Am J Physiol Lung Cell Mol Physiol 289: L1011–L1018, 2005.
First published July 8, 2005; doi:10.1152/ajplung.00250.2005.
Pathways for clearance of surfactant protein A from the lung
Deepika Jain, Chandra Dodia, Aron B. Fisher, and Sandra R. Bates
Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania
Submitted 9 June 2005; accepted in final form 4 July 2005
PULMONARY SURFACTANT FORMS a thin film at the air-liquid
interface of the alveoli, contributing to alveolar stability and
immune functions. The chemical composition of lung surfactant is highly conserved among species and consists of 80 –
90% phospholipids, 3–10% neutral lipids (primarily cholesterol), and 5–10% surfactant-associated proteins (10). The
extracellular metabolic pathway is only partially understood
and includes morphological transformations of surfactant
forms within the liquid phase (31, 40), as well as clearance of
spent surfactant. Possible mechanisms for clearance of surfactant components from the alveoli include extracellular degradation within the air spaces, uptake by various cell types,
swallowing after movement up the airways, and removal
through the air-blood barrier (49). Previous studies have demonstrated that uptake by type II cells and alveolar macrophages
is responsible for the major fraction of surfactant phospholipid
and protein removed from the alveolus (5, 12, 18, 20).
Surfactant protein A (SP-A), the most abundant protein in
pulmonary surfactant, is an ⬃700-kDa oligomeric hydrophilic
glycoprotein belonging to the collectin family. Monomeric
SP-A varies from 26 to 36 kDa depending on the degree of
glycosylation. Although SP-A is synthesized, secreted, and
recycled primarily by type II pneumocytes (30, 47), it also has
been immunohistochemically identified in other cell types of
the lung, including macrophages (45, 49) and nonciliated
bronchial (Clara) cells (45) and cells of the tracheal and
bronchial glands (34). SP-A is thought to be secreted constitutively by type II cells into the alveolar lumen, where it
associates with surfactant lipid (24).
Several in vitro studies have identified important functions
of SP-A in the lung, such as formation of tubular myelin (36),
enhancement of the rate of surface adsorption of the lipids (41),
prevention of inactivation of the biophysical properties of
surfactant by plasma (15), and modulation of various immunologic functions of the surfactant (50). With regard to regulation of extracellular surfactant metabolism, it is well established that SP-A plays an important role in surfactant phospholipid secretion, reuptake, and recycling (18, 20, 27). SP-A
has been shown to enhance uptake of phospholipid liposomes
by type II cells (3, 43), macrophages (2, 52), and intact lung
(25), to modulate the activity of the lysosomal-type phospholipase A2, which degrades dipalmitoylphosphatidylcholine
(16), and to inhibit phospholipid secretion (7, 51). Secretagogues that enhance phospholipid secretion and uptake also
enhance Ca2⫹-dependent SP-A binding by the type II cell and
recruitment of SP-A receptors to the type II cell membrane
(13). However, the exact mechanism for uptake of SP-A is not
well defined.
Mammalian cells are known to internalize particles through
a number of processes collectively termed “endocytosis.” Endocytic pathways include clathrin-dependent receptor-mediated uptake and clathrin-independent endocytosis, along with
other mechanisms such as macropinocytosis, phagocytosis, and
caveolae-dependent endocytosis (1, 32). Clathrin is a heteromeric protein that forms a basketlike framework of coated pits
on the cytoplasmic face of the membrane. Internalization of
clathrin-coated vesicle and its contents occurs in response to
receptor cross-linking. Considering that the major endocytic
pathways of the type II cell would involve clathrin and/or actin,
we previously used specific inhibitors of clathrin and actin to
identify the mechanism whereby phospholipid liposomes and
phospholipids in natural surfactant were incorporated into the
lung and type II pneumocytes (18, 27). We found evidence for
participation of clathrin-dependent and clathrin-independent
pathways with actin polymerization necessary for uptake of
lipids via the clathrin-mediated pathway. Previous studies have
provided evidence that SP-A uptake is mediated by clathrin
(39, 42, 55), but a possible role for nonclathrin endocytosis has
not been evaluated. In the present study, we examined this
possibility for uptake and whether actin plays a role in the
clathrin-dependent uptake of SP-A. In addition, we have compared the effect of inhibitors on clearance of SP-A by cultured
cells in vitro and in the intact lung.
Address for reprint requests and other correspondence: S. R. Bates, Institute
for Environmental Medicine, Univ. of Pennsylvania School of Medicine, 1
John Morgan Bldg., 3620 Hamilton Walk, Philadelphia, PA 19104-6068
(e-mail: [email protected]).
