Surfactant metabolism in SP-D gene-targeted mice

Machiko Ikegami1, Jeffrey A. Whitsett1, Alan Jobe1, Gary Ross1, James Fisher2, and Thomas Korfhagen1

1 Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039; and 2 Pulmonary Section/Critical Care, Denver Health Medical Center, University of Colorado Health Sciences Center, Denver, Colorado 80204-4507


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mice with surfactant protein (SP)-D deficiency have three to four times more surfactant lipids in air spaces and lung tissue than control mice. We measured multiple aspects of surfactant metabolism and function to identify abnormalities resulting from SP-D deficiency. Relative to saturated phosphatidylcholine (Sat PC), SP-A and SP-C were decreased in the alveolar surfactant and the large-aggregate surfactant fraction. Although large-aggregate surfactant from SP-D gene-targeted [(-/-)] mice converted to small-aggregate surfactant more rapidly, surface tension values were comparable to values for surfactant from SP-D wild-type [(+/+)] mice. 125I-SP-D was cleared with a half-life of 7 h from SP-D(-/-) mice vs. 13 h in SP-D(+/+) mice. Although initial incorporation and secretion rates for [3H]palmitic acid and [14C]choline into Sat PC were similar, the labeled Sat PC was lost from the lungs of SP-D(+/+) mice more rapidly than from SP-D(-/-) mice. Clearance rates of intratracheal [3H]dipalmitoylphosphatidylcholine were used to estimate net clearances of Sat PC, which were approximately threefold higher for alveolar and total lung Sat PC in SP-D(-/-) mice than in SP-D(+/+) mice. SP-D deficiency results in multiple abnormalities in surfactant forms and metabolism that cannot be attributed to a single mechanism.

dipalmitoylphosphatidylcholine; surfactant protein A; surfactant protein B; surfactant protein C; surfactant protein D


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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SURFACTANT PROTEIN (SP) D is a 43-kDa member of the collectin family of proteins that shares structural homology with SP-A and mannose binding protein (3). In contrast to SP-A, SP-D does not interact with the major surfactant phospholipids and is not associated with lamellar bodies or tubular myelin. Based on in vitro studies, SP-D was thought to be primarily a host defense protein, and there was no direct evidence that SP-D contributed to surfactant function or homeostasis (19). SP-D gene-targeted [(-/-)] mice survive and breed normally under laboratory conditions (2, 18); however, their lungs have marked increases in tissue and alveolar surfactant phospholipids. SP-D(-/-) mice also have increased numbers of enlarged foamy alveolar macrophages, changes in the structure of extracellular surfactant, enlarged type II cells with increased lamellar body number and size, and enlarged distal air spaces.

The pool sizes of surfactant lipids and proteins are regulated by the net contributions from synthesis, secretion, uptake and catabolism by macrophages, and reuptake by type II cells that recycle or catabolize surfactant components (35). The factors that influence alveolar and tissue pools of surfactant components are poorly understood. The important role of granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling was demonstrated in GM-CSF- and common beta -chain receptor-deficient mice that had an alveolar proteinosis characterized by increases in surfactant lipids and proteins, with a selective increase in SP-D (26). Likewise, increased expression of interleukin-4 (IL-4) caused increased surfactant lipid and SP-A, SP-B, and SP-C content in transgenic mice. The largest change in the IL-4 mice was a 90-fold increase in SP-D (10). In contrast, surfactant lipids were markedly increased in the lungs of SP-D(-/-) mice without substantial changes in the amounts of SPs relative to those in wild-type mice (18). To better define the role of SP-D in surfactant homeostasis, we measured SP-A, SP-B, and SP-C, evaluated surfactant forms, and characterized the metabolism of Sat PC and SP-D in SP-D wild-type [(+/+)] and SP-D(-/-) mice in vivo.


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
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Mice. NIH Swiss black SP-D(+/+) and SP-D(-/-) mice were generated as previously described (18) and carried as breeding colonies in the vivarium at Children's Hospital (Cincinnati, OH) according to protocols approved by the Institutional Animal Care and Use Committee. Genotyping was performed by DNA blot analysis. All of the studies were performed with 7- to 9-wk-old mice except as indicated for selected experiments. Processing of tissue and biochemical determinations in SP-D(+/+) and SP-D(-/-) mice were performed simultaneously.

