Distinct changes in pulmonary surfactant homeostasis in common beta -chain- and GM-CSF-deficient mice

Jacquelyn A. Reed1, Machiko Ikegami1, Lorraine Robb2, C. Glenn Begley2, Gary Ross1, and Jeffrey A. Whitsett1

1 Children's Hospital Medical Center, Cincinnati, Ohio 43229-3039; and 2 Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Victoria 3050 Australia


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Pulmonary alveolar proteinosis (PAP) is caused by inactivation of either granulocyte-macrophage colony-stimulating factor (GM-CSF) or GM receptor common beta -chain (beta c) genes in mice [GM(-/-), beta c(-/-)], demonstrating a critical role of GM-CSF signaling in surfactant homeostasis. To distinguish possible phenotypic differences in GM(-/-) and beta c(-/-) mice, surfactant metabolism was compared in beta c(-/-), GM(-/-), and wild-type mice. Although lung histology in beta c(-/-) and GM(-/-) mice was indistinguishable, distinct differences were observed in surfactant phospholipid and surfactant protein concentrations and clearance from lungs of beta c(-/-) and GM(-/-) mice. At 1-2 days of age, lung saturated phosphatidylcholine (Sat PC) pool sizes were higher in wild-type, beta c(-/-), and GM(-/-) mice compared with wild-type adult mice. In wild-type mice, Sat PC pool sizes decreased to adult levels by 7 days of age; however, Sat PC increased with advancing age in beta c(-/-) and GM(-/-) mice. Postnatal changes in Sat PC pool sizes were different in GM(-/-) compared with beta c(-/-) mice. After 7 days of age, the increased lung Sat PC pool sizes remained constant in beta c(-/-) mice but continued to increase in GM(-/-) mice, so that by 56 days of age, lung Sat PC pools were increased three- and sixfold, respectively, compared with wild-type controls. After intratracheal injection, the percent recovery of [3H]dipalmitoylphosphatidylcholine and 125I-recombinant surfactant protein (SP) C was higher in beta c(-/-) compared with wild-type mice, reflecting decreased clearance in the receptor-deficient mice. The defect in clearance was significantly more severe in GM(-/-) than in beta c(-/-) mice. The ratio of SP Sat PC to SP-A, -B, and -C was similar in bronchoalveolar lavage fluid (BALF) from adult mice of all genotypes, but the ratio of SP-D to Sat PC was markedly increased in beta c(-/-) and GM(-/-) mice (10- and 5-fold, respectively) compared with wild-type mice. GM-CSF concentrations were increased in BALF but not in serum of beta c(-/-) mice, consistent with a pulmonary response to the lack of GM-CSF signaling. The observed differences in surfactant metabolism suggest the presence of alternative clearance mechanisms regulating surfactant homeostasis in beta c(-/-) and GM(-/-) mice and may provide a molecular basis for the range in severity of PAP symptoms. surfactant metabolism; alveolar macrophage; granulocyte-macrophage colony-stimulating factor


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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INACTIVATION OF THE granulocyte-macrophage colony-stimulating factor (GM-CSF) gene in mice caused alveolar proteinosis with pathological findings similar to the human disorder known as pulmonary alveolar proteinosis (PAP) (36). Histological findings in the lungs of GM-CSF-deficient [GM(-/-)] mice included eosinophilic material in the alveolar spaces, perivascular and peribronchiolar mononuclear cell infiltrates, and foamy, lipid-laden alveolar macrophages (9, 15, 16, 32). Metabolic studies demonstrated that the four- to fivefold increase in phospholipid pool size in GM(-/-) mice was due at least in part to a defect in catabolism or clearance of surfactant, demonstrating the critical role that GM-CSF signaling plays in surfactant clearance (19, 20).

Gene targeting of the beta c-chain of the GM-CSF receptor also caused alveolar proteinosis in mice, although the biochemical and physiological mechanisms underlying the histological abnormalities have not been clarified in the beta c-deficient [beta c(-/-)] mice (26, 28). Histological findings in the lungs of beta c(-/-) mice were similar to those observed independently in GM(-/-) mice (9, 32). The beta c-chain is shared with interleukin-3 (IL-3) and IL-5 receptor complexes in which heterodimers of alpha - and beta c-subunits form high-affinity binding sites (31). The alpha -subunit of each receptor complex confers ligand specificity and constitutes a low-affinity binding site. The alpha -subunit is converted to a high-affinity binding site when bound to the beta c-chain. In the mouse, two distinct beta -subunits, originally known as AIC2A and AIC2B, have been identified. AIC2B, or mouse beta c, is the homolog of the human beta -chain and mediates IL-3, IL-5, and GM-CSF binding and signaling (13). AIC2A, or mouse beta IL3, shares 96% homology with mouse beta c but is specific for binding the IL-3 receptor alpha -subunit (IL-3Ralpha ) only. The two mouse beta -subunits (beta c and beta IL3) are transcribed from separate genes and interact with the IL-3Ralpha with equal affinities. The IL-3Ralpha -beta IL3 receptor complex cross-competes with IL-3Ralpha -beta c complexes for IL-3 binding but has no effect on GM-CSF or IL-5 binding. Thus GM-CSF and IL-5 signaling are disrupted in beta c(-/-) mice, whereas IL-3 signaling is intact.