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.
clathrin-mediated uptake; actin; type II cells; macrophages; degradation; perfused lung
http://www.ajplung.org
1040-0605/05 $8.00 Copyright © 2005 the American Physiological Society
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Jain, Deepika, Chandra Dodia, Aron B. Fisher, and Sandra R.
Bates. Pathways for clearance of surfactant protein A from the lung.
Am J Physiol Lung Cell Mol Physiol 289: L1011–L1018, 2005. First
published July 8, 2005; doi:10.1152/ajplung.00250.2005.—Uptake
and degradation of 125I-surfactant protein A (SP-A) over a 1-h period
was studied in alveolar cells in culture and in isolated perfused lungs
to elucidate the mechanism for clearance of the protein from the
alveolar space. Specific inhibitors of clathrin- and actin-dependent
endocytosis were utilized. In type II cells, uptake of SP-A, compared
with controls, was decreased by 60% on incubation with clathrin
inhibitors (amantadine and phenylarsine oxide) or with the actin
inhibitor cytochalasin D. All agents reduced SP-A metabolism by
alveolar macrophages. Untreated rat isolated perfused lungs internalized 36% of instilled SP-A, and 56% of the incorporated SP-A was
degraded. Inhibitors of clathrin and actin significantly reduced SP-A
uptake by ⬃54%, whereas cytochalasin D inhibited SP-A degradation. Coincubation of agents did not produce an additive effect on
uptake of SP-A by cultured pneumocytes or isolated perfused lungs,
indicating that all agents affected the same pathway. Thus SP-A clears
the lung via a clathrin-mediated pathway that requires the polymerization of actin.
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CLEARANCE OF SURFACTANT PROTEIN A FROM THE LUNG
MATERIALS AND METHODS
AJP-Lung Cell Mol Physiol • VOL
RESULTS
SP-A association with type II cells. Microscopy and biochemical analysis provided evidence that SP-A is found in
association with coated pits and is internalized into type II cells
(39, 42, 55). We previously showed that PAO, an agent that
prevents formation of clathrin-coated vesicles, inhibited the
uptake of SP-A by type II cells in culture, suggesting a role for
clathrin-coated pits in internalization of SP-A (8). To confirm
and extend our data, the present experiments used an additional
clathrin inhibitor that works downstream of PAO, i.e., amantadine, a cationic amphiphilic agent that prevents budding of
clathrin-coated vesicles. In addition, the effect of actin inhibition on incorporation of SP-A into type II cells was examined
by treating the cells with CytoD, an agent that destabilizes the
actin cytoskeleton.
Control cells not exposed to agents showed that SP-A
binding was similar at 30 and 60 min (Fig. 1A). Neither of the
inhibitors had a significant effect on binding of SP-A to type II
cells (Fig. 1A). Uptake of SP-A by the pneumocytes increased
linearly with time during a 60-min incubation (Fig. 1B). In
contrast to the effect on binding, blocking clathrin- or actinmediated processes resulted in inhibition of SP-A internaliza-
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Sprague-Dawley pathogen-free male rats weighing ⬃200 –250 g
were obtained from Charles River Breeding Laboratories (Kingston,
NY). Animal procedures were approved by the Institutional Animal
Care and Use Committee of the University of Pennsylvania. Phenylarsine oxide (PAO), cytochalasin D (CytoD), amantadine HCl, and
bovine serum albumin (BSA) were purchased from Sigma (St. Louis,
MO); fatty acid-free BSA from Roche Diagnostics (Indianapolis, IN);
and 125I from Amersham Biosciences (Piscataway, NJ).
Isolation of surfactant and purification of SP-A. SP-A was purified
from bronchoalveolar lavage (BAL) fluid of alveolar proteinosis
patients after therapeutic lavage at the Hospital of the University of
Pennsylvania. Whole surfactant was isolated by density gradient
centrifugation of cell-free lavage fluid. The surfactant fraction was
dialyzed and lyophilized as described previously (3). SP-A was
extracted from whole surfactant using 1-butanol and ␤-D-glucopyranoside (23). Rat lung surfactant was isolated after intratracheal
lavage of rat lungs followed by NaCl-NaBr gradient centrifugation of
the cell-free lavage fluid (20).
Iodination. Iodination of SP-A was performed using Iodogen
(Pierce, Rockford, IL) according to the manufacturer’s directions.
Iodinated protein was dialyzed against 5 mM Tris buffer for 24 h with
frequent buffer changes to remove free 125I. Specific activity for all
preparations was ⬃200 – 400 cpm/ng protein. The trichloroacetic acid
(TCA) precipitability was 76 –98%. The iodinated proteins were
stored at 4°C and used within 2–3 wk. Storage of SP-A did not
appreciably affect TCA precipitability.