Sat PC pool sizes. Total lung Sat PC pools (calculated as µmol/kg body wt) were measured in 7 SP-D(+/+) and 11 SP-D(-/-) mice fetuses 16.5 days after conception. Measurements of Sat PC pool sizes were also taken in 27 SP-D(+/+) and 35 SP-D(-/-) mice from 4 litters/genotype at 2 days of age and in separate groups of 8 mice aged 7-140 days. Sat PC in alveolar lavage fluid and lung tissue (after alveolar lavage) was measured at 21, 56, and 140 days of age. Each mouse was deeply anesthetized with intraperitoneal pentobarbital sodium, and the distal aorta was cut to exsanguinate the animal (11). After the chest was opened, a 20-gauge blunt needle was tied into the trachea, and 0.9% NaCl was flushed into the airway until the lungs were fully expanded. The fluid was then withdrawn by syringe three times for each aliquot. The saline lavage was repeated five times and the samples were pooled, and the volume was measured. The lavaged lung tissue was homogenized in 0.9% NaCl. Aliquots of alveolar lavage fluid and the lung homogenates were extracted with chloroform-methanol (2:1), and Sat PC was isolated with the technique of Mason et al. (20). The amount of Sat PC was measured by phosphorus assay (1).

SP pool sizes. The SPs in fluid from the alveolar washes and large-aggregate surfactant were analyzed by Western blot in six mice from each genotype. Samples containing 1.35 nmol of Sat PC were used for analysis of SP-A, and samples containing 2.7 nmol of Sat PC were used for analysis of SP-C. Proteins were separated by SDS-PAGE in the presence of beta -mercaptoethanol. For SP-B analysis, aliquots containing 0.27 nmol of Sat PC were electrophoresed under nonreducing conditions. SP-A was separated on 8-16% acrylamide gels with Tris-glycine buffer. SP-B and SP-C samples were separated on 10-20% acrylamide gels with Tricine buffer (Novex, San Diego, CA). After electrophoresis, proteins were transferred to nitrocellulose paper (Schleicher & Schuell, Keene, NH) for SP-A or to polyvinylidene difluoride paper (Bio-Rad, Hercules, CA) for SP-B and SP-C. Immunoblot analysis was carried out with the following dilutions of antisera: 1:25,000 for guinea pig anti-rat SP-A; 1:10,000 for rabbit anti-bovine SP-B; and 1:25,000 for rabbit anti-recombinant human SP-C (21, 30, 34). Appropriate peroxidase-conjugated secondary antibodies were used at 1:10,000 dilutions. Immunoreactive bands were detected with enhanced chemiluminescence (ECL) reagents (Amersham, Chicago, IL). Protein bands were quantitated by densitometric analyses with Alpha Imager 2000 documentation and analysis software (Alpha Innotech, San Leandro, CA).

Precursor incorporation into Sat PC and secretion. SP-D(+/+) and SP-D(-/-) mice were given intraperitoneal injections of 8 µl saline/g body wt containing 0.5 µCi [3H]palmitic acid/g body wt or 0.3 µCi [14C]choline chloride/g body wt (American Radiolabeled Chemicals, St. Louis, MO) (11). The palmitic acid was stabilized in solution with 5% human serum albumin (17). Groups of six to nine mice were killed at preselected times, and alveolar lavage fluid was recovered from each animal. Lung tissue was placed in 0.9% NaCl. Sat PC was isolated from the alveolar lavage fluid and homogenized lung tissue as described in Sat PC pool sizes, and radioactivity was measured. The percent secretion of labeled Sat PC was calculated as the percent of radioactivity in alveolar Sat PC relative to the total radioactivity in the alveolar lavage fluid plus lung tissue.

Clearance of dipalmitoylphosphatidylcholine. Mice were anesthetized with methoxyflurane and orally intubated with a 25-gauge animal feeding needle. Each mouse received 60 µl of saline containing 0.5 µCi of [3H]palmitoyl-labeled dipalmitoylphosphatidylcholine (DPPC; American Radiolabeled Chemicals), 1.5 µg of DPPC, and 3.3 µg of lipid-extracted sheep surfactant suspended by the use of glass beads (11). The phospholipids given by intratracheal injection were 2% of the alveolar pool size for SP-D(+/+) mice and 0.5% of the alveolar pool size for SP-D(-/-) mice. Groups of four to six SP-D(+/+) and SP-D(-/-) mice were killed 10 min and 3, 8, 16, 24, 48, and 72 h after intratracheal injection, and 3H was measured in the lipid extracts and cell pellets of the alveolar lavage fluid and in the lung tissue. To recover the cell pellets, alveolar lavage fluid was layered over a 0.8 M sucrose in 0.9% NaCl cushion and centrifuged at 500 g for 15 min. More than 90% of the cell pellets were macrophages.

Clearance of SP-D. Mice deficient for both GM-CSF and SP-A accumulate large amounts of SP-D in their air spaces, providing a source for the isolation of SP-D that is not contaminated with SP-A. Mouse SP-D was isolated from alveolar lavage fluid of the GM-CSF- and SP-A-deficient mice by the methods described by Persson et al. (22), with the use of an affinity column of mannose-Sepharose 6B in the presence of Ca2+. Mouse SP-D was iodinated with the Bolton-Hunter reagent (Amersham Life Science, Arlington Heights, IL) with the same techniques that were used for SP-A (11). 125I-SP-D was injected intratracheally as described in Clearance of dipalmitoylphosphatidylcholine. Groups of five to seven SP-D(+/+) and SP-D(-/-) mice were killed 0.5, 2, 8, 24, and 48 h after injection, and 125I was measured in the alveolar lavage fluid and lung tissues.

