Surfactant protein D influences surfactant ultrastructure and uptake by alveolar type II cells
Machiko Ikegami,
Cheng-Lun Na,
Thomas R. Korfhagen, and
Jeffrey A. Whitsett
Division of Pulmonary Biology, Cincinnati Childrens Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio
Submitted 15 April 2004
; accepted in final form 10 November 2004
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ABSTRACT
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Surfactant protein D (SP-D) is a member of the collectin family of the innate host defense proteins. In the lung, SP-D is expressed primarily by type II cells. Gene-targeted SP-D-deficient [SP-D(/)] mice have three- to fivefold higher surfactant lipid pool sizes. However, surfactant synthesis and secretion by type II cells and catabolism by alveolar macrophages are normal in SP-D(/) mice. Therefore, we hypothesized that SP-D might regulate surfactant homeostasis by influencing surfactant structure, thereby altering its uptake by type II cells. Large (LA) and small aggregate (SA) surfactant were isolated from bronchoalveolar lavage fluid (BALF) from SP-D(/), wild-type [SP-D(+/+)], and transgenic mice in which SP-D was expressed under conditional control of doxycycline in alveolar type II cells. Uptake of both LA and SA isolated from SP-D(-/-) mice by normal type II cells was decreased. Abnormally dense lipid forms were observed by electron microscopy of LA from SP-D(/) mice. SA from SP-D(/) mice consisted of atypical multilamellated small vesicles. Abnormalities in surfactant uptake by type II cells and in surfactant ultrastructure were corrected by conditional expression of SP-D in vivo. Preincubation of BALF from SP-D(/) mice with SP-D changed surfactant ultrastructure to be similar to that of SP-D(+/+) mice in vitro. The rapid changes in surfactant structure, increased uptake by type II cells, and decreased pool sizes normally occurring in the postnatal period were not seen in SP-D(/) mice. SP-D regulates uptake and catabolism by type II cells and influences the ultrastructure of surfactant in the alveolus.
saturated phosphatidylcholine; collectins; surfactant catabolism
SURFACTANT PROTEIN D (SP-D) is a 43-kDa member of the collectin family of calcium-dependent lectins whose host defense function has been well recognized (4). The critical role of SP-D in pulmonary homeostasis was not apparent until the SP-D gene-targeted mouse was developed (2, 17). Targeted inactivation of the SP-D gene in mice [SP-D(/)] caused two major unexpected abnormalities in the lung: 1) marked accumulation of tissue and alveolar surfactant phospholipids (13) and 2) inflammation and emphysema associated with increased numbers of activated alveolar macrophages (30, 35). Genetic experiments in which SP-D was replaced with mutant SP-D protein demonstrated that phospholipid abnormalities were modulated independently of lung inflammation and emphysema (22, 36, 37). In SP-D(/) mice, the normal reduction in alveolar and lung saturated phosphatidylcholine (Sat PC) pools that occurs from newborns to adults was not observed. Lung Sat PC concentrations were increased three- to fourfold in SP-D(/) mice at all ages (13). Surfactant phospholipids and proteins are synthesized, stored, secreted, and recycled in type II cells (33). Alveolar type II epithelial cells and alveolar macrophages contribute equally to surfactant phospholipid catabolism (6). In healthy individuals, alveolar and lung tissue surfactant pool sizes are tightly regulated. We previously demonstrated that Sat PC synthesis, secretion by alveolar type II cells, and catabolism by alveolar macrophages were normal in SP-D(/) mice (9). Therefore, lipid abnormalities in SP-D(/) mice might relate primarily to changes in surfactant uptake, catabolism, and/or recycling by type II cells. The ultrastructure of surfactant from SP-D(/) mice contained abnormal dense large aggregate (LA) forms (17). We hypothesized that SP-D might regulate surfactant homeostasis in the lung, at least in part by influencing the physical structure of the phospholipid-rich aggregates that, in turn, might influence their uptake and catabolism by type II epithelial cells (31). In the present study, effects of SP-D on surfactant ultrastructure and uptake by type II cells were assessed. Because surfactant pool sizes change during normal development, we also determined the role of SP-D in the modulation of surfactant ultrastructure and pool sizes during the postnatal period.
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MATERIALS AND METHODS
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Mice.