Isolation and culture of type II cells. Alveolar type II pneumocytes
were isolated from 200- to 250-g Sprague-Dawley rats as previously
described (3). Rat lungs were perfused and subjected to endotracheal
lavage before instillation of elastase (Sigma). The lobes were minced
with a tissue chopper, and the lysate was sequentially filtered through
160-, 37-, and 10-␮m mesh. Macrophages were removed from the
resulting cell suspension by plating on 100-mm dishes precoated with
rat immunoglobulin G (Sigma) in serum-free medium for 1 h at 37°C.
Nonadherent cells were resuspended in Eagle’s minimal essential
medium (MEM) containing 10% fetal calf serum and plated on
35-mm plastic tissue culture dishes (Costar, Cambridge, MA) for 24 h
at 37°C in a humidified incubator with 5% CO2 in air.
SP-A uptake in cultured alveolar pneumocytes. After overnight
culture, cells were washed twice with MEM and once with MEM
containing 0.1% fatty acid-free BSA. Cells then were incubated for 30
min in MEM in the presence or absence of inhibitors (2.5 mM
amantadine, 2 ␮M PAO, and 10 ␮M CytoD). The concentration of
inhibitors was determined previously and represents the levels necessary for maximum effect without toxicity for the uptake of liposomes
(38) and SP-A (data not shown). 125I-SP-A was added to each of the
dishes at a final concentration of 1 ␮g/ml, and the dishes were
incubated for 1 h. After 125I uptake, cells were washed once with
MEM ⫹ 0.3% fatty acid-free BSA, twice with MEM ⫹ 0.1% fatty
acid-free BSA, and twice with PBS. Cells were then removed from the
dishes by trypsinization. SP-A binding was assessed by measuring
trypsin-releasable 125I-SP-A, and uptake represented trypsin-insensitive disintegrations per minute. After one wash with PBS, the cell
pellet was finally suspended in 1 ml of PBS and sonicated. A portion
of the sonicate was used to estimate total protein (11), and the
remaining sonicate was counted in a gamma counter (Beckman).
Values were corrected for TCA precipitability of the iodinated SP-A
preparation. We did not examine SP-A degradation, inasmuch as
available evidence indicates that type II cells in culture do not
catabolize SP-A (5, 27).
SP-A uptake by alveolar macrophages. Alveolar macrophages,
obtained from lung BAL of pathogen-free male Sprague-Dawley rats
(500 g), were plated in 35-mm plastic dishes (Costar, Cambridge,
MA) at 37°C in serum-free MEM. After 1 h, the cells were washed
three or more times to remove red blood cells and other nonadherent
cells; 99% of the cells that adhered to the dish were alveolar macrophages. Experiments on the uptake of 125I-SP-A followed the protocol
used for type II cells. Macrophages were solubilized using 0.2 N
NaOH, and an aliquot was used for determination of total protein by
the method of Lowry et al. (29); another aliquot was counted for
radioactivity. To estimate degradation of SP-A by macrophages,
medium was collected from the dishes at the end of the experiment
and analyzed for the presence of TCA-soluble SP-A as previously
described (5).
SP-A uptake in isolated perfused lung. Rats were anesthetized with
pentobarbital sodium (50 mg/kg body wt ip). A tracheal catheter was
used for instillation of 7.5 ␮g of 125I-SP-A in 100 ␮l of PBS into the
lungs. This procedure ensured uniform distribution of the labeled
SP-A throughout the lung. Of the total SP-A recovered after instillation, 24, 14, 16, 38, and 9% were recovered from the lower-left,
middle-left, upper-left, lower-right, and upper-right portions of the
lung, respectively; these recoveries are consistent with the sizes of the
lobes. Lungs were cleared of blood by perfusion with 5 mM KrebsRinger bicarbonate buffer containing 3% BSA and moved to an
isolated organ perfusion system (19). Perfusion buffer consisted of
Krebs-Ringer bicarbonate with 3% fatty acid-free BSA and 10 mM
glucose. Perfusate was constantly gassed with 5% CO2 in air. Lungs
were ventilated at 60 cycles/min, 2 ml tidal volume, and 2 cmH2O
end-expiratory pressure. Lungs were perfused for 1 h with buffer
alone (control) or with buffer containing 2 ␮M PAO, 5 mM amantadine, or 10 ␮M CytoD, and 1-ml aliquots of the perfusate were
collected every 10 min. At the end of the 1-h experimental perfusion,
lungs were lavaged five times with 10-ml aliquots of ice-cold Ca2⫹and Mg2⫹-free PBS. Lung tissue was homogenized in PBS on ice
with a Polytron homogenizer and then with a motorized mortar and
pestle. Perfusate, lavage, and lung homogenate were counted for total
radioactivity as well as TCA precipitability (5). Results are expressed
as a percentage of the total 125I-SP-A counts recovered, which
represents ⬃83 ⫾ 0.7% (n ⫽ 22) of the total 125I-SP-A instilled.
