Surfactant metabolic consequences of overexpression of GM-CSF in the epithelium of GM-CSF-deficient mice

Machiko Ikegami1, Alan H. Jobe1, Jacquelyn A. Huffman Reed2, and Jeffrey A. Whitsett2

1 Department of Pediatrics, University of California Los Angeles School of Medicine, Harbor-University of California Los Angeles Medical Center, Torrance, California 90502; and 2 Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Granulocyte macrophage colony-stimulating factor (GM-CSF) is a regulator of surfactant metabolism because GM-CSF-deficient mice have abnormally slow clearance and catabolism of saturated phosphatidylcholine (Sat PC) and surfactant protein (SP)-A in airspaces and lung tissue. Overexpression of GM-CSF only in respiratory epithelial cells of mice deficient in GM-CSF using the SP-C promotor (GM-/-,SP-C-GM+/+) resulted in increased type II cell numbers and normalization of alveolar Sat PC pool sizes. Metabolic measurements demonstrated that incorporation of radiolabeled choline and palmitate was increased more than twofold, but the amount of radiolabeled Sat PC that accumulated in airspaces relative to the amount incorporated was decreased by 50% relative to normal GM+/+ mice. The clearance of dipalmitoylphosphatidylcholine and SP-B from the airspaces was more rapid for GM-/-,SP-C-GM+/+ mice than for GM+/+ mice. Loss of Sat PC and SP-B from the lungs (alveolar plus lung tissue) was similar in the two strains of mice. The normal surfactant pools in the GM-/-,SP-C-GM+/+ mice were achieved by the net effects of increases in type II cell numbers, increased incorporation, decreased accumulation, and increased reuptake rates for surfactant components, demonstrating the multiple effects of GM-CSF on surfactant metabolism.

dipalmitoylphosphatidylcholine; surfactant protein B; alveolar proteinosis; granulocyte macrophage colony-stimulating factor

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

GRANULOCYTE MACROPHAGE colony-stimulating factor (GM-CSF) is a 25-kDa glycoprotein normally expressed in a variety of hematopoietic and nonhematopoietic cells (5). GM-CSF was identified as a potent stimulator of granulocyte and macrophage precursors, and treatment of animals and humans with recombinant GM-CSF stimulates the bone marrow production of both red blood cell and white blood cell lineages (17). Nevertheless, GM-CSF null mutant mice (GM-/-) had normal steady-state hematopoiesis, probably because of redundancies in cytokine and growth factor regulatory pathways (8, 26). These GM-/- mice developed a progressive accumulation of lipid and protein in their airspaces that anatomically was similar to alveolar proteinosis in humans (8, 26). There is a single case report of the efficacy of GM-CSF for the treatment of alveolar proteinosis in humans (25).

In the GM-/- mice, the type II cells were normal in number and general appearance and had normal amounts of mRNA for the surfactant proteins (SP) SP-A, SP-B, and SP-C. However, alveolar and lung tissue pools of saturated phosphatidylcholine (Sat PC) were increased 10-fold, and alveolar pools of the surfactant proteins were increased severalfold (8). The major metabolic abnormalities were large decreases in the rates of alveolar clearance and catabolism of Sat PC and SP-A in the GM-/- mice (14). These results identified surfactant catabolic pathways as the major sites of abnormality induced by a systemic lack of GM-CSF. Both type II cells and macrophages normally participate in the catabolism of surfactant components (24, 28), and the site of the catabolic abnormality was not identified in the GM-/- mice (14). Type II cells normally express GM-CSF (4), and Huffman Reed et al. (12) recently reported that overexpression of GM-CSF only in respiratory epithelial cells using the SP-C promotor in GM-/- mice resulted in a correction of the lung abnormalities in that alveolar surfactant phospholipid and protein pools were normal. The overexpression of GM-CSF by the SP-C promotor could correct the abnormalities in surfactant clearance and catabolism or could alter other metabolic pathways to achieve a net effect of normal alveolar pools of surfactant. In the companion paper, Huffman Reed et al. (12) report that overexpression of GM-CSF caused proliferation of normal-appearing type II cells and an increase in alveolar macrophages. Therefore, to begin to explore how cytokines regulate surfactant pool sizes, we asked how overexpression of GM-CSF in respiratory epithelial cells influenced surfactant homeostasis. We have compared metabolic measurements of the surfactant components Sat PC and SP-B in normal mice, GM-CSF-deficient mice, and mice that overexpress GM-CSF only in respiratory epithelial cells.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Mice. The transgenic GM-CSF-deficient mice (referred to as GM-/-) were bred at the University of Cincinnati from C57BL/6/129SV F2 homozygous GM-CSF-deficient mice as described previously (11, 14). The comparison normal mice were C57 black mice (referred to as GM+/+). Two strains of mice overexpressing GM-CSF under control of the promotor for the human SP-C gene in respiratory epithelial cells only were previously described (11, 12). Strain 59, which overexpressed GM-CSF in the GM-CSF-deficient background, was used for all studies reported here (referred to as GM-/-,SP-C-GM+/+). All mice were bred at the University of Cincinnati and were housed under identical conditions at Harbor-University of California Los Angeles until study at 7-9 wk of age. The GM+/+ and GM-/- mice were from the same breeding colonies as previously used for studies of surfactant metabolism and for the description of the GM-CSF overproducing strains (11, 14).

