EDITORIAL FOCUS
GM-CSF regulates protein and lipid catabolism by alveolar
macrophages
Mitsuhiro
Yoshida,
Machiko
Ikegami,
Jacquelyn A.
Reed,
Zissis C.
Chroneos, and
Jeffrey A.
Whitsett
Division of Pulmonary Biology, Children's Hospital Medical Center,
Cincinnati, Ohio 45229-3039
 |
ABSTRACT |
Metabolism of surfactant protein (SP) A and
dipalmitoylphosphatidylcholine (DPPC) was assessed in alveolar
macrophages isolated from granulocyte-macrophage colony-stimulated
factor (GM-CSF) gene-targeted [GM(
/
)] mice, wild-type mice, and
GM(
/
) mice expressing GM-CSF under control of the SP-C promoter
element (SP-C-GM). Although binding and uptake of 125I-SP-A
were significantly increased in alveolar macrophages from GM(
/
)
compared with wild type or SP-C-GM mice, catabolism of 125I-SP-A was markedly decreased in GM(
/
) mice.
Association of [3H]DPPC with alveolar macrophages
from GM(
/
), wild-type, and SP-C-GM mice was similar; however,
catabolism of DPPC was markedly reduced in cells from GM(
/
) mice.
Fluorescence-activated cell sorter analysis demonstrated decreased
catabolism of rhodamine-labeled dipalmitoylphosphatidylethanolamine
by alveolar macrophages from GM(
/
) mice. GM-CSF deficiency was
associated with increased SP-A uptake by alveolar macrophages but with
impaired surfactant lipid and SP-A degradation. These findings
demonstrate the important role of GM-CSF in the regulation of alveolar
macrophage lipid and SP-A catabolism.
pulmonary alveolar proteinosis; granulocyte-macrophage
colony-stimulating factor
 |
INTRODUCTION |
GRANULOCYTE-MACROPHAGE
colony-stimulating factor (GM-CSF) is a 23-kDa protein initially
identified as a factor mediating the proliferation and differentiation
of hematopoietic cells in the granulocyte-macrophage lineage (see Ref.
27 for a review). GM-CSF binds to a dimeric receptor
complex consisting of
- and common
-chains, the latter shared
with interleukin-3 and interleukin-5 signaling pathways. Targeted
ablation of either GM-CSF [GM(
/
)] or the common
-chain
of the GM-CSF receptor caused pulmonary alveolar proteinosis (PAP)
associated with a marked accumulation of surfactant protein (SP) A,
SP-B, SP-C, SP-D, and lipids in the alveolar spaces of the lungs in
mice (7, 11, 17, 22, 25). Although metabolic studies
(11) demonstrated that the synthesis of surfactant
components was relatively unperturbed in the lungs of GM(
/
) mice,
clearance of SPs and lipids was markedly decreased, suggesting
that either the uptake, catabolism, or recycling of surfactant
components by alveolar epithelial cells or alveolar macrophages was
regulated by GM-CSF (11). Abnormalities in alveolar
macrophage function and morphology in the GM-CSF and common
-chain
gene-targeted animals were noted primarily in the lung, GM(
/
) mice
being more susceptible to pulmonary infection and developing a marked
accumulation of surfactant in alveolar spaces (7, 11, 16, 17, 22,
25). Although systemic administration of GM-CSF did not correct
the increased surfactant concentrations, expression of recombinant
GM-CSF in the lungs of GM(
/
) mice corrected surfactant homeostasis
in vivo whether GM-CSF was delivered by aerosol (20) or
adenoviral gene transfer vector (30) or whether GM-CSF was
expressed under control of the SP-C promoter element (SP-C-GM) in
transgenic mice (10). Common
-chain-deficient mice also
developed PAP that was substantially ameliorated by transplantation of
hematopoietic precursors expressing the common
-chain receptor
(5), supporting the concept that dysfunction of cells of
hematopoietic origin contributed to the pulmonary abnormalities
observed in the common
-chain-deficient mice.
Surfactant homeostasis is maintained by mechanisms controlling
surfactant synthesis, secretion, uptake, catabolism, and recycling. Surfactant lipids are synthesized, secreted, catabolized, and recycled
by type II epithelial cells. Catabolism of surfactant lipids is also
mediated by alveolar macrophages that account for the clearance of
~20% of the phospholipids from the air spaces in normal adult
rabbits (21). Because GM-CSF affects both proliferation and differentiation of type II cells and alveolar macrophages (10), it remains unclear whether the PAP associated with
the deficiency of GM-CSF signaling is mediated primarily by changes in
respiratory epithelial or alveolar macrophage cell function in vivo.
