Macrophages primed by overnight culture demonstrate a marked
stimulation of surfactant protein A degradation
Sandra R.
Bates,
Jin
Xu,
Chandra
Dodia, and
Aron B.
Fisher
Institute for Environmental Medicine, School of Medicine,
University of Pennsylvania, Philadelphia, Pennsylvania 19104-6068
 |
ABSTRACT |
The current study
examined whether long-term culture of macrophages affects their
metabolism of surfactant components. Compared with freshly isolated
resting macrophages in culture for 1 h, macrophages attached to plastic
dishes for 24 h showed evidence of conversion to a "primed" state
with 1) an altered morphology characterized by a larger size, ruffled membranes, lamellipodia, and a
"foamy" appearance after attachment to glass and
2) a fivefold greater respiratory
burst in response to phorbol 12-myristate 13-acetate stimulation. On
incubation with iodinated surfactant protein A (SP-A), the 24-h
alveolar or tissue macrophages showed a 5- or a 23-fold greater
increase in SP-A degradation, respectively, than macrophages cultured
for 1 h. Conditioned media experiments demonstrated that the elevated
rate of SP-A degradation after prolonged culture was not a result of
proteases secreted by the macrophages. Incubation of cells with
NH4Cl reduced the degradation of
SP-A to a similar extent (to 33% of control values) in resting and
primed tissue macrophages. On the other hand, length of time of cell
culture did not affect macrophage uptake and degradation of
[3H]dipalmitoylphosphatidylcholine
in mixed unilamellar liposomes. Thus freshly isolated resting tissue
and alveolar macrophages can be primed to specifically increase their
rate of SP-A degradation. Activation of macrophages associated with
lung disease may be important for SP-A metabolism and surfactant
function.
lung; human alveolar proteinosis; reduced oxygen species; phospholipid; pulmonary surfactant
 |
INTRODUCTION |
THE ALVEOLAR MACROPHAGE is important in maintaining the
ability of the lung to preserve sterility and to respond to injury. These macrophages are derived ultimately from peripheral blood monocytes that have been recruited into the lung tissue by
chemoattractants. Once in the lung, cytokines promote their maturation
into interstitial macrophages, which can then move into the alveolar
space in response to appropriate signals (for review see Ref. 25).
Alveolar macrophages release a variety of products, including
cytokines, enzymes, biologically active lipids, and oxygen metabolites,
and have >50 ligand-specific membrane receptors on the cell surface
(for review see Ref. 41).
In addition to host defense, evidence is accumulating that alveolar
macrophages also play an important role in the turnover of surfactant
through uptake and degradation of surfactant components. In vivo
studies have documented the presence of tubular myelin figures and
surfactant protein A (SP-A) within macrophages lining the alveolar
space (36, 45). In vitro studies have demonstrated the ability of
alveolar macrophages isolated by lung lavage to degrade phospholipid
(32, 48), surfactant protein B (5), and SP-A (6, 48). Macrophage
surface receptors specific for SP-A have been described (14, 37, 38)
and recently isolated (13). Because SP-A has been shown to modulate
phospholipid uptake (4, 43, 47), secretion (18, 40), and catabolism
(21) by type II cells, macrophage degradation of SP-A may contribute to
the regulation of surfactant levels in the alveoli.
On injury to the lung, the inflammatory cell population of the lung
increases (3) and surfactant components are altered (16). Although
alveolar macrophages provide the first line of defense in the lower
airways, when the system is overwhelmed, materials may pass through a
damaged epithelial barrier into the interstitium, where they may be
engulfed by interstitial macrophages (for review see Ref. 10).
Pulmonary macrophage appearance can be dramatically changed from small,
round cells to large "foamy" cells, depending on the etiology of
lung damage (44). Such morphological changes may reflect alterations in
cell biochemistry that would affect the metabolism of surfactant by
these primed or "activated" macrophages and provide a possible
mechanism whereby changes in surfactant levels occur. To convert a
nonresponsive macrophage to a responsive or "primed" state in
vivo, macrophages are elicited by injection of thyoglycollate or
endotoxin (26). In vitro, adherence of human macrophages to
protein-coated surfaces primed the macrophages for a massive
respiratory burst on exposure to cytokines (35), whereas macrophages
adhered to plastic produced less reactive oxygen species (ROS). Thus
resting and primed macrophages can be activated on
stimulation, but the degree of responses differs quantitatively. We
noted that alveolar and tissue macrophages markedly altered their
morphological appearance after 24 h of attachment to a culture dish and
centrifugation onto glass slides. The cells became large and vacuolated
with many pseudopodia, physical characteristics of active macrophages.
