1 Division of Pulmonary and Critical Care Medicine, University of Michigan, Ann Arbor 48109; and 2 Pulmonary Section, Department of Veterans Affairs Medical Center, Ann Arbor, Michigan 48105
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ABSTRACT |
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We hypothesized that pulmonary
granulocyte-macrophage colony-stimulating factor (GM-CSF) is
critically involved in determining the functional capabilities of
alveolar macrophages (AM) for host defense. To test this hypothesis,
cells were collected by lung lavage from GM-CSF mutant mice
[GM(/
)] and C57BL/6 wild-type mice. GM(
/
) mice yielded almost
4-fold more AM than wild-type mice. The percentage of cells positive
for the
2-integrins CD11a and CD11c was reduced
significantly in GM(
/
) AM compared with wild-type cells, whereas
expression of CD11b was similar in the two groups. The phagocytic
activity of GM(
/
) AM for FITC-labeled microspheres was impaired
significantly compared with that of wild-type AM both in vitro and in
vivo (after intratracheal inoculation with FITC-labeled beads).
Stimulated secretion of tumor necrosis factor-
(TNF-
) and
leukotrienes by AM from the GM(
/
) mice was greatly reduced compared
with wild-type AM, whereas secretion of monocyte chemoattractant
protein-1 was increased. Transgenic expression of GM-CSF exclusively in
the lungs of GM(
/
) mice resulted in AM with normal or supranormal
expression of CD11a and CD11c, phagocytic activity, and TNF-
secretion. Thus, in the absence of GM-CSF, AM functional capabilities
for host defense were significantly impaired but were restored by
lung-specific expression of GM-CSF.
lung; inflammation; growth factors; transgenic/knockout; granulocyte-macrophage colony-stimulating factor
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INTRODUCTION |
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THE PULMONARY ALVEOLAR SPACE is the largest interface of the host with the external environment. Alveolar macrophages (AM) are the resident inflammatory cells in the alveolar space (9, 11). These mobile cells reside in close proximity to alveolar epithelial cells. AM form the first line of defense against inhaled or aspirated microbes, engulfing and killing organisms that enter the lung in low numbers (3). However, AM also serve as sentinel cells, initiating a cascade of mediators to recruit and activate additional inflammatory cells (18).
AM are highly differentiated cells of monocyte lineage that differ from
peripheral blood monocytes and from tissue macrophages from other sites
(3). Complex factors in the alveolar environment are
likely to be responsible for determining the particular functional characteristics of AM. A candidate molecule that is likely to play a
major role in determining AM activity is granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF has important effects on
mature mononuclear cells. It is both mitogenic (1, 17) and
chemotactic (20) for AM and can activate macrophages to increase antimicrobial activity against a variety of pathogens (7, 13, 24). The lung is a rich source of GM-CSF. Alveolar epithelial cells, interstitial cells, and macrophages themselves all
can produce GM-CSF upon appropriate stimulation (10).
Recent studies have demonstrated that GM-CSF has an important role in homeostasis in the normal lung. Mice with a targeted deletion of the
GM-CSF gene [GM(/
) mice] have abnormalities in the turnover of
pulmonary surfactant that eventually lead to a histological picture
resembling human alveolar proteinosis (8). This defect is
corrected by expression of GM-CSF solely in the lung (12, 33) or by chronic inhalation of recombinant GM-CSF
(22).
Emerging evidence indicates that GM-CSF plays an important role in
pulmonary host defense. Recent reports from this laboratory and
elsewhere have described impaired host defense of GM(/
) mice
against pneumonia as a result of two specific pathogens, Pneumocystis carinii (21) and group B
streptococci (13). GM-CSF has multiple effects on
inflammatory and parenchymal cells and is likely to participate in
determining the activity of cells in the alveolar environment. The
present study has been undertaken to more fully determine the
functional characteristics of AM from mice congenitally deficient in
GM-CSF and of AM from mice in which GM-CSF is expressed exclusively in
the lung.
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MATERIALS AND METHODS |
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Animals.