There is considerable heterogeneity in the severity and clinical course of PAP in humans, and the role of GM-CSF signaling in the pathogenesis of PAP remains poorly understood. An association between hematological malignancies, including acute myelogenous leukemia and PAP, has been observed clinically (2, 14). Recent studies have identified patients diagnosed with PAP in association with loss of GM-CSF production or inhibition of GM-CSF activity. Tchou-Wong et al. (34) isolated alveolar macrophages from a PAP patient and treated them with lipopolysaccharide. Lipopolysaccharide treatment stimulated transcription of GM-CSF mRNA, but, in contrast to alveolar macrophages from normal controls, no GM-CSF was secreted into the culture medium. Tanaka and colleagues (33) isolated a GM-CSF-binding factor from bronchoalveolar lavage fluid (BALF) samples obtained from 11 PAP patients. The factor specifically bound GM-CSF and neutralized its growth-promoting activity. These latter studies support the hypothesis that the GM-CSF signaling pathway is intact in some individuals but that GM-CSF production or availability is impaired, causing PAP. Impaired function of the common beta -chain was observed in several patients with PAP, suggesting that lesions affecting the GM-CSF receptor or signaling pathway are involved in the pathogenesis of PAP in a subset of individuals (8). Studies of PAP patients insensitive to GM-CSF stimulation were reported by Seymour and colleagues (30). More recently, Dirksen et al. (7) demonstrated a loss of GM-CSF receptor on alveolar macrophages obtained from several patients diagnosed with both acute myelogenous leukemia and PAP. After a course of chemotherapy eliminating leukemic cells, GM-CSF receptor expression was restored in alveolar macrophages, and PAP symptoms were resolved. Findings of PAP in association with impairment of GM-CSF receptor signaling provide a basis for further characterizing surfactant homeostasis in beta c(-/-) mice. Earlier studies of beta c(-/-) and GM(-/-) mice reported similar lung morphology, suggesting that the phenotypes were indistinguishable (9, 26, 28, 32). However, in the present study, differences in surfactant phospholipid content and metabolism were noted in beta c(-/-) and GM(-/-) mice.


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Animals. beta c(-/-) mice were generated by Robb et al. (28) by targeted insertion of the neomycin-resistant gene into the beta c gene locus, as described previously. GM(-/-) mice used were generated by targeted ablation of the GM-CSF gene locus, kindly provided by Dranoff et al. (9). The beta c(-/-) mutation was maintained in a C57BL/ 6-129Sv strain background and GM(-/-) in C57BL/6-FVB/N. Both colonies of mice were bred and housed in microisolator cages at the Cincinnati Children's Hospital Research Foundation animal facility and required no special care. C57BL/6 wild-type control mice were obtained from Harlan (Indianapolis, IN).

Processing and staining of tissues for immunohistochemistry. Mice were anesthetized with intraperitoneal pentobarbital sodium. Lungs were inflation fixed with 4% paraformaldehyde in PBS, pH 7.4, for 24 h as described previously (12). Tissues were then washed in PBS, dehydrated in a series of alcohols, and embedded in paraffin. Paraffin sections (5 µm) sampling all five lobes of lung tissue were stained for surfactant protein-B (SP-B) and SP-D. SP-B was detected using an anti-SP-B (number 28031) rabbit anti-bovine polyclonal antibody that recognizes mature SP-B described previously (12). SP-D was detected using a rabbit anti-rat SP-D antiserum, which was a kind gift from Dr. Frances X. McCormack (University of Cincinnati) (10). Primary antibody was detected with the Vectastain ABC goat anti-rabbit immunohistochemical horseradish peroxidase kit from Vector Laboratories (Burlingame, CA). Tissues were counterstained with Tris-cobalt and nuclear fast red and qualitatively assessed for relative SP-B immunostaining. Type II cells were counted with paraffin sections stained with anti-proSP-C rabbit serum (number 68514) and goat anti-rabbit immunohistochemical horseradish peroxidase, as previously described (35). Sections were cut from distinct distal and proximal regions of all five lobes. Eight consecutive fields of 2 × 104 µm2 contained within a larger grid were counted in each tissue section (>= 100 fields counted/mouse) and counted at a magnification of ×40.