We compared freshly isolated resting macrophages with 24-h macrophages
to determine whether priming by this mechanism alters the uptake and
degradation of surfactant components.
 |
METHODS |
Lung cell preparation and staining.
Alveolar macrophages were isolated from pathogen-free male
Sprague-Dawley rats (500 g) by lung lavage, as described previously (5). The alveolar macrophages (4 × 106) were plated in 35-mm
plastic dishes (Costar, Cambridge, MA) for 1 h at 37°C in minimal
essential medium (MEM) with 10% fetal calf serum (FCS). After 1 h, the
cells were washed three or more times to remove red blood cells and
other nonadherent cells. Ninety-nine percent of the adherent cells were
alveolar macrophages (5). The cells were then used for study or
refed MEM containing 10% FCS and were examined 24 h later.
Interstitial macrophages were isolated from minced rat lungs. The lungs
were perfused, lavaged eight times with 7 ml of calcium-free
phosphate-buffered saline containing 10 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 0.2 mM ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, and 6 mM glucose, minced with a tissue chopper, shaken in a
37°C water bath for 3 min, filtered through nylon mesh, and centrifuged (7). The tissue macrophages (4 × 106) attached to 35-mm plastic
dishes (Costar) after 1 h of incubation at 37°C in MEM with 10%
FCS. The plates then were washed at least three times to remove
nonadherent cells, including red blood cells. The resultant preparation
contained <15% nonmacrophage cell types, with lymphocytes as the
principal contaminating cell. The cells were studied immediately or
refed MEM containing 10% FCS and were studied 24 h later. After 24 h,
~75% of the alveolar and tissue macrophages had detached from the
plastic, and these cells then were washed off the dishes. Only
macrophages that remained attached to the dishes were used in
experiments, and the viability of these cells was >98% by vital dye
exclusion. For light-microscopic examination, the macrophages were
scraped from the dish, attached to a glass slide using a cytospin
(Shandon, Pittsburgh, PA), stained using a Kwik-Diff stain kit and
visualized by microscopy under oil.
FCS was used without heat inactivation, since the manufacturer has not
found any complement components after the necessary processing of the
FCS before delivery. In addition, repeated freezing and thawing
irreversibly denatures complement factor C1q. To ensure that possible
residual complement in the serum would not affect the results,
macrophages were incubated in MEM with either 10% heat-inactivated
(56°C for 30 min) or unheated FCS, and SP-A metabolism and ROS
generation were measured. Heating the serum did not significantly affect the cell association or degradation of SP-A with either alveolar
or tissue macrophages or with time of incubation; values for cells in
heated serum were 106 ± 5% (SD) of values for cells in unheated
serum (n = 4). The generation of ROS
by 1- or 24-h macrophages incubated in heated serum did not differ from
that by macrophages incubated in unheated serum. ROS production by unstimulated macrophages in heated serum was 99.5 ± 4% (SD) of that for cells in unheated serum (n = 6). In addition, phorbol 12-myristate 13-acetate (PMA) stimulation of
ROS production was not influenced by culture of the macrophages for 1 or 24 h in heated serum; values for cells in heated serum were 97 ± 4% (SD) of values for cells in unheated serum
(n = 5).
Type II cells were isolated from the lungs of male Sprague-Dawley rats
by methods previously described (12). Briefly, after clearance of blood
by perfusion, the lavaged lungs were digested with elastase and minced.
The crude cell preparation was panned on immunoglobulin G-coated
bacteriological plates and plated overnight on 35-mm plastic tissue
culture dishes (Costar) at 3 × 106 cells/dish in MEM with 10%
FCS at 37°C. Purity of the final preparation was >90%, with
macrophages being the primary contaminating cell type.
Surfactant and SP-A isolation.
Bronchoalveolar lavage fluid was obtained from normal (slaughterhouse)
bovine lungs or patients with alveolar proteinosis. Surfactant was
isolated from an NaCl-NaBr gradient after centrifugation (20). The
bovine and human SP-A were purified from surfactant according to the
butanol extraction method described by Hawgood et al. (24). SP-A was
iodinated using Iodo-gen (Pierce, Rockford, IL), as described
previously (6), and was dialyzed against tris(hydroxymethyl)aminomethane-buffered saline to remove free Na125I. An aliquot of
125I-SP-A was analyzed for protein
concentration and trichloroacetic acid (TCA) precipitability. The
specific activity of the radioiodinated SP-A preparations was
200-2,000 cpm/ng protein, and 97% of the labeled SP-A was
precipitable with TCA. The ligand was stored and used within 2 wk of
iodination.