Furthermore, it is unclear whether binding, uptake, or degradation of
surfactant components by alveolar macrophages are influenced by GM-CSF.
Therefore, we hypothesized that GM-CSF plays a role in the regulation
of binding, uptake, and catabolism of protein and lipids by alveolar macrophages.
 |
METHODS |
Transgenic mice.
GM(
/
) mice were generated by gene-targeted ablation of the GM-CSF
locus previously described by Dranoff et al. (7) and have
been maintained in the C57BL/6 background for several years. Bitransgenic mice bearing a chimeric gene consisting of the 3.7-kb human SP-C gene promoter that drives expression of mouse GM-CSF were
maintained in the GM(
/
) mutant background (SP-C-GM), generating mice in which GM-CSF was selectively expressed at increased levels in
the lungs of GM(
/
) mice, the PAP in the GM(
/
) mutant being corrected by the SP-C-GM transgene (10). Control C57BL/6
mice, termed wild type (WT), were purchased from Jackson Laboratories (Bar Harbor, ME). All of the mice used were housed and studied in the
Animal Facility of the Children's Hospital Research Foundation (Cincinnati, OH) under approved procedures of the Institutional Animal
Care and Use Committee. Mice were used between 8 and 10 wk of age. The
animals were maintained in a pathogen-free barrier facility, and
analysis of sentinel mice revealed no evidence of pathogens.
Alveolar macrophage isolation.
Alveolar cells were obtained by bronchoalveolar lavage by instilling
ten 1-ml aliquots of phosphate-buffered saline (PBS) containing 0.5 mM
EDTA. Alveolar cells obtained from two to eight mice from each group
were pooled and used for analysis. After centrifugation at 1,000 g for 5 min, the cell pellet was resuspended in 15% bovine
serum albumin (BSA), PBS, and 10 mM EDTA followed by centrifugation for
removal of the surfactant lipid and SPs (8, 23). The cells
were then resuspended in Dulbecco's modified Eagle's medium (DMEM)
containing 0.1% BSA, cultured at a density of 1 × 105 cells/well in flat-bottom, 96-well tissue culture
plates, and allowed to adhere for 1 h at 37°C. The nonadherent
cells were removed, and the adherent cells were washed three times with
DMEM containing 0.1% BSA. Peritoneal macrophages were isolated by
peritoneal lavage in GM(
/
), SP-C-GM, and WT mice.
SP-A binding, uptake, and degradation by macrophages.
SP-A (a generous gift from Dr. G. Ross, Children's Hospital,
Cincinnati, OH) was isolated from the lung lavage fluid of patients with alveolar proteinosis (9). The SP-A used here had no
detectable endotoxin (<0.06 endotoxin unit/ml) as tested with the
Limulus amebocyte lysate assay (Sigma, St. Louis, MO). SP-A
was iodinated with chloramine T (3). The specific activity
of the SP-A preparations was between 400 and 700 counts · min
1 · ng
protein
1, and >92% of the radioactive protein was
precipitable with trichloroacetic acid (TCA). 125I-SP-A was
stored at 4°C and used within 3 days of iodination.
To measure binding of 125I-SP-A, the cells were incubated
for 3 h at 4°C with various concentrations from 0.5 to 16 µg/ml of 125I-SP-A. Each well contained at least 2 × 104 counts/min. To terminate the experiment, the medium
was removed and the cells were washed three times with DMEM containing
0.1% BSA. The cells were then dissolved in 0.2 N NaOH, and the
radioactivity was measured.
To determine degradation of SP-A, the cells were incubated for 4 h
at 37°C with 1 µg/ml of 125I-SP-A. Initial time-course
studies of the degradation demonstrated that catabolism was linear for
1-4 h under these conditions. The supernatants were then collected
and assayed for TCA-soluble, 125I-labeled degradation
products according to the procedure previously described
(29). The cells were washed with sodium acetate buffer to
remove the surface-bound 125I-SP-A and then washed three
times with the medium. Finally, the cells were lysed in 0.2 N NaOH, and
the radioactivity was counted to assess intracellular
125I-SP-A. Total uptake was calculated from the sum of
degradation and cell-associated label. Background counts, measured in
the absence of cells, associated with empty wells, or found in the medium as degradation products, were subtracted from the
cell-associated radioactivity.
