit.

Data Analysis

All results are means ± SE. Statistical analysis was done by paired and unpaired t-tests. For multiple comparisons, analysis of variance with subsequent Fisher's protected least significant difference test was used. The level of significance was set at P < 0.05. The number of experiments and significance are indicated in RESULTS as well as in Figs. 1-9.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of Intracellular Endocytic Compartments

Early endosomal compartments. As shown in Fig. 1, in type II cells, antibodies against Rab5 (red), which is involved in transport from the plasma membrane to early endosomes, colocalized extensively with antibodies against the early endosomal marker EEA1 (green) within large peripheral vesicles (Fig. 1A). Antibodies against CD63, a component of late endosomes, lysosomes, and lamellar bodies in type II cells, colocalized with LysoTracker (1 µM), which labels acidified vesicles (results not shown). As reported before, 5 min after the start of internalization, endocytosed FITC-SP-A colocalized with lipid (Fig. 1B). At this time point, it also colocalized with the early endosomal marker EEA1 in large peripheral vesicles as previously described (29). Also, almost all of the internalized SP-A colocalized with Rab5 (Fig. 1D), again arguing for the presence of the SP-A-lipid complex in early endosomes. However, neither SP-A nor lipid at this early time point colocalized with 3C9, an antibody recognizing a component of the limiting membrane of classic lamellar bodies (Fig. 1C). In type II cells, the early endosomal marker EEA1 and the lamellar body marker 3C9 do not colocalize (results not shown). This therefore suggests that the compartment, entered early (<= 5 min) by endocytosed SP-A and lipid, is the early endosome, which is distinct from classic lamellar bodies.

SP-A partially colocalized with compartments positive for Rab4, a GTPase associated with vesicles recycling from the early endosomal compartment to the plasma membrane (Fig. 1E). SP-A did not colocalize with Rab11 at any time point after internalization was tested (5-30 min). As in other cells, Rab11, which is associated with the slower RRC, is typically found in type II cells in a pericentriolar location (Fig. 1F). This suggests that SP-A is recycled via rapidly recycling vesicles and does not pass through the slower recycling so-called RRC.

Late endosomal, lysosomal, and lamellar body compartments. As previously described (29, 42), after 5 min of uptake and 10 min of chase in DMEM, surfactant components did no longer colocalize with EEA1-positive compartments (results not shown). At this time point, in the great majority of cells, SP-A and LysoTracker Red or CD63 did not colocalize (Fig. 2A). In a few cells, at late time points (>15 min after the start of internalization), a very small and variable amount of SP-A could be found colocalized with LysoTracker Red- or CD63-positive compartments. Most of the SP-A was found in vesicles distinct from those labeled by these markers. On the other hand, internalized lipid colocalized extensively with LysoTracker Green at this time point (Fig. 2B). As expected, antibodies against CD63, a component of late endosomes, lysosomes, and lamellar bodies in type II cells, colocalized extensively with LysoTracker (1 µM; results not shown).

Although SP-A at no time point colocalized with Rab7 (Fig. 2C) or with 3C9-positive compartments (Fig. 2E), lipid taken up via the SP-A-mediated pathway after 5 min of uptake and 10 min of chase did colocalize with Rab7 (Fig. 2D) and with 3C9-positive compartments (Fig. 2F).

To verify that our in vitro findings corresponded to the in vivo situation, FITC-labeled SP-A and rhodamine-PE-labeled lipid were instilled in vivo into rat lungs 7 min before snap-freezing of the lung. After in situ instillation with FITC-SP-A and rhodamine-PE liposomes in rat lungs, as shown in Fig. 3, sections of these lungs showed that FITC-SP-A (green) did not colocalize with 3C9-positive compartments (red) of type II cells (Fig. 3A, zoom 4), whereas lipid (red) did colocalize with 3C9-positive compartments (green) in type II cells (Fig. 3B, zoom 4), supporting our in vitro findings.

These results therefore suggest that there is sorting and segregation of SP-A and lipid, most likely at the level of the early endosome. Lipids but not SP-A are then transported toward lamellar bodies.

Effect of Drugs on Type II Cell Metabolism

The effect of selected drugs on intracellular surfactant transport pathways was tested. Viability of the cells was tested by dye exclusion before and after addition of the drugs. Dye was excluded in 96 ± 0.6% of the untreated cells. With W-7 at 200 µM, 96 ± 0.7% of the cells excluded dye vs. 94 ± 1.2% with bafilomycin A1 (1 µM). Lactate dehydrogenase release was <2% in all experiments.