Aggregate forms of alveolar surfactant. Large- and small-aggregate surfactants were isolated from the alveolar lavage fluid of eight SP-D(+/+) and six SP-D(-/-) mice by centrifugation at 40,000 g for 15 min over a 0.8 M sucrose in 0.9% NaCl cushion (13). The large-aggregate surfactant was collected from the 0.8 M sucrose interface. The supernatant contained small-aggregate surfactant. The amount of Sat PC was measured for both large- and small-aggregate surfactant, and the percent of Sat PC in large-aggregate surfactant in the total alveolar Sat PC was calculated. The Sat PC was also measured in the cell pellets at the bottom of the tubes under the sucrose step gradient.

Conversion of large-aggregate to small-aggregate surfactant. Large-aggregate surfactant (0.1 µmol of Sat PC) was mixed with 2 ml of Tris buffer in a capped 12 × 75-mm polystyrene tube, and surface area cycling was performed as previously described (7, 12). Briefly, tubes were attached to the disk of Rototorque rotators (Cole-Parmer Instruments, Chicago, IL) in a 37°C incubator. Five tubes each for SP-D(+/+) and SP-D(-/-) mice were rotated for 6 h at 40 rotations/min to change the surface area from 1.1 to 9.0 cm2 two times per cycle. One tube for each group was incubated for the same time period without rotation. The large- and small-aggregate surfactants were isolated by centrifugation at 40,000 g for 15 min. The large-aggregate surfactant in the pellet and the residual small-aggregate surfactant in the supernatants were measured by phosphorus assay (1).

Surface activity. The surface properties of three pools (3 mice/pool) of large-aggregate surfactant isolated from SP-D(+/+) and SP-D(-/-) mice were measured with the captive bubble surfactometer (31). Aliquots of the surfactant pools were organic solvent extracted, and Sat PC concentrations were measured as described in Sat PC pool sizes. The concentration of each pool was adjusted to 3 µmol Sat PC/µl, and 3 µl of the native surfactant were applied to the air interface of a 25-µl bubble. The surface tension was measured every second for 5 s and at 10, 20, 30, and 60 s, and bubble pulsation was started. The minimum surface tensions after 65% volume reductions of the bubbles were measured for 5th and 10th pulsations.

Data analysis. All values are given as means ± SE. Differences between the two groups were determined by a two-tailed Student's t-test. The between-group comparisons were made by analysis of variance followed by the Student-Newman-Keuls multiple comparison procedure. Curves were fit with the use of linear regression.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Sat PC pool sizes. Sat PC (µmol Sat PC/kg) was similar in lungs from fetal (16.5 days after conception; term = 18 days) SP-D(-/-) and SP-D(+/+) mice (Fig. 1A). In SP-D(+/+) mice, Sat PC decreased with advancing age after birth. In contrast, Sat PC content did not decrease with age in SP-D(-/-) mice. The amount of Sat PC in lungs from SP-D(-/-) mice increased three- to fourfold compared with SP-D(+/+) mice after 21 days of age. Alveolar Sat PC pool sizes were measured in SP-D(+/+) and SP-D(-/-) mice at 21, 56, and 140 days of age (Fig. 1B), and alveolar Sat PC was increased approximately fourfold in SP-D(-/-) mice compared with levels in SP-D(+/+) mice.


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Fig. 1.   A: total lung saturated phosphatidylcholine (Sat PC) pool sizes for surfactant protein (SP)-D wild-type [(+/+)] and SP-D gene-targeted [(-/-)] fetal mice 16.5 days postconception and from mice at indicated ages. Total lung Sat PC pools for the SP-D(-/-) group did not decrease with age as is normal for mice. B: alveolar Sat PC pool sizes in SP-D(-/-) mice lungs were increased ~4-fold over those from SP-D(+/+) mice. * P < 0.001 vs. SP-D(+/+) mice.

SP pool sizes. The amounts of the SPs in the fluid from the alveolar washes from SP-D(-/-) mice were estimated by Western blot (Fig. 2A). In SP-D(-/-) mice, SP-A content was decreased to 12% of normal values. SP-B content was increased twofold, and SP-C was unchanged. Because alveolar Sat PC was increased 2.3-fold in the SP-D(-/-) mice used for these measurements, the contents of the SPs are also expressed relative to Sat PC (Fig. 2B). In SP-D(-/-) mice, the amount of SP-A was decreased to 6 ± 3% of the normal value, the amount of SP-B was unchanged, and there was 52 ± 7% of the normal amount of SP-C relative to Sat PC.


