Incorporation of biotinylated SP-A into rat
lung surfactant layer, type II cells, and Clara
cells
Jordan
Savov1,
J. R.
Wright2, and
S. L.
Young1
1 Department of Medicine, Veterans Affairs Medical Center
and Duke University Medical Center, and 2 Department of Cell
Biology, Duke University Medical Center, Durham, North Carolina
27705
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ABSTRACT |
The goal of this study was to
compare the functions of Clara and type II cells during alveolar
clearance and recycling of surfactant protein (SP) A, a secretory
product of both cell types. We examined the incorporation of instilled
biotinylated SP-A (bSP-A) into rat lung type II and Clara cells as a
measure of clearance and recycling of the protein. Ultrastructural
localization of bSP-A was accomplished by an electron-microscopic
immunogold technique at 7, 30, and 120 min after intratracheal
instillation. Localization of bSP-A was quantitatively evaluated within
extracellular surfactant components (lipid-rich forms: myelin figures,
vesicles, and tubular myelin; and lipid-poor hypophase) and in
compartments of type II and Clara cells. bSP-A was incorporated into
myelinic and vesicular forms of extracellular surfactant, but tubular
myelin and hypophase had little bSP-A. Lamellar bodies of type II cells
demonstrated a significant time-dependent increase in their
incorporation of bSP-A. There was a concentration of bSP-A in the
secretory granules and mitochondria of Clara cells, but no Clara cell
compartment showed a pattern of time-dependent change in
immunolabeling. Our immunolabeling data demonstrated a time-dependent
movement of exogenous SP-A from extracellular components into type II
cells and their secretory granules. Clara cells did not demonstrate a
time-dependent incorporation of bSP-A into their secretory granules during the period of this study. If Clara cells recycle SP-A, they must
reach a steady state very quickly or very slowly.
surfactant recycling; surfactant subfraction; immunocytochemistry; electron microscopy
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INTRODUCTION |
PULMONARY
SURFACTANT is a complex mix of lipids and lung-specific
surfactant-associated proteins. Surface tension-lowering functions as
well as host defense functions have been attributed to lung surfactant.
Surfactant has an extracellular metabolic pathway that is only
partially understood and that includes morphological transformations of
surfactant forms within the alveolar liquid phase (13,
17). There are several possible pathways of surfactant clearance from the alveolar space: degradation within alveoli or
airways, uptake by several cell types, movement up the airways, or
removal through the air-blood barrier (25). Internalized surfactant components could undergo either recycling or degradation. Although many details of intracellular metabolism remain to be discovered, recycling within alveolar type II cells may be an important
part of surfactant metabolism. Both surfactant phospholipid and
surfactant-associated protein are synthesized as well as recycled by
alveolar type II cells (6, 25,
28).
Surfactant protein (SP) A is a major protein of the alveolar fluid and
is the most extensively characterized of the four known surfactant-associated proteins. It is synthesized, secreted, and recycled by type II alveolar cells. It is found in alveolar
macrophages, but it is not synthesized by them and is degraded by
macrophages after phagocytosis (25). Nonciliated
bronchiolar (Clara) cells are another source of SP-A production in the
lung (1, 2, 21). Humans produce
SP-A not only in type II and Clara cells but also in the cells of
tracheal and bronchial glands (10, 14).
Whether Clara cells have the ability of type II cells not only to
produce but also to recycle SP-A is a subject of this report on the
distribution of biotinylated SP-A (bSP-A) instilled into rat lungs.
The majority of extracellular SP-A is evidently bound with the alveolar
lipids, with <1% reported as free in the surfactant hypophase
(3). We also report herein on the distribution of exogenously administrated bSP-A within extracellular surfactant compartments.
Several in vitro studies identified important functions of SP-A in the
regulation of extracellular surfactant metabolism. SP-A inhibits lipid
secretion by type II cells (5) and enhances lipid uptake
by type II cells (27). SP-A participates in the formation
of tubular myelin (TM) (15, 20), increases
the rate of surface adsorption of surfactant lipids (18),
enhances the resistance of surfactant to inactivation by pulmonary
edema fluid (4), and plays a role in lung host defense
(26). Because of these properties, SP-A may be a potential
component of surfactant replacement therapy. Information about the
handling of instilled SP-A, therefore, may have practical value.
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MATERIALS AND METHODS |
Purification of SP-A.