The effect of bafilomycin A1 on intracellular vesicle acidification was verified by fluorescence microscopy after the cells were loaded with acridine orange (1 µM for 10 min at 37°C) in the presence and absence of 250 nM, 500 nM, or 1 µM of bafilomycin A1. In untreated cells, acridine orange accumulated in vesicular compartments as expected, whereas complete loss of vesicle staining was seen in the presence of 1 µM bafilomycin A1 (results not shown), compatible with the reported inhibition of vesicle acidification by bafilomycin A1. This concentration of bafilomycin A1 was therefore used in all subsequent experiments.

SP-A uptake. Type II cells were incubated for 10 min at 37°C in the presence and absence of drugs with disulfide biotin-labeled SP-A (3 µg/106 cells) and liposomes. Then the cells were washed and processed as described in METHODS.

W-7 (200 µM) was included in the medium during internalization inhibited SP-A uptake to 90% (P < 0.0001). Bafilomycin A1 had a less pronounced effect on SP-A uptake (60% inhibition; P = 0.0371; Fig. 4).

SP-A binding. To exclude that the differences found in the previous experiment were due to changes in binding affinity or receptor number, specific high-affinity SP-A binding was calculated with slope peeling after incubation of the cells in the presence and absence of drugs at 37°C for 60 min as previously described (11, 29). Half-maximal binding was 5.4 × 10-10 M in untreated cells vs. 4.9 × 10-10 M in W-7-treated cells and 5.5 × 10-10 M in bafilomycin A1-treated cells. Differences were not significant.

SP-A-mediated lipid uptake. To test the effect of the drugs chosen on lipid uptake, type II cells were incubated for 10 min at 37°C in the presence and absence of drugs and 4 µg SP-A/106 cells as well as of [3H]DPPC-labeled liposomes as described in METHODS. For time = 0 min, the cells were incubated at 3°C with the above-mentioned components and then immediately centrifuged, washed, and processed as described in METHODS.

At time = 0 min, no difference in lipid label association to the cells could be found among all three groups. The values at this time point were therefore pooled and averaged, and the obtained value was subtracted from the values obtained at later time points.

Treatment of the cells with W-7 (200 µM) inhibited SP-A-mediated lipid uptake after 10 min by 80% (P < 0.0001 vs. control), whereas bafilomycin A1 (1 µM) inhibited uptake by 55% (P = 0.0011 vs. control; Fig. 5). Differences between drugs were not significant (n = 3 experiments).

The effect of W-7 on lipid uptake was concentration dependent. At 10 µM, inhibition was 18%, at 50 µM 27% (not significantly different from control cells), and at 200 µM 60% (P = 0.0002 vs. untreated control cells; n = 3 experiments). In the assays described above, the drug used was added to the cells at the same time as the surfactant components.

The effect of pretreatment of the cells with W-7 for 10 min at 37°C before addition of the lipid and SP-A was not significantly different from that found in cells that had not been pretreated (inhibitory effect of the drug on lipid uptake at 10 µM 33%, at 50 µM 48%, and at 200 µM 69%; not significantly different from no pretreatment).

SP-A resecretion. After 10 min of uptake and 30 min of chase in medium under normal conditions, i.e., without added drugs (control), 9 ± 2% of the internalized SP-A label remained intracellularly. This result is consistent with a previously published work (42). With W-7 (200 µM), 64 ± 14% of the label remained in the cells at this time point (P = 0.0034 vs. control cells) vs. 17 ± 4% with bafilomycin A1 (1 µM; not significant; Fig. 6). We therefore conclude that SP-A resecretion is calmodulin dependent. In contrast, inhibition of vesicle acidification had no effect on intracellular trafficking of internalized SP-A.

Lipid resecretion. All drugs used inhibited lipid resecretion. In untreated cells, after 10 min of uptake (time = 0 min) and 30 min of chase in medium, 43 ± 2.9% of the internalized lipid was resecreted (P < 0.0001 vs. time = 0 min); i.e., 57.5 ± 3% of the internalized lipid remained cell associated. After treatment with W-7, 80 ± 4% of the lipid remained in the cells (P = 0.0245 vs. time = 0 min), and with bafilomycin A1, 76.1 ± 1.2% remained intracellularly (P = 0.0130 vs. time = 0 min; Fig. 7).