Separate wells containing identical aliquots of the cells without
radiolabeled SP-A were utilized to determine the amount of cellular
protein per well in each experiment. The cellular protein was measured
by the Bradford (2) method, and the results are expressed
as nanograms of SP-A per microgram of cellular protein. The average
total protein contents per 105 cells from WT, SP-C-GM, and
GM(
/
) mice were similar: 14.8, 16.9, and 15.5 µg, respectively.
Analysis of binding data.
Binding data were analyzed graphically according to Scatchard analysis
or the Hughes-Klotz equation (19) to estimate the number
of SP-A binding sites per cell and the affinity constant or multimeric
dissociation constant (Kd) for SP-A binding to
macrophages. An estimated molecular weight of 650,000 for SP-A and
100,000 cells/100-µl assay were used in the calculations. The binding constants for each experiment (n = 5-6) were
obtained separately.
Dipalmitoylphosphatidylcholine association, uptake, and
degradation by alveolar macrophages.
Natural surfactant was isolated from normal mouse lung lavage fluid
(11). The surfactant was resuspended in PBS and stored in
aliquots at
20°C. The natural surfactant was labeled by mixing with
dipalmitoylphosphatidylcholine
[1,2-dipalmitoyl-L-3-phosphatidyl[N-methyl-3H]choline
([3H]DPPC), Amersham, Arlington Heights, IL]. The final
suspension in the medium contained 100 µg/ml of phospholipid
with 10 µCi/ml of [3H]DPPC radioactivity. To
measure the binding of [3H]DPPC, the cells
were incubated for 4 h at 4°C with the medium containing the
labeled natural surfactant. After incubation, the supernatant was
removed, and the cells were lysed with radioimmunoprecipitation assay
buffer (50 mM Tris · HCl, pH 8, 150 mM NaCl, 1% Nonidet P-40,
0.1% sodium dodecyl sulfate, 0.1% sodium deoxycholate, and 5 mM EDTA)
to measure the cell-associated radioactivity. To determine the
degradation of [3H]DPPC, the cells were incubated for 1, 3, and 5 h at 37°C with the [3H]DPPC-labeled
surfactant in culture medium. At each time point, the supernatant was
collected, and the cells were washed three times with DMEM containing
0.1% BSA followed by cell lysis with radioimmunoprecipitation assay
buffer. The lipid and aqueous fractions were extracted from both the
supernatants and cells according to Bligh and Dyer (1).
The degradation of [3H]DPPC by alveolar macrophages was
estimated by measuring the generation of radioactive products
partitioning in the water-methanol phase during the extraction.
Background counts in the absence of the cells were subtracted. Results
are expressed as counts per minute per microgram of cellular protein.
Fluorescently labeled surfactant uptake by macrophages.
N-rhodamine dipalmitoylphosphatidylethanolamine
(R-DPPE; Avanti Polar Lipids, Alabaster, AL) was mixed with
natural surfactant at a ratio of 1:1 (wt/wt). The final suspension in
the medium contained 100 µg/ml of phospholipid. Adherent alveolar
macrophages were incubated with the fluorescently labeled surfactant at
37°C. After a 30-min incubation, the supernatant was removed and the cells were washed three times with DMEM containing 0.1% BSA. The cells
were then harvested with trypsin-EDTA treatment for
fluorescence-activated cell sorter (FACS) analysis. The fluorescence
intensity associated with the cells after trypsinization was regarded
as lipid uptake by the macrophages. The cells in a separate culture
well were incubated for another 4 h, trypsinized, and assessed by
FACS analysis. The cells incubated with 100 µg/ml of nonlabeled
natural surfactant were used as controls. Samples of labeled cells were
also imaged by fluorescence microscopy and photographed.
Statistical analyses.
Results are expressed as means ± SE and evaluated for
significance by analysis of variance. The level of significance was taken at P < 0.05.
 |
RESULTS |
Binding, uptake, and degradation of 125I-SP-A.
Binding of 125I-SP-A to purified alveolar macrophages from
GM(
/
), SP-C-GM, and WT mice was compared in vitro. Binding was time dependent, complete by 3-4 h of incubation, and saturable.
Unexpectedly, binding of 125I-SP-A to alveolar macrophages
from GM(
/
) mice was significantly greater than that in WT mice,
whereas the number of SP-A binding sites on alveolar macrophages from
SP-C-GM mice was significantly less than that in WT mice (Fig.