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Fig. 2.   A: pool sizes for SP-A, SP-B, and SP-C in alveolar lavage fluid estimated by Western blot and normalized to the quantity of each protein (which was given the value of 1.0) in fluid from alveolar washes done in SP-D(+/+) mice. In SP-D(-/-) mice, SP-A decreased to 12%, SP-B was increased 2-fold, and SP-C was unchanged. B: amounts of SPs (which were normalized to a value of 1.0) in alveolar lavage fluid relative to the amount of Sat PC in the fluid of SP-D(+/+) mice. The SP-D(-/-) mice had 2.3 times more Sat PC in the alveolar lavage fluid than did the SP-D(+/+) mice. Relative to Sat PC, SP-A was decreased, SP-B was unchanged, and SP-C was decreased in SP-D(-/-) mice. * P < 0.05 vs. SP-D(+/+) mice. ** P < 0.01 vs. SP-D(+/+) mice.

Precursor incorporation into Sat PC. [3H]palmitic acid labeling of Sat PC was measured for 72 h after precursor injection into SP-D(+/+) and SP-D(-/-) mice (Fig. 3). Three and eight hours after administration of the radiolabel, [3H]palmitic acid incorporation into Sat PC was similar in SP-D(-/-) and SP-D(+/+) mice. These time points best indicate net incorporation of the precursor into Sat PC. As anticipated, the amount of radiolabeled Sat PC decreased to 58% of the average of the 3- and 8-h values in the lungs of SP-D(+/+) mice by 72 h, indicating the anticipated catabolism of the labeled Sat PC synthesized by the lung. There was only a 36% decrease in labeled Sat PC in total lungs in SP-D(-/-) mice between the average of the 3- and 8-h values and the 72-h value, demonstrating a slower turnover of labeled Sat PC and suggesting less catabolism of endogenously synthesized Sat PC in SP-D(-/-) mice.


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Fig. 3.   [3H]palmitic acid labeling of lung Sat PC for the total lung (A) and for alveolar lavage fluid (B). CPM, counts/min. At 3 and 8 h after radiolabeling, the net incorporations were similar for SP-D(-/-) and SP-D(+/+) mice. Endogenously synthesized Sat PC was lost from the lungs of SP-D(+/+) but not from the lungs of SP-D(-/-) mice. Radiolabeled Sat PC decreased in alveolar lavage fluid from SP-D(+/+) but not from SP-D(-/-) mice. * P < 0.05 vs. SP-D(+/+) mice.

Labeling of Sat PC also was measured for 72 h after administration of [14C]choline (Fig. 4). [14C]Choline incorporation into lung Sat PC was similar in SP-D(-/-) and SP-D(+/+) mice 3, 8, and 16 h after precursor injection. Choline-labeled Sat PC decreased significantly after 16 h in SP-D(+/+) mice as anticipated. However, in SP-D(-/-) mice, the amount of choline-labeled Sat PC increased by 60% from 16 to 72 h. This latter result indicates that radiolabeled choline entered the lung Sat PC pool over many hours and in excess of any catabolic activity that may have occurred. A more efficient recycling of choline in SP-D(-/-) mice could also contribute to the increased choline labeling.


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Fig. 4.   [14C]choline labeling of Sat PC in the total lung (A) and in alveolar lavage fluid (B). The net incorporations at 3, 8, and 16 h are similar in total lung for SP-D(-/-) and SP-D(+/+) mice. The amounts of radioactive Sat PC increased in alveolar and total lung Sat PC in SP-D(-/-) mice and decreased in SP-D(+/+) mice. * P < 0.05 vs. SP-D(+/+) mice.

The percentages of radiolabeled Sat PC secreted to the air spaces and recovered by alveolar washes were very similar for the first 16 h in SP-D(+/+) and SP-D(-/-) mice (Fig. 5). The absolute values for percent secretion were higher for choline than for palmitic acid labels. Subsequently, the amount of choline- or palmitic acid-labeled Sat PC in alveolar lavage fluid decreased for SP-D(+/+) mice as anticipated (Figs. 3B and 4B). In contrast, the amount of labeled Sat PC in fluid from alveolar washes did not decrease in the SP-D(-/-) mice. By 72 h, ~23% of the estimated amount of palmitic acid incorporated into Sat PC (an average of the 3- and 8-h values in total lung Sat [3H]PC activity) was recovered by lavage. The amount of choline-labeled Sat PC in alveolar lavage fluid at 72 h was 34% of the total lung choline-labeled Sat PC at 72 h.