Characterization of animals. Five mice from each group were killed with intraperitoneal pentobarbital sodium. After body weight and lung weight measurements, the lungs were homogenized in 4 ml of 0.9% NaCl, and aliquots were used for DNA and protein measurements according to Hill and Whatley (10) and Lowry et al. (19), respectively. Another 10-13 mice for each of three groups were used for recovery of surfactant by alveolar lavage. A 20-gauge blunt needle was tied into the proximal trachea after opening the chest, and five aliquots of 0.9% NaCl were flushed into the lungs and were withdrawn by syringe three times for each aliquot (14). Aliquots of alveolar lavage were extracted with chloroform-methanol (2:1), and Sat PC was isolated followed by phosphorous measurement (1, 20). GM-CSF was measured using alveolar lavage samples with the GM-CSF mini kit enzyme-linked immunosorbent assay (Endogen, Boston, MA) with a detection limit of <5 pg/ml (12).

Precursor incorporation into Sat PC and secretion. The three groups of mice were given an intraperitoneal injection with 13 µl/g body wt containing 0.013 µCi/µl [3H]choline chloride (NEN, Boston, MA) and 0.03 µCi/µl [14C]palmitic acid (American Radiolabeled Chemicals, St. Louis, MO). The palmitic acid had been stabilized in solution with 5% human serum albumin. Four or five mice from each group were killed with intraperitoneal pentobarbital at five preselected times from 3 to 48 h after isotope injection (14). For alveolar lavage, the chest of each animal was opened, and alveolar lavage with 0.9% NaCl was recovered as described above. The lavaged lung tissue was weighed and homogenized in 4 ml of 0.9% NaCl. Alveolar lavage and aliquots of the lung homogenates were extracted with chloroform-methanol (2:1), and Sat PC was isolated (20). The Sat PC was divided for measurement of phosphorous (1) and radioactivity. The total radioactivity recovered in Sat PC for the alveolar lavage and lung tissue of each animal was calculated. Percent accumulation of labeled Sat PC in alveolar lavage was calculated as the radioactivity in alveolar Sat PC divided by the total radioactivity in the alveolar lavage plus lung tissue times 100 (14). Pool sizes of Sat PC in alveolar lavage and lung tissue were calculated as micromoles per kilogram body weight.

Clearance of dipalmitoylphosphatidylcholine and SP-B. The three groups of mice were given intratracheal injections with 50 µl saline that contained 0.25 µCi [3H]choline-labeled dipalmitoylphosphatidylcholine (DPPC) and 0.02 µCi 125I-labeled SP-B. [3H]DPPC was purchased from Amersham (Arlington Heights, IL), and mouse SP-B was isolated from large-aggregate surfactant from alveolar lavages of GM-/- mice according to the method of Beers, Bates, and Fisher (3). Purified SP-B was iodinated with Bolton-Hunter reagent (Amersham) as described previously (27). The injection mixture was prepared by rotary evaporation from chloroform of [3H]DPPC, 125I-SP-B, and unlabeled DPPC (27). The DPPC and SP-B were resuspended with glass beads in 0.9% NaCl to achieve a final DPPC concentration of 0.1 µmol/ml. Iodinated SP-B and SP-B labeled in vivo with amino acids had similar clearance rates in adult rabbits, indicating that the iodinated protein can be used to study SP-B metabolism (27).