TCA-soluble counts in the instilled SP-A (2–24% of total) were
subtracted from TCA-soluble counts in the lung fractions.
Statistical analysis. Values are means ⫾ SE unless otherwise
stated. Results were analyzed statistically by t-test or paired t-test
using SigmaStat for Windows (Jandel, San Rafael, CA), where statistical significance is taken as P ⬍ 0.05. Multiple-group comparisons
were done by one-way ANOVA.
CLEARANCE OF SURFACTANT PROTEIN A FROM THE LUNG
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Fig. 1. Effect of inhibitors of clathrin [amantadine HCl (Amant), 2.5 mM] or
actin [cytochalasin D (CytoD), 10 ␮M] on time course of binding (A) and
uptake (B) of surfactant protein A (SP-A) by alveolar type II cells in culture.
Isolated type II cells were preincubated with or without inhibitors for 30 min
before addition of 125I-labeled SP-A (1 ␮g/ml). Cells were harvested by
trypsinization. Radioactivity in trypsin-releasable (surface-bound) SP-A and in
cell sonicates (trypsin-insensitive, internalized SP-A) was counted. Values
(means ⫾ SE of 5– 6 experiments) are expressed as percentage of uptake of
control cells at 30 min. *P ⬍ 0.05 vs. inhibitors. Neither inhibitor had an effect
on SP-A binding. Amantadine and CytoD reduced SP-A uptake after 60 min.
tion by type II cells at the initial time point (30 min), with a
significant reduction in cellular incorporation of SP-A after 1 h
of intervention (Fig. 1B). After 1 h of incubation with 125ISP-A, PAO decreased SP-A uptake in type II cells to ⬃50% of
the control value (Fig. 2), confirming previous results (8).
Amantadine and CytoD were as effective as PAO in inhibiting
SP-A uptake by type II cells in culture (Fig. 2). Treatment of
type II cells with a combination of actin and clathrin inhibitors
did not further reduce SP-A incorporation by the cells, indicating a single clathrin-dependent pathway that requires actin
polymerization.
Effect of lipid on SP-A uptake. SP-A in lung surfactant in
vivo is associated with phospholipid, especially phosphatidylcholine. To elucidate whether association of SP-A with lipid
has an effect on the mechanism of uptake, whole rat surfactant
isolated from lung lavage or Survanta, a commercially available SP-A-free surfactant preparation, was mixed with iodinated SP-A in the ratio of 9:1 (9 ␮g phospholipid/␮g 125I-SP-A).
Isolated type II cells preincubated with or without inhibitors for
AJP-Lung Cell Mol Physiol • VOL
30 min were further incubated with this SP-A-phospholipid
mixture for 1 h. With rat surfactant, results were corrected for
the presence of endogenous SP-A, estimated to be 1.5 ␮g of
SP-A in 9 ␮g of surfactant phospholipid (44). The presence of
rat surfactant phospholipid resulted in 1.9-fold augmentation of
SP-A uptake (Fig. 3). As with lipid-free SP-A, uptake of the
SP-A-phospholipid complex was blocked by clathrin and actin
antagonists. Addition of a combination of agents had no further
effect (Fig. 3). Similar results were seen with addition of
Survanta phospholipid (data not shown), demonstrating that
the inhibitory effect of clathrin antagonists and CytoD was the
same, irrespective of differences in the source of phospholipid.
SP-A metabolism by alveolar macrophages in culture. Alveolar macrophages have been shown to endocytose and de-
Fig. 3. Effect of exogenous phospholipids on SP-A uptake by type II cells.
Isolated type II cells were incubated with inhibitors for 30 min before addition
of 1 ␮g/ml of 125I-labeled SP-A and 9 ␮g/ml of rat surfactant. Uptake was
measured after 1 h of incubation. Values are means ⫾ SE of 3 experiments.
*P ⬍ 0.05 vs. control without surfactant. #P ⬍ 0.05 vs. control with surfactant.
Addition of exogenous surfactant enhanced SP-A uptake 2-fold. Clathrin and
actin inhibitors had a pronounced negative effect on internalization of SP-A,
irrespective of the presence of surfactant.