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Fig. 5.   The percent secretion of endogenously labeled Sat PC (A: [3H]palmitic acid; B: [14C]choline) was similar for SP-D(-/-) and SP-D(+/+) mice. Percent secretion was calculated as radiolabeled Sat PC in alveolar lavage fluid divided by (radiolabeled Sat PC in alveolar lavage fluid plus lung tissue) multiplied by 100.

Clearance of DPPC. Percent recoveries of labeled DPPC after intratracheal injection are shown in Fig. 6 for alveolar lavage fluid, alveolar cell pellets (primarily macrophages), lung tissues after lavage, and total lungs (the sums of all the fractions). There was exponential loss of labeled Sat PC from the alveolar washes, with a lower recovery of 0.7% at 40 h in SP-D(+/+) mice compared with the 7.7% recovery measured for SP-D(-/-) mice. However, because alveolar pool sizes were fourfold larger in SP-D(-/-) than in SP-D(+/+) mice, net losses were 0.2 µmol/h for SP-D(+/+) and 0.6 µmol/h for SP-D(-/-) mice. The cell pellet, containing primarily macrophages, initially contained ~5% of the labeled Sat PC for both strains of mice, and the percent decreased exponentially, with more rapid loss in SP-D(+/+) than in SP-D(-/-) mice. However, the ratio of macrophage-labeled to alveolar-labeled Sat PC recovered was constant at ~0.08, demonstrating no accumulation of labeled Sat PC in macrophages. The percent of the radiolabel associated with the lung tissue after lavage was remarkably similar for the SP-D(+/+) and SP-D(-/-) mice. The exponential loss of labeled Sat PC from the total lungs measured net catabolic activity within the lungs. Although the loss of labeled Sat PC was somewhat slower in SP-D(-/-) mice, the increased Sat PC pool sizes in these animals resulted in net losses of 0.6 µmol/h for SP-D(+/+) and 1.9 µmol/h for SP-D(-/-) mice.


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Fig. 6.   Clearances of labeled dipalmitoylphosphatidylcholine (DPPC) given with a trace amount of surfactant by intratracheal injection. Recovery of the radiolabeled DPPC was measured in alveolar lavage fluid (A), tissue after alveolar wash (B), the macrophage fraction from alveolar lavage fluid (C), and total lung (D), which were the sums of all the fractions. Curves were fit by linear regression. * P < 0.05 vs. SP-D(+/+) mice. ** P < 0.01 vs. SP-D(+/+) mice.

Clearance of SP-D. The clearance of mouse 125I-SP-D from alveolar lavage fluid and total lungs of SP-D(-/-) mice was slower than in their SP-D(+/+) counterparts (Fig. 7). By 48 h, 21.7% of the labeled SP-D was recovered from the SP-D(-/-) lungs, a value higher than the 11.8% recovered from SP-D(+/+) mice (P < 0.01). The half-life of 17 h for SP-D in the total lungs of SP-D(-/-) mice was somewhat longer than the half-life of 13 h for SP-D(+/+) mice.


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Fig. 7.   Percent recovery of 125I-SP-D given by intratracheal injection to groups of 5-7 SP-D(+/+) and SP-D(-/-) mice. A: loss of radiolabeled SP-D from total lungs was exponential with time and less rapid in SP-D(-/-) mice. B: alveolar clearances were slower in the SP-D(-/-) mice than in SP-D(+/+) mice at 24 and 48 h. * P < 0.05 vs. SP-D(+/+) mice.

To assess whether 125I-SP-D associated with large-aggregate surfactant after intratracheal administration, the percent recovery of 125I-SP-D in large-aggregate surfactant, supernatant, and cell pellets was measured 48 h after intratracheal injection of 125I-SP-D in SP-D(+/+) mice. 125I-SP-D was found primarily in supernatant (84 ± 6%), and less was associated with the cell pellet (5 ± 3%) or the large-aggregate surfactant (11 ± 5%).

Surfactant aggregates and conversion rates. The percent Sat PC in large-aggregate form relative to the total Sat PC in alveolar lavage fluid was significantly lower in lungs from SP-D(-/-) mice than in those from SP-D(+/+) mice (Fig. 8A). Because the Sat PC alveolar pool sizes were three- to fourfold higher in SP-D(-/-) than in SP-D(+/+) mice, the amount of Sat PC in large-aggregate surfactant of SP-D(-/-) mice was increased about twofold compared with that in SP-D(+/+) animals. The cell pellets from the alveolar washes recovered after centrifugation over 0.8 M sucrose contained 1.0 ± 0.2% of the alveolar Sat PC in SP-D(+/+) and 1.2 ± 0.3% of the Sat PC in SP-D(-/-) mice. Although the amounts were small, the pellets from lavages of SP-D(-/-) mice contained about four times more Sat PC than did the pellets of SP-D(+/+) mice.