Mice were sedated with intraperitoneal ketamine, and the trachea of each mouse was exposed through a 0.5-cm midline skin incision in the neck (14). The radiolabeled surfactant was injected using a 1-ml syringe with a 30-gauge needle. Three minutes after injection, five mice from each of the three groups were killed with intraperitoneal pentobarbital sodium, and alveolar lavages were collected as described above. The lavaged lung tissue was weighed and homogenized in 0.9% NaCl. Other groups of four to six mice received alveolar lavages at times up to 40 h after tracheal injection. The amount of 125I in alveolar lavages and lung homogenates was measured. The alveolar lavages and lung homogenates subsequently were used for lipid extraction, Sat PC isolation (20), and quantification of recoveries of [3H]DPPC in association with Sat PC. The recoveries were normalized to the mean total recoveries for the mice that were killed 3 min after the tracheal injections. In a group of animals studied 6 h after intratrachel injection of [3H]DPPC, alveolar macrophages and other cells in the alveolar lavages were separated from surfactant by centrifugation of alveolar lavages layered over 0.8 M sucrose in 0.9% NaCl for 15 min at 500 g (24).

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

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Description of animals and pool sizes. The lungs of the GM-/-,SP-C-GM+/+ mice were heavier in absolute weight and relative to body weight (Table 1). The GM-/- mice also had an increased lung weight-to-body weight ratio. The lungs from the GM-/-,SP-C-GM+/+ mice also contained more protein and DNA. Alveolar lavages of the GM-/-,SP-C-GM+/+ mice contained 104 ± 16 ng/kg GM-CSF, and no GM-CSF was detected in alveolar lavages from the other genotypes. Although the GM-/-,SP-C-GM+/+ mice had a 50% increase in the amount of Sat PC in lung tissue relative to the control GM+/+ mice, the alveolar pool sizes were the same (Fig. 1). Relative to GM+/+ mice, the amounts of Sat PC were increased >10-fold in alveolar lavages and >4-fold in the total lungs of GM-/- mice.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Characterization of animals


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1.   Amount of saturated phosphatidylcholine (Sat PC) in alveolar lavages, lung tissue after alveolar lavage, and total lung (alveolar + tissue) of mice used for the incorporation studies. There are 25 mice for each genotype. Sat PC was increased in alveolar and tissue pools for granulocyte macrophage colony-stimulating factor (GM-CSF)-deficient mice (GM-/- mice). The amount of Sat PC in lung tissue also was increased for mice deficient in GM-CSF that express GM-CSF in lung epithelium with the surfactant protein (SP)-C promoter (GM-/-,SP-C-GM+/+ mice) relative to control mice (GM+/+). * P < 0.05 vs. GM+/+ and tt P < 0.01 vs. GM-/-.

Precursor incorporation and accumulation in alveolar lavage. The incorporations of radiolabeled palmitic acid and choline, which were given as body weight-adjusted doses, were measured as total incorporations into Sat PC and not as specific activities, because lung Sat PC pool sizes were different in the three genotypes. Incorporations of [14C]palmitate and [3H]choline into Sat PC were increased in the GM-/-,SP-C-GM+/+ mice at 3, 8, and 15 h after precursor injection relative to control GM+/+ and GM-/- mice (Figs. 2 and 3). At later times after precursor injection, increased radiolabeled Sat PC was present in the GM-/- mice because of the decreased catabolic rates (15). Accumulation of radiolabeled Sat PC in alveolar lavages calculated as percent total radioactivity in the alveolar lavages at times to 24 h was equivalent for the GM+/+ mice and GM-/- mice, and ~40% of the total radioactivity was recovered by alveolar lavage at 15 h (Fig. 4). In contrast, the percentage of radiolabeled Sat PC that accumulated in the airspace of GM-/-,SP-C-GM+/+ mice was decreased by ~50% relative to the other two genotypes. Because of increased incorporation into lung Sat PC, the total amount of radiolabeled Sat PC recovered by alveolar lavage was similar for the GM-/-,SP-C-GM+/+ mice and for the other genotypes at times from 3 to 15 h (Figs. 2 and 3).