Bronchoalveolar lavage, tissue processing, and saturated phosphatidylcholine measurement. Mice were given intraperitoneal pentobarbital sodium to achieve deep anesthesia, and the distal aorta was cut to exsanguinate each animal. The chest of the animal was opened, a blunt needle was tied into the proximal trachea, and five aliquots of 0.9% NaCl were flushed into the lungs to achieve full inflation (about 1 ml) and withdrawn by syringe three times for each aliquot (20). A 22-gauge needle was used to lavage 7- and 15-day-old animals, and a 20-gauge needle was used for older animals. Total BALF for each animal was pooled, measured for volume, and divided into aliquots for analyses of saturated phosphatidylcholine (Sat PC) as described previously (20) or proteins as described in Western blotting. Lung tissue was weighed and homogenized in 0.9% NaCl for Sat PC measurement as described previously (20). Lungs of mice 1- or 2 days old were not lavaged before homogenization.

Phospholipid precursor incorporation into Sat PC. Mice were given an intraperitoneal injection with 8 µl/g body wt containing 0.45 µCi/g [3H]choline chloride (DuPont-NEN; Boston, MA) and 0.27 µCi/g [14C]palmitic acid (American Radiolabeled Chemicals; St. Louis, MO). The palmitic acid was stabilized in solution with 2.5% human serum albumin. Mice were killed with intraperitoneal pentobarbital sodium at 3, 8, 15, 24, or 48 h after isotope injection. Bronchoalveolar lavage, lung homogenization, and Sat PC measurement were performed as described in Bronchoalveolar lavage, tissue processing, and saturated phosphatidylcholine measurement.

Clearance of DPPC and SP-C. Mice were given intratracheal injections of 50 µl of saline that contained 0.3 µCi of [3H]choline-labeled dipalmitoylphosphatidylcholine (DPPC, Amersham; Arlington Heights, IL) and 0.15 µCi 125I-labeled recombinant human SP-C (rSP-C). The rSP-C (a gift from Byk Gulden; Constance, Germany) is a peptide consisting of the 34-amino acid human sequence. The rSP-C was iodinated with 125I-labeled Bolton-Hunter reagent as previously reported (17). Previous studies demonstrated that metabolism of 125I-rSP-C in rabbit and mouse lungs was similar to that of the native SP-C peptide (17). The [3H]DPPC and 125I-rSP-C were mixed with a chloroform-methanol extract of mouse surfactant, dried under N2, and resuspended in 0.9% NaCl by brief sonication. The tracer dose represents a phospholipid dose of 0.1 µmol Sat PC/kg body wt and therefore is unlikely to perturb endogenous pools (15 µmol Sat PC/kg) in the mice. Mice were sedated with isofluorane, and the trachea was exposed through a 0.5-cm midline skin incision. Radiolabeled surfactant was injected using a 1-ml syringe with a 30-gauge needle. At 3 min, 24 h, or 48 h after injection, four to six mice from each genotype group were killed with intraperitoneal pentobarbital sodium. Lungs were lavaged and homogenized for analysis of Sat PC or recovery of [3H]DPPC and 125I-rSP-C. Values for mice killed 3 min after the intratracheal injections were set at 100% and used to calculate the percentage recovery at 24 and 48 h.

Western blotting. Bronchoalveolar lavage samples containing 1 µg of Sat PC were subjected to SDS-PAGE in the presence of beta -mercaptoethanol for analysis of SP-A, -C, and -D. For SP-B analysis, aliquots containing 0.2 µg of Sat PC were electrophoresed under nonreducing conditions. SP-A and -D were separated on 8-16% acrylamide gel with Tris-glycine buffer; SP-B and -C samples were separated on 10-20% acrylamide gel with Tricine buffer (Novex; San Diego, CA). After electrophoresis, proteins were transferred to nitrocellulose paper (Schleicher & Schuell; Keene, NH) for SP-A and -D or to polyvinylidene difluoride paper (Bio-Rad; Hercules, CA) for SP-B and -C. Immunoblot analysis was carried out with the following dilutions of antisera: SP-A, 1:25,000 guinea pig anti-rat SP-A; SP-B, 1:10,000 rabbit anti-bovine SP-B; SP-C, 1:25,000 rabbit anti-recombinant human SP-C; and SP-D, 1:10,000 rabbit anti-rat SP-D (10, 11, 17, 35). The rabbit anti-rat SP-D antiserum was a kind gift from Dr. Frances X. McCormack (University of Cincinnati). Appropriate peroxidase-conjugated secondary antibodies were used at 1:10,000 dilutions. Immunoreactive bands were detected using enhanced chemiluminescence reagents (Amersham; Chicago, IL). Semiquantitation of protein bands was determined using a Molecular Dynamics phosphorimaging system and ImageQuant analysis software.