Morphology. To obtain morphological confirmation of our biochemical assays, CLSM was used.

As seen in Fig. 8A, under the influence of 200 µM W-7, SP-A (green) bound to the cell membrane but was not detected intracellularly. Intracellular EEA1-positive peripheral vesicles (red), corresponding to early endosomes, could easily be detected. Essentially the same results were seen with KN-62 (10 µM), another calmodulin inhibitor, and with bafilomycin A1 (1 µM; results not shown). Lipid uptake was also severely inhibited (results not shown). These results therefore confirmed those obtained with the biochemical assays, which had indicated that calmodulin inhibitors and bafilomycin A1 inhibit SP-A and lipid uptake by type II cells.

The effects of the drugs on resecretion were studied with CLSM by following the changes of the lipid-to-protein fluorescence intensity ratio. In the absence of drugs (control), the ratio of fluorescence intensities of lipid to protein was 1 ± 0.01 after 5 min of internalization of both labels. Lengthening the uptake period did not change the relative fluorescence intensity ratio. After 10 min of uptake, the ratio was 0.9 ± 0.03. After 10 min of resecretion, the ratio had increased to 1.7 ± 0.1. At the same time, the fluorescence intensity of the SP-A had decreased by 55%. In comparison, the fluorescence intensity of the lipid label had only decreased by 25%. After a subsequent 30-min chase period in the absence of drugs, the internalized labeled components did not colocalize at all anymore. At this point in time, the fluorescence intensity ratio of lipid to SP-A had increased to 2.85 ± 0.06. Little FITC-SP-A could still be found within the cells (Fig. 8B) and did not colocalize with CD63-positive compartments (Fig. 8C), in contrast to the lipid label that did colocalize extensively with CD63 (results not shown).

In the presence of drugs added during the resecretion period, significant differences in the distribution and intensity of the label were seen.

In the presence of W-7 (200 nM), the ratio of lipid to SP-A fluorescence after 30 min of resecretion did not change versus the starting point of the resecretion period (0.94 ± 0.13; not significantly different from the end of the uptake period). This correlates with the biochemical assays where a pronounced inhibitory effect of W-7 on both SP-A and lipid resecretion was found.

The morphology of the label-containing vesicles after 30 min of resecretion in the presence of W-7 was different from that in control cells. With W-7, the labels colocalized in large peripheral EEA1-positive vesicles, corresponding to the early endosomal vesicles found at the beginning of the resecretion period (i.e., the end of the uptake period, 5 min after the start of internalization). However, these vesicles were approximately twice the size of the corresponding vesicles in control cells (Fig. 8D).

In contrast to the findings with W-7, after 10 min of chase in the presence of bafilomycin A1 (1 µM), the ratio of lipid to SP-A fluorescence intensity had increased to 2.54 ± 0.2, further increasing to 4.31 ± 0.18 after 30 min of chase, suggesting preferential accumulation of lipid. After 30 min of chase in bafilomycin A1, the lipid label was found in large EEA1-positive vesicles (Fig. 8F) in which the location corresponded to the early endosomal vesicles described in control cells but which were approximately three times the size of the corresponding organelles in control cells. They tended to localize in the central part of the EEA1-positive vesicle population. We could not detect endocytosed lipid in CD63-positive compartments (results not shown). A few small vesicles, positive for SP-A but not for lipid, could be found (Figs. 8E and 9F). These results are compatible with the biochemical assays showing inhibition by bafilomycin A1 of lipid but not of SP-A resecretion.

Imaging of Surfactant Uptake and Resecretion Processes in Living Single Type II Cells Trapped in a Field Cage

By trapping freshly isolated type II cells in an electromagnetic field cage and combining this with CLSM, it was possible to visualize intracellular trafficking and resecretion processes of endocytosed surfactant components by living type II cells. The experimental setup, showing a trapped type II cell between four of the eight electrodes is shown in Fig. 9A. A timed series of images taken from one representative cell is presented in Fig. 9, B-E.