Wild-type C57BL/6 mice were obtained from Jackson Laboratory (Bar
Harbor, ME). GM(/
) mice were generated by Dranoff et al. (8) by targeted interruption of the GM-CSF gene and
expressed no detectable GM-CSF. These mice have been extensively
backcrossed against C57BL/6 mice. GM(
/
) mice were used at 6-8
wk of age. Bitransgenic mice, in which GM-CSF is expressed exclusively
in the lung, were generated from GM(
/
) mice by transgenic
expression of a chimeric gene containing GM-CSF under the surfactant
protein C (SP-C) promoter (SP-C-GM mice; see Ref. 12). The
specificity of the SP-C promoter results in targeted expression of
GM-CSF exclusively by type II alveolar epithelial cells of these
SP-C-GM mice. Founder GM(
/
) and SP-C-GM mice were kindly provided
by Dr. J. Whitsett (Children's Hospital, Cincinnati, OH). All mice were housed in microisolator cages under laminar flow hoods in an
isolation room of the Animal Care Facilities at the University of
Michigan and the Ann Arbor Veterans Affairs Medical Center. Mice were
supplied with autoclaved bedding, food, and water. Sentinel mice
cohoused with experimental mice were necropsied periodically for
detection of opportunistic pathogens. All procedures were approved by
the Animal Care Committees at the University of Michigan and the Ann
Arbor Veterans Affairs Medical Center.
Whole lung lavage and differential cell counting. AM were recovered by whole lung lavage, as described previously (21). Mice were killed with intraperitoneal pentobarbital sodium. The trachea was cannulated, and the lungs were lavaged with a total of 5 ml of PBS (containing 0.5 mM EDTA) in aliquots of 0.5 ml. The lavage aliquots for each animal were pooled, and the cell pellet was collected by centrifugation. Differential cell counts were performed on >200 cells/mouse stained with hematoxylin and eosin, as described previously (2).
AM adherence.
AM were collected from wild-type, GM(/
), and SP-C-GM mice
by whole lung lavage and were suspended in DMEM (GIBCO BRL)
supplemented with penicillin-streptomycin (1 × 106
cells/ml). The cells were plated in four-well Lab-Tek tissue culture-treated plastic chamber slides (Nunc, Naperville, IL) at
2.5 × 105 cells/well. After 30 min, the wells were
washed three times with warm PBS, fixed with paraformaldehyde (0.5%),
and then viewed on a phase-contrast microscope (×20 objective). The
adherent cells were counted in eight random fields.
Flow cytometry of AM.
AM were collected from groups of three wild-type, GM(/
), and
SP-C-GM mice and pooled. The cell pellets were washed three times with
PBS. After each wash, the cells were collected by centrifugation for 5 min at 400 g. The cells were suspended in staining buffer (PBS with 2% FCS and 0.1% sodium azide) at 1 × 106
cells/ml. Aliquots containing 1 × 105 cells were
processed as follows. All incubations were for 30 min at 4°C on ice.
Cells were blocked with anti-murine Fc
III/II (CD16/CD32; PharMingen,
San Diego, CA) and then incubated with primary antibodies,
including phycoerythrin-conjugated rat anti-murine CD11a and
rat anti-murine CD11b (both 4 µg/ml), FITC-conjugated hamster
anti-murine CD11c (5 µg/ml), and isotype-matched controls. In
separate experiments, lymphocytes were identified by staining with rat
anti-murine CD19 (pan B cell), rat anti-murine CD3 (T cells), or rat
anti-murine Gr-1 (Ly-6G, staining neutrophils). All antibodies were
from PharMingen. In selected experiments, cell viability was
determined by flow cytometric analysis of cells stained with propidium
iodide (0.25 µg/1.5 × 105 cells in 100 µl;
PharMingen) to identify dead cells. Cells also were stained with
FITC-conjugated Bandeiraea simplicifolia (BS-I) lectin
(Sigma, St. Louis, MO). After being stained, the cells were
washed two times in FA buffer (Difco, Detroit, MI) and then fixed with
0.5% paraformaldehyde. Flow cytometry was performed on a
FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Data were
analyzed using the Cell Quest software package (Becton Dickinson).
Thresholds for positive staining were determined from the
isotype-matched control samples.
In vivo and in vitro phagocytosis assays.
For the in vitro assay, AM first were obtained from wild-type C57BL/6,
GM(/
), and SP-C-GM mice by whole lung lavage. AM (105/well in DMEM without FCS) were adhered for 30 min in
wells of eight-well tissue culture-treated plastic slides (Lab-Tek).