RNA isolation. RNA was isolated by a modification of the guanidinium thiocyanate method described by Chomczynski and Sacchi (3). Briefly, tissues were homogenized in guanidinium thiocyanate. A series of phenol-chloroform extractions was followed by precipitation in isopropanol as described in the RNA isolation protocol package insert for Phase-Lock Gel IIA Heavy Tubes (5 Prime right-arrow 3 Prime; Boulder, CO).

S1 nuclease protection analysis. Surfactant protein mRNAs were subjected to S1 nuclease protection analysis as described previously (9). Two micrograms of total lung RNA were used for analysis of SP-A, -B, and -C, and 10 µg were used for SP-D. Plasmids containing probe sequences for SP-A, -B, -C, and -D, and L32 were described previously (9, 21). Bands were quantitated using a Molecular Dynamics phosphorimaging system and ImageQuant analysis software.

Measurement of GM-CSF and IL-5 by ELISA. GM-CSF was measured in BALF and serum using Endogen GM-CSF Minikit ELISA (Endogen; Cambridge, MA) as described previously (15). The lower limit of detection is <5 pg/ml. IL-5 was measured by ELISA using a mouse IL-5 kit (Endogen; Cambridge, MA) following the manufacturer's protocol.

Data analysis. All values are reported as means ± SE. Differences between groups were tested by two-tailed Student's t-test. When more than two comparisons were made, ANOVA followed by the Student-Newman-Keuls multiple comparison procedure was used. Significance was accepted at P < 0.05.


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Wild-type, beta c(-/-), and GM(-/-) mice had similar body weights at all ages analyzed. Mice of all three genotypes produced litters with similar numbers and survival rates. No abnormalities were observed in the lungs or other organs of the animals. There was no evidence of concurrent infection in the colonies as assessed by serology and necropsy of sentinel mice.

Surfactant protein immunostaining of lung sections from beta c(-/-) and GM(-/-) mice. Although anti-SP-B staining was observed only in alveolar type II and bronchiolar epithelial cells in sections from wild-type mice, intense staining was noted in the alveolar spaces of lungs from beta c(-/-) and GM(-/-) mice at 7-9 wk of age (Fig. 1). SP-B staining was increased in alveolar type II cells and macrophages in lung sections from beta c(-/-) and GM(-/-) mice compared with wild-type mice. SP-D immunostaining was similar to that of SP-B, with intense staining in the alveolar spaces of lungs from beta c(-/-) and GM(-/-) mice but not in those of wild-type mice (data not shown).


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Fig. 1.   Immunohistochemical staining of surfactant protein B (SP-B) in lung sections from wild-type (A), common beta -chain deficient [beta c(-/-)] (B), and granulocyte-macrophage colony-stimulating factor-deficient [GM(-/-)] (C) mice. Representative lung sections from wild-type, beta c(-/-), and GM(-/-) mice 7-9 wk of age (n = 6) were stained with antibodies to SP-B and examined by light microscopy. Anti-SP-B staining was observed only in alveolar type II and bronchiolar epithelial cells in lung sections from wild-type mice, whereas sections from beta c(-/-) and GM(-/-) mice also contained alveolar material that was heavily stained.

To determine whether numbers of type II cells were altered in beta c(-/-) mice, sections from wild-type, beta c(-/-), and GM(-/-) mice were immunostained with antibodies to pro SP-C (data not shown). Numbers of type II cells within a grid area of 2 × 104 µm2 were counted in each group of animals and compared with wild-type mice. The average number of type II cells in lungs from beta c(-/-) (7.1 ± 0.2) and GM(-/-) (6.7 ± 0.3) mice did not differ from wild-type control mice (7.3 ± 0.2, n = 4).

Total lung and alveolar phospholipid pool sizes. Total lung Sat PC pool sizes were highest (148 ± 8 µmol/kg body wt) in wild-type mice at birth and decreased by 7 days of age to the levels found in older mice (Fig. 2A). The total lung Sat PC in beta c(-/-) mice (151 ± 10 µmol/kg) was not different from that of wild-type mice at 1-2 days but was increased threefold by day 7 and thereafter. In GM(-/-) mice, total lung Sat PC was somewhat lower (112 ± 3 µmol/kg) than in wild-type mice at birth but subsequently increased so that by 56 days of age it was 4- to 6-fold that of wild-type and ~50% higher than that of beta c(-/-) mice (P < 0.001). The total lung Sat PC was similar in all animals at 1-2 days of age, suggesting that the PAP seen in older beta c(-/-) and GM(-/-) mice is caused by postnatal changes in surfactant homeostasis.