The purity of the SP-A preparation was monitored using sodium dodecyl
sulfate-polyacrylamide gel electrophoresis according to the method of
Laemmli (27), as described previously (6). Autoradiographic analysis of
radiolabeled 125I-SP-A showed no
evidence of degradation products or contaminating proteins.
Iodinated SP-A was added to surfactant with gentle mixing and placed on
ice for 30 min before use. The
125I-labeled bovine SP-A was
reconstituted with surfactant at a labeled protein-to-surfactant
phospholipid ratio of 1:100 and a labeled SP-A protein-to-unlabeled
SP-A protein in surfactant ratio of 1:17, assuming that the amount of
SP-A in bovine surfactant was 40% of total protein (6).
Interaction of SP-A with cells.
Cells were incubated with radiolabeled SP-A for the indicated time
period at 37°C. To terminate the experiment, the medium was removed
and processed as outlined below. The cells were washed three times with
MEM and twice with phosphate-buffered saline to remove unbound ligand.
The cells were then dissolved in 0.2 N NaOH (total cell-associated
SP-A). Aliquots were taken to measure radioactivity and protein (30).
The medium from the experiment was centrifuged to remove any cells and
precipitated with cold 10% TCA. Insulin was added to serve as a
carrier protein. The intact precipitated protein was removed by
centrifugation, and the supernatant was treated with KI and
H2O2
and was extracted with chloroform to remove any free labeled iodide, as
described previously (6). An aliquot of the aqueous fraction of the
125I-labeled TCA-soluble,
chloroform-extracted supernatant containing 125I-labeled amino acids
and/or small protein fragments was counted (5, 23). This
material represents SP-A degradation products released from the cell
into the culture media (23), as outlined in detail previously (5). SP-A
protein association or degradation by the cells was calculated from the
radioactivity and protein in the cell extract and the specific activity
of the SP-A. Medium with
125I-SP-A was
incubated on empty dishes in each experiment, and any 125I counts were subtracted from
the radioactivity found in the presence of cells. Radioactive iodine
counts from empty dishes found in the NaOH were <10%, and those in
the media as degradation products were <20%. Separate dishes of
cells were used to determine the amount of cellular protein per dish
present at the initiation of each experiment and served as the
denominator in the quantitation of the amount of SP-A degraded per
milligram of protein, since experimental manipulations caused
detachment of a fraction of the cells. Because of the variation in
cellular metabolism between experiments, the extent of cell association
and degradation (as ng SP-A/mg cell protein) at a specified
concentration of SP-A or time of experiment was set equal to 100%
(control) and the remaining data were expressed as a percentage of
control. This allowed comparison of data from several experiments.
For conditioned media experiments, macrophages were incubated with
medium for 2 h, the medium was centrifuged to remove detached cells,
and the conditioned medium was transferred to empty plastic dishes (35 mm; Costar). Fresh medium was added to the macrophages and to other
empty dishes. 125I-labeled bovine
SP-A (8.6 µg/ml in surfactant) was added to all dishes, and the cells
were incubated for 2 h. Then the medium was removed and assayed for
SP-A degradation products. Degradation that occurred with fresh medium
on empty dishes was subtracted from the data.
Measurement of ROS production.
ROS generation was followed using the fluorescent detection of
dichlorofluorescein (DCF), formed by the oxidation of the
nonfluorescent precursor DCF (DCFH) according to the method described
by Cathcart et al. (11). 2',7'-Dichlorofluorescin diacetate
(1 mM; Eastman Kodak, Rochester, NY) in ethanol was hydrolyzed to DCFH
with 0.01 N NaOH at room temperature for 30 min and neutralized by a
1:40 dilution in Krebs-Ringer buffer. Macrophages were incubated for 20 min in 2 ml of Krebs-Ringer buffer containing 5 µM DCFH, 12.5 µg/ml
horseradish peroxidase (Sigma Chemical, St. Louis, MO), and 1 mM
glucose with or without PMA at 50 ng/ml. DCF fluorescence was measured
on a Hitachi fluorescence spectrophotometer using the excitation and
emission wavelengths of 490 and 530 nm, respectively. By use of
H2O2
as a standard, the assay was linear from 100 nM to 1 µM
H2O2.