1). Scatchard analysis revealed the
presence of a single class of binding sites in WT mice (Fig. 2), with a Kd of
14.8 ± 3.4 nM (Table 1). The number
of binding sites per cell (1.1 × 106 ± 0.12 × 106) estimated from the Scatchard plot (Table
1) in macrophages from the WT mouse was similar to the value of
0.97 × 106 ± 0.29 × 106
previously reported in rat alveolar macrophages (3). Human SP-A bound to alveolar macrophages from SP-C-GM mice with a similar Kd; however, the number of binding sites per
cell was significantly decreased in SP-C-GM (~60%) mice compared
with that in WT mice (Table 1). In contrast to findings in WT and
SP-C-GM mice, the binding constants for SP-A binding to alveolar
macrophages from GM(
/
) mice could not be reliably estimated by
Scatchard analysis because the shape of this curve was nearly flat
(Fig. 2A). To further evaluate the binding characteristics
of SP-A to macrophages, the data were analyzed according to the
Hughes-Klotz double-reciprocal equation (19). As shown in
Fig. 2B, the binding data from both WT and GM(
/
) mice
were fit to a straight line. The Kd and the number of binding sites estimated by the Hughes-Klotz equation for
binding of SP-A to WT macrophages were similar to the results obtained
by Scatchard analysis (Table 1). Analysis of the data from the
GM(
/
) macrophages revealed a large number of low-affinity binding
sites, with a Kd of 167 ± 62.5 nM and
4.54 × 106 ± 1.46 × 106
sites/cell in this model. Interestingly, the data from SP-C-GM macrophages were not well fit to a straight line (Fig. 2B).
The convex shape of this curve may indicate negative cooperativity or
multiple classes of binding sites in the cells from SP-C-GM mice.
Because alveolar macrophages from GM(
/
) mice express a single class
of low-affinity binding sites, it is suggested that the high-affinity
binding site in WT alveolar macrophages may be distinct from those
affinity sites or perhaps is composed of more than one subunit in which
affinity is influenced by GM-CSF. The uptake of 125I-SP-A
by alveolar macrophages was significantly increased in cells from
GM(
/
) mice compared with that in WT mice and significantly decreased in cells from SP-C-GM mice, suggesting a relationship between
internalization and the density of binding sites for SP-A on the
macrophage surface (Fig. 3).

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Fig. 1.
Binding of surfactant protein (SP) A by alveolar
macrophages. Alveolar macrophages (1 × 105/well) were
incubated with various concentrations of 125I-SP-A at 4°C
for 3 h. , Granulocyte-macrophage
colony-stimulating factor (GM-CSF)-deficient [GM( / )]
mice; , wild-type (WT) mice; ,
GM( / ) mice expressing GM-CSF under control of the SP-C promoter
element (SP-C-GM). Values are means ± SE; n = 6 experiments. SP-A binding to alveolar macrophages from GM( / ) mice
was significantly greater than that in WT mice, whereas SP-A binding to
alveolar macrophages from SP-C-GM mice was significantly less than that
in WT mice. *P < 0.05 compared with WT mice.
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Fig. 2.
Analysis of 125I-SP-A binding data. The
binding data from the saturation curves depicted in Fig. 1 were
analyzed according to Scatchard analysis (A) and the
Hughes-Klotz equation (B). The binding constants for binding
of SP-A to alveolar macrophages from the WT ( ) and
SP-C-GM ( ) mice shown in Table 1 were derived from the
slopes and intercepts of the linear curves in A. The binding
constants for binding to alveolar macrophages from the GM( / ) mice
( ) were derived from the linear curve in B
(19). Values are means ± SE; n = 6 experiments.
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Fig. 3.
Uptake of SP-A by alveolar macrophages. Alveolar
macrophages (1 × 105/well) were incubated with 1 µg/ml of 125I-SP-A at 37°C for 4 h. Total uptake
was calculated from the sum of degradation products in the medium and
cell-associated radioactivity. Values are means ± SE;
n = 6 experiments. SP-A uptake to alveolar macrophages
from GM( / ) mice was significantly greater than that in WT mice,
whereas SP-A binding to alveolar macrophages from SP-C-GM mice was
significantly less than that in WT mice. * P < 0.05 compared with WT mice.
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Although the number of SP-A binding sites was markedly increased in the
GM(
/
) mice, the degradation of 125I-SP-A as determined
by the generation of TCA-soluble fragments at 37°C was markedly
decreased in alveolar macrophages from the GM(
/
) mice (Fig.