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Fig. 2.   Surfactant saturated (Sat) phosphatidylcholine (PC) pool sizes during postnatal development. A: Sat PC was measured in total lungs of wild-type, beta c(-/-), and GM(-/-) mice (n = 4-6). At 1-2 days, total lung Sat PC pool in wild-type and beta c(-/-) mice was approximately double that of wild-type adults. Total lung Sat PC in 1- to 2-day-old GM(-/-) mice was also increased compared with wild-type adults but was slightly lower than that of 1- to 2-day-old wild-type and beta c(-/-) mice. In wild-type mice, total lung Sat PC was similar to that in adult by 7 days of age. In contrast, total lung Sat PC in beta c(-/-) mice was increased at 7 days (~3 times that of 7-day wild type) and remained at that level through 56 days of age. In GM(-/-) mice, total lung Sat PC continued to increase during postnatal period, so that by 56 days of age concentration was ~6 times that of wild type. B: Sat PC was also measured in bronchoalveolar lavage fluid (BALF) from lungs (alveolar) of wild-type, beta c(-/-), and GM(-/-) mice (n = 4-6). At all ages Sat PC was markedly increased in beta c(-/-) and GM(-/-) mice compared with age-matched wild-type control mice, with Sat PC pool sizes higher in GM(-/-) than in beta c(-/-) mice. * P < 0.01 vs. wild-type. t P < 0.01 vs. GM(-/-).

Alveolar Sat PC pool sizes were also measured in the wild-type, beta c(-/-), and GM(-/-) animals at 7, 15, 28, and 56 days old (Fig. 2B). Alveolar Sat PC pool sizes of 40.1 ± 6.1 and 45.4 ± 7.0 µmol/kg in beta c(-/-) and GM(-/-) mice at 7 days of age, respectively, were approximately 10-fold higher than those in wild-type mice, 4.4 ± 0.3 µmol/kg. By 15 days of age, alveolar Sat PC pool in the beta c(-/-) mice was increased sevenfold compared with wild-type controls and did not change thereafter. In contrast to findings in beta c(-/-) mice, Sat PC continued to increase in the airways of GM(-/-) mice so that it was twice that of beta c(-/-) mice by 28 days of age.

Precursor incorporation into Sat PC. More labeled palmitic acid and choline were incorporated into the total lung Sat PC of beta c(-/-) mice than of wild-type mice at all time points (Fig. 3, A and B). The differences between labeled Sat PC recovered in the alveolar wash from beta c(-/-) mice and wild-type mice increased with time. Accumulation of [14C]palmitate- and [3H]choline-labeled Sat PC in the alveolar compartment of beta c(-/-) mice was consistent with decreased catabolism of Sat PC, increased recycling, or both.


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Fig. 3.   Radiolabeled precursor incorporation into Sat PC. Radiolabeled Sat PC was measured in BALF (alveolar) and lung tissue from wild-type and beta c(-/-) mice after intraperitoneal injections of [14C]palmitic acid and [3H]choline (n = 4-6). A: increased levels of 14C-labeled Sat PC were measured in BALF by 8 h and at all time points in tissue from beta c(-/-) mice compared with wild type control mice. B: 3H-labeled Sat PC was increased in BALF by 15 h and by 8 h in lung tissue from beta c(-/-) mice compared with wild type. Increased levels of 14C- and 3H-labeled Sat PC measured in lungs of beta c(-/-) mice at 24 and 48 h are consistent with decreased phospholipid catabolism, increased recycling, or both. Significantly different from wild-type control mice: * P < 0.01; ** P < 0.05.

Previous studies of [14C]palmitate and [3H]choline incorporation in lungs of GM(-/-) mice demonstrated patterns similar to those presently observed in beta c(-/-) mice (19, 20). To directly compare findings in beta c(-/-) and GM(-/-) mice, [3H]choline was administered to beta c(-/-) and GM(-/-) mice (n = 8). Alveolar tissue and total lung 3H-labeled Sat PC were measured at 8 h, and no significant differences in recovery of radiolabeled Sat PC were found between beta c(-/-) and GM(-/-) mice, suggesting that precursor incorporation into lung Sat PC was similar in mice of these two genotypes.

Clearance of [14C]DPPC and 125I-rSP-C. Recovery of intratracheally instilled [14C]DPPC and 125I-rSP-C was measured in lung tissues and alveolar wash from wild-type, beta c(-/-), and GM(-/-) mice (Figs. 4, A and B). Most of the [14C]DPPC was cleared from the alveolar compartment of control mice by 24 and 40 h, with only ~18 and ~5% recovered from the alveoli at these time points, respectively. Recovery of [14C]DPPC from airways of beta c(-/-) mice (~30%) did not differ significantly from wild-type controls at 24 h. However, the amount of radiolabel recovered from alveoli of beta c(-/-) mice at 40 h was still ~30%, significantly higher than in the control mice at 40 h and consistent with decreased clearance of the rSP-C. Recovery of [14C]DPPC from lungs of GM(-/-) mice (~52% alveolar) was significantly higher at 24 h compared with wild-type mice. The pattern of [14C]DPPC recovered in total lung was similar to that of the alveolar recovery in each genotype. Recoveries of 125I-rSP-C from alveolar wash and total lung in each genotype group were similar to those of [14C]DPPC. Previous studies demonstrated increased recovery of [14C]DPPC, 125I-SP-A, and 125I-SP-B in alveolar wash and total lungs of GM(-/-) mice (19, 20). Thus in beta c(-/-) and GM(-/-) mice, clearance of both [14C]DPPC and 125I-rSP-C from the airways was impaired. beta c(-/-) and GM(-/-) mice accumulated more label in both alveolar and lung tissue compartments than did wild-type mice; however, accumulation of labeled lipids in the alveolar compartment was greater in GM(-/-) than in beta c(-/-) mice, consistent with the greater increase in alveolar and Sat PC pool sizes in the GM(-/-) mice.