Swiss black wild-type [SP-D(+/+)] and SP-D(/) mice and conditional SP-D mice, a triple transgenic mouse in which rat SP-D is expressed in response to doxycycline (Dox) [Clara cell secretory protein (CCSP)-reverse tetracycline transactivator transcription factor (rtTA+), (tetO)7·rat SP-D+, mouse SP-D(/)], were generated as previously described (17, 38). Breeding colonies are maintained in the vivarium at Cincinnati Childrens Hospital Medical Center according to protocols approved by the Institutional Animal Care and Use Committee. Genotyping was performed by PCR analysis (13). Expression of rat SP-D was induced in triple transgenic mice (conditional SP-D mice) by treatment from embryonic day 4 with Dox-containing food pellets (625 mg/kg; Harlan Tekland, Madison, WI) (23, 29). Expression of SP-D was reversed when the mice were removed from Dox (Off Dox) at 6 wk of age (38). Another group of triple transgenic mice was fed Dox at 6 wk of age (On Dox). LA surfactant and small aggregate (SA) surfactant were isolated from bronchoalveolar lavage fluid (BALF) obtained from these groups of mice.
Isolation of LA and SA.
Mice were anesthetized by intraperitoneal injection of pentobarbital. Mice were exsanguinated after we severed the distal aorta. A 20-gauge blunt needle was tied into the proximal trachea. Bronchoalveolar lavage was performed five times with enough volume (
0.81 ml) of 15 mM NaCl to fully inflate the lung. BALF was centrifuged at 4,000 g over 0.8 M sucrose cushion for 15 min. LA surfactant was collected from the interface, diluted with 15 mM NaCl, and centrifuged again at 40,000 g for 15 min (13). The supernatant from the 40,000 g centrifugation that contained SA surfactant was pelleted with an ultracentrifuge at 100,000 g for 48 h.
Because BALF could not be recovered from newborn mice, their surfactant was isolated from lung minces (1, 19). Surfactant ultrastructure was assessed in the normal lungs from SP-D(+/+) mice at 2, 4, and 21 days of age and from SP-D(/) mice at 14 days (n = 3 pools from 15 mice/pool). Lung tissue was cut into small (
1 mm3) pieces and slowly stirred in 15 mM NaCl with a magnetic stirrer for 20 min at 2°C. The lung tissue was removed from the suspension by pouring through two layers of gauze, and aliquots of the filtered suspension were used for Sat PC and SP-D analyses. LA and SA were isolated from the suspension as described for adult BALF.
Ultrastructure of surfactant.
LA and SA were isolated from three pooled murine BALF samples (n = 5/pool) and processed for electron microscopy using a modified protocol from Palaniyar et al. (22). LA and SA were fixed with 2% paraformaldehyde (EM grade; Electron Microscopy Sciences, Fort Washington, PA), 2% glutaraldehyde (EM grade; Electron Microscopy Sciences), and 0.1% CaCl2 (Sigma, St. Louis, MO) in 0.1 M sodium cacodylate buffer (SCB), pH 7.3, at 4°C for 30 min, followed by postfixation with fresh fixative at 4°C. Pellets obtained from LA and SA were washed with 0.1 M SCB, pH 7.3, incubated with 1% osmium tetroxide (Electron Microscopy Sciences) and 1.5% potassium ferrocyanide (Sigma) in 0.1 M SCB at room temperature for 2 h, and stained en bloc with 4% aqueous uranyl acetate (Electron Microscopy Sciences) at 4°C overnight. They were dehydrated through a series of graded alcohol solutions and embedded with EMbed 812 resin (Electron Microscopy Sciences). To minimize bias, the samples were coded so that the investigator was unaware of study groups. Ultrathin sections sampled from duplicated LA and SA blocks were examined with or without uranyl acetate and lead citrate poststains using a JEOL 1230 transmission electron microscope (JEOL USA, Peabody, MA). Electron micrographs of LA and SA were photographed using Kodak 4489 EM films (Kodak, Rochester, NY) or acquired digitally using an AMT Advantage Plus 2kx2k TEM charge-coupled device camera (AMT, Danvers, MA).
Sizes (diameter and surface area) of lamellated structures in LA from conditional SP-D mice, SP-D(/) BALF incubated with SP-D in vitro, and normal SP-D(+/+) mice (newborn to adulthood) were determined. The ultrathin sections (120-nm thick) were cut from duplicated epon blocks (n = 4) from each group and transferred to 200 mesh copper grids. Five to ten electron micrographs were randomly taken, one section per block. Each electron micrograph was coded according to the groups and digitally acquired at a final magnification of x15,000. Measurements of surface area and diameter of the lamellated structure were determined using the area and Ferets diameter routines in Image J for OS X (a public domain image analysis program developed by Wayne Rasband at the National Institutes of Health).