After incubation of the cell at 3°C in medium with rhodamine-PE-labeled lipid and FITC-labeled SP-A, as described in METHODS, the cells were allowed to warm up to 26°C (ambient temperature). Thirteen minutes after the start of the warmup, both labels were predominantly colocalized (Fig. 9B). After 14 min, both labels started to segregate (Fig. 9C) and to be secreted by the cells. Most of the FITC label and a substantial part of the lipid label left the cells in small droplets after 15 min, either by themselves or combined, as seen in Fig. 9D. Note that the dielectric field will tend to trap large particles (>= 1 µm) such as lamellar bodies, which in most of the pictures show up as prominent budlike structures sticking to the outside of the cell membrane. After 16 min, very little intracellular SP-A label remained intracellularly, and the cells contained almost exclusively lipid label (Fig. 9E). Extracellular label that has just left the cell can be recognized as red streaks in Fig. 9, C and D.

Figure 9F shows a representative sample from similar experiments in which the cells were treated with bafilomycin A1 before and during the uptake and resecretion periods. In contrast to normal untreated cells (Fig. 9, B-E), in these cells after initial colocalization and subsequent segregation of the two labels, the lipid label accumulates and remains in large irregular structures throughout the cell. No loss of lipid label (red) could be observed. SP-A (green), on the other hand, segregates into small vesicular structures before leaving the cell, confirming the results of the biochemical assays as well as those with CLSM on fixed cells (see Fig. 8E).

Agonist-Mediated Stimulation of Secretion of Internalized Lipids

In previous work, Wissel et al. (42) had found that type II cells could resecrete endocytosed surfactant lipid components rapidly. They had also found that a considerable amount of lipid remained in the cells after 30 min. They asked whether this remaining lipid was lamellar body associated and whether it could be recruited for secretion or whether this pool was distinct from the lamellar body pool.

To answer these questions, we used two different approaches. First, in CLSM experiments, we looked at various time points after uptake, that is, before and after 30 min of resecretion, and after an additional incubation for 30 min with the secretagogues ATP and PMA, we looked at the association of the endocytosed lipid with the lamellar body marker 3C9. We found that before the start of the resecretion period, most of the endocytosed lipid colocalized with 3C9 (Fig. 2F) and after 30 min of resecretion, colocalization had decreased. Now a considerable fraction of the endocytosed lipid was in 3C9-negative vesicles. This would suggest that a major fraction of the endocytosed lipid, which is not resecreted rapidly, is not lamellar body associated. This was then confirmed by additional biochemical assays. An assay to measure secretagogue-stimulated surfactant secretion was developed. Briefly, after 10 min of uptake of labeled SP-A and lipid, the type II cells in solution were washed and material adhering to the membrane was removed as described earlier (41) (time = 0 min, lipid label = 100%), and then the cells were chased in medium for 30 min at 37°C (time = 30 min). After another wash, the cells were then incubated with drugs for another 30 min (time = 60 min). At all time points, aliquots of the cells and medium were taken for the determination of radioactivity and protein content.

We found that after 30 min of resecretion in the absence of drugs, 46 ± 5.3% of the internalized lipid was resecreted, similar to the results previously reported (42). After 60 min, 46 ± 4.8% of the lipid was resecreted, i.e., 54% of the lipid remained within the cells. After stimulation of the cells with PMA (10-8 M), 60.6 ± 3.1% of the lipid was secreted, corresponding to an additional 14.6 ± 3% of the internalized lipid. In comparison, ATP (10-4 M) caused an additional 10 ± 3.2% secretion and 10.9 ± 4% combined with the calcium ionophore A-23187 (2 µM). The effect of the combination PMA and ATP was additive (19 ± 3%), and the effect of the combination of ATP and A-23137 was slightly less pronounced (18 ± 2%). These results, therefore, again suggest that only a fraction (~20% of the initial amount taken up) of the lipid remaining in the cells can be recruited by secretagogues.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

After secretion, SP-A and surfactant lipids are reinternalized by type II cells (9, 32, 45). In vitro, SP-A and lipid are internalized together via the coated-pit pathway. At early time points, both components reside in the same early endosomal compartments (29). Internalized surfactant components can then be rapidly resecreted by the type II cell (42).

The regulatory mechanisms as well as the pathways and vesicles involved in this resecretion pathway are not well defined. To clarify the intracellular transport routes leading to resecretion of SP-A and lipid, in this study, we combined immunocytochemical and biochemical methods. We used the specific localization of several different molecules to track the pathways of surfactant components through the cell.