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 2.   Recoveries of [14C]Sat PC at intervals to 48 h after ip [14C]palmitate given as a weight-adjusted dose. Recoveries are given for alveolar lavages (A), lung tissue (B), and total lungs (C). * P < 0.05 vs. GM+/+, ** P < 0.01 vs. GM+/+, t P < 0.05 vs. GM-/-, and tt P < 0.01 vs. GM-/-. DPM, disintegrations/min.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 3.   Recoveries of [3H]Sat PC at intervals to 48 h after ip [3H]choline given as a weight-adjusted dose. Recoveries are given for alveolar lavages (A), lung tissue (B), and total lung (C). * P < 0.05 vs. GM+/+, ** P < 0.01 vs. GM+/+, t P <0.06 vs. GM-/-, and tt P < 0.01 vs. GM-/-.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4.   Accumulation in airspaces of Sat PC radiolabeled with [14C]palmitic acid and [3H]choline. Accumulation was calculated as percent radioactivity recovered in alveolar lavage relative to total radioactivity in the total lungs of each animal. Curves for GM+/+ and GM-/- mice were not different. A lower percent of total lung radioactivity was recovered from the GM-/-,SP-C-GM+/+ mice. * P < 0.05 vs. GM+/+, ** P < 0.01 vs. GM+/+, t P <0.05 vs. GM-/-, and tt P < 0.01 vs. GM-/-.

Clearance of DPPC. Radiolabeled DPPC given via the trachea was cleared rapidly from the airspaces of the control GM+/+ mice such that 5.4 ± 1.4% was left at 40 h (Fig. 5). The expression of GM-CSF in lung epithelial cells in the GM-/-,SP-C-GM+/+ mice resulted in very rapid clearance of radiolabeled DPPC from the airspaces of these mice (96% loss vs. 78% loss for GM+/+ mice at 15 h). However, overall loss of radiolabeled DPPC from the total lungs was not different for GM+/+ and GM-/-,SP-C-GM+/+ mice. The percent of Sat PC in the macrophage and cell fraction of the alveolar lavages was 1.1 ± 0.2% for GM+/+ mice (n = 8) and 9.4 ± 1.7% for GM-/-,SP-C-GM+/+ mice (n = 6; P < 0.01). The recoveries of [3H]DPPC in macrophages as a percent of the total [3H]DPPC in alveolar lavages 6 h after intratracheal injection of [3H]DPPC were 2.6 ± 0.4 in GM+/+ mice and 10.7 ± 2.5 in GM-/-,SP-C-GM+/+ mice (P < 0.01).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5.   Recovery of [3H]DPPC after intratracheal administration in alveolar lavages and in total lung. * P < 0.05 vs. GM +/+ and ** P < 0.01 vs. GM +/+.

Clearance of SP-B. The loss of 125I-SP-B from the airspaces was increased in GM-/-,SP-C-GM+/+ mice relative to the control GM+/+ (Fig. 6), and these curves were similar to those for DPPC (Fig. 5 vs. Fig 6). The loss of labeled SP-B from the total lung was similar for the GM+/+ and GM-/-,SP-C-GM+/+ mice, and the curves were similar to those for DPPC.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6.   Recovery of 125I-labeled SP-B after intratracheal administration in alveolar lavages and in total lung. * P < 0.05 vs. GM +/+ and ** P < 0.01 vs. GM +/+.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Measurements of different components of surfactant metabolism in vivo demonstrate that the alveolar Sat PC pool size is normal in GM-/-,SP-C-GM+/+ mice despite increased precursor incorporation, decreased percent accumulation of radiolabeled Sat PC in airspaces, and increased clearance of Sat PC from the airspaces but not the total lung compartment. Assuming that the animal needs to maintain an acceptable surfactant homeostasis to survive, obligate abnormalities resulting from GM-CSF overexpression in respiratory epithelial cells must be compensated for if the animal is to survive. In this example, the compensation is almost complete in that the alveolar Sat PC pool size is normal, but Sat PC in lung tissue is increased. Mechanisms by which the normal lung regulates surfactant pool sizes are unknown (28). In the developing lung at term, expanded pool sizes are accomplished primarily by a decreased catabolic rate and increased efficiency of recycling (16). Agents such as amiodarone and chronic corticosteroid exposure also can chronically elevate surfactant pool sizes in animals, perhaps by interfering with lysosomal catabolic pathways (18, 29). Acute increases in surfactant pools follow stimulation of secretion by secretagogues or by exercise (6, 21). The effect of exercise is probably a combined response of type II cells to secretory agonists such as catecholamines and stretch of the alveolar walls with hyperventilation.