SP determination.
The content of SP-A, SP-B, SP-C, and SP-D in BALF was analyzed by Western blot. Sat PC was measured as previously described (13). Samples containing 1.25 nmol of Sat PC were used for SP-A and SP-D, 0.22 nmol of Sat PC were used for analysis of SP-B, and 0.65 nmol of Sat PC were used for SP-C. Antisera were from Chemicon (Temecula, CA), and immunoreactive bands were quantitated by densitometric analyses as described previously (13). For LA surfactant from newborn and adult SP-D(+/+) mice, samples containing 0.4 nmol of Sat PC were used for Western blot analysis of SP-A and SP-D.
Preincubation of mouse SP-D with BALF from SP-D(/) mice.
Double transgenic mice lacking both SP-A and granulocyte/macrophage colony-stimulated factor (GM-CSF) [SP-A(/), GM-CSF(/)] were developed in our laboratory. Surfactant phospholipid pool sizes and SP-D were increased approximately fivefold and lacked SP-A, facilitating purification of mouse SP-D. Mice were anesthetized with pentobarbital injection. The neck was shaved followed by sterilization of the surgical area with iodine and 70% isopropyl alcohol before trachea tube insertion. SP-D was isolated from BALF from SP-A(/), GM-CSF(/) mice using a maltosyl/agarose (Sigma) column (28) with sterile procedures used throughout the procedure. Endotoxin concentrations in the resultant SP-D preparations were not detectable by Limulus amebocyte lysate assay (Sigma). The purity of SP-D was confirmed by SDS electrophoresis with silver staining and Western blot.
BALF was recovered from SP-D(/) mice using buffer containing 10 mM Tris·HCl/2.5 mM CaCl2/1 mM MgSO4/0.1 mM EDTA/15 mM NaCl, pH 7.0. Mouse SP-D (1.5% of total phospholipid) was added to BALF from SP-D(/) mice and incubated at 37°C for 2 h with slow rotation. LA and SA were isolated as described above, and ultrastructure was determined.
Uptake of surfactant by murine type II cells.
Type II cells were isolated from SP-D(+/+) and SP-D(/) mice and cultured as previously reported (27). Briefly, after lungs were perfused with 1020 ml of sterile 15 mM NaCl via the pulmonary artery, dispase (Collaborative Research, Bedford, MA) was rapidly instilled into the lungs followed by 1% low-melt agarose warmed to 45°C. Lungs were immediately cooled with ice for 2 min and incubated in 1 ml of dispase for 45 min at room temperature. Lungs were subsequently transferred to a culture dish containing 100 U/ml of DNase I (Sigma). The tissue was gently teased from the bronchi. Cell suspensions were filtered and collected by centrifugation and placed on prewashed 100-mm tissue culture plates that had been coated with CD45 and CD32 antibodies (Pharmingen, San Diego, CA). After incubation for 2 h at 37°C in 5% CO2, combined type II cells from four mice were resuspended and seeded into 12 wells. Type II cells were cultured on Matrigel matrix in bronchial epithelial cell growth media containing 5% charcoal-stripped FBS and 10 ng/ml of keratinocyte growth factor. The purity of type II cell preparations was >90% as assessed by modified Papanicolaous stain (5). Under this condition, it was recently shown that after 3 days in culture, murine type II cells actively synthesize and secrete surfactant for at least 7 days (27).
The rate of LA or SA uptake by type II cells was studied after 56 days in culture. LA and SA containing 15 nmol of phospholipid were labeled by mixing with 4.8 µg/well of N-rhodamine dipalmitoylphosphatidylethanolamine (Avanti Polar Lipids, Alabaster, AL) (8, 34). Fluorescently labeled LA and SA isolated from transgenic mice were added to the cultured type II cells from SP-D(+/+) mice and SP-D(/) mice. After a 0.5-, 2-, and 4.5-h incubation, cells were carefully washed five times with PBS and extracted with methanol containing 1 mg/ml of dipalmitoylphosphatidylcholine from the wells, and fluorescence measured.
Assessment of direct effects of SP-D on surfactant uptake.
To see whether SP-D directly stimulated surfactant uptake with type II epithelial cells, mouse SP-D (100 ng) or media were added to type II cells from SP-D(/) mice. After 1 h of incubation, SP-D was removed by washing five times with PBS. Uptake of fluorescently labeled SA from SP-D(/) mice was then determined as described above.
Data analysis.