Up to now, it has been technically difficult to demonstrate by electron-microscopic techniques that the lipid in the lamellae of the lamellar body are of endocytic origin because lipids tend to be lost during the preparatory fixation steps. For this reason, we opted to use CLSM combined with specific markers for intracellular organelles, in particular, lamellar bodies in vitro and in vivo. In addition, we combined CLSM techniques after in vivo or in vitro marking SP-A and lipid with a DFC setup on living freshly isolated cells, which enabled us to follow in real time the endocytosis, intracellular transport, and secretion of surfactant components.

Overall, the pathways used in early endocytic events of SP-A and lipid seem to be the same as those described for various early systems. As in many other systems, after endocytosis, SP-A-lipid complexes are transported via clathrin-coated vesicles toward early endosomes. In this process, the small GTP binding protein Rab5 (Fig. 1D) as well as its effector EEA1 (Fig. 1A) is involved (20).

In the early endosome, internalized molecules are sorted into different intracellular trafficking routes. Material destined for the recycling pathway is transported via recycling vesicles back to the plasma membrane. The GTPase Rab4 is involved in the regulation of this recycling back from early endosomes to the plasma membrane (27). In a previous paper, Wissel et al. (42) reported that SP-A could be resecreted rapidly within minutes after internalization. In accord, we found that 5 min after internalization, Rab4 already colocalized with internalized SP-A to some extent (Fig. 1E) but not with lipid (results not shown). This therefore suggests that SP-A is recycled to the plasma membrane and/or extracellular space via the fast recycling pathway through recycling vesicles in a fashion similar to that described for the fast recycling of the transferrin receptor (19).

A second recycling pathway for endocytosed receptors with somewhat slower kinetics involves a separate compartment, the so-called RRC. Another GTPase, Rab11, is associated with this compartment (33). Part of the internalized transferrin receptors will recycle via this pathway (19). In type II cells, such a compartment seems to exist in its typical pericentriolar location as suggested in Fig. 1F. However, we could not detect internalized SP-A or lipid colocalizing with Rab11 either after 5 or after 30 min of uptake (Fig. 1F). Therefore, the RRC does not seem to play a role in the recycling of SP-A or lipid.

Although early endocytic events in type II cells thus seem to follow the classic pathways described for most cells, later events somewhat deviate from the general scheme. Transfer of material from early endosomes to the degradative compartment (late endosomes or lysosomes) in most cells involves the dissociation of vesicular elements from the early endosome and subsequent transport toward later compartments. Thereby, these vesicles gradually acquire late endosomal/lysosomal components, likely via fusion with primary lysosomes. Late endosomes have been shown to be associated with lysosomal markers such as CD63 and LAMP-1 as well as with the GTPase Rab7 (8). Also in the course of this transformation, the content of the vesicles acidifies through the action of a vacuolar proton pump.

In type II cells, internalized lipid enters a compartment with late endosomal characteristics as shown by its labeling with LAMP-1 and CD63 (results not shown) as well as with Rab7 (Fig. 2D) and by its acidified interior, labeling strongly with the pH-sensitive LysoTracker dye (Fig. 2B). However, this compartment is not really degradative as shown in previous work from our own group as well as that from other laboratories. Internalized surfactant phospholipids entering these organelles are not degraded but remain essentially unmodified as shown by phospholipid analysis of subcellular fractions (32, 41, 44) as well as of resecreted surfactant (42). This compartment most likely corresponds to lamellar bodies, which are known to possess characteristics of both the de novo synthetic pathway as well as the endocytic or degradative pathway. They contain newly synthesized surfactant lipids as well as SP-B and SP-C and at the same time carry late endosomal or lysosomal markers such as Rab7 (40), LAMP-1, and CD63 (37). Also, they have an acidic interior (4). The compartment identified by us, which contains endocytosed lipid, is positive for Rab7 and CD63 and reacts positively with LysoTracker. In addition, it labels positively with the monoclonal antibody 3C9, which is specific for the limiting membrane of lamellar bodies (47) (Figs. 2F and 3C). Therefore, we feel confident that this compartment indeed corresponds to lamellar bodies and that therefore internalized lipids are transported from early endosomes to lamellar bodies before resecretion.