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Fig. 4.   Recovery of radiolabeled dipalmitoylphosphatidylcholine (DPPC) and recombinant SP-C (rSP-C). [14C]DPPC and 125I-rSP-C were instilled into lungs of wild-type, beta c(-/-), and GM(-/-) mice by intratracheal injection. Percentage recovery of [14C]DPPC (A) and 125I-rSP-C (B) was measured in BALF (alveolar) and total lung (n = 8). At 24 h, recovery of [14C]DPPC and 125I-rSP-C from alveolar and total lung tissue was increased in beta c(-/-) and GM(-/-) mice compared with wild-type controls. By 40 h, recovery of [14C]DPPC and 125I-rSP-C from alveolar wash and total lung was significantly increased in beta c(-/-) mice compared with wild type. * P < 0.05 vs. wild type.

Selective increase in SP-D in both beta c(-/-) and GM(-/-) mice. Surfactant proteins were analyzed by Western blotting after normalization to Sat PC content (Fig. 5). Relative amounts of proteins were obtained by phosphorimaging. The ratios of SP-A, -B, or -C to Sat PC were similar in all animals, although the total amount of each surfactant protein was markedly increased in the airways of beta c(-/-) and GM(-/-) mice compared with wild-type controls. In contrast, SP-D was selectively increased in beta c(-/-) and GM(-/-) mice compared with controls, with the ratio of SP-D to Sat PC increasing 10- and 5-fold, respectively. Because alveolar Sat PC pools in beta c(-/-) and GM(-/-) mice were increased three- and sixfold, respectively, the total alveolar SP-D content in both beta c(-/-) and GM(-/-) mice was increased ~30-fold overall compared with wild-type control mice.


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Fig. 5.   Semiquantitation of surfactant proteins relative to phospholipids. Bronchoalveolar lavage samples from wild-type, beta c(-/-), and GM(-/-) mice 7-9 wk old (n = 6) were normalized by phospholipid content, separated by SDS-PAGE, and transferred to filters. Filters were hybridized with antibodies specific for SP-A, -B, -C, or -D and quantitated. Although total amounts of SP-A, -B, and -C were markedly increased in beta c(-/-) and GM(-/-) compared with wild-type mice, ratios of these surfactant proteins to phospholipid concentration were similar in all three genotypes. In contrast, ratio of SP-D to phospholipid was increased ~10-fold in beta c(-/-) and ~6-fold in GM(-/-) compared with wild-type control mice. * P < 0.05 compared with wild-type mice.

Surfactant protein mRNA concentrations in total lung. Total lung RNA from wild-type, beta c(-/-), and GM(-/-) mice at 7-9 wk of age was analyzed by S1 nuclease protection assay (Fig. 6). SP-A, -B, and -C mRNA content was similar in wild-type and beta c(-/-) mice, consistent with previous studies demonstrating that SP-A, -B, and -C mRNA levels in GM(-/-) mice did not differ from those in wild-type mice (9, 15). SP-D mRNA was slightly increased in beta c(-/-) mice but was indistinguishable in GM(-/-) and wild-type mice. Thus changes in SP-A, -B, -C, or -D mRNA synthesis or accumulation were negligible and not likely to account for the increased surfactant proteins in beta c(-/-) and GM(-/-) mice.


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Fig. 6.   S1 nuclease protection assay of surfactant protein mRNAs. Surfactant protein mRNAs in total lung RNA from wild-type, beta c(-/-), and GM(-/-) mice were assayed by S1 nuclease protection and quantitated (n = 9). Despite increased levels of SP-A, -B, -C, and -D proteins in the lungs of beta c(-/-) mice, SP-A, -B, and -C mRNAs were not increased and SP-D mRNA was increased only slightly (P = 0.055). SP-D mRNA concentration was not increased in GM(-/-) mice. Data are representative of 3 separate experiments. L32 is ribosomal RNA used as loading control for S1 nuclease protection assay.