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Fig. 8.   Surfactant aggregate sizes. A: the percent large-aggregate surfactant in alveolar lavage fluid was lower for SP-D(-/-) than for SP-D(+/+) mice. B: the percent Sat PC that remained as large-aggregate surfactant after 6 h of surface area cycling was lower for large-aggregate surfactant from SP-D(-/-) than that from SP-D(+/+) mice. C: amount of SPs relative to the amount of Sat PC in large-aggregate surfactant is normalized to a value of 1 for SP-D(+/+) mice. There was less SP-A and SP-C in large-aggregate surfactant from SP-D(-/-) than in that from SP-D(+/+) mice. * P < 0.05 vs. SP-D(+/+) mice. ** P < 0.01 vs. SP-D(+/+) mice.

The large aggregates from SP-D(-/-) mice converted with surface area cycling to small-aggregate surfactant faster than did the surfactant from SP-D(+/+) mice. Eighty-nine percent of the large-aggregate surfactant from SP-D(-/-) mice converted to small-aggregate forms after 6 h of surface area cycling (Fig. 8B). The large aggregates were not converted to small aggregates (>96% recovery) when the tubes were kept at 37°C for 6 h without cycling. The relative amounts of SPs were estimated in large-aggregate surfactants by Western blot (Fig. 8C). The concentrations of SP-A and SP-C were markedly decreased in large-aggregate surfactant from SP-D(-/-) mice. There were no differences in concentration of SP-B relative to Sat PC.

Surface activity. Large-aggregate surfactants from SP-D(+/+) and SP-D(-/-) mice reached equilibrium surface tension within 10 s. The equilibrium surface tension was 21.6 ± 0.30 mN/m (n = 3 pools of surfactant) for SP-D(+/+) mice and 22.8 ± 0.2 mN/m (n = 3 pools) for SP-D(-/-) mice. The minimum surface tensions on the 5th pulsation were 4.9 ± 2.6 mN/m for surfactant from SP-D(+/+)and 4.0 ± 1.0 mN/m for surfactant from SP-D(-/-) mice. Minimum surface tensions for the 10th pulsations were similar to those for the 5th pulsations. Therefore, surfactants isolated from both SP-D(-/-) and SP-D(+/+) mice were highly surface active. The lower concentrations of SP-A and SP-C did not alter the surface activity of the surfactant isolated from SP-D(-/-) mice.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We found a number of changes in alveolar surfactant that may contribute to the altered homeostasis in SP-D(-/-) mice. The age-independent three- to fourfold increase in both alveolar and total lung Sat PC is associated with an increased relative amount of small-aggregate surfactant. The large-aggregate surfactant fraction converts more rapidly to small-aggregate surfactant when the surface area is cycled. The ratios of SP-A and SP-C to Sat PC are decreased, and the ratio of SP-B to Sat PC is not changed in the large-aggregate surfactant of SP-D(-/-) relative to that in SP-D(+/+) control mice. Lower SP-A might result in increased small-aggregate surfactant, which is the form preferentially taken up by type II cells (9). We interpret the metabolic data based on the assumption that precursor pools for SP-D(+/+) and SP-D(-/-) mice are the same. The incorporation of palmitic acid and choline into Sat PC and the initial secretion of endogenously synthesized radiolabeled Sat PC in SP-D(+/+) and SP-D(-/-) mice are similar, indicating comparable synthetic and secretory rates for Sat PC. There is less alveolar clearance and degradation of the endogenously synthesized radiolabeled Sat PC. The biological half-lives of exogenously administered DPPC and SP-D are longer in SP-D(-/-) mice than in their SP-D(+/+) counterparts; however, the pool sizes are larger, resulting in a net increased catabolism of Sat PC.

These results extend the descriptions of the first two reports (2, 18) of separately generated SP-D-deficient mice with the use of comparable transgenic technologies. The only substantive difference between the mouse described by Botas et al. (2) and the mouse that was reported by Korfhagen et al. (18) was the content of SPs in alveolar lavage fluid. They (18) found a modest decrease in SP-A mRNA and decreased total SP-A in fluid from alveolar washes. Botas et al. (2) reported a 2.4-fold increase in SP-A, a 6-fold increase in SP-B, and no change in mRNAs for the SPs in animals of a similar age of 8 wk. Therefore, we further evaluated the amounts of the SPs in alveolar lavage fluid and related those amounts to the amount of Sat PC. Relative to Sat PC, SP-A in lavage fluid of SP-D(-/-) mice was only 6% of the normal amount, SP-B was present in normal amounts, and SP-C was decreased by 50%. This analysis emphasizes the ratio of the SPs to Sat PC because surfactant function is presumably dependent on the SP-to-lipid ratios (25, 32). Because the small-aggregate surfactant does not contain SPs in appreciable amounts and the small-aggregate pool is increased in SP-D(-/-) mice relative to that in SP-D(+/+) mice, we further evaluated the ratio of SPs to Sat PC in the large-aggregate surfactant. The large-aggregate surfactant is the biophysically active fraction of surfactant and the presumed precursor pool for the surface film (36). In large-aggregate surfactant, the SP-A- and SP-C-to-Sat PC ratios are 25 and 30%, respectively, of the control values in SP-D(-/-) mice. The ratio is unchanged for SP-B.