4). Degradation of 125I-SP-A
by alveolar macrophages from GM(
/
) mice was approximately fivefold
less than that in either WT or SP-C-GM mice. There were no significant
differences in the degradation of 125I-SP-A by alveolar
macrophages from WT mice compared with that in SP-C-GM mice (Fig. 4).

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Fig. 4.
Degradation of SP-A by alveolar macrophages. Alveolar
macrophages (1 × 105/well) were incubated with 1 µg/ml of 125I-SP-A at 37°C for 4 h. TCA-soluble
degradation products were measured in the medium. Values are means ± SE; n = 6 experiments. Degradation of
125I-SP-A by alveolar macrophages from GM( / ) mice was
significantly less than that in WT or SP-C-GM mice.
* P < 0.05 compared with WT mice.
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Cell association and degradation of DPPC by alveolar macrophages.
To assess whether GM-CSF influenced cell association or uptake or
degradation of surfactant lipids, natural surfactant lipids were
labeled with tracer quantities of [3H]DPPC. Initial
studies determining the association of the labeled lipid with alveolar
macrophages at 4°C in vitro showed that [3H]DPPC was
associated with alveolar macrophages in a time-dependent manner, which
plateaued by 4 h. The amount of cell-associated [3H]DPPC was similar in alveolar macrophages from WT and
SP-C-GM mice. Association of [3H]DPPC with alveolar
macrophages from GM(
/
) mice at 4°C was significantly decreased
compared with that in WT or SP-C-GM mice, although these differences
were relatively small (Fig. 5). As seen
in the studies with 125I-SP-A, the catabolism
of [3H]DPPC by alveolar macrophages from GM(
/
)
mice was markedly decreased compared with that in WT or
SP-C-GM mice (Fig. 6). Degradation of
[3H]DPPC by alveolar macrophages from the GM(
/
)
mice was approximately fourfold less than that from WT mice. The
degradation of [3H]DPPC by alveolar macrophages from
SP-C-GM transgenic mice was significantly increased compared with that
in WT mice (Fig. 6).

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Fig. 5.
Binding of dipalmitoylphosphatidylcholine (DPPC) by
alveolar macrophages. Alveolar macrophages (1 × 105/well) were incubated with the natural surfactant
suspension containing 100 µCi/ml of phospholipid with 10 µCi/ml of
[3H]DPPC activity at 4°C for 4 h. cpm, Counts/min.
Values are means ± SE; n = 6 experiments. DPPC
binding to alveolar macrophages from GM( / ) mice was significantly
but modestly decreased compared with that in WT mice, whereas DPPC
binding to alveolar macrophages from SP-C-GM mice was similar to that
in WT mice. * P < 0.05 compared with WT mice.
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Fig. 6.
Degradation of [3H]DPPC by alveolar
macrophages. Alveolar macrophages (1 × 105/well) were
incubated with the natural surfactant suspension containing 100 µg/ml
of phospholipid with 10 µCi/ml of [3H]DPPC activity at
37°C for 1-5 h. Degradation of [3H]DPPC by
alveolar macrophages was estimated by measuring the generation of
radioactive products partitioning in the water-methanol phase during
the extraction from both the supernatants and cells. ,
GM( / ) mice; , WT mice; , SP-C-GM
mice. Values are means ± SE; n = 6 experiments.
Degradation of [3H]DPPC by alveolar macrophages from
GM( / ) mice was significantly less than that in WT mice, whereas
lipid degradation was significantly increased in SP-C-GM than in WT
mice. * P < 0.05 compared with WT mice.
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DPPC degradation by peritoneal macrophages.
To determine whether the observed catabolic defect in alveolar
macrophages from GM(
/
) mice was restricted to the lung, catabolism of [3H]DPPC by peritoneal macrophages was compared in
GM(
/
), WT, and SP-C-GM mice. In WT mice, degradation of
[3H]DPPC by peritoneal macrophages was less than that by
alveolar macrophages (Fig. 7).
Degradation of [3H]DPPC by peritoneal macrophages
from both GM(
/
) and SP-C-GM mice was approximately twofold less
than that by peritoneal macrophages from WT mice (Fig. 7).
Interestingly, there were no differences in the degradation of
[3H]DPPC by peritoneal macrophages from GM(
/
) mice
compared with that in SP-C-GM mice, supporting the concept that local
production of GM-CSF influences DPPC degradation by macrophages.

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Fig. 7.