The cells were washed gently, and latex microbeads labeled with FITC (1.7 µm in diameter; Polysciences) were added to the wells (8 × 106 beads/well). After 1 h of incubation at 37°C,
the wells were washed gently with PBS, fixed with methanol at
20°C
for 20 min, and washed extensively. The cells were viewed by a blinded
observer using a Nikon Labphot 2 microscope equipped with
epifluorescence. In each well, the fraction of cells containing labeled
beads and the phagocytic index were determined by microscopy counting
of at least 200 cells in random high-power fields. The phagocytic index
(PI) was calculated as follows
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Measurement of AM leukotriene production in vitro.
AM were collected by whole lung lavage of wild-type, GM(/
), and
SP-C-GM mice by instilling 1-ml aliquots of PBS containing 5 mM EDTA to
a total volume of 5 ml. Cells were pooled from three mice in each
instance. The cells were plated in triplicate at 0.2 × 106/200 µl in 96-well plates for 1 h. The
nonadherent cells were washed gently, and the adherent cells were
stimulated with A-23187 (1 µM) for 30 min. The culture supernatants
were harvested and frozen at
70°C for future analysis for
peptidoleukotriene content by enzyme immunoabsorbant assay (EIA; Cayman
Chemical, Ann Arbor, MI).
ELISAs for tumor necrosis factor- production by
AM in vitro and monocyte chemoattractant protein-1 in
bronchoalveolar lavage fluid.
AM were obtained from the lungs of wild-type C57BL/6, GM(
/
), and
SP-C-GM mice by whole lung lavage as described above and placed in
culture in 96-well plates (105 cells/well). After the AM
had adhered for 30 min, the plates were washed gently, and
lipopolysaccaride (LPS; Escherichia coli 026:B6; Sigma) was
added to the wells in DMEM with or without 10% FCS. The cells were
incubated for 24 h at 37°C, the medium was harvested, and the
concentration of tumor necrosis factor-
(TNF-
) or monocyte
chemoattractant protein (MCP)-1 in the cell-free supernatant was
determined using ELISA kits (both from R&D Systems, Minneapolis, MN)
following the manufacturer's recommendations. For each data point,
supernatants from quadruplicate wells obtained from a single animal
[GM(
/
) or SP-C-GM mice] or pooled from two to three mice
(wild-type mice) were measured.
Statistical methods. Data are expressed as means ± SE and were compared by one-way ANOVA with the Tukey-Kramer multiple comparisons test using the InStat software program (version 3.01 for Windows 95; GraphPad Software, San Diego, CA). Data were considered statistically significant if P values were <0.05.
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RESULTS |
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Number and appearance of alveolar cells recovered by
BAL.
To examine the effects of congenital absence of GM-CSF on the
population of alveolar inflammatory cells, BAL was performed on
wild-type C57BL/6 mice and GM(/
) mice aged 6 wk. Alveolar cells
also were harvested from SP-C-GM mice, which lack GM-CSF except for
expression in type II alveolar epithelial cells. GM-CSF is expressed in
higher concentrations in the lungs of SP-C-GM mice than in wild-type
mice (12, 21). Significantly more cells per mouse were
recovered by lung lavage from GM(
/
) mice and SP-C-GM mice than from
wild-type controls (Fig. 1). Differential cell counting of Diff-Quik-stained BAL fluid cells identified 89%
of the cells from wild-type mice as AM, with 8.5% lymphocytes and
1.2% neutrophils. Cells from GM(
/
) mice and SP-C-GM mice were
>99% monocyte/macrophage in appearance (Fig.
2). To better characterize these cells,
forward and side light scatter of alveolar cells collected by BAL were
determined by flow cytometry. As shown in Fig.
3, there was a slight increase in the
number of smaller cells from the GM(
/
) mice compared with wild-type
mice. Trypan blue exclusion demonstrated >95% viability of BAL fluid
cells from all three strains of mice. In selected experiments, the
fraction of cells stained with propidium iodide was determined by flow cytometry as an indication of cell death. Only 1-4% of the BAL fluid cells from GM(
/
) mice stained with propidium iodide,
confirming results from trypan blue exclusion.
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Decreased in vitro adhesion of AM from
GM(/
) mice.