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Fig. 2. Clathrin and actin inhibitors reduce SP-A uptake by alveolar type II
cells. Type II cells in culture were incubated with or without inhibitors before
addition of 1 ␮g of 125I-labeled SP-A. Uptake was measured in cell sonicates
after 1 h of incubation with SP-A. PAO, phenylarsine oxide. Values are
means ⫾ SE of 5– 6 experiments. *P ⬍ 0.05 vs. control. Disruption of clathrin
as well as actin inhibited SP-A uptake by ⬃50%. Addition of a combination of
2 agents had no further effect.
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CLEARANCE OF SURFACTANT PROTEIN A FROM THE LUNG
Fig. 4. SP-A metabolism by alveolar macrophages. Alveolar macrophages
were isolated from bronchoalveolar lavage and cultured on plastic dishes. After
addition of 1 ␮g/ml 125I-labeled SP-A in the presence or absence of inhibitors,
adherent cells were solubilized in 0.2 N NaOH, and radioactivity was counted
in cell lysates for SP-A uptake. Medium was analyzed for TCA-soluble
degradation products of SP-A (B). SP-A uptake (A) is the sum of intact SP-A
in cell lysates and degraded products in the medium. Values are means ⫾ SE
of 3–5 different experiments. *Significantly different from control (P ⬍ 0.05).
All disrupting agents had a significant inhibitory effect on SP-A uptake by
alveolar macrophages. SP-A uptake in control cells ⫽ 217 ⫾ 55 (SE) ng
SP-A/mg cell protein (n ⫽ 5).
AJP-Lung Cell Mol Physiol • VOL
demonstrated a minor (30%) effect on the extent of SP-A
degradation once SP-A was taken up by the cells (Fig. 4B),
indicative of a possible effect on an intracellular degradative
process.
Metabolism of SP-A in isolated perfused lung. Because in
vitro cell culture may result in alteration of metabolic pathways, extrapolation to results in vivo can be problematic. To
examine the in vivo situation more closely, we utilized the
isolated perfused lung model. Iodinated SP-A was instilled into
lungs before they were removed from the chest, then the lungs
were perfused for 1 h in the presence or absence of inhibitors
(see MATERIALS AND METHODS). Uptake of SP-A from the alveolar space into the lung cells was defined as the sum of the
TCA-precipitable SP-A products recovered from lung tissue
and the TCA-soluble SP-A recovered from the lung homogenate, lavage, and perfusate. There was no TCA-precipitable
SP-A in the perfusate. Total recovery of instilled 125I-SP-A
was ⱖ85% after 1 h of perfusion.
Catabolism of SP-A by the lung, as indicated by the appearance of TCA-soluble 125I-labeled amino acids in the perfusate,
was fairly rapid (Fig. 5A). SP-A degradation products appeared
in the perfusate within the initial time period (10 min) and
accumulated linearly over the subsequent 60-min perfusion
period. At 1 h after instillation, significant uptake of SP-A, as
measured by the sum of the TCA-precipitable 125I disintegrations per minute in the lung, together with the TCA-soluble
counts in the lavage, perfusate, and lung, had occurred. Uptake
of SP-A into control lungs was 2.63 ⫾ 0.18 ␮g of SP-A or 36%
of SP-A instilled. Amantadine- and PAO-treated lungs internalized 1.02 ⫾ 0.03 and 1.07 ⫾ 0.13 ␮g of SP-A, respectively,
⬃40% of control values. Uptake of SP-A by CytoD-treated
lungs was 1.07 ⫾ 0.14 ␮g (P ⬍ 0.05 vs. control). Addition of
PAO ⫹ CytoD had no significant further effect on SP-A uptake
over either of the inhibitors alone (Fig. 5B).
To determine whether 125I-SP-A reached type II cells and
alveolar macrophages via the intratracheal instillation procedure, type II cells (isolated as described in MATERIALS AND
METHODS) and alveolar macrophages (isolated from the lung
lavage fluid) were obtained from rat lungs 1 h after instillation
of 125I-SP-A. A direct comparison of the quantity of SP-A
associated with the two cell types is not meaningful because of
differences in the protocols. The isolation of type II cells from
the rat lungs took several hours, and the cells were treated with
enzymes at 37°C during the procedure, whereas the isolation of
macrophages was performed quickly and at cold temperatures.
However, 125I-SP-A was associated with both cell types (43 ⫾
6 and 751 ⫾ 158 ng SP-A/mg cell protein for type II cells and
macrophages, respectively, n ⫽ 3), demonstrating that 125ISP-A reached type II cells and macrophages via our instillation
procedure.