Degradation of [3H]DPPC by peritoneal
macrophages. Peritoneal macrophages and alveolar macrophages (1 × 105/well) were incubated with a natural surfactant
suspension containing 100 µg/ml of total phospholipid with 10 µCi/ml of [3H]DPPC at 37°C for 5 h. The
degradation of [3H]DPPC by macrophages was estimated by
measuring the generation of radioactive products partitioning in the
water-methanol phase during the extraction from both the supernatants
and cells. Extent of degradation was calculated as percent degradation
in alveolar macrophages from WT mice [=100% (control)]. Values are
means ± SE; n = 6 experiments. Degradation of
[3H]DPPC by peritoneal macrophages from GM( / ) and
SP-C-GM [in GM( / ) background] mice was significantly less than
that in WT control mice. * P < 0.05 compared with
WT mice.
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Uptake and degradation of R-DPPE.
Uptake and degradation of fluorescence R-DPPE surfactant by alveolar
macrophages were studied by FACS and fluorescence microscopy. Images
observed by fluorescence microscopy showed that there was cellular
heterogeneity in the level of uptake by alveolar macrophages but that
the amount of uptake of R-DPPE by alveolar macrophages from GM(
/
),
WT, and SP-C-GM mice was similar (Fig.
8). Likewise, FACS analysis consistently
demonstrated that the fluorescence intensity of labeled alveolar
macrophages 30 min after incubation with the labeled lipid was similar
in all strains of mice. However, after 4.5 h of continued
incubation at 37°C, fluorescence of R-DPPE was decreased in alveolar
macrophages from WT and SP-C-GM mice but persisted in cells from
GM(
/
) mice, consistent with the observed decreased catabolism of
the radiolabeled lipid by alveolar macrophages from GM(
/
) mice
(Fig. 9).

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Fig. 8.
Fluorescence microscopy of rhodamine-labeled lipid uptake by
alveolar macrophages. Alveolar macrophages were incubated with natural
surfactant containing rhodamine-labeled
dipalmitoylphosphatidylethanolamine (R-DPPE) at 37°C for 30 min.
After incubation, the cells were harvested with trypsin-EDTA,
centrifuged, and observed by fluorescence microscopy. Alveolar
macrophages from WT (A), SP-C-GM (B), and
GM( / ) (C) mice took up similar amounts of
R-DPPE.
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Fig. 9.
Fluorescence-activated cell sorter (FACS) analysis of
fluorescence-labeled lipid uptake and degradation by alveolar
macrophages. Alveolar macrophages from WT (A), SP-C-GM
(B), and GM( / ) (C) mice were incubated with
natural surfactant labeled with R-DPPE at 37°C for 30 min. The cells
incubated with nonlabeled natural surfactant were used as controls.
After incubation, the cells were harvested with trypsin-EDTA and
assessed by FACS analysis. The cells were also incubated for 4 more h
and then analyzed. The shift in labeled cells indicated a loss of
labeled R-DPPE that was evident in WT and SP-C-GM mice. Labeled R-DPPE
persisted in alveolar macrophages from GM( / ) mice. Results are
representative of at least 3 separate experiments. Thick line, 30 min;
thin line, 4.5 h; dotted line, control (autofluorescence).
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 |
DISCUSSION |
Catabolism but not binding and uptake of SP-A and phospholipid was
markedly impaired in alveolar macrophages from GM-CSF(
/
) mice.
Prolonged local expression of GM-CSF in the lungs of SP-C-GM mice restored catabolic activity in alveolar macrophages but
not in peritoneal macrophages, demonstrating a critical role for local GM-CSF in the regulation of surfactant degradation by alveolar macrophages. Unexpectedly, SP-A uptake was significantly increased in
the alveolar macrophages from GM(
/
) mice and was reduced in SP-C-GM
mice compared with WT control mice. The affinity and number of SP-A
binding sites on the alveolar macrophages were also influenced by
GM-CSF genotype. Taken together, the present findings support the
concept that the decreased clearance of SPs and lipids in GM(
/
)
mice is mediated, at least in part, by the decreased catabolism of
surfactant components by alveolar macrophages.