Normal AM readily adhere to tissue culture-treated plastic. The
adhesion of AM from GM(
/
) mice was significantly less than that of
AM from wild-type mice (Fig. 4).
Interestingly, AM from SP-C-GM adhered to plastic dishes at a rate
equivalent to that of wild-type AM. Thus, in the absence of GM-CSF,
there was defective AM adherence to tissue culture-treated plastic, and
this defect was reversed when GM-CSF was expressed in the lungs of
otherwise GM-CSF-deficient mice.
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Expression of 2-integrins is altered in
AM from GM(
/
) mice.
Macrophage interactions with endothelial and epithelial cells are
mediated in part by
2-integrins expressed on the AM
surface. AM from GM(
/
), wild-type, and SP-C-GM mice were collected
by BAL and stained for the
2-integrins CD11a, CD11b, and
CD11c. The proportion of cells staining positively for these integrins was determined by flow cytometry (Fig.
5). The fractions of AM expressing CD11a
and CD11c were reduced significantly in AM from GM(
/
) mice compared
with those from wild-type mice. In contrast, expression of CD11b was
similar in AM from GM(
/
) and wild-type mice. Interestingly, AM from
SP-C-GM mice demonstrated higher expression of all three integrins
compared with either GM(
/
) or wild-type mice. Thus GM-CSF has
pronounced effects on integrin expression by AM, with diminished
expression in the absence of GM-CSF and supranormal expression when
GM-CSF was overexpressed in the lung.
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Impaired phagocytic activity of AM from
GM(/
) mice in vitro and in vivo.
To directly explore the consequences of congenital absence of GM-CSF
for AM function, FITC-labeled latex microspheres were incubated with AM
collected by BAL from the three strains of mice. After 1 h, the
cells were fixed, and the phagocytic activity of the AM was determined
by microscopy counting of microspheres associated with AM. AM from
GM(
/
) mice were significantly less effective in
binding/phagocytosis of microspheres compared with wild-type AM. Both
the percentage of AM that had bound/internalized beads and the
phagocytic index were reduced in the GM(
/
) AM (Fig. 6). Long-term exposure to GM-CSF in the
lung reversed this deficit as shown by the enhanced activity of AM from
SP-C-GM mice. To extend these in vitro observations to the intact host,
mice of all three strains were inoculated intratracheally with
FITC-labeled latex microspheres. After 1 h, AM were recovered by
BAL. Binding and phagocytosis of microspheres by AM then were assessed
by microscopic counting. In GM(
/
) mice, both the percentage of AM
that contained latex microspheres (Fig.
7A) and the phagocytic index
(Fig. 7B) were reduced significantly compared with those in
wild-type mice and SP-C-GM mice. These data demonstrate that phagocytic
activity of AM is impaired in GM-CSF(
/
) mice and that phagocytic
function is in fact greater than normal in the setting of targeted
overexpression of GM-CSF in the lung.
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Impaired production of leukotrienes by AM from
GM(/
) mice.
AM are an important source of leukotrienes in the context of host
defense. Inhibition of endogenous leukotriene synthesis has resulted in
diminished AM phagocytosis of bacterial pathogens. To determine whether
the impaired macrophage inflammatory activity of GM(
/
) AM extended
to leukotriene secretion, AM were collected from wild-type and
GM(
/
) mice and stimulated in vitro with calcium ionophore. Total
peptidoleukotrienes (leukotrienes C, D, and E4) were
measured in the culture supernatants by EIA. As anticipated, wild-type
AM released abundant leukotrienes (2,830 ± 528 pg/ml). However,
leukotriene secretion was completely suppressed in the GM(
/
) AM
(22.6 ± 4.5 pg/ml; P < 0.01 vs. wild type).
Interestingly, leukotriene secretion in AM from SP-C-GM mice (496 ± 45.6) was greater than that from GM(
/
) AM but was not restored
to the level of wild-type mice. Thus congenital absence of GM-CSF
resulted in the loss of normal leukotriene synthesis by AM. This
deficit was only partially corrected by exclusive expression of GM-CSF in the alveolar space.
Diminished TNF- expression by AM
from GM(
/
) mice in response to LPS.