Total degradation of SP-A was taken as the sum of TCAsoluble 125I-SP-A disintegrations per minute recovered from
the homogenate, lavage, and perfusate. Approximate recoveries of degraded SP-A, expressed as a percentage of the total
TCA-soluble 125I disintegrations per minute, were 20% from
the lung homogenate, 33% from the BAL, and 47% from the
perfusate. The location for recovery of the degraded SP-A
products did not change on treatment with agents. Of the total
radioactivity recovered from control lungs after 1 h of perfusion, ⬃44% was associated with TCA-precipitable protein
(intact SP-A), and the remaining radioactivity (56%) was in the
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grade SP-A (4, 5, 53). Although macrophages have receptors
for SP-A (14) and would be expected to utilize a clathrincoated pit pathway for internalization, macrophages are also
active phagocytes, a process requiring actin polymerization.
Clathrin or actin antagonists were utilized to determine possible effects on the uptake of SP-A by macrophages. In addition,
alterations in macrophage degradation of SP-A were followed.
Alveolar macrophages were isolated from lung lavage, and
cells were preincubated with the inhibitors before addition of
125
I-SP-A to the medium. As seen for type II cells, clathrin
inhibitors and CytoD inhibited total uptake of SP-A (intact
SP-A ⫹ degradation products) by macrophages in culture (Fig.
4A). Exposure to clathrin and actin inhibitors was not additive,
which is indicative of a single clathrin-dependent uptake pathway requiring actin activity. Macrophages without agents degraded SP-A rapidly, with ⬃85% of the total SP-A recovered
from the culture as TCA-soluble products (Fig. 4B). All agents
CLEARANCE OF SURFACTANT PROTEIN A FROM THE LUNG
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filaments in the subsequent transport of the internalized SP-A
to the site of degradation.
DISCUSSION
TCA-soluble fraction. Because all the agents inhibited the
uptake of SP-A into the lung, the extent of degradation of SP-A
(as ␮g SP-A/lung) also was reduced by the drugs (Fig. 5B).
However, when SP-A degradation is expressed as a percentage
of uptake (Fig. 5C), alteration of the actin cytoskeleton with
CytoD resulted in a reduction of SP-A degradation in the
absence or presence of PAO, whereas perfusion of the lungs in
the presence of the clathrin inhibitors PAO and amantadine did
not affect degradation (Fig. 5C), implying a role for actin
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Fig. 5. SP-A uptake and degradation in isolated perfused lung. Rats were
anesthetized, and lungs were instilled with 7.5 ␮g/ml 125I-labeled SP-A in
saline. A: time course of appearance of TCA-soluble SP-A in perfusate.
Samples were collected from perfusate every 10 min and analyzed for the
presence of TCA-soluble radioactivity, which represents degradation products
of SP-A. Values are means ⫾ SE (n ⫽ 15). B: uptake and degradation of SP-A.
After instillation of SP-A, lungs were perfused for 1 h in the absence [control
(Cont)] or presence of amantadine, PAO, CytoD, or PAO ⫹ CytoD. Perfusate,
lavage, and lung homogenates were assessed for the presence of TCAprecipitable intact SP-A and TCA-soluble degradation products. SP-A uptake
is defined as the sum of intact SP-A recovered from lung tissue ⫹ degradation
products recovered from perfusate, lavage, and homogenate. Values are
means ⫾ SE (n ⫽ 3–20). *P ⬍ 0.05 vs. control. Control lungs internalized
28% of total SP-A recovered. Clathrin antagonists, amantadine and PAO, as
well as the actin destabilizer CytoD significantly attenuated SP-A uptake in
intact lung but showed no synergistic effects. C: degradation as a percentage
of uptake. Data in B are expressed as percentage of SP-A taken up by the lung,
which was recovered as TCA-soluble disintegrations per minute. *Significantly different from control. #Significantly different from PAO.
Previously, using radioactive liposomes, we evaluated the
endocytic pathways involved in the uptake of surfactant phospholipid and determined that the incorporation of surfactant
lipid into the lung and type II cells was sensitive to inhibitors
of clathrin-coated pit formation and to the polymerization of
actin fibers (38). Our results indicated two distinct pathways
for endocytosis of liposomes, clathrin dependent and clathrin
independent, with some involvement of actin filaments in the
clathrin-mediated uptake. To confirm that SP-A uptake is
strictly clathrin dependent and that this is the case for pneumocytes in culture as well as for the lung in vivo, we have
performed a parallel experiment designed to study the mechanisms utilized for endocytosis of SP-A. Our data indicate that
SP-A uptake in vitro in primary cultures of type II pneumocytes and alveolar macrophages and, importantly, in vivo in the
isolated perfused lung occurred only through a single clathrinmediated pathway that required a functioning actin polymerization process.