Results are given as means ± SE. Two group comparisons were carried out by unpaired Students t-tests. Comparisons among groups were made by ANOVA with Tukeys tests used for post hoc analyses. Significance was accepted at P < 0.05.
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RESULTS
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Ultrastructure of LA and SA from SP-D(/) and SP-D(+/+) mice.
Ultrastructure of LA and SA isolated from SP-D(+/+)and SP-D(/) mice was determined by electron microscopy (Fig. 1, AD). Lamellated lipid structures in LA from normal [SP-D(+/+)] mice are shown. The LA fraction from SP-D(/) mice contained abnormally dense lipid forms (Fig. 1B). The structure of SA was also abnormal, consisting of atypical multilamellated small vesicles (Fig. 1D). The lamellated vesicles in SA from SP-D(/) mice were approximately one-third the size of lamellar bodies seen in normal LA.

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Fig. 1. Ultrastructure of large aggregate (LA) and small aggregate (SA) isolated from surfactant protein D-deficient [SP-D(/)] and SP-D(+/+) mice. A: LA from normal SP-D(+/+) mice. Normal lamellar bodies are shown (filled arrowheads). B: LA from SP-D(/) mice contained abnormal large and dense lipid forms (open arrowheads). C: SA in SP-D(+/+) mice. D: SA in SP-D(/) mice was also abnormal, consisting of atypical multilamellated small vesicles (arrows). The lamellated vesicles in SA from SP-D(/) mice were approximately one-third the size of lamellar bodies seen in normal SP-D(+/+) mice LA. Photomicrographs are representative of samples pooled from 5 mice of each genotype.
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Effects of SP-D on surfactant structure in vivo.
In the absence of Dox, surfactant ultrastructure from the conditional SP-D(/) mice was similar to that in SP-D(/) mice (Fig. 1, B and D and Fig. 2, A and C). Surfactant structures in the conditional SP-D(/) mice were changed toward that seen in SP-D(+/+) mice by day 14 of Dox treatment (Fig. 2B). Likewise, the small lamellated vesicles typical of SA surfactant from SP-D(/) mice decreased during continued treatment with Dox (Fig. 2D). The ultrastructure of LA and SA surfactant from conditional SP-D(/) mice that were continuously treated with Dox from embryonic day 4 to 6 wk of age was similar to that isolated from SP-D(+/+) mice (Fig. 3, A and C). When Dox was discontinued at 6 wk of age, abnormally dense LA forms typical of SP-D deficiency reappeared after 14 days (Fig. 3, B and D). After removal from Dox for 14 days (Fig. 3D), the ultrastructure of SA had changed to that typical of SP-D(/) mice (Fig. 1D). Thus the removal of SP-D influenced ultrastructure of LA and SA forms in adult mice.

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Fig. 2. Reversible effects of SP-D on surfactant ultrastructure in vivo. Conditional SP-D mice were fed doxycycline (On Dox) at 6 wk of age to induce expression of rat SP-D. Surfactant in SP-D(/) mice before treatment with Dox is shown in A and C; surfactant after treatment with Dox is shown in B and D. In the absence of Dox, LA and SA surfactant ultrastructures were similar to that in SP-D(/) mice (A and C). Fourteen days (d) after exposure to Dox, the lamellar and tubular myelin structures in LA were changed toward that of SP-D(+/+) mice (B). The abundance of lamellated small vesicles (arrows in C) decreased in SA during continued treatment with Dox (D). Abnormalities in surfactant ultrastructure were changed after 14 days of Dox (B and D). A normal lamellar body in LA is shown (filled arrowhead in B). Abnormally large and dense lipid forms are shown by open arrowheads in A.
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Fig. 3. Changes in surfactant ultrastructure following conditional expression of SP-D. Expression of rat SP-D was induced in conditional SP-D(/) mice lung by continuous treatment with Dox from embryonic day 4. Mice were removed from Dox (Off Dox) at 6 wk of age. During continuous treatment with Dox (A and C), ultrastructures of LA and SA were similar to that in SP-D(+/+) mice. Normal lamellar bodies in LA are shown by filled arrowheads (A). Fourteen days after removal of Dox, abnormally dense lipid forms (open arrowheads) were observed in LA (B) while tubular myelin structures persisted. Abnormally small lamellated vesicles (arrow) were observed in SA (D) after removal of Dox. Thus ultrastructural abnormalities in LA and SA were partially corrected by conditional expression of SP-D in vivo.
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SP-D reverses surfactant ultrastructure in vitro.