The rate of ROS formation was continuously measured over a 20-min
period by analysis of 0.3 ml of media taken at several time points. The
rate of ROS production was calculated from the linear portin of the
curves following the initial lag phase and was expressed in arbitrary
fluorescence units.
Preparation of liposomes.
Lipids were obtained from Avanti (Birmingham, AL), and tracer
radiolabeled
[3H-methyl]choline
dipalmitoylphosphatidylcholine (DPPC) was from New England Nuclear. The
liposomes were composed of a mixture of lipids combined in the ratio of
0.5 mol of DPPC, 0.25 mol of egg phosphatidylcholine (PC), 0.15 mol of
cholesterol, and 0.1 mol of egg phosphatidylglycerol and tracer
[3H]DPPC (specific
activity = 4,400 degradations · min
1 · nmol
1).
The lipids were dried under nitrogen and were resuspended in buffer.
Liposomes were prepared by extrusion through polycarbonate membranes,
as previously described (19), and were stored overnight at 4°C.
Uptake and degradation of DPPC by macrophages.
Macrophages were incubated with
[3H]DPPC-labeled
liposomes at 80 µM DPPC (120 µM total PC) for 2 h at 37°C.
Cells then were washed three times with MEM, harvested with trypsin
(0.05% trypsin in 0.02% EDTA), and pelleted twice by centrifugation.
Aliquots were taken for protein, total degradations per minute by
scintillation counting, and lipid extraction using the method of Bligh
and Dyer (8). The aqueous fraction was analyzed for degradations per minute. One aliquot of the lipid fraction was subjected to thin-layer chromatography on silica gel G plates run in the solvent system CHCl3-CH3OH-NH4OH-H2O
(65:35:2.5:2.5 by volume) to separate lysophosphatidylcholine from PC
(19, 33). The bands were identified by exposure to I2 vapor, scraped, and counted for
degradations per minute. Another aliquot of the lipid fraction was
osmicated and chromatographed on neutral alumina columns to isolate
disaturated PC (31). Incorporation into the different fractions was
calculated on the basis of the specific activity of DPPC. Unsaturated
PC was determined by subtraction of DPPC from total PC (19).
Statistical analysis.
Values are means of at least two samples in each experiment and are
presented as means ± SE for the number of experiments indicated
(n) or means ± range when the
number of experiments was two. Each experiment used a different
preparation of macrophages. Statistical analysis was performed using
the t-test with SigmaStat for Windows
(Jandel, San Rafael, CA), where significance is set at
P < 0.05. When SE bars are not
visible on Figs. 2 and 3, they are within the symbols.
 |
RESULTS |
Light microscopy.
Alveolar and tissue macrophages cultured for 1 or 24 h were cytospun
onto glass slides, and stained preparations were visualized by light
microscopy (Fig. 1). Most of the 1-h
alveolar and tissue macrophages (Fig. 1,
A and
B) were circular, compact cells with darkly staining nuclei, distinct smooth plasma membranes, and few
pseudopodia. On the other hand, most of the 24-h macrophages (Fig. 1,
C and
D) showed morphological indications
of activation, such as an enlargement of the cytoplasmic area with many
filopodia or lamellipodia, or a vacuolated appearance with a large,
irregular, flattened shape and pale nuclei.

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Fig. 1.
Light micrograph of alveolar (A and
C) and tissue
(B and
D) macrophages after 1 (A and
B) or 24 (C and
D) h of culture. Alveolar and tissue
macrophages were placed in culture for 1 or 24 h, scraped from dish,
cytospun onto glass slides, stained, and visualized by microscopy under
oil. Scale bar, 20 µm.
|
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ROS release.
One measure of the responsive state of macrophages is an increased rate
of release of superoxide and
H2O2
in primed cells compared with quiescent cells when challenged with PMA
(26). To examine whether the 24-h macrophages were "primed," the
freshly isolated alveolar macrophages (1-h cells) or the macrophages
cultured overnight (24-h cells) were incubated without or with PMA (50 ng/ml) in the presence of 5 µM DCFH. The 1-h macrophages showed a low
rate of ROS production that was stimulated over threefold by the
addition of PMA (Table 1). Incubation of
the 24-h macrophages with PMA resulted in a sevenfold increase in ROS
production, which was significantly (5-fold) higher than that found
after PMA exposure of 1-h macrophages. Similar results were seen with
tissue macrophages, although the production of ROS was attenuated
compared with the alveolar macrophages. PMA stimulation of the tissue
macrophages resulted in a significant enhancement of ROS levels by the
24-h macrophages (2-fold) over that produced by 1-h macrophages.