In addition to their important role as phagocytes, AM function as
sentinel cells, amplifying the innate immune response through the
initiation of a cascade of inflammatory mediators. TNF-
is a
critical component of this inflammatory cascade. AM from GM(
/
) and
wild-type mice were collected by BAL and placed in culture in DMEM with
or without 10% FCS. LPS was added to the medium at increasing
concentrations. After 24 h, TNF-
antigen was measured in the
culture supernatants (Fig. 8). AM from
wild-type mice released abundant TNF-
after LPS stimulation. As
anticipated, the response to low-dose (1-10 ng/ml) LPS was greater
in the presence of serum than under serum-free conditions, although, at
higher doses of LPS, large amounts of TNF-
were released under
either condition. In contrast, AM from GM(
/
) mice expressed no
detectable TNF-
at baseline, with very little TNF-
induced after
exposure to LPS in the presence or absence of serum. AM from SP-C-GM
mice consistently secreted at least as much TNF-
in response to LPS as wild-type AM. Thus, in the absence of GM-CSF, AM secretion of
TNF-
in response to LPS is greatly impaired, whereas expression of
GM-CSF solely in the lung is sufficient to reverse this abnormality.
|
Increased MCP-1 expression in lung lavage fluid and
AM from GM(/
) mice.
Our initial studies demonstrated increased numbers of AM recovered by
lung lavage from GM(
/
) mice compared with wild-type controls. One
possible explanation for this increased number of cells in the absence
of GM-CSF would be that these cells are recruited to the lung by
increased expression of a monocyte chemoattractant. Therefore, we
determined the expression of MCP-1, a monocyte chemoattractant that is
produced by a number of different cells in the alveolar space, in BAL
fluid. MCP-1 protein was not detected in BAL fluid from normal
wild-type mice (limit of detection 15.6 pg/ml). In contrast, there was
abundant MCP-1 (654.5 ± 186 pg/ml, n = 3) in the
BAL fluid from GM(
/
) mice. To determine whether AM themselves might
be one source of increased MCP-1 expression in these mice, AM from
GM(
/
) and wild-type mice were exposed to LPS (10 ng/ml) in the
presence of 10% FCS. There was significant MCP-1 in the culture
supernatants from GM(
/
) AM (189 ± 67 pg/ml), whereas MCP-1
was not detectable in undiluted supernatants from similarly treated
wild-type AM (<16 pg/ml).
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DISCUSSION |
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Previously, it has been recognized that GM-CSF is produced in
abundance in lung tissue after inflammatory stimulation
(6) and plays an important role in regulation of pulmonary
surfactant (23). More recently, based on studies using
mice congenitally deficient in GM-CSF, it has become clear that GM-CSF
plays an important role in pulmonary host defense. GM(/
) mice
demonstrate enhanced susceptibility to pneumonia resulting from group B
streptococci (13) or P. carinii
(21). We now provide new information concerning the
specific consequences of the absence of GM-CSF for AM function. We
found that GM(
/
) mice had increased numbers of AM in BAL fluid
compared with wild-type mice. Associated with this increased number of
AM was increased expression of MCP-1 in BAL fluid from GM(
/
) mice
compared with wild-type mice. Although more abundant, AM from GM(
/
)
mice demonstrated a diminished capacity to perform a number of classic
functions of AM. In particular, they were less effective than AM from
wild-type mice in binding and phagocytosis of latex microspheres both
in vitro and in vivo. In contrast to normal AM, AM from GM(
/
) mice
produced very little TNF-
or peptidoleukotrienes upon stimulation in
vitro. AM from GM(
/
) mice also demonstrated an altered pattern of
cell surface expression of
2-integrins and diminished
BS-I lectin binding compared with wild-type AM. Finally, with the
exception of leukotriene expression, these deficiencies could be
corrected by expression of GM-CSF exclusively in the alveolar space.
AM are critically involved in pulmonary host defense, both as effector
cells that bind and engulf pathogens and as sentinel cells that secrete
inflammatory mediators to recruit and activate inflammatory cells.
Studies in which AM have been specifically depleted by tracheal
instillation of liposomes containing dichloromethylene diphosphonate have confirmed the importance of AM for host defense against gram-negative pathogens (5) and against P. carinii (14, 15). AM from GM(/
) mice evidenced
defects in binding and phagocytosis of latex beads that were apparent
not only during in vitro studies but also when the beads had entered
the alveolar space in vivo. The defect in binding/phagocytosis observed
in the GM(
/
) mice would significantly impair the ability of these AM to engulf and kill pathogens that enter the lung either by aspiration of oropharyngeal contents or by aerosol deposition, rendering the animals susceptible to pneumonia in response to small
numbers of organisms.