The ultrastructure of LA and SA forms was assessed after preincubation of purified SP-D with BALF from SP-D(/) mice (Fig. 4). Addition of mouse SP-D in vitro changed the abnormal large-size surfactant lipid lamellar structures typical of LA from SP-D(/) mice to normal-appearing lamellar bodies (Fig. 4B). Likewise, the structure of SA was partially corrected by incubation with SP-D (Fig. 4D).

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Fig. 4. Preincubation with SP-D reverses surfactant ultrastructure in vitro. Mouse SP-D (1.5% of total phospholipid) was added to bronchoalveolar lavage fluid (BALF) from SP-D(/) mice and incubated at 37°C for 2 h. LA and SA were then isolated. Addition of SP-D to BALF from SP-D(/) mice [SP-D(/)+SP-D] changed the ultrastructure of surfactant from SP-D(/) mice in vitro (B, D). Addition of buffer lacking SP-D to BALF from SP-D(/) mice did not alter the ultrastructure of the surfactant (A, C). Data are representative of 3 separate experiments. Open arrowhead (A) marks abnormally dense lipid forms of LA. Normal lamellar bodies in LA are shown by filled arrowheads (B). Abnormal, small lamellated vesicles typical of SA are shown by the arrow in C.
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SP-D influences the size of lamellated structures in LA in vivo and in vitro.
The surface area and diameter of lamellar bodies in LA were analyzed before and after On or Off Dox. BALF from SP-D(/) mice was incubated with exogenous SP-D (Fig. 5). Surface area and diameter of lamellar bodies were decreased by replacement of SP-D [from 014 days On Dox group in vivo (Fig. 5, A and B)]. Addition of SP-D to BALF from SP-D(/) mice in vitro also corrected lamellar body sizes (Fig. 5, C and D). Consistent with these findings, lamellar body size increased when Dox treatment was discontinued for 14 days (Fig. 5, A and B).

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Fig. 5. Quantitative analysis of lamellar body size. The sizes of lamellated structures (LB) in LA were determined. Surface area (A) and diameter (B) of LB were increased from 0 days (n = 63) before Dox treatment was discontinued to 14 days Off Dox (n = 122). In contrast, LB size was decreased from 0 days (n = 106), no Dox treatment to 14 days On Dox (n = 146). Addition of exogenous SP-D to BALF from SP-D(/) decreased lamellar body size (n = 120) (C and D). The decreased size (surface area and diameter) of LB by the presence of SP-D was demonstrated in vivo and in vitro. LA from normal newborn [2 and 4 days of age SP-D(+/+)] mice contained larger LB compared with normal adult mice (E and F). In normal newborn lungs, LB size decreased with age. AD: *P < 0.001 vs. 0 days; n = number of LB analyzed. E and F: *P < 0.001 vs. 2 and 4 days.
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Association of SP-D with SA.
SP-D was not detectable in LA from SP-D(+/+) mice by Western blot. In contrast, high levels of SP-D were present in the isolated SA pellet recovered after 48 h of centrifugation at 100,000 g (Fig. 6). In previous studies, 56% of total Sat PC in BALF was demonstrated to be associated with SA in SP-D(+/+) mice (13). Therefore, the mean SP-D level relative to Sat PC in isolated SA was 60% higher than that in BALF from SP-D(+/+) mice. Most of the SP-D in BALF was associated with SA, and the SP-D recovered in SA from BALF from SP-D(+/+) mice was similar to that in total BALF. SA contained none or a trace amount of SP-A, SP-B, and SP-C (32), whereas almost all the SP-D in BALF was associated with SA.

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Fig. 6. Quantitation of SP-D following conditional expression. Top: representative Western blot. BALF SP-D levels (n = 3/group) are normalized to those for SP-D(+/+) mice (value of 1) shown at bottom. BALF SP-D was similar in SP-D(+/+) mice and in conditional transgenic mice after 21 days On Dox. SA is SP-D in small aggregate surfactant.
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SP levels in conditional SP-D mice.
The content of SP-A, SP-B, and SP-C was estimated in BALF from conditional SP-D(/) mice by Western blot (Fig. 7). In the absence of Dox, SP-D was undetectable and increased dramatically after treatment with Dox. After 21 days On Dox, SP-D levels in BALF were similar to those in BALF from SP-D(+/+) mice (Fig. 6). SP-B and SP-C levels were increased twofold in the SP-D(/) mice as previously observed (13). SP-B and SP-C content was restored when the mice were treated with Dox. SP-A content was not influenced by Dox, thus correction of SA ultrastructure was mediated by changes in SP-D rather than SP-A. When adult conditional SP-D(/) mice were removed from Dox, SP-D content decreased (Fig. 8). SP-A decreased and SP-B significantly increased after removal of Dox. Thus effects of SP-D on SP concentrations were reversed when the conditional SP-D(/) mice were removed from Dox.