The structure of the large-aggregate surfactant from SP-D(-/-) mice was previously described as having enlarged and dense phospholipid arrays with less tubular myelin compared with that from SP-D(+/+) mice (18). These structures are similar to those described for SP-A(-/-) mice except that those mice have no tubular myelin (12). Although SP-D can cause the formation of tubular myelin-like structures with surfactant lipids in vitro (24), the abnormal structures probably result from the low SP-A-to-Sat PC ratios. The functional consequences are an increased rate of conversion from large-aggregate to small-aggregate surfactant with surface area cycling. Conversion rates increased in surfactant that lacked SP-A (12), and conversion rates decreased when excess SP-A was added to rabbit surfactant (8). Therefore, it is reasonable to attribute the increased conversion from large-aggregate to small-aggregate surfactant to the very low SP-A content of large-aggregate surfactant from SP-D(-/-) mice. Despite the low SP-A- and SP-C-to-Sat PC ratios, surface activity of large-aggregate surfactant isolated from SP-D(-/-) mice was similar to surfactant from normal SP-D(+/+) mice. The normal SP-B-to-Sat PC ratio in large-aggregate surfactant from SP-D(-/-) mice was sufficient for normal surface activity. SP-C is not required for surface activity if SP-B is present in normal amounts (29).

Our studies of the metabolism of Sat PC demonstrate very complex alterations in SP-D(-/-) mice. On the anabolic side of the equation, type II cell numbers are probably similar in SP-D(-/-) and SP-D(+/+) mice, although formal morphometrics have not been performed. The mice that we studied at ~8 wk of age do not have much of the emphysema that develops markedly after 12 wk of age (33). Incorporation of the precursors palmitic acid and choline into lung Sat PC over the initial hours after precursor administration was similar for SP-D(-/-) and SP-D(+/+) animals. If it is assumed that precursor pools are similar, this measurement is the in vivo equivalent of comparable synthetic rates for Sat PC. Similarly, the initial percent secretion of the newly synthesized radiolabeled Sat PC is comparable for the mice despite the description of enlarged lamellar bodies in some type II cells in SP-D(-/-) mice (2). The incorporation and secretion data for the first 16 h after precursor administration indicate comparable anabolism in SP-D(+/+) and SP-D(-/-) mice.

As anticipated, palmitic acid-labeled Sat PC is lost from the alveolar lavage fluid and total lungs, indicating catabolism of endogenously synthesized Sat PC in SP-D(+/+) mice. This result has been replicated by multiple measurements (10, 13). In contrast, palmitic acid-labeled Sat PC continues to accumulate in alveolar washes in SP-D(-/-) mice. There also is less loss of palmitic acid from the lungs of SP-D(-/-) mice. This abnormality is magnified with the choline label, where choline labeling of Sat PC in both alveolar lavage fluid and total lungs continues to increase for up to 72 h. The mice were given the precursor by intraperitoneal injection because we found more uniform labeling between animals injected in this fashion than those measured with intravascular precursor injections (13), and intraperitoneal labeling was used previously for similar metabolic studies in mice by Gross et al. (6). However, intraperitoneal labeling will result in less rapid clearance of the intravascular label and preferential labeling of the liver. Some label will enter the lungs at later times, and the stability and pool sizes of choline precursors in the lung are not known. In wild-type mice, catabolism of the initially synthesized Sat PC is in excess of any new label entering the lungs, and net catabolic activity is measured. If it is assumed that new label entering the lungs is comparable in SP-D(+/+) and SP-D(-/-) mice, the conclusion is that there is less catabolism of endogenously synthesized Sat PC in SP-D(-/-) than in SP-D(+/+) mice because labeled Sat PC continues to accumulate. The persistence of the radiolabel in Sat PC suggests that endogenously synthesized Sat PC is degraded more slowly in SP-D(-/-) mice than in their SP-D(+/+) counterparts. The caveat is that the total lung Sat PC pool size is three to four times higher in SP-D(-/-) than in SP-D(+/+) mice. The larger pool sizes may promote an increased catabolic rate by mass action. If this endogenously synthesized pool mixes uniformly with the total pool, the net catabolic rate (in µmol Sat PC/kg) may not be very different.