SP-A metabolism.
The cell association and degradation of SP-A by alveolar macrophages
either freshly isolated (1 h) or in culture for 24 h are shown in Fig.
2. Cells were incubated for 3 h at 37°C
with increasing concentrations of
125I-SP-A. The media then were
removed and assayed for SP-A degradation products. The cells were
harvested by dissolution in 0.2 N NaOH to determine the total SP-A
associated with the cell, which includes the SP-A bound to the surface
of the cell and that internalized into the cell during the 3-h
incubation period. The extent of SP-A cell association and degradation
for the 1- and 24-h macrophages varied with the SP-A concentration in
the medium, as has been reported previously for binding of SP-A to
freshly isolated alveolar macrophages (38). Cell association of SP-A,
binding plus uptake, was similar for 1- and 24-h alveolar
macrophages, whereas degradation of SP-A was significantly enhanced
approximately fivefold with overnight culture for all concentrations of
SP-A tested. For tissue macrophages (Fig.
3), the cell association of SP-A by the
24-h macrophages was similar to that of freshly isolated cells.
However, there was a striking 20-fold increase in the amount of SP-A
degradation by tissue macrophages cultured for 24 h compared with
macrophages in culture for 1 h.

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Fig. 2.
Effect of time of cell culture on metabolism of surfactant protein A
(SP-A) by tissue macrophages. Tissue macrophages in culture for 1 h
( ) or 24 h ( ) were incubated for 3 h with increasing
concentrations of bovine SP-A. Cell association
(A) and degradation
(B) of SP-A were measured. Values
are means ± SE of 5 experiments at 0.5 µg SP-A/ml and means ± range of 2 experiments at 1-2 µg SP-A/ml. Where SE or range bars
are not visible, they are within the symbol. At 0.5 µg SP-A/ml,
degradation of SP-A was 20-fold higher
(P < 0.01) by 24-h than by 1-h
macrophages.
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Fig. 3.
Effect of time in culture on metabolism of SP-A by alveolar
macrophages. Alveolar macrophages were placed in culture for 1 h ( )
or 24 h ( ) and then were incubated for an additional 3 h with
increasing concentrations of bovine SP-A. Cell-associated
(A) and degraded
(B) SP-A were measured. Values are
means ± SE of 2-5 experiments. Extent of cell association and
degradation was calculated as percentage of cell association of SP-A to
1-h macrophages at 5 µg SP-A/ml = 100% (control). Control value was
516 ± 91 (SE) ng SP-A/mg cell protein
(n = 5). Cell association of SP-A for
24-h macrophages was significantly different
(P < 0.05) from that for 1-h
macrophages at 3, 7, and 9 µg SP-A/ml and for degradation of SP-A at
all media concentrations of SP-A.
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To directly compare the metabolism of SP-A by alveolar and tissue
macrophages with time in culture, the cell association, degradation,
and total metabolism of SP-A at one concentration in the media (0.5 µg SP-A/ml) were analyzed with the two types of macrophages present
in the same experiment. Incubating the macrophages on culture dishes
for 24 h resulted in a 40% decrease (P < 0.05) or a 70% increase
(P = 0.063) in the cell association of
SP-A to alveolar or tissue macrophages, respectively, but had a marked
stimulatory effect on the rate of SP-A degradation by these cells.
There was a 5-fold (alveolar macrophages) or a 23-fold (tissue
macrophages) increase in the catabolism of SP-A, resulting in a 3- to
11-fold increase in the total SP-A metabolism (binding, uptake, and
degradation) of these cells. Exposure of the 1-h tissue and alveolar
macrophages to SP-A reconstituted with surfactant slightly enhanced the
degradation of SP-A, as indicated by the increase in the the ratio of
SP-A degradation to total SP-A metabolism (cf. Tables
2 and 3).