The GM-CSF-deficient mouse has normal-appearing type II cells but 10-fold increases in lung tissue and alveolar surfactant pools (11, 14). The increased pool sizes were explained by decreased alveolar clearance and lung catabolism of Sat PC and SP-A (14). In the GM-/-,SP-C-GM+/+ line 59 mouse, GM-CSF is overexpressed only by lung epithelial cells, and multiple effects on surfactant metabolism occur. In the companion paper, it was demonstrated that line 59 has normal-appearing but increased numbers of type II cells (12), and an increase in alveolar macrophages was reported previously (11). These changes in cell numbers are consistent with our findings of heavier lungs that contain more protein and DNA (Table 1). The changes in cell numbers complicate the interpretation of the metabolic data. Macrophages can take up and degrade Sat PC, SP-A, and SP-B (2, 16, 27). In the rabbit, ~20-30% of the catabolic activity for Sat PC can be attributed to macrophages, and 70-80% occurs within type II cells in lysosomal elements (24). Similar measurements are not available for the mouse. However, total Sat PC content of macrophages is not influencing alveolar pool size measurements very much because the macrophages in the lavage from GM+/+ mice and GM-/-,SP-C-GM+/+ mice contained 1.1 and 9.4% of lavage Sat PC, respectively. Measurements of the clearance of radiolabeled Sat PC and SP-B demonstrate more rapid loss of the surfactant components from the airspaces of the GM-/-,SP-C-GM+/+ mice than the GM+/+ mice. Therefore, uptake is accelerated, perhaps because more cells are available to take up a similar amount of alveolar surfactant per cell per unit time. However, the overall loss from the lungs is not changed, indicating equivalent net catabolic rates for the lungs and therefore lower catabolic rates for each cell. A conclusion therefore is that, although GM-CSF deficiency results in decreased catabolic rates, GM-CSF overexpression in type II cells does not result in increased catabolism of alveolar surfactant. Another result is that the initial rapid rate of clearance of Sat PC and SP-B at 3 and 8 h after intratracheal injection of radiolabeled surfactant had decreased by 24 and 48 h, a curve shape consistent with an increased recycling efficiency of the surfactant components (16). Macrophages rapidly degrade surfactant components (2, 25), and the lack of increased net loss of Sat PC and SP-B suggests that the increased clearance was to type II cells and not macrophages.

The results for the choline and palmitate incorporation studies suggest a somewhat different scenario. Incorporation of both precursors is increased more than twofold at 3 and 8 h after precursor injection. Assuming that there were similar precursor pools in type II cells of GM+/+ and GM-/-,SP-C-GM+/+ mice, then the increased incorporation may reflect the increased numbers of type II cells incorporating precursors at a similar rate per cell. Huffman Reed et al. (12) found a fourfold increase of type II cells in the lungs of 4- to 5-mo-old GM-/-,SP-C-GM+/+ mice, and these metabolic studies were performed in 2-mo-old mice that probably had fewer type II cells in the lungs. There was less accumulation of de novo synthesized Sat PC in airspaces. At early times after precursor injection, most of the radiolabeled Sat PC recovered by alveolar lavage will represent secretion. At later times, the amount of secreted radiolabeled Sat PC will be depleted by loss of components to recycling and catabolism. Therefore, we have used the term accumulation to refer to the net amount of radiolabeled Sat PC recovered by lavage after incorporation of precursors. The rate of loss of the radiolabeled Sat PC from lung tissue also was increased relative to the control mice. For example, the total amount of palmitate in Sat PC in the lungs decreased to 49% of the 8-h value at 40 h in GM+/+ mice and to 24% of the 8-h value at 40 h in GM-/-,SP-C-GM+/+ mice. This result suggests increased overall catabolism in GM-/-,SP-C-GM+/+ mice, a conclusion in conflict with the data for the overall catabolism of radiolabeled Sat PC and SP-B originating from the airspace. Sat PC is in type II cells and other lung structures in pathways not associated with surfactant (28). These separate data sets for precursor incorporation and for the fate of airspace surfactant components are measuring different aspects of Sat PC metabolism.