SP-A was purified from human alveolar proteinosis fluid by a butanol
extraction method as previously described (27). Briefly, lavage fluid from alveolar proteinosis patients was allowed to settle
overnight at 4°C, and the pellet was resuspended in a minimal amount
of water and frozen in aliquots. The thawed pellet was injected into
butanol to extract hydrophobic proteins. The butanol extract was
centrifuged, and the resulting pellet was reextracted, recentrifuged,
and dried under a stream of nitrogen gas. The dried pellet was
extracted twice with octylglucoside and sodium chloride to remove serum
proteins; the resulting pellet was treated with octylglucoside and
polymyxin B-agarose to remove bacterial endotoxin and dialyzed against
sterile 5 mM Tris-buffered water, pH 7.4. After dialysis, residual
polymyxin, lipid, and insoluble proteins were removed by centrifugation.
Labeling of SP-A with biotin.
SP-A was biotinylated at low pH according to the procedure described by
Ryan et al. (16). Sulfo-NHS-biotin (Pierce, Rockford, IL)
was added to SP-A in a 2:1 weight ratio, and the mixture, pH 6.3, was
incubated 20 min at room temperature with rotation. The reaction was
stopped by the addition of glutamine to a final concentration of 20 mM
(to react with free NHS-biotin), and then the resulting mixture was
placed on ice for 10 min and dialyzed against Tris-buffered water (2 liters × 3 changes) to remove excess biotin and glutamine. bSP-A
purity was checked by SDS-PAGE and Western blot with
streptavidin-labeled horseradish peroxidase as a probe.
Dose preparation.
We mixed 1 µl of 10% bovine serum albumin (BSA), 2 µl of 1% Evans
blue dye, both in phosphate-buffered saline (PBS), and 22 µl of
additional PBS. The dye was used to determine the lung location of the
instillate; the ratio of dye to albumin was chosen to minimize any free
dye that might bind to bSP-A. Then bSP-A was added to the mixture to
obtain 58.5 µg of SP-A in each 50-µl dose. The instillate did not
contain lipids. The instilled dose of protein was subsequently
localized in a part of only one lobe, usually in the right lung.
Animals.
Eight specific pathogen-free male Sprague-Dawley rats (Charles River,
Raleigh, NC) were used. The animals were lightly anesthetized with
halothane (Fluothane) vaporized in a large glass vessel and intratracheally intubated by the transoral insertion of a 16-gauge blunt steel needle. A polyethylene tube (Clay Adams PE-10, Intramedic) was passed through the steel needle until slight positive resistance was felt, and then 50 µl of the mixture described in Dose
preparation were instilled and the animal was extubated. A
period of hyperpnea occurred during the next 2-4 min, after which
the animal appeared recovered.
The animals were killed at times from 1 min to 2 h after
instillation of the bSP-A. After anesthesia with 100 mg/kg of
intraperitoneal pentobarbital sodium, the trachea was cannulated, the
rats were exsanguinated, and the pulmonary artery was cannulated with
PE-250 tubing. Vascular perfusion of the lungs was begun with a
solution of 0.9 NaCl containing 0.3% NaNO3 and 100 U/ml of
heparin. Fixation proceeded immediately with a mixture of 2%
glutaraldehyde and 1% paraformaldehyde buffered at pH 7.4 with 85 mM
sodium cacodylate buffer. After 30 min, areas of interest were selected
by identifying the region colored by Evans blue, and small tissue
blocks were cut. Specimens were fixed by immersion for 30 min in the
same fixative mixture.
Tissue processing.
After primary fixation, the samples were washed in 85 mM sodium
cacodylate buffer 3 times for 10 min and then postfixed in 2% osmium
tetroxide in the same buffer at pH 7.4 for 1 h. Then tissues were
washed (3 × 10 min) with distilled H2O and stained with 2% uranyl acetate in distilled H2O for 90 min at room
temperature. Finally, the tissues were washed again with 85 mM sodium
cacodylate buffer and dehydrated through graded ethanols. An unselected
sample of eight tissue blocks was infiltrated in an ethanol-LR White resin (Polysciences, Warrington, PA) mixture and embedded and polymerized in LR White at 50-55°C for 72 h. Three of the
eight blocks were prepared for thin sectioning by sequential cutting on
a Reichert-Jung ultratome with a glass knife. The surface of these
blocks was examined for the presence of small airways and the absence
of large airways and vessels.
Ultrathin sections were cut on the ultratome. The sections were picked
up on Formvar-coated 200-mesh nickel grids and air-dried for 30 min.
Immunogold labeling.