SP-A, however, follows a different route. A tiny amount of SP-A was found associated with CD63-positive compartments with an acidic LysoTracker-positive interior only in very few cells (Figs. 2A and 8C). This confirms findings from previous biochemical assays by Stevens et al. (29) and Wissel et al. (42) as well as from work by others (16, 46) that there is very little, if any, degradation of SP-A in type II cells, at least at early time points after internalization. Also, SP-A was not found associated with Rab7 (Fig. 2C). Furthermore, SP-A never colocalized with the lamellar body-specific antibody 3C9 (Figs. 2E and 3A). As shown in Figs. 6, 8E, and 9E, its resecretion is not inhibitable by bafilomycin A1, an inhibitor of the vacuolar proton pump, whereas that of lipid is clearly inhibited (Figs. 7, 8E, and 9F), further strengthening our argument that endocytosed SP-A does not enter the classic lamellar body.

An important concern involving the use of an in vitro model such as ours is that intracellular pathways may be different from the in vivo situation. To investigate this possibility, we instilled labeled SP-A and lipids into the lungs of living animals before processing the lungs for confocal microscopy. As shown in Fig. 3, in these lungs, internalized SP-A was found in type II cells in small vesicles distinct from lamellar bodies. In contrast, lipid was found colocalized with lamellar bodies. Although not absolute proof of identity, these findings nevertheless strongly suggest that the intracellular pathways used in our freshly isolated type II cells are not different from those used in vivo.

A major finding in this study is that SP-A and lipid are segregated after uptake. The mechanisms involved in this sorting process are as yet only partially understood. We have identified two factors contributing to this mechanism, calmodulin and intravesicular acidification.

Calmodulin is a calcium-sensor molecule, transducing the calcium signal in different cellular processes. It has been shown to be involved in coated-pit formation (39). The calmodulin inhibitor W-7 causes misassembly of clathrin lattices, an important structural component of coated pits and vesicles, leading to decreased uptake via this pathway. As shown in Figs. 4 and 5 as well as in Fig. 8A, in our system, inhibition of calmodulin by W-7 blocked surfactant uptake at the cell membrane level, which would be compatible with the above-mentioned influence on early endocytosis. Another point of interaction between calmodulin and endocytotic transport is at the early endosomal level. The Rab5 effector molecule EEA1, which is associated with early endosomes, contains a calmodulin-binding motif, which also explains part of the effect of calmodulin on endocytosis and early endosomal transport (20).

In our system, the vacuolar pump inhibitor bafilomycin A1 inhibited uptake. This is compatible with the concept that the activity of these proton pumps, most likely at the endosomal level, is necessary for full function of the internalization pathway as has been suggested in other systems. For example, internalization of Semliki Forest virus, bound to its receptor, occurs via coated vesicles, which then mature to endosomes. Bafilomycin A1 prevents acidification of the endosomes and stops internalization of the virus (22, 23).

It is conceivable that drug treatment could change the affinity or number of binding sites for SP-A on the cell exterior. We have found no evidence for a changed affinity of the binding sites for SP-A on the cell membrane with drug treatment. The estimates of relative dissociation constant values were within the range of published values for SP-A binding to type II cells (29, 31). Also, the critical step, at least for W-7, seems to be the actual internalization event and not the binding to the receptor itself. As seen in Fig. 8A, SP-A did bind to the cell but was unable to enter, even after long incubation times.

In contrast to their similar effect on the uptake process, the two drugs have different effects on the resecretion pathways for both surfactant components followed.

Although approx 90% of the internalized SP-A was resecreted under control conditions within 30 min, after treatment with W-7, only approx 35% of the SP-A was resecreted. On the other hand, with bafilomycin A1, approx 80% had left the cells after 30 min (Fig. 6). Similarly, in normal cells after SP-A-mediated lipid uptake, approx 45% of the internalized lipids was resecreted; i.e., 55% of the internalized lipid remained in the cell. With W-7 added after the internalization period, approx 20% and with bafilomycin A1, approx 25% of the internalized lipids were resecreted (Fig. 7). Therefore, although calmodulin affects resecretion of both SP-A and lipid, bafilomycin A1 treatment affects resecretion of lipid only.

These results were confirmed by our microscopic assays (see RESULTS and Figs. 8, D-F, and 9F). W-7 and bafilomycin A1 inhibited SP-A-mediated lipid resecretion, whereas bafilomycin A1 did not affect SP-A resecretion but arrested the intracellular transport of lipids.