The multiple alterations in the surfactant system resulting from GM-CSF overexpression that yield as a net effect a normal alveolar surfactant pool size suggest as a working hypothesis that other cytokines or factors are acting to compensate for any negative physiological effect of the GM-CSF overexpression. GM-CSF is the first cytokine to be identified that can influence alveolar surfactant pool sizes chronically. Systemic deletion results in increased alveolar and tissue pools of surfactant (alveolar proteinosis), and replacement of GM-CSF only in bone marrow-derived cell lines by bone marrow transplantation or by cell-specific GM-CSF transgene expression in lung epithelial cells corrects the alveolar proteinosis (12, 22). Other factors such as keratinocyte growth factor and hepatocyte can increase type II cell number in adult animals and increase Sat PC pools in developing animals (13, 23). There are other models of dysregulation of surfactant in mice. The beige mouse model of Chediak-Higashi syndrome has modestly decreased alveolar pools of Sat PC that are maintained by altered Sat PC metabolism (9). Severe combined immunodeficient mice have about threefold increases in surfactant pools (15). There may be two levels of regulation or control of the surfactant system. GM-CSF is an example of a factor that seems to proportionately influence all of the major components of surfactant. Another level of regulation may be specific for different components of surfactant. For example, SP-B deficiency results in lethal respiratory failure at birth with disruption of the surfactant processing pathways but no alteration in the incorporation of precursors into Sat PC (7). The challenge for the future is to elucidate what factors are working in concert to regulate surfactant pool sizes at steady state.

    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants HD-11932 and HL-28623.

    FOOTNOTES

Address for reprint requests: M. Ikegami, Children's Hospital Medical Center, Division of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039.

Received 30 December 1996; accepted in final form 28 May 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Bartlett, G. R. Phosphorus assay in column chromatography. J. Biol. Chem. 234: 466-468, 1959[Free Full Text].

2.   Bates, S. R., M. F. Beers, and A. B. Fisher. Binding and uptake of surfactant protein B by alveolar type II cells. Am. J. Physiol. 263 (Lung Cell. Mol. Physiol. 7): L333-L334, 1992[Abstract/Free Full Text].

3.   Beers, M. G., S. R. Bates, and A. B. Fisher. Differential extraction for the rapid purification of bovine surfactant protein B. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L773-L778, 1992[Abstract/Free Full Text].

4.   Blau, H., S. Riklis, V. Kravtsov, and M. Kalina. Secretion of cytokines by rat alveolar epithelial cells: possible regulatory role for SP-A. Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L148-L155, 1994[Abstract/Free Full Text].

5.   Burgess, A. W., J. Camakaris, and D. Metcalf. Purification and properties of colony-stimulating factor from mouse lung-conditioned medium. J. Biol. Chem. 252: 1998-2003, 1977[Abstract].

6.   Chander, A., and A. B. Fisher. Regulation of lung surfactant secretion. Am. J. Physiol. 258 (Lung Cell. Mol. Physiol. 2): L241-L253, 1990[Abstract/Free Full Text].

7.   Clark, J. C., T. E. Weaver, H. S. Iwamoto, M. Ikegami, A. H. Jobe, W. M. Hull, and J. A. Whitsett. Decreased lung compliance and air trapping in heterozygous SP-B deficient mice. Am. J. Respir. Cell Mol. Biol. 16: 46-52, 1997[Abstract].

8.   Dranoff, G., A. D. Crawford, M. Sadelain, B. Ream, A. Rashid, R. T. Bronson, G. R. Dickersin, C. J. Bachurski, E. L. Mark, J. A. Whitsett, and R. C. Mulligan. Involvement of granulocyte-macrophage colony-stimulating factor in pulmonary homeostasis. Science 264: 713-716, 1994[Medline].

9.   Gross, N. J., E. Barnes, and K. R. Narine. Recycling of surfactant in black and beige mice: pool sizes and kinetics. J. Appl. Physiol. 64: 2017-2025, 1988[Abstract/Free Full Text].

10.   Hill, B. T., and S. Whatley. A simple, rapid microassay for DNA. FEBS Lett. 56: 20-23, 1975[Medline].

11.   Huffman, J. A., W. M. Hull, G. Dranoff, R. C. Mulligan, and J. A. Whitsett. Pulmonary epithelial cell expression of GM-CSF corrects the alveolar proteinosis in GM-CSF-deficient mice. J. Clin. Invest. 97: 649-655, 1996[Abstract/Free Full Text].

12.   Huffman Reed, J. A., W. R. Rice, Z. Zengellér, S. E. Wert, G. Dranoff, and J. A. Whitsett. GM-CSF enhances lung growth and causes alveolar type II epithelial cell hyperplasia in transgenic mice. Am. J. Physiol. 273 (Lung. Cell. Mol. Physiol. 17): L715-L725, 1997[Medline].