Tissue sections on grids were stained at room temperature by immersion
(section side down) in 30-µl drops on dental wax in the following
sequence: 1) PBS plus 1% BSA for 10 min; 2) 5%
normal rabbit serum for 30 min; 3) primary antibody (goat
anti-biotin; Calbiochem, La Jolla, CA) in 1:120 PBS-Tween for 6 h
at room temperature in a moisture chamber; 4) PBS for three
washes of 10 min each; 5) secondary antibody (10-nm
gold-conjugated rabbit anti-goat IgG; Sigma, St. Louis, MO) in 1:120
PBS-Tween for 4 h also at room temperature in a moisture chamber;
6) PBS for three washes of 10 min each; and 7)
three washes with double-distilled H2O for 10 min each.
Grids were air-dried overnight. Then they were stained with lead
citrate for 4 min and viewed in a JOEL 1200 electron microscope.
Several controls were performed: lung tissue without bSP-A taken from
the contralateral lung, omission of primary antibody, or its
substitution with nonspecific serum or irrelevant goat IgG.
Morphometry (quantitative immunolabeling analysis).
All extracellular surfactant compartments as well as all type II and
Clara cell profiles that were in contact with immunoreactive bSP-A (the
instilled dose) were photographed at ×6,000 magnification. Four
hundred twenty-six micrographs were printed on 11 × 14-inch paper
at ×21,000 magnification. Two hundred sixty micrographs of Clara cells
and type II cells were made; this resulted in ~16 micrographs per
cell type per animal or between 60 and 100 micrographs per cell type
per time point. Point counting volumetry was done by using a plastic
grid overlay of 464 1-cm lines in a pattern described by Weibel
(22). The end of each line was considered to be a point,
giving a total of 928 points/micrograph. Point counting was employed to
calculate the volume density of different compartments at high
magnification (×21,000).
Three different extracellular surfactant compartments were quantified.
Two of them represented a lipid-rich part of the surfactant layer: TM
plus two vesicular forms [unilamellar and multilamellar, also called
myelin figures (MFs)]. The third compartment quantified was the
lipid-poor liquid hypophase. Large vesicle-like structures found within
the surfactant layer and having any diameter of 0.5 µm or more were
omitted, and their area was not included in the calculations. Such
structures are of uncertain origin, are uncommon, and are not present
in undisturbed lungs fixed in a similar manner. Inclusion of these
would have a disproportionate effect on the calculated areas of the
typical small vesicular pool.
The volume density of the cytoplasm, nuclei, and mitochondria of all
type II and Clara cell profiles was also measured. For type II cells,
lamellar bodies (LBs) were also quantified. Clara cell granules were
distinguished from the cytoplasm and quantified. Enough total points
falling over the profiles of interest were counted to reduce to <5%
the estimated error of the smallest compartment measured
(23).
Because gold particles presumably demonstrate biotin-immunoreactive
sites (gold identifies location of bSP-A), the gold particles overlying
each of the above-mentioned extra- and intracellular compartments were
counted. The density of labeling is reported as the number of gold
particles divided by the number of points overlying each extracellular
form and cellular organelle. These ratios were calculated for every
type II or Clara cell profile as well as for each extracellular
surfactant compartment found on the same electron-microscopic
micrograph. Average values for every animal were calculated, and then
the mean results for each experimental group were determined and are
presented as the number of gold particles per square micrometer of
measured area.
Statistics.
ANOVA followed by pairwise comparisons with Scheffé's correction
procedures was done with commercial software (SAS, Cary, NC)
(19).
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RESULTS |
The human bSP-A migrated on SDS gels as expected for the protein
(Fig. 1). bSP-A instilled into rat lungs
was found within the bronchiolar mucous layer and was also incorporated
into alveolar surfactant forms. Because we examined only those areas
where we found the instilled bSP-A in close contact with alveolar type II or bronchiolar Clara cells, bSP-A should have had an opportunity to
be recognized and internalized by the epithelial cells studied.

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Fig. 1.
Surfactant protein (SP) A was biotinylated (bSP-A) as
described in MATERIALS AND METHODS and analyzed by
SDS-PAGE. SP-A was stained with Coomassie blue for detection of total
protein. bSP-A was transferred to nitrocellulose and probed with
streptavidin-conjugated horseradish peroxidase followed by enhanced
chemiluminescence. Nos. on left, molecular mass markers in
kDa. Both methods of detecting the biotinylated protein demonstrated a
major band between 35 and 40 kDa and a minor band at about twice that
molecular mass. The gels do not align precisely because of swelling of
the Coomassie blue-stained gel during the destaining process.
