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 |
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 |
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 |
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 |
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.

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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).

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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.
|
|

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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 / .
|
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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).

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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.

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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 |
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.
 |
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