The precise receptors or mechanisms mediating SP and lipid binding,
uptake, and catabolism remain relatively poorly understood. The
increased binding and uptake of SP-A observed in the alveolar macrophages from GM(
/
) mice were associated with changes in both
the affinity and increased number of SP-A binding sites, supporting a
role for GM-CSF-dependent pathways in the regulation of SP-A binding
sites on alveolar macrophages. These findings support previous
observations (4) that binding of SP-A decreased during
GM-CSF-induced differentiation of monocytes in vitro. Although Scatchard analysis suggests the presence of a single class of SP-A
binding sites on alveolar macrophages from WT mice, heterogeneity and/or changes in cooperativity were observed in binding studies with
cells from GM(
/
) mice. An increased number of relatively low-affinity 125I-SP-A binding sites was correlated with
increased SP-A uptake by alveolar macrophages from GM(
/
) mice,
suggesting a relationship between binding site number and the uptake
process. Because prolonged expression and administration of GM-CSF were
required for correction of PAP in GM(
/
) mice, changes in surfactant
homeostasis by GM-CSF are likely dependent on the effects of GM-CSF in
alveolar macrophage differentiation that, in turn, influence SP-A
binding and surfactant catabolism. GM(
/
) mice develop severe PAP
associated with marked increases in alveolar SP-A concentrations
[increased >10-fold in adult GM(
/
) mice] in the absence of
changes in SP-A mRNA, supporting an important role for surfactant
clearance in the pathogenesis of the disorder (7). Thus
alveolar macrophages in GM(
/
) mice have been exposed to high
prevailing concentrations of SPs and lipids that may also influence
SP-A binding site number and affinity. It is intriguing to speculate
that the increased number of binding sites may represent a response to
the increased concentration of SP-A in the air spaces of GM(
/
)
mice. The number of binding sites and affinity for SP-A observed in the
GM-CSF-replete alveolar macrophages in the present study are consistent
with those in previous studies (3, 18). Whether the
characteristics of the SP-A binding sites detected in the present study
represent single or multiple classes of SP-A receptor(s) or binding
sites and whether the SP-A binding sites represent clearance receptors, lack high-affinity binding signaling receptors, or both remain unclear.
Alveolar macrophages are highly responsive to SP-A, the polypeptide
influencing phagocytosis, viral and bacterial clearance, oxidant burst,
and cytokine production in vivo (12-15). The present findings that the local expression of GM-CSF in SP-C-GM mice reduced the number of SP-A binding sites on alveolar macrophages provide support for the concept that GM-CSF or GM-CSF-dependent signaling pathways regulate SP-A binding sites on alveolar macrophages. Interpretation of quantitative data is complicated by differences in
binding site affinity, cell size, and cell adherence in the assays and
imprecision regarding the precise size of SP-A oligomers binding to the
cell surface.
Consistent with the increased number of SP-A binding sites on alveolar
macrophages, 125I-SP-A uptake by alveolar macrophages from
GM(
/
) mice was significantly greater than that in WT or SP-C-GM
mice. Although SP-A binding sites were increased in cells
from GM(
/
) mice, substantive differences in
[3H]DPPC binding by alveolar macrophages from
GM(
/
), WT, and SP-C-GM mice were not observed. FACS studies also
demonstrated that the uptake of R-DPPE-labeled surfactant by alveolar
macrophages from mice of all genotypes was approximately similar. Thus
despite increased binding or uptake activity of surfactant components by alveolar macrophages, catabolism of both 125I-SP-A and
[3H]DPPC was markedly impaired in alveolar macrophages
from GM(
/
) mice. Differences in uptake and degradation were not
likely related to cell viability because the cells were isolated by
adherence to plastic and cell viability was similar in cells from each genotype.
The production of GM-CSF in the lungs of GM(
/
) mice with the
SP-C-GM transgene corrected alveolar proteinosis in transgenic mice in
vivo, demonstrating the importance of local expression of GM-CSF
(10). Consistent with those findings, the present data
demonstrate that degradation of SP-A and lipids by alveolar but not by
peritoneal macrophages was restored in SP-C-GM mice. With advancing
age, alveolar macrophages from both GM(
/
) and common
-chain-deficient mice develop an increasingly foamy appearance associated with an accumulation of both lipids and SPs in the air
spaces and within alveolar macrophages. Although phagocytosis of group
B streptococcus was unaltered, production of superoxide radicals was
markedly deficient in macrophages from GM(
/
) mice (16). The present findings are consistent with these
observations and demonstrate the ability of alveolar macrophages from
GM(
/
) mice to bind and take up but not to degrade surfactant
components. Increased lipid content and a marked decrease in clearance
of DPPC from the lungs of GM(
/
) mice are likely to be caused, at least in part, by a deficiency of GM-CSF-dependent pathways controlling phospholipid catabolism by alveolar macrophages.