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Fig. 7. SP levels after conditional expression of SP-D in vivo. SP levels were expressed relative to mice exposed to Dox from 0 to 21 days. SP-D was not detectable in the absence of Dox (0 days). Dox induced SP-D from 7 to 21 days after treatment. SP-A levels were not altered by Dox. SP-B and SP-C increased 2-fold in SP-D(/) mice compared with SP-D(+/+) mice. Dox normalized SP-B and SP-C levels. *P < 0.05 vs. 0 days.
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Fig. 8. Reversibility of changes in SPs after removal from Dox. SPs are expressed relative to controls (0 days). SP-D decreased following discontinuance of Dox. Likewise, SP-A decreased after discontinuing Dox. Levels of SP-B and SP-C increased to near normal levels. *P < 0.01 vs. 0 days.
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Surfactant LA and SA uptake by type II cells.
Uptake of LA and SA surfactant increased with time of incubation (Fig. 9). The uptake of SA surfactant was generally greater than that of LA forms. Surfactant uptake was consistently influenced by the origin of the surfactant. In type II cells from SP-D(/) and SP-D(+/+) mice, uptake of LA and SA surfactant isolated from SP-D(/) mice was significantly less than that of surfactant from SP-D(+/+) mice. Differences in the uptake of surfactant between type II cells from SP-D(+/+) mice and SP-D(/) mice were detected. The uptake of normal SA by SP-D(+/+) type II cells was greater than by SP-D(/) type II cells. In contrast, uptake of normal LA was greater in SP-D(/) type II cells. Thus rates of surfactant uptake are influenced by the SP-D genotype from which the type II cells were isolated.

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Fig. 9. Decreased uptake of LA and SA from SP-D(/) mice by type II cells in vitro. Fractional uptake of LA and SA by type II cells was determined by fluorescence intensity and expressed relative to the total label added to the culture wells as described in MATERIALS AND METHODS. Fractional uptake of LA and SA isolated from SP-D(/) mice by wild-type and SP-D(/) mice type II cells was significantly less than LA and SA isolated from SP-D(+/+) mice. N = 4 separate studies/group. *P < 0.05 vs. SP-D(+/+) mice. tP < 0.05 vs. SP-D(+/+) type II cells.
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The uptake of fluorescently labeled surfactant by type II cells, not associated on the cell surface, was confirmed morphologically by fluorescent microscopy as reported in our previous studies (7). Representative fluorescence micrographs of cultured SP-D(+/+) type II cells after incubation with fluorescently labeled SA are shown in Fig. 10. Uptake of fluorescently labeled normal SA from SP-D(+/+) mice was greater than that of SA from SP-D(/) mice.

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Fig. 10. Representative fluorescence micrographs of type II cells incubated with SA from SP-D(+/+) mice (A and B) and SP-D(/) mice (C and D) for 4.5 h. The quantitative results are shown in Fig. 7. Fluorescence-labeled surfactant was not localized on the cell surface and was detected within type II cells. Increased fluorescence with SA from SP-D(+/+) mice was detected in the type II cells compared with SA from SP-D(/) mice. Micrographs are representative of 3 separate experiments/group.
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Effects of SP-D on surfactant uptake by type II cells.
Because SA surfactant was preferably taken up by type II cells, the uptake of SA isolated from conditional SP-D(/) mice was studied. Before treatment with Dox and 14 days after removal from Dox, uptake of SA isolated from conditional SP-D(/) mice was similar to that in SP-D(/) mice (Fig. 11). Fourteen days after treatment with Dox, uptake of surfactant by type II cells from SP-D(+/+) mice was increased more than twofold. Thus conditional replacement of SP-D in vivo restored surfactant ultrastructure and its uptake by type II cells.

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Fig. 11. Conditional expression of SP-D reverses uptake of SA by type II cells. SA was isolated from SP-D(/) mice conditionally expressing SP-D. Uptake was assessed in type II cells from SP-D(+/+) mice in vitro. Uptake was significantly less in mice lacking SP-D (14 days Off Dox and before treatment with Dox for 0 days On Dox). Abnormalities in SA uptake were corrected by expression of SP-D (0 days Off Dox and 14 days On Dox). *P < 0.05 vs. 0 days. N = 4 separate experiments/group.