Resecretion is a complex process involving different intracellular steps. Using our markers for intracellular compartments, we identified the organelle from which the drugs used blocked transport. We found that with both drugs, after internalization had occurred, the drugs blocked further transport from EEA1-positive compartments, presumably early endosomes, as shown for bafilomycin A1 in Fig. 8F. The effect seen after inhibition of calmodulin is not unexpected. Calmodulin has been reported to be involved in recycling in several systems. Calmodulin antagonists inhibit IgA receptor recycling in Madin-Darby canine kidney (MDCK) cells (15) as well as transferrin receptor recycling in rat reticulocytes (13) and recycling of the Ricinus toxin to the apical plasma membrane in MDCK cells (17). The results with SP-A and lipid therefore fit with the general role described for calmodulin.

Although inhibition of calmodulin will trap both surfactant components within the early endosome, bafilomycin A1 will not affect the exit of SP-A from this compartment. Our results suggest that bafilomycin A1 blocks the transport from early endosomes to later compartments. It is likely that acidification of the organelle is essential for its function. It could that, as has been shown for other systems, acidification is needed for formation of endosomal carrier vesicles between early and late endosomes (5). Because bafilomycin A1 does not block the exit of SP-A from this compartment, we think it is unlikely that the dissociation itself of SP-A from lipid is pH dependent.

Our results seem in contradiction with previous work from other groups (32, 45), suggesting that endocytosed SP-A reaches lamellar bodies. Several explanations for this discrepancy are possible. Major methodological differences exist between our system and those of other groups, such as the use of freshly isolated versus adherent type II cells, the lack of distinction between endogenous SP-A and endocytosed SP-A in some previous studies, and different labeling techniques and markers for identification of endocytic compartments. Also, in all studies that used subcellular gradients for isolation of lamellar bodies, contamination of the lamellar body fraction by nonlamellar body organelles, such as early endosomes and recycling vesicles, was not excluded.

Young et al. (45) found endocytosed SP-A in what morphologically corresponds to lamellar bodies. In a previous work, Stevens et al. (29) demonstrated the existence of intracellular organelles structurally resembling lamellar bodies, which contain endocytosed SP-A but are negative for LAMP-1 and CD63, lysosomal markers shown to be associated with classic lamellar bodies. This would suggest that there is heterogeneity within the lamellar body compartment, which was identified up to now mostly by structural morphological criteria only (i.e., multilamellar lipid-rich large vesicles). With lamellar body-specific markers becoming available, the issue of whether there are distinct organelles, e.g., along a maturing endocytic pathway within this compartment, can be approached.

A previous work (42) suggested that there could be more than one pathway for SP-A and lipid resecretion. The results of this study add to our understanding of this differential resecretion and would suggest that there is segregation of SP-A and lipid at the early endosomal level, i.e., at or after the common EEA1-positive compartment. Integrating our new results with previous findings into one concept, we would predict that subgroups of lamellar bodies exist, some of which will contain both SP-A and endocytosed lipid but are not yet LAMP-1 positive and at least one other, containing only internalized lipid and no SP-A (anymore), which is LAMP-1, CD63 positive, and labels with 3C9.

In summary, the present study demonstrates that SP-A and lipid uptake are calmodulin and vacuolar proton pump dependent. At early time points after internalization (<5 min), SP-A and lipid colocalize within intracellular vesicles carrying early endosomal markers (EEA1 and Rab5). Within the early endosome, SP-A and lipid dissociate. Ninety percent of the SP-A is rapidly resecreted via a Rab4-associated and calmodulin-sensitive pathway, presumably so-called recycling vesicles. It does not enter classic lamellar bodies. Lipid, in contrast, is transported from the early endosome to late endosomes or lamellar bodies via a calmodulin- and bafilomycin A1-sensitive process.


    ACKNOWLEDGEMENTS

This work was supported by Deutsche Forschungsgemeinschaft Grant Ste 459/4-2 and 4-3 (to P. A. Stevens) and by German Ministry for Research and Technology Grant 01 ZZ 9511.


    FOOTNOTES

Address for reprint requests and other correspondence: P. A. Stevens, Clinic of Neonatology, CCM, Univ. Hospital Charité, Humboldt-Univ. Berlin, Schumannstrasse 20-21, 10098 Berlin, Germany (E-mail: paul_a.stevens{at}charite.de).

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. Section 1734 solely to indicate this fact.

Received 14 November 2000; accepted in final form 14 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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