However, even in the presence of surfactant, the macrophages in culture
for 24 h continued to demonstrate a 6- to 10-fold higher rate of SP-A
degradation than the freshly isolated cells. The metabolism of SP-A by
tissue and alveolar macrophages was similar, although the 1-h tissue
macrophages showed a statistically significantly lower cell association
and degradation of SP-A than the alveolar macrophages (Table 2), which
was not seen in the presence of surfactant (Table 3). Transition of
macrophages from a quiescent to a responsive state with regard to
enhancement of SP-A degradation required more than activation of the
macrophages. Freshly isolated (1 h) alveolar macrophages were incubated
with PMA (50 ng/ml) and 125I-SP-A
(0.5 µg/ml) for 3 h. PMA-exposed macrophages showed a slightly lower
cell association of SP-A (81 ± 6%) than control cells, whereas degradation of SP-A was reduced to 41 ± 5% (SE) of control values (n = 3).
SP-A degradation by alveolar macrophages in culture for 1 h was
previously determined to be intracellular (6). To examine whether this
was also the case with tissue macrophages and was independent of
macrophage time in culture, we determined the effect of a
lysosomotrophic agent and evaluated the possible presence of
proteolytic activity in conditioned culture medium. The pH within
acidic organelles was raised by addition of
NH4Cl (10 mM) to the incubation
medium. This treatment did not markedly alter the cell association of
125I-SP-A (in surfactant, 8.6 µg
SP-A/ml) to the 1- or 24-h tissue macrophages (106 ± 2 and 122 ± 37% of control values, respectively) but inhibited degradation
of SP-A by 64 ± 7% with freshly isolated tissue macrophages and 72 ± 5% with those cultured for 24 h (means ± range of 2 experiments performed in duplicate). Medium conditioned by incubation
with 24-h alveolar macrophages did not degrade SP-A, similar to the
results with freshly isolated macrophages (7). Conditioned medium was
obtained after a 2-h incubation with the 24-h macrophages, as described
previously (7), and placed on empty dishes. Fresh medium was placed on
empty dishes, and the macrophages were refed fresh medium. After an
additional 2-h incubation with
125I-labeled bovine SP-A in
surfactant (8.6 µg/ml), SP-A degradation was quantitated and
corrected for degradation that occurred with fresh medium on the empty
dishes. The extent of SP-A catabolism by conditioned medium was
negligible [0.3 ± 0.1% (SE),
n = 3] compared with the
catabolism of SP-A by the 24-h macrophages over the 2-h time period
(100%). We previously found that medium conditioned by freshly
isolated macrophages also did not catabolize SP-A (6). These data
indicated that possible secretion of enzymes into the media did not
play a role in the enhanced protein degradation rate observed with the
24-h macrophages.
Phospholipid metabolism.
Although the 24-h macrophages demonstrated an enhanced SP-A
degradation, the phospholipid metabolism by these cells was not affected by time in culture. Macrophages were incubated for 2 h with
liposomes containing
[3H]DPPC, and the
uptake and degradation of the DPPC were evaluated (Table
4). The incorporation of DPPC by alveolar
and tissue macrophages was similar and was unaffected by time in
culture. The macrophages degraded approximately one-half of the
internalized DPPC during a 2-h incubation regardless of the source of
macrophages or the time in culture. The principal radiolabeled
metabolites of choline-labeled DPPC were recovered in the aqueous
soluble portion with a small fraction in lysophosphatidylcholine (Table
5). There were virtually no
3H-labeled unsaturated
phospholipids in the cells, indicating that the macrophages did not
reutilize the
[3H]choline for
unsaturated PC synthesis, unlike the findings with type II pneumocytes
(19). After 2 h, the incubation media contained <0.5% of
radioactivity in the aqueous soluble choline-containing products.
 |
DISCUSSION |
Alveolar macrophages freshly isolated from the lung by lavage have
demonstrated the ability to bind, incorporate, and degrade SP-A (6, 37,
38, 48). The present study demonstrates that alveolar and tissue
macrophages that have been in culture for 24 h have characteristics of
primed macrophages with altered morphology and enhanced respiratory
burst on stimulation. Furthermore, the 24-h macrophages show a
substantially elevated rate of SP-A degradation compared with the fresh
macrophages because of intracellular degradation of SP-A. On the other
hand, macrophage uptake and degradation of phospholipid liposomes were
not altered by prolonged culture time, showing differential regulation
of catabolic function.