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SP-D does not directly alter surfactant uptake.
To test whether SP-D directly interacts with type II cells to alter surfactant uptake, mouse SP-D was added to cultured type II cells from SP-D(/) mice and incubated for 1 h. SP-D was removed by washing, and uptake of SA surfactant from SP-D(/) mice was assessed in wild-type type II cells. The percentage of fluorescently labeled SA taken up and recovered in type II cells was not altered by preincubation with SP-D. After a 4.5-h incubation with SA, uptake was 2.8 ± 0.2% (means ± SE) with preincubation with SP-D and uptake was 2.9 ± 0.3% without preincubation (P > 0.5, n = 3 separate experiments/group). Thus SP-D did not directly interact, whether via signaling or intracellular routing, with type II epithelial cells to alter uptake of the abnormal SA from SP-D(/) mice.
Surfactant ultrastructure and uptake by type II cells in normal newborn mice.
Ultrastructure of LA surfactant for SP-D(+/+) mice was assessed at 2, 4, and 21 days of age. Lamellar bodies in LA surfactant were larger in newborns than in adults. The enlarged lamellated structures seen in LA from 2- and 4-day-old mice (Fig. 12, A and B) were similar to those seen in adult SP-D(/) mice (Fig. 1, B and D). Surfactant ultrastructure was similar to that in adults by 21 days of age (Fig. 12E). Lamellated structures were seen in SA from normal newborn mice (Fig. 12, C and D, arrows). The surface area and diameter of lamellar bodies in LA from newborn SP-D(+/+) mice were larger. Lamellar body size decreased to the adult size by 21 days of age (Fig. 5, E and F).

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Fig. 12. Developmental changes in surfactant ultrastructure. LA from normal neonatal mice at postnatal day 2 (A) and postnatal day 4 (B) consisted of dense large lipid forms (open arrowheads), whereas SA consisted primarily of lamellated structures (C and D, arrows). Surfactant ultrastructure changed with age and was similar to that seen in normal adults by 21 days of age (E). Ultrastructure of surfactant isolated from SP-D(/) mice was similar at all ages. LA structures from SP-D(/) mice at 14 days of age are shown (F). Thus SP-D influences the normal changes in surfactant ultrastructure that occurs following birth. Filled arrowheads in E, normal lamellar bodies in LA; open arrowheads in F, abnormally dense lipid forms in LA.
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The larger lamellar body size characteristic of newborn mice was not associated with decreased concentrations of SP-A or SP-D. SP-D and SP-A levels, relative to Sat PC, were higher in newborn than in adult lung (Fig. 13A). As shown in Fig. 12, ultrastructure of surfactant was similar in newborn SP-D(+/+) and SP-D(/) mice. Uptake of newborn SA by type II cells was decreased compared with that from normal adults (Fig. 13B). This low surfactant uptake by type II cells was associated with higher surfactant pool size seen in normal newborn lungs (13). In SP-D(/) mice, the normal developmental change in surfactant ultrastructure and uptake by type II cells do not occur. Thus SP-D is required for the normal postnatal changes in surfactant ultrastructure and metabolism that occurs following birth.

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Fig. 13. A: levels of SP-A and SP-D were determined relative to saturated phosphatidylcholine (Sat PC) by Western blot analysis in BALF at 2 days of age (lanes 1 and 2), 4 days of age (lanes 3 and 4), and in normal adult (lanes 5, 6, and 7) mice. SP-A and SP-D levels in BALF from normal neonatal mice were higher than in adult mice. Although the ultrastructure of LA from neonatal mice was similar to that seen in SP-D(/) mice, SP-D levels relative to Sat PC were higher in the neonate than in adult mice. B: uptake of surfactant by type II cells was determined. Uptake of SA from newborn mice by wild-type type II cells was significantly decreased compared with that from adult SP-D(+/+) mice. Uptake of LA by type II cells was less than SA and similar between adult and newborn surfactant. Uptake of newborn surfactant and adult surfactant was similar. *P < 0.05 vs. adult surfactant; n = 4 separate experiments/group.