We began by examining the role of clathrin and actin
integrity in internalization of SP-A by alveolar type II cells in
vitro. SP-A has been morphologically localized in clathrincoated pits in type II cells by microscopic studies (39, 42). We
reported previously the involvement of clathrin-coated pits in
SP-A internalization by type II cells in culture (8). In the
present study, we have extended our observations about clathrin-mediated SP-A uptake by using two functionally different
clathrin inhibitors, amantadine and PAO. Amantadine is a
cationic amphiphilic drug that stabilizes clathrin-coated vesicles and prevents their budding, whereas PAO blocks clathrinmediated endocytosis by cross-linking clathrin and preventing
the formation of clathrin-coated vesicles. In addition, we
blocked actin-mediated SP-A uptake by using a broad-spectrum disrupter of actin polymerization, CytoD, which caps
F-actin filaments and prevents actin polymerization. We found
that both classes of clathrin inhibitors blocked SP-A uptake in
cultured type II cells by ⬃50%. These results confirm previous
studies which showed that intracellular potassium depletion
inhibits SP-A uptake, compatible with a clathrin-mediated
process (42). As for the clathrin-independent, actin-dependent
pathway, we saw that the actin inhibitor CytoD also inhibited
uptake by 50% in type II cells. However, when the cells were
treated with a combination of clathrin and actin inhibitors,
there was no cumulative effect, showing that each of these
inhibitors acted via the same pathway. Thus there was no
evidence for SP-A endocytosis independent of clathrin. The
observation that an intact actin cytoskeleton plays an important
role in the clathrin-mediated uptake of SP-A by pneumocytes
supports several recent reports that actin assembly has a critical
role in clathrin-coated vesicle formation and receptor-mediated
endocytosis (28, 54). The results add emphasis to several
reports that found an important contribution of actin assembly
to coated pit-dependent uptake processes (9, 21, 28).
The SP-A used in this study was isolated from alveolar
proteinosis patients because of the limited amounts of SP-A
available from the lavage of rat lungs. Previous studies have
shown no qualitative differences in binding rat vs. human SP-A
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CLEARANCE OF SURFACTANT PROTEIN A FROM THE LUNG
AJP-Lung Cell Mol Physiol • VOL
Therefore, we define total uptake as the sum of intact SP-A
associated with lung tissue and degraded SP-A products recovered from lung homogenate, alveolar lavage, and perfusate. As
in isolated alveolar cells in culture, uptake of SP-A by intact
lung was sensitive to clathrin (Amant, PAO) and actin (CytoD)
inhibitors. Addition of inhibitors together failed to produce an
additive effect, suggesting that SP-A is internalized mainly via
a clathrin-dependent pathway and that actin plays a pivotal role
in the clathrin-coated pit mechanism of endocytosis in the lung,
as was found for the pneumocytes in primary culture. Thus we
can distinguish between SP-A incorporation into the lung and
incorporation of surfactant phospholipids, since the latter
showed an additive effect of clathrin inhibitors and CytoD
(38).
Once SP-A was internalized, the intact lung rapidly degraded the surfactant protein. Control lungs catabolized ⬃56%
of the internalized SP-A during 1 h of perfusion. Although
treatment of the lungs with clathrin or actin inhibitors blocked
uptake of SP-A, the ensuing degradation of the internalized
SP-A, expressed as a percentage of uptake, was only affected
by actin inhibition. Thus movement of the internalized SP-A to
the degradation compartment seems to require a functioning
cytoskeleton. However, treatment with PAO or amantadine
reduced SP-A degradation by macrophages, indicating that
macrophages are not the only cell type involved in SP-A
catabolism in the lung, leaving open the question as to the role
of type II cells in this process.
A substantial portion of SP-A uptake (⬃40%) was insensitive to the effects of clathrin and actin inhibitors in pneumocytes in culture and in the isolated lung. This internalization of
SP-A, despite the presence of inhibitors, could be due to the lag
time necessary for the inhibitor to reach the critical cells in the
lung or to take effect in intracellular compartments of the cells
in vivo or in vitro. Other possible explanations include the
following: 1) PAO inhibited formation of clathrin-coated pits
but allowed internalization of pits that had formed before PAO
exposure. 2) Amantadine disrupted clathrin function by preventing budding of the clathrin-coated vesicles. However,
SP-A may have bound to membrane invaginations that were
inaccessible to the cell-washing procedures. This residual uptake might represent SP-A bound to a trypsin-insensitive site
on the cell surface or just inside the cell or SP-A transported
utilizing an alternative internalization pathway (see explanation 5). 3) Loss of cytoskeletal integrity by disruption of actin
with CytoD might result in nonspecific internalization of SP-A.