Transgenic mice lacking biological activity of GM-CSF can be generated
by targeted deletion either of GM-CSF or of its receptor. Transgenic
mice with a targeted deletion of the -chain of this receptor develop
pulmonary pathology resembling that found in GM(
/
) mice (19,
25). In vitro studies using macrophages from these
receptor-deficient mice demonstrate diminished adherence and variable
defects in phagocytosis with impaired phagocytosis of colloidal carbon.
However, AM from these receptor-deficient mice demonstrate preserved
phagocytosis of latex beads (28). The reasons for
discrepancies between the GM(
/
) mice and
-chain-deficient mice
are not yet clear (27).
AM are an abundant source of inflammatory mediators. The defect in
binding and phagocytosis identified in AM from GM(/
) mice would be
less critical if these AM were still able to vigorously recruit and
activate neutrophils and other inflammatory cells in response to
invading pathogens. However, we identified profound abnormalities in
expression of both TNF-
and leukotrienes by GM(
/
) AM in response
to standard stimuli. Among the many AM secretory products, we chose to
investigate TNF-
because it is a classic early-response cytokine
that is produced in response to a broad array of microbial products. In
turn, TNF-
stimulates macrophage production of C-X-C
chemokines to recruit neutrophils (31). Other cellular
sources may provide TNF-
in the lung in GM(
/
) mice
(13; Paine, unpublished observations). However, the profound decrease in TNF-
expression by GM(
/
) AM greatly impairs the ability of these resident cells to initiate a vigorous inflammatory response.
The effect of GM-CSF on leukotriene expression is quite striking. In
the absence of GM-CSF, the production of all of the peptidoleukotrienes is greatly reduced. This defect appears to be the result of a diminished content of two proteins that are critical for leukotriene production. Expression of 5-lipoxygenase, the rate-limiting enzyme for
leukotriene synthesis, and of 5-lipoxygenase-activating protein are
greatly reduced in AM from GM(/
) mice compared with AM from wild-type mice (Coffey, unpublished observation). Stimulated
leukotriene secretion by AM from SP-C-GM mice remains significantly
reduced compared with that of AM from wild-type mice, demonstrating
that these AM are not equivalent to those from wild-type mice. This is
in contrast to the normalization of other functional capabilities in AM
from mice expressing GM-CSF exclusively in alveolar epithelial cells.
The absence of GM-CSF during monocyte development in the bone marrow
may contribute to this failure of SP-C-GM AM to secrete normal levels
of leukotrienes. However, AM themselves are normally a source of
GM-CSF. It is possible that normal leukotriene expression by these
cells requires autocrine expression of this growth factor by AM.
The mechanisms that lead to impaired AM function in the GM(/
) mice
have not been defined. It is possible that the aberrant phenotype of
the AM from GM(
/
) mice is a consequence of loss of expression of a
series of GM-CSF-responsive genes such as TNF-
(26). It
is also possible that a number of the functional capabilities of the
mature AM are interdependent. For instance, expression and binding of
2-integrins can be critical for phagocyte adherence, phagocytosis, and respiratory burst (29). Thus
significantly reduced expression of CD11a/CD18 and CD11c/CD18 by the
GM(
/
) AM is likely to contribute to the functional deficiencies of
these cells.
The increased phospholipid and protein content in the alveolar space of
GM(/
) mice could contribute to some of the abnormalities in AM
function. Excess cell surface SP-A has been shown to impair AM
secretion of TNF-
(4, 16). However, we found that
washing AM from GM(
/
) mice with EGTA to remove surface-bound SP-A
did not restore TNF-
secretion to that found in wild-type cells. Similarly, sedimentation of AM through albumin to remove excess surfactant phospholipid did not correct the defect in TNF-
expression. Importantly, there were characteristics that were increased
in the GM(
/
) mice compared with controls. First, cell surface
expression of CD11b was increased in AM from GM(
/
) mice compared
with wild-type AM, perhaps suggesting that in the absence of GM-CSF,
alveolar mononuclear cells have a less mature phenotype. Second, MCP-1 secretion in response to LPS in vitro was increased in GM(
/
) AM
compared with wild-type AM. This clearly indicates that the loss of
TNF-
production is not simply a manifestation of a global defect in
cytokine production. Thus, although we cannot exclude some effect of
excess surfactant components on macrophage function, it is likely that
the major mechanism accounting for impaired AM function in the
GM(
/
) mice is the absence of GM-CSF.