13.   Ikegami, M., A. H. Jobe, and A. M. Havill. Keratinocyte growth factor increases surfactant pool sizes in premature rabbits. Am. J. Respir. Crit. Care Med. 155: 1155-1158, 1997[Abstract].

14.   Ikegami, M., T. Ueda, W. Hull, J. A. Whitsett, R. C. Mulligan, G. Dranoff, and A. H. Jobe. Surfactant metabolism in transgenic mice after granulocyte macrophage-colony stimulating factor ablation. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14): L650-L658, 1996[Abstract/Free Full Text].

15.   Jennings, V. M., D. L. Dillehay, S. K. Webb, and L. A. S. Brown. Pulmonary alveolar proteinosis in SCID mice. Am. J. Respir. Cell Mol. Biol. 13: 297-306, 1995[Abstract].

16.   Jobe, A. H., and M. Ikegami. Surfactant metabolism. Clin. Perinatol. 20: 683-696, 1993[Medline].

17.  Jones, T. C. Future uses of granulocyte-macrophage colony stimulating factor (GM-CSF). Stem Cells 12, Suppl. 1: 229-240, 1994.

18.   Kodavanti, U. P., and H. M. Mehendale. Amiodarone- and desethylamiodarone-induced pulmonary phospholipidosis, inhibition of phospholipases in vivo, and alteration of [14C]amiodarone uptake by perfused lung. Am. J. Respir. Cell Mol. Biol. 4: 369-378, 1991[Medline].

19.   Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951[Free Full Text].

20.   Mason, R. J., J. Nellenbogen, and J. A. Clements. Isolation of disaturated phosphatidylcholine with osmium tetroxide. J. Lipid Res. 17: 281-284, 1976[Abstract].

21.   Nicholas, T. E., J. H. T. Power, and H. A. Barr. Surfactant homeostasis in the rat during swimming exercise. J. Appl. Physiol. 53: 1521-1528, 1982[Abstract/Free Full Text].

22.   Nishinakamura, R., R. Wiler, U. Dirksen, Y. Morikawa, K. Arai, B. S. Miyajima, and R. Murray. The pulmonary alveolar proteinosis in granulocyte macrophage colony-stimulating factor/interleukins 3/5 beta c receptor-deficient m is reversed by bone marrow transplantation. J. Exp. Med. 183: 2657-2662, 1996[Abstract].

23.   Panos, R. J., P. M. Bak, W. S. Simonet, J. S. Rubin, and L. J. Smith. Intratracheal instillation of keratinocyte growth factor decreases hyperoxia-induced morality in rats. J. Clin. Invest. 96: 2026-2033, 1995[Medline].

24.   Rider, E. D., M. Ikegami, and A. H. Jobe. Localization of alveolar surfactant clearance in rabbit lung cells. Am. J. Physiol. 263 (Lung Cell. Mol. Physiol. 7): L201-L209, 1992[Abstract/Free Full Text].

25.   Seymour, J. F., J. M. Vincent, J. J. Presneill, and M. C. Pain. Efficacy of GM-CSF in acquired alveolar proteinosis (Abstract). N. Engl. J. Med. 335: 1924, 1996[Free Full Text].

26.   Stanley, E., G. J. Lieschke, D. Grail, D. Metcalf, G. Hodgson, J. A. M. Gall, W. Darryl, J. Cebon, V. Sinickas, and A. R. Dunn. Granulocyte/macrophage colony-stimulating factor-deficient mice show no major perturbation of hematopoiesis but develop a characteristic pulmonary pathology. Proc. Natl. Acad. Sci. USA 91: 5592-5596, 1994[Abstract].

27.   Ueda, T., M. Ikegami, M. Henry, and A. H. Jobe. Clearance of surfactant protein B from rabbit lungs. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L636-L641, 1995[Abstract/Free Full Text].

28.   Wright, J. R. Clearance and recycling of pulmonary surfactant. Am. J. Physiol. 259 (Lung Cell. Mol. Physiol. 3): L1-L12, 1990[Abstract/Free Full Text].

29.   Young, S. L., and R. Silbajoris. Dexamethasone increases adult rat lung surfactant lipids. J. Appl. Physiol. 60: 1665-1672, 1986[Abstract/Free Full Text].


AJP Lung Cell Mol Physiol 273(4):L709-L714
1040-0605/97 $5.00 Copyright © 1997 the American Physiological Society