GM-CSF and IL-5 in BALF. GM-CSF and IL-5 were measured by ELISA in the BALF from wild-type, beta c(-/-), and GM(-/-) mice. IL-5 was not detectable in BALF from any mice. GM-CSF concentrations in BALF from wild-type mice were below the limits of detection by ELISA (<5 pg/ml). As expected, GM-CSF was not detected in BALF from GM(-/-) mice. However, GM-CSF was markedly increased in BALF from beta c(-/-) mice (161 ± 30 pg/ml BALF, n = 6). Despite the increased concentration of GM-CSF in the pulmonary compartment, GM-CSF was undetectable in the serum of beta c(-/-) mice.


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REFERENCES

Inactivation of the common beta -chain [beta c(-/-)] or GM-CSF [GM(-/-)] gene in mice disrupted pulmonary surfactant homeostasis, resulting in surfactant accumulation in lung tissue and airways. Lung histology in beta c(-/-) and GM(-/-) adult mice was perturbed in a like manner; however, surfactant Sat PC pool sizes and clearance were different in the two mutant genotypes. Although lung Sat PC pool sizes were increased in both beta c(-/-) and GM(-/-) mice, the increase was consistently greater in GM(-/-) than in beta c(-/-) mice. Likewise, whereas decreased clearance of DPPC was seen in both beta c(-/-) and GM(-/-) mice, the metabolic abnormality was more severe in GM(-/-) than beta c(-/-) mice. GM-CSF was increased in the BALF of beta c(-/-) mice but not in serum, suggesting a lung-selective response to inactivation of beta c. Decreased surfactant clearance and increased surfactant pool sizes were observed with both gene-targeted mice; however, differences in severity of the accumulation in GM(-/-) and beta c(-/-) mice suggest the presence of alternative metabolic pathways that influence surfactant metabolism in these two models.

Lung histology of beta c(-/-) and GM(-/-) mice was indistinguishable, consistent with earlier findings in several laboratories (9, 15, 16, 26, 28, 32). However, the present study demonstrated differences in the pattern of lung Sat PC pool sizes in beta c(-/-) and GM(-/-) mice from birth to adulthood, suggesting that distinct pathological alterations in surfactant homeostasis occurred postnatally. Total lung Sat PC pool size was similarly increased in all newborn animals of beta c(-/-), GM(-/-), and wild-type genotypes compared with wild-type adults. In control mice, lung Sat PC pool size was decreased to adult levels by 7 days of age. This rapid decrease in Sat PC pool size from the neonatal period to adulthood is consistent with earlier studies in rabbits, sheep, and humans (22). In contrast, Sat PC pool sizes increased in beta c(-/-) and GM(-/-) mice by 7 days of age, suggesting that GM-CSF signaling is required for establishment of normal surfactant homeostasis in the early postnatal period. The patterns of Sat PC pool sizes differed in GM(-/-) and beta c(-/-) mice with subsequent development. The increased total lung and alveolar Sat PC pool sizes in beta c(-/-) mice remained stable from 7 through 56 days. In contrast, total and alveolar Sat PC progressively accumulated in lungs of GM(-/-) mice over the same time period. The intermediate level of Sat PC accumulation seen in beta c(-/-) mice suggests that Sat PC concentrations in beta c(-/-) mice were influenced in a manner distinct from that in GM(-/-) mice.

Exogenous radiolabeled DPPC and rSP-C were rapidly removed from the airways of wild-type mice. In contrast, clearance of radiolabeled DPPC and rSP-C from the airways was delayed in beta c(-/-) and GM(-/-) mice, and the defect in clearance was more severe in the GM(-/-) mice. Previous studies demonstrated that little exogenous radiolabeled DPPC, SP-A, or SP-B was cleared from the airways of GM(-/-) mice by 40 h. In contrast, the half-life for surfactant proteins and lipids in the normal mouse lung was ~12 h (18-20). Clearance of Sat PC in beta c(-/-) mice was intermediate compared with wild-type and GM(-/-) mice, consistent with the greater increase in steady-state Sat PC pools seen in GM(-/-) mice.

Phospholipid precursor incorporation and accumulation were increased similarly in both beta c(-/-) and GM(-/-) mice (19, 20). The pattern of accumulation in the airways of beta c(-/-) and GM(-/-) mice reflects a decreased loss of labeled Sat PC compared with wild-type control mice, consistent with impaired degradation and/or increased recycling. Accumulation of Sat PC in the alveoli of beta c(-/-) mice and GM(-/-) mice is also consistent with the observed decreased clearance of radiolabeled DPPC and rSP-C.