Because alveolar macrophages in GM(
/
) mice become progressively
foamy with age, it is possible that secondary abnormalities in
macrophage function may contribute to the defect in surfactant catabolism in GM(
/
) mice. To determine whether the defect in surfactant degradation by alveolar macrophages from GM(
/
) mice was
limited to the lung or was influenced by accumulation of surfactant components in the cells, surfactant lipid degradation was compared in
peritoneal macrophages isolated from GM(
/
), WT, and SP-C-GM mice.
Degradation of [3H]DPPC by peritoneal macrophages was
also impaired in GM(
/
) and SP-C-GM mice. Because GM-CSF expression
in SP-C-GM mice [these mice are GM(
/
)] is restricted to the lung,
these findings demonstrate that local production of GM-CSF is required
for correction of the defect in lipid catabolism in GM(
/
) mice.
Interestingly, although the present study demonstrates a defect in
lipid catabolism in peritoneal macrophages, phenotypic changes in organ
pathology are restricted to the lung in GM(
/
) mice. These findings
support the concept that GM-CSF is required for DPPC clearance by
macrophages in general but that the marked PAP seen in GM(
/
) mice
reflects the unique role played by alveolar macrophages in phospholipid homeostasis in the lung.
Although cell association of labeled lipid was approximately similar in
alveolar macrophages from all genotypes, the catabolism of surfactant
lipids by alveolar macrophages from SP-C-GM mice was significantly
increased compared with that in WT mice. However, the uptake of R-DPPE
as assessed by fluorescence microscopy and FACS analysis demonstrated
some heterogeneity in the ability of cells to take up and degrade
labeled lipids. Differences in lipid uptake or degradation in WT,
GM(
/
), and SP-C-GM-replete mice may reflect a change in the
proportion of cells able to rapidly take up and degrade lipids. The
findings that the percentage of cells actually degrading surfactant
lipids is increased in the GM-CSF-sufficient mice supports the concept
that GM-CSF influences cell differentiation of alveolar macrophages (or
progenitors), producing cells with an increased ability to catabolize
surfactant components. Because chronic expression of GM-CSF in the
lungs also increased the number of alveolar macrophages in vivo
(10), changes in the absolute number of active macrophages
may also influence surfactant catabolism, providing yet another
mechanism by which steady-state lipid and surfactant concentrations may be modulated by GM-CSF in vivo.
Human PAP is a heterogeneous disorder of acquired or genetic etiology.
The pathogenesis of most cases is uncertain. Previous reports suggested
that some cases of PAP were caused by defects in GM-CSF signaling
(6, 24) or decreased production or secretion of GM-CSF
(28). A recent study (26) demonstrated that
PAP may also be caused by factors that neutralize GM-CSF activity in
bronchoalveolar lavage fluid.
GM-CSF influences binding, uptake, and degradation of SPs and lipids by
alveolar macrophages, likely reflecting the importance of GM-CSF in the
differentiation of alveolar macrophages in vivo. The striking defect in
SP-A and lipid catabolism observed in alveolar macrophages from
GM(
/
) mice was restored by chronic, local replacement of GM-CSF.
The present findings are consistent with the importance of alveolar
macrophage differentiation and function in the pathogenesis of PAP and
support previous studies (5, 10, 20, 30) that demonstrated
that bone marrow transplantation of normal hematopoietic cell
precursors improved PAP in common
-chain-deficient mice in vivo.
There is a possibility that GM-CSF also affects surfactant metabolism
by type II cells. Type II cells express GM-CSF receptors, and GM-CSF
enhances type II cell proliferation in vivo (10). It
remains unclear whether PAP in GM-CSF deficiency is caused primarily by
alterations in the activity of type II cells or alveolar macrophages or
by contributions from both cell types. Because PAP in humans has been
associated with defects in GM-CSF production, function, or receptor
activity, strategies focused on enhancing mechanisms by which alveolar
macrophages degrade surfactant may provide new therapeutic approaches
for the life-threatening disorder PAP.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Gary Ross for providing purified surfactant protein A,
Dr. Tomoyuki Imagawa and Wei Lu for technical assistance, and Ann Maher
for assistance with manuscript preparation.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grants HL-28623 (to J. A. Whitsett), HL-56387, HL-61646 (to M. Ikegami), and HL-07752 (to J. A. Reed) and a Parker B. Francis
Award (to Z. C. Chroneos).
Address for reprint requests and other correspondence: J. A. Whitsett, Children's Hospital Medical Center, Division of
Neonatology and Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH
45229-3039 (E-mail: jeff.whitsett{at}chmcc.org).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 February 2000; accepted in final form 31 July 2000.
 |
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