Pulmonary macrophages are distinguished primarily by their anatomic
location in the lung. The alveolar macrophage is found on the surface
of the alveoli in contact with the epithelial cells lining the airway
and can be isolated by lavage of the alveolar compartment. The
interstitial macrophage resides within the lung parenchyma, where it
undergoes maturation and differentiation before migrating into the
alveoli and becoming the alveolar macrophage. The interstitial
macrophage is isolated by mechanical disruption of lavaged lung with or
without enzymatic digestion of lung slices. It remains controversial as
to whether the postlavage tissue macrophages represent subpopulations
of alveolar macrophages or are, in fact, interstitial macrophages
analogous to those of other organs because of the lack of
distinguishing characteristics between the two cell types. The alveolar
and interstitial macrophages share most of the same functional
properties of phagocytic activity (28) and fibroblast growth factor
production (1), although they exhibit quantitative differences in some
functions, such as cytokine and oxygen radical production (29). In the
present study, macrophages isolated from lung minced tissue without
enzymatic digestion after extensive lung lavage are referred to as
tissue macrophages. Quantitative but not qualitative differences in
SP-A metabolism and ROS production were noted between the tissue and
alveolar macrophages that would indicate that they were different cell
types or were at a different stage of maturation. Freshly isolated
alveolar macrophages took up slightly more SP-A than tissue
macrophages, whereas alveolar macrophages degraded two to three times
as much SP-A. Although both types of pulmonary macrophages increased
their rates of SP-A degradation after activation by prolonged time in
culture, tissue macrophages demonstrated a greater change with time in
culture (23-fold) than the alveolar macrophages (5-fold). On the other hand, ROS production stimulated by PMA was higher in alveolar than in
tissue macrophages. However, the phospholipid metabolism of the
pulmonary macrophages was quite similar, as reflected in the rates of
uptake and degradation of DPPC.
Macrophages are usually studied as freshly isolated cells in suspension
or allowed to attach to a dish for a short period of time. The priming
of pulmonary macrophages by prolonged culture has not been reported
directly, although Prokhorova et al. (39) noted a greater sensitivity
to lipopolysaccharide and interferon-
stimulation of nitric oxide
production after overnight culture of interstitial macrophages than in
freshly isolated cells, data that would be consistent with an increase
in reactive state. In addition, a 24-h exposure of macrophages to serum
proteins causes rabbit alveolar macrophages to release reactive oxygen
intermediates (22), induces maturation of human monocytes as assessed
by increases in intracellular enzymatic activity (34), and stimulates
peritoneal macrophage spreading (9). Primed and activated macrophages demonstrate a greater rate of cell metabolism than resting cells, as
exemplified by a more rapid spreading on glass, higher glucose oxidation, more effective killing of microorganisms, and elevated content of lysosomal enzymes (42). In this study, we have shown that
alveolar and tissue macrophages show morphological and biochemical evidence of priming when placed in tissue culture overnight on plastic
dishes. After 24 h in culture and centrifugation onto glass slides, the
cells were spread, contained empty vacuoles resulting in a foamy
appearance, and had many pseudopodia. In addition, on stimulation with
phorbol ester, the macrophages released elevated amounts of reactive
oxygen species.
The mechanism for macrophage priming by overnight culture is not known.
Because only a portion of the cells remain attached to the dishes after
24 h of culture, it is possible that the culture conditions select for
cells primed during the 1-h incubation and that 24 h of culture are not
necessary to convert the cells to a more responsive state. Another
possibility is that the macrophages during 24 h of culture act as
frustrated phagocytes, in that they try to "ingest" the tissue
culture dish, which they recognize as foreign material. Several pieces
of evidence support the conclusion that prolonged culture is priming
the cells, and that, rather than selection, is responsible for their
increased activity. Light microscopy indicated that the 24-h
macrophages, particularly the alveolar macrophages, have a morphology
entirely different from that of the 1-h cells and were typified by
enlarged, foamy cells with many lamellipodia. Second, a 75% loss of
macrophages from the 1-h cell population during 24 h of culture could
potentially result in a fourfold increase in catabolism of SP-A on a
per milligram of cell protein basis, assuming that all the lost cells
were nonresponsive. Although this is close to the 5-fold increase
observed for alveolar macrophages, it could not account for the 10- to
23-fold increase in degradation seen with the tissue macrophages.
Finally, the data on the production of ROS provide additional evidence
that priming of the cells has occurred during 24 h of culture. If the macrophage population at 1 h contained one-fourth primed cells (equivalent to the 24-h cells) and three-fourths resting cells, then
PMA stimulation should have increased ROS production 7.3-fold for 1-h
macrophages, which is equivalent to 24-h macrophages. However, the 1-h
cells were stimulated only 3.8-fold by PMA exposure.