The amounts of SP-A, -B, and -C increased in proportion to the increased Sat PC pool sizes in BALF from beta c(-/-) and GM(-/-) mice. This observation is consistent with previous studies in which SP-A, SP-B, and phospholipids increased in a similar manner in sheep with silica-induced alveolar proteinosis (24). Despite the marked increase in protein, SP-A, -B, and -C mRNA levels were similar in wild-type, beta c(-/-), and GM(-/-) mice. Thus impaired clearance rather than increased surfactant synthesis likely caused the accumulations of surfactant proteins in lungs of beta c(-/-) and GM(-/-) mice.

The ratio of SP-D protein to Sat PC was markedly increased in BALF from both beta c(-/-) and GM(-/-) mice. These findings were consistent with earlier observations in humans, where SP-D was disproportionately elevated in PAP compared with other surfactant proteins (5). Alveolar and tissue Sat PC pool sizes were markedly increased in SP-D gene-targeted mice in the absence of significant changes in SP-A, -B, or -C concentrations, suggesting that SP-D plays an important and selective role in surfactant phospholipid metabolism (1, 23). SP-D mRNA levels were similar in GM(-/-) and wild-type mice and increased only slightly in beta c(-/-) mice. The slight (~25%) increase in SP-D mRNA in lungs from beta c(-/-) mice was statistically significant but is unlikely to account for the ~30-fold increase in overall SP-D concentration seen in both GM(-/-) and beta c(-/-) mice. The disproportionately high SP-D concentrations in the lungs of beta c(-/-) and GM(-/-) mice may reflect a compensatory response to the high-surfactant lipid concentrations.

Increased GM-CSF was measured in BALF but not in serum from beta c(-/-) mice, suggesting a local response of pulmonary tissues to the lack of beta c signaling in the lung. Metcalf and colleagues (25) observed that the histological findings of alveolar proteinosis were not altered in double-transgenic beta c(-/-) mice expressing high levels of systemic GM-CSF. Although there is no direct evidence that the increased concentrations of GM-CSF in the airways had a direct effect on metabolism or steady-state concentrations of surfactant in beta c(-/-) mice, we speculate that distinct clearance pathways exist in the beta c(-/-) compared with GM(-/-) mice. Although it is generally accepted that the alpha -subunit of the GM-CSF receptor (GMRalpha ) does not directly participate in GM-CSF signaling, previous studies in Xenopus oocytes demonstrated that the human GMRalpha activates glucose transport in the absence of the beta -subunit (6). Ding et al. (6) hypothesized that multiple intracellular signaling pathways are activated by the GM-CSF receptor, with specific events, such as some types of transport signaling machinery, being modulated by the GMRalpha alone. This was in contrast to findings by Scott et al. (29), in which hematopoietic cells from beta c(-/-) mice did not take up 2-[3H]deoxy-D-glucose when stimulated with GM-CSF but did so in control experiments with IL-3 (29).

Nishinakamura et al. (27) transplanted normal [beta c(+/+)] mouse bone marrow into lethally irradiated beta c(-/-) mice to study the role of alveolar macrophages in PAP. Significant numbers of donor macrophages derived from the engrafted beta c(+/+) bone marrow were found in the lungs of the beta c(-/-) mice within 8-12 wk after transplantation. Alveolar proteinosis was substantially improved in beta c(-/-) mice receiving beta c(+/+) bone marrow cells, although peribronchiolar or perivascular mononuclear cell infiltrates and mildly elevated SP-B levels in the BALF persisted (4). The residual infiltrates and excess SP-B concentrations suggested that engrafted alveolar macrophages increased catabolic functions in a compensatory manner in the lungs of beta c(-/-) mice but that the contribution of other cell types, i.e., type II cells, may also influence surfactant homeostasis.

The present study shows that surfactant clearance is impaired in beta c(-/-) and GM(-/-) mice. However, biochemical and metabolic differences in beta c(-/-) and GM(-/-) mice were observed, and similar pathological mechanisms may underlie the varying severity of PAP seen in humans and the individual responses to therapeutic lavage. Studies demonstrating both similar and distinct features of surfactant metabolism in beta c(-/-) and GM(-/-) mice provide a rationale to more precisely define the pathogenesis of various forms of PAP in humans.


    ACKNOWLEDGEMENTS

We thank Wei Lu, Dr. Cindy Bachurski, Dr. Susan Wert, and Sherri Profitt for technical assistance and Dr. Alan Jobe for helpful discussions of this work.


    FOOTNOTES

This work was supported in part by National Heart, Lung, and Blood Institute (NHLBI) Grant POI-HL-61646 (M. Ikegami), NHLBI Specialized Center of Research Grant HL-56387 (J. A. Whitsett and M. Ikegami), and NHLBI Training Grant HL-07752 (J. A. Reed).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. A. Whitsett, Div. of Neonatology and Pulmonary Biology, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229 (E-mail: whitj0{at}chmcc.org).

Received 8 September 1999; accepted in final form 7 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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