Expression of GM-CSF exclusively in the lung in the absence of GM-CSF during monocyte development in the bone marrow is sufficient to induce expression of most of the characteristics of the mature AM phenotype in SP-C-GM mice. AM are differentiated cells (3) that differ from blood monocytes or tissue macrophages at other sites. Our data now indicate that GM-CSF plays a complex role in coordinating the maturation of AM in the alveolar space. In the absence of GM-CSF, both signaling and effector functions of AM are severely disrupted, whereas pulmonary expression of GM-CSF promotes expression of features of the fully differentiated AM phenotype. These data support the hypothesis that pulmonary GM-CSF acts as a local regulator of AM maturation.
The manner in which the number of AM in the normal lung is sensed and
the means by which this number is controlled are poorly understood.
Increased numbers of AM were recovered from both SP-C-GM and GM(/
)
mice compared with wild-type mice. In the SP-C-GM mice, this increase
may be attributable to the mitogenic effect of GM-CSF for macrophages
(32) or to the chemotactic activity of GM-CSF for AM
(20). Our finding that MCP-1 was expressed in lung lavage
fluid from GM(
/
) mice but not in normal controls is consistent with
the hypothesis that monocyte chemoattractants are induced in the
absence of GM-CSF in an attempt to regulate AM activity in the alveolar
space by increasing the recruitment of monocytes to the lung.
Our studies broaden the concept of pulmonary GM-CSF as a regulator of
alveolar homeostasis. Previous studies have defined a role for GM-CSF
on AM in pulmonary surfactant homeostasis and have demonstrated that
GM(/
) mice are more susceptible to pneumonia because of two
specific pathogens, group B streptococcus (13) and
P. carinii (21). Our findings indicate that
GM(
/
) AM have a global defect affecting both phagocytosis and
expression of pivotal inflammatory signals. Based on these functional
deficits, one would predict that GM(
/
) mice would be susceptible to
pulmonary infections with a wide range of pathogens, from routine
bacteria to eukaryotes. Thus one may think of pulmonary GM-CSF as
playing a "homeostatic" role in host defense in the lung by
providing an environment in which AM are equipped to provide an optimal innate immune response to invading pathogens.
In conclusion, we have found that GM(/
) mice have increased numbers
of AM but that these AM demonstrate a broad-based abnormality in host
defense function compared with AM from wild-type mice. In particular,
AM adherence and expression of
2-integrins are diminished, phagocytosis of latex microbeads in vivo and in vitro is
impaired, and secretion of TNF-
and leukotrienes is greatly diminished. Interestingly, targeted expression of GM-CSF exclusively in
the alveolar space is sufficient to restore most aspects of AM
function, with the exception of leukotriene production. These studies
provide further evidence that GM-CSF in the alveolar space is a
critical component of the pulmonary innate immune response and can
determine the adequacy of the innate immune response to the initial
entry into the lung of noxious infectious agents.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Jeffrey Whitsett and Jacqueline Reed, Children's
Hospital, Cincinnati, OH, for the gift of the GM(/
) and SP-C-GM founder mice.
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FOOTNOTES |
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This work was supported by a Merit Review Award from the Medical Research Service of the Department of Veterans Affairs (R. Paine), National Heart, Lung, and Blood Institute Grant HL-64558 (R. Paine) and Specialized Center of Research Grant 1P50-HL-56402 (R. Paine, B. B. Moore, and G. B. Toews), and Research Enhancement Award Program funds from the Department of Veterans Affairs. R. Paine is a Career Investigator of the American Lung Association.
Address for reprint requests and other correspondence: R. Paine III, Pulmonary Section (111G), VAMC, 2215 Fuller Rd., Ann Arbor, MI 48105 (E-mail: rpaine{at}umich.edu).
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 28 November 2000; accepted in final form 20 July 2001.
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