Prolonged culture of macrophages did not substantially affect the cell
association of SP-A but markedly enhanced the degradation of SP-A. The
fact that binding plus uptake was not altered suggests that the
substrate for the degradative enzymes did not change, and thus the
catabolic activity must have increased. Because the results are
expressed on a "per milligram of cell protein" basis, the
increase in cell size means an increase in enzymatic activity per cell.
Such a rise in catabolic activity could occur through several
mechanisms, including an increase in enzymatic activity, a change in
kinetic properties of degradative enzymes, an augmentation in the
amount of enzyme, a change in the type of enzymes, or an alteration in
intracellular compartmentation. Activated macrophages have been shown
to have enhanced antimicrobial activity (2) and an elevation in the
content of lysosomal enzymes (for review see Ref. 41). An enrichment in
the level of degradative enzymes would be consistent with the enhanced
ability to degrade SP-A shown in our data. The increase in degradative
ability required prolonged time in culture, since activation of
alveolar macrophages with PMA for 3 h served to reduce, not enhance,
SP-A degradation.
Our previous studies using freshly isolated alveolar macrophages (6)
indicated that the degradation of SP-A occurred within the cell. The
present work determined that this was the case regardless of the state
of activation. When the pH within intracellular acidic organelles was
raised with a lysosomotropic weak base, breakdown of SP-A was inhibited
in all the macrophage populations, supporting an important role for
such sites in SP-A degradation by pulmonary macrophages. Furthermore,
as the external concentration of SP-A was raised, the extent of SP-A
degradation was parallel to the increase in cell association of SP-A,
demonstrating the dependence of degradation on the amount of
cell-associated substrate. Finally, extracellular enzymes that may have
been released by primed macrophages (41) did not participate in SP-A
catabolism, since medium conditioned by 24-h macrophages did not
degrade SP-A.
It was of interest that, whereas SP-A degradation was markedly altered
with time in culture, phospholipid degradation was not affected. Such
data imply that, once internalized, degradation of surfactant
phospholipid and proteins occurs in separate cellular compartments or
that only one set of degradative enzymes was activated. Segregation of
DPPC and SP-A after uptake has been noted in the isolated, perfused
lung (20) and in morphological (48) and biochemical studies (42) of
isolated type II cells. The metabolism of DPPC liposomes by macrophages
shares some but not all characteristics of type II cells. Table
6 compares the uptake, reutilization, and
degradation of phospholipid liposomes by the two cell types. Whereas
type II cells incorporated two times as much PC as macrophages, both
cell types actively degraded the phospholipid. Sixty-five percent of
the PC incorporated into type II cells was recovered as aqueous
degradation products, lysophosphatidylcholine, or unsaturated PC,
whereas macrophages degraded 50%. Consistent with the expected low
rate of macrophage PC synthesis, there was no evidence of reutilization
of the liberated
[3H]choline by
macrophages, whereas type II cells readily resynthesize unsaturated PC
from the choline initially associated with the DPPC. The 20% choline
"reutilization" by the type II cells shown in Table 6 represents
a lower limit, since it does not include choline reincorporated into
DPPC.
In conclusion, we have shown that freshly isolated interstitial and
alveolar macrophages were converted to a responsive state during
prolonged culture. These macrophages produced ROS and degraded SP-A,
but not phospholipid, at an elevated rate. In several
pathophysiological states, including acute endotoxemia, pulmonary
interstitial and alveolar macrophages demonstrate enhanced phagocytosis
and production of reactive oxygen intermediates (45) and could be more
active in their metabolism of SP-A. Although possible effects of
various pulmonary diseases on the rate of surfactant lipid and protein turnover by macrophages in intact tissue remain to be determined, the
present study demonstrates that the capacity of pulmonary macrophages
to degrade SP-A depends on their state of activation.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Qiping Chen for the iodination of the SP-A,
to Dr. Michael Beers for the donation of lavage fluid from patients
with alveolar proteinosis, and to Dr. Henry Shuman and Kathy
Notarfrancesco for light microscopy and photography.
 |
FOOTNOTES |
This research was supported by National Heart, Lung, and Blood
Institute Grant HL-19737.
Address for reprint requests: S. R. Bates, Institute for Environmental
Medicine, University of Pennsylvania, 36th and Hamilton Walk, 1 John
Morgan Bldg., Philadelphia, PA 19104.
Received 15 October 1996; accepted in final form 10 June 1997.
 |
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