Alveolar clearance and catabolism also were measured after intratracheal injection of radiolabeled DPPC. These measurements avoided the problem of the radiolabel entering the lungs at late times but depended on the tracers mixing with the alveolar pool (14). In normal animals, metabolic variables estimated by precursor measurements and from intratracheal injections of labeled DPPC are similar (15). Alveolar clearance of DPPC was slower in SP-D(-/-) mice than in SP-D(+/+) mice, with biological half-life values of 5.1 and 2.8 h, respectively. If it is assumed that the clearance of the tracer and of the total alveolar pool are the same, discounting recycling, and noting that the alveolar Sat PC pool size is three to four times larger in SP-D(-/-) mice, the amount of Sat PC cleared would be 0.2 µmol/h for SP-D(+/+) and 0.6 µmol/h for SP-D(-/-) mice. Therefore, the SP-D(-/-) mice clear more Sat PC per unit time than SP-D(+/+) mice. Loss of the radiolabel to macrophages is a relatively constant percentage of the alveolar pool, demonstrating no accumulation of DPPC in macrophages. This labeling pattern was described previously in rabbits and provides no information about the catabolic activity of the macrophages (28). Alveolar clearance would appear delayed if a high percentage of the DPPC were recycled. The recycling efficiency in wild-type mice was estimated to be ~50% (6).

Total lung catabolic activity can be estimated by the loss of DPPC from the lungs. Intact DPPC does not leave the lungs in appreciable amounts (23). The biological half-life of 15 h for SP-D(-/-) mice was somewhat longer than the 9 h measured for SP-D(+/+) mice. However, the three- to fourfold larger Sat PC pool sizes in SP-D(-/-) compared with SP-D(+/+) mice resulted in catabolic rates of 1.9 and 0.6 µmol/h, respectively, demonstrating threefold higher catabolic activity in the SP-D(-/-) group.

These metabolic studies resulted in a conundrum because anabolism (incorporation and secretion) is comparable in SP-D(+/+) and SP-D(-/-) mice, and endogenously synthesized Sat PC likely is catabolized more slowly in SP-D(-/-) than in SP-D(+/+) animals. In contrast, the intratracheal clearance studies indicate more rapid net catabolism of the larger pools in the SP-D(-/-) mice. These measurements have not considered recycling. The accumulation of lipids in the air spaces of SP-D(-/-) mice is not uniform, based on histopathology, nor is the distribution of cells with large lamellar bodies (2, 18). It is possible that the intravascular precursors are selectively evaluating different cell populations and that intratracheal DPPC is measuring clearance and catabolism for only part of the alveolar pool.

The contribution of recycling to the increased surfactant pool sizes is not known, but increased recycling might occur because of the increased alveolar small-aggregate surfactant pool. The small-aggregate surfactant thought to be the catabolic form for the phospholipids is preferentially taken up by type II cells in vitro (9). Further studies will be required to sort out the relationship between the endogenous incorporation data and the enlarged lamellar bodies in type II cells. The increased numbers of lipid-laden macrophages will also no doubt contribute to the overall metabolic abnormalities in these mice. Alveolar macrophages contribute ~20% to the overall catabolic rate of surfactant in adult rabbit lungs (27), but the macrophage contribution in mice is not known.

Animal models of increased surfactant phospholipid and increased SP pool sizes include silica and mineral dust exposure (4), the severe-combined immunodeficient mouse (16), gene-targeted disruption of the GM-CSF or GM-CSF receptor beta /C gene (13, 26), and overexpression of IL-4 (10). Increased surfactant phospholipid in SP-D(-/-) mice was not associated with increased amounts of SPs, distinguishing this phenotype from the alveolar lipoproteinosis syndromes. Each of the models of alveolar proteinosis has different alterations in metabolic pathways that yield a similar phenotype. For example, GM-CSF deficiency and GM-CSF receptor inactivation result primarily from a decrease in phospholipid and SP catabolism (13, 26). Mice with defective GM-CSF signaling also accumulate large amounts of SP-D in the air spaces. In contrast, SP-D was cleared relatively rapidly in SP-D(-/-) mice. Those that overexpress IL-4 have alveolar proteinosis primarily because of increased precursor incorporation and secretion rates (10). In these models, the ratio of SPs to Sat PC is normal or increased. The SP-D(-/-) phenotype is unique because of the decreased SP-A and SP-C relative to Sat PC.

An absolute SP-D(-/-) deficiency results in multiple abnormalities in the surfactant system. In contrast, SP-D(+/-) mice have normal surfactant lipid pool sizes, and mice that overexpress SP-D also have normal surfactant pool sizes, indicating that the phenotype is sensitive to the amount of SP-D (5). SP-D deficiency alters surfactant lipid, SP-A, and SP-C pools and metabolism by unknown mechanisms.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-63329-01 and HL-61646.


    FOOTNOTES

Address for reprint requests and other correspondence: M. Ikegami, Children's Hospital Medical Center, Division of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: machiko.ikegami{at}chmcc.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. §1734 solely to indicate this fact.

Received 16 February 2000; accepted in final form 12 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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