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DISCUSSION
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Our previous metabolic and genetic studies showed that SP-D regulates surfactant pool size by influencing surfactant catabolism in type II epithelial cells rather than in alveolar macrophages (13). In SP-D(/) mice, surfactant phospholipid synthesis, secretion by type II cells, and catabolism of alveolar macrophages were normal (9, 13). The present study demonstrates that uptake of surfactant by type II cells is closely associated with changes in structure of LA and SA surfactant that are dependent on SP-D. Effects of surfactant structure, stoichiometry, and their uptake by type II cells were reversible in vivo. Preincubation of BALF with SP-D changed the structure of SP-D(/) surfactant toward that which is typical of SP-D(+/+) surfactant. Preincubation of type II cells with SP-D did not change surfactant uptake. Thus SP-D plays a critical role in maintaining normal surfactant ultrastructure in adult mice that in turn influences surfactant homeostasis. Together, surfactant is present in distinct physical forms that determine, at least in part, its homeostasis in the lung.
The present study demonstrated the strong influence of SP-D on surfactant ultrastructure. Preincubation of BALF from SP-D(/) mice with SP-D restored surfactant ultrastructure. The mechanisms by which SP-D affects surfactant structure are unclear but may be dependent on the interaction of SP-D with phosphatidylinositol and fatty acids (21, 24). Addition of SP-D to phospholipid vesicles containing phosphatidylinositol and SP-B reconstituted highly ordered tubular arrays in vitro (25).
Alveolar type II cells and alveolar macrophages equally contribute to surfactant phospholipid catabolism (6). Surfactant taken up by type II cells were either recycled or catabolized, whereas all surfactant taken up by macrophage were catabolized. In the gene-targeted mouse models of GM-CSF-deficient mice (11), GM common receptor
-chain-deficient mice (26), and acid sphingomyelinase-deficient mice lung (12), surfactant Sat PC was increased four- to eightfold in the lung. Defects in surfactant pool sizes in these mice were caused by defects in surfactant catabolism by alveolar macrophages. Although the levels of accumulation of surfactant phospholipids in the lungs of those models were similar to that seen in SP-D(/) mice, the mechanisms by which surfactant pools were increased are distinct. Rates of surfactant uptake and catabolism by alveolar macrophages were normal in SP-D(/) mice (9). Whereas surfactant uptake by alveolar macrophages was not dependent on physical forms of surfactant or the presence of SPs (20), its uptake by type II cells was more selective. In studies using electron-dense tracers of different molecular sizes and charges, including native (anionic) ferritin, cationic ferritin, 70-kDa dextran, and colloidal carbon, type II cells only took up cationic ferritin (31). Endocytosis of particles by alveolar type II cells was selective and affected by charge and size (18, 31).
SP-A levels are consistently decreased in SP-D(/) mice (13, 17) and conditional SP-D mice following discontinuance of Dox. Whereas SP-A played a critical role in the formation of tubular myelin, surfactant metabolism was normal in SP-A(/) mice (16), and changes in SP-A do not account for changes in ultrastructure or uptake seen in SP-D(/) mice. Restoration of SP-D in the conditional SP-D(/) mice did not normalize levels of SP-A after 21 days on Dox, a time when surfactant ultrastructure and uptake were fully corrected. The present and previous (38) studies demonstrated that the replacement of SP-D in vivo normalized Sat PC levels within 14 days.
Surfactant pool sizes are highest in newborn animals (9, 15) and decrease dramatically with advancing age to the normal adult level. Mechanisms accounting for the postnatal adjustment of surfactant pool sizes are not known. Slower clearance and longer half-life of secreted surfactant in newborn lung compared with adult lung have been shown previously in rabbit and sheep in vivo (10, 14). Lower uptake of newborn surfactant by type II cells demonstrated in the present studies may contribute to slower surfactant clearance and higher surfactant pool sizes in the newborn lung. SP-D levels are higher in newborn mice than in adult mice, perhaps indicating a role for SP-D in developmental adjustment of surfactant uptake. The structural forms of surfactant also change in the postnatal period (3). Paradoxically, ultrastructure of surfactant from newborns is similar to that from SP-D(/) mice. The ultrastructure of surfactant rapidly changes postnatally and occurs in temporal association with decreased surfactant pool size. Neither surfactant pool size nor surfactant ultrastructure matures in SP-D(/) mice, but both are rapidly normalized by replacement of SP-D in adult mice, demonstrating that the normal maturation of surfactant pool sizes and ultrastructure are dependent on SP-D.
In summary, SP-D determines the normal ultrastructure of surfactant aggregates in the alveoli, influencing uptake of surfactant by type II cells that in turn determine pulmonary surfactant pool sizes.
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GRANTS
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-63329.
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FOOTNOTES
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Address for reprint requests and other correspondence: M. Ikegami, Cincinnati Childrens Hospital, Division of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: machiko.ikegami{at}cchmc.org)
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.
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