4) The pharmacological agents used in this study may not
completely inhibit cell processes. 5) It is tempting to speculate
that the newly described pathway utilizing uncoated nonclathrin caveolae-independent endocytic vehicles might play a role
in the uptake of SP-A (26). Our previous study on the uptake
of surfactant-like liposomes showed that the inhibition of the
incorporation of lipids into the lung by these same agents also
was incomplete. In that study, uptake via fusion of the phospholipid liposomes with the plasma membrane or via exchange
of phospholipids was a possibility, which would not be the case
with SP-A. Thus the newly described endocytic vehicle pathway might transfer protein and lipid from the alveolar space
into the lung.
This study was not designed to address the relative contributions of macrophages and type II cells to the processing of
SP-A in the intact lung. Using a nondegradable analog of
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by rat alveolar cells in vitro (55). Several reports from our
laboratory and others have shown that iodination of purified
SP-A retains its biological activity as assessed by its ability to
inhibit phosphatidylcholine secretion by type II cells (7) and to
induce chemotaxis of macrophages in vitro (48), thereby making it a convenient marker to study uptake in the present
experiment.
Because phospholipid uptake occurred via a clathrin-dependent pathway and lipids are not known to employ any receptors, it was hypothesized that phospholipid transport utilizes
SP-A receptors (38). Our present study supports the suggestion
that SP-A could facilitate internalization of the SP-A-lipid
complex through the clathrin-mediated pathway. The pathway
of SP-A incorporation into type II cells was not affected by the
presence of surfactant isolated from rat lung or Survanta,
suggesting that the mechanism of SP-A uptake was not influenced by the presence of lipid. Phospholipid uptake also
utilized a non-clathrin, actin-dependent pathway. It was possible that SP-A might bind to surfactant phospholipids and be
carried into the type II cells via this non-receptor-mediated
pathway. That this might be the case is demonstrated by the
slightly greater, but significantly different, inhibitory effect of
CytoD over amantadine on SP-A uptake in the presence of
surfactant.
Available evidence indicates that alveolar macrophages
make a major contribution to the degradation of SP-A (5, 22,
49). SP-A uptake by alveolar macrophages is mainly via the
clathrin-coated pit pathway (2). Because phagocytosis by alveolar macrophages occurs through clathrin-mediated processes (33), our study using the inhibitors of clathrin (amantadine and PAO) on the metabolism of SP-A by alveolar
macrophages in culture cannot differentiate between coated
pit- and phagocytosis-dependent pathways. However, as with
type II cells, amantadine and PAO significantly inhibited
internalization of SP-A by macrophages. Interestingly, CytoD
also reduced uptake by ⬃60% and had no additive effect with
clathrin inhibitors, indicating an important role for actin in
coated pit movement in macrophages as well. Once SP-A was
internalized, all these agents had a slight, but significant,
inhibitory effect on degradation of SP-A. Thus clathrin and
actin may play a role in the transfer of SP-A to macrophagedegradative compartments.
Most studies of the trafficking of SP-A have primarily
utilized isolated type II cells or alveolar macrophages in cell
culture systems that serve as in vitro models of the lung. To
more closely approximate the physiological environment in the
lung, we have utilized an isolated perfused lung model. The
advantages of this system are that the functional and structural
properties of the organ are preserved and cell-cell interactions
are maintained (25, 38). With this system, whole organ metabolism can be examined under controlled conditions (17). To
study the metabolism of SP-A by the isolated perfused lung,
iodinated SP-A was instilled intratracheally, and rat lungs were
perfused in the presence or absence of inhibitors. The amount
of instilled SP-A, representing ⬃10% of the endogenous SP-A
(44), distributed evenly to the lung lobes. Thus we assume that
the instilled SP-A mixed with the endogenous pool and,
thereby, represents the turnover of SP-A in natural surfactant.
After uptake and degradation of SP-A by pneumocytes, the
degraded products may remain in the lung or appear in the
alveolar space (lavage fluid) or the circulation (perfusate).
CLEARANCE OF SURFACTANT PROTEIN A FROM THE LUNG
ACKNOWLEDGMENTS
We thank Michelle Sperry, Jian-Qin Tao, and Kathy Notafrancesco for
excellent technical assistance.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-19737.
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