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Controls.
Sections made from blocks free of dose (contralateral lung) and those
prepared from blocks containing the instilled bSP-A but incubated
without primary antibody did not react with the secondary antibody. The
extracellular surfactant on those sections was nearly free of
gold particles or had very low levels (Table 1). Very little labeling of control
sections was observed over type II or Clara cell profiles (Tables
2 and 3).
Regions of sections free of tissue (including the air spaces and empty
capillaries) were not labeled. Lung from the contralateral side not
instilled with bSP-A had no recognizable labeling even with primary and secondary antibodies.
Extracellular surfactant compartments.
Lipid-rich components of alveolar surfactant were composed of
unilamellar vesicles, multilamellar (myelin) figures, and other lipid
lamellae including the unique TM figures. Viewed at low power, the
air-liquid interface of dye-stained lung was densely labeled with gold
particles. Lipid-rich vesicles and MFs were strongly gold labeled (Fig.
2a), with an average gold
grain density significantly higher than the labeling of hypophase and
TM profiles at the earliest time period (Fig. 2, b and
c). These differences remained throughout the study period
because there was no significant change in gold density over the
surfactant compartment within the 2-h period. Occasional large
vesicle-like structures were not quantified due to their large size,
uncertain nature, unusual occurrence, and lack of any internal
structure.

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Fig. 2.
Extracellular surfactant compartments after immunogold
reaction. a: 7 min after intratracheal instillation of
biotin-labeled SP-A. The lipid-rich vesicular compartment contains
unilamellar vesicles (arrowheads) seen beneath the air-liquid interface
and multilamellar vesicles (*) situated deeper in the hypophase (H).
Both vesicular figures are gold labeled. C, capillary. Dashed lines,
basement membrane. b: 7 min after intratracheal instillation
of biotin-labeled SP-A. The density of gold grains over the tubular
myelin (TM) profiles is considerably less compared with the lipid-rich
vesicular forms (arrowheads). Hypophase contained few gold grains.
c: 30 min after intratracheal instillation of bSP-A. The
air-liquid interface is almost continuously marked with gold (arrows);
the region closely related to TM is gold free. Gold grains could be
seen connected with lipid-rich vesicular surfactant forms (arrowheads).
Bars, 0.5 µm.
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We found gold clusters (groups of
3 particles lying within 30 nm of
each other) over lipid lamellae or close to the lipid vesicles and
speculated that clustered particles might indicate clustering of
antigen. With the two-step immunogold method that we were using, an
ideal geometry would be realized only when a gold particle projected
directly over the antigen and therefore accurately marked the actual
location of the antigen (bSP-A). In principle, there is always a
possibility that a gold particle might project away from the antigen
(maximum distance with this assay would be two immunoglobulin lengths,
~18-20 nm) (8). We did not assume that gold
clusters represented the exact location of clustered antigen molecules
in the underlying structure.
TM consisted of intersecting lipid layers with 50 nm spacing. TM
profiles had a gold grain density three- to fourfold lower than other
lipid-rich compartments. Single gold particles were found over the TM
lattice in no specific order, and no clusters were observed. Regions
from the air-liquid interface that contained TM profiles situated
immediately underneath the interface monolayer were free of gold (Fig.
2c). No significant time-dependent change of gold grain
density over TM was apparent at the 2-h point compared with the earlier periods.
The lipid-poor liquid hypophase also had a very low gold label density
compared with the lipid-rich vesicular components. Especially low were
the number of particles found at the earliest time point. Gold
particles were scattered randomly, and no clusters were observed. There
was a threefold increase in the average number of gold particles after
30 min and 2 h compared with that after the 7-min period. This was
not significant. The total number of gold grains over the hypophase
represented ~2% of all gold grains found over surfactant lipid
profiles in the same set of micrographs. Table 1 shows the average
density of gold labeling (number of gold particles/volume of the
compartment) of extracellular surfactant compartments.
Type II cells.
We studied type II cells only from alveolar spaces that contained
extracellular lipid forms immunostaining for bSP-A. At the early time
periods (7 and 30 min), gold particles were associated with cellular
organelles (nucleus, cytoplasm, mitochondria, and LBs), but no
significant difference in subcellular distribution of the
immunolabeling was found (Fig. 3).

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Fig. 3.
Type II cell 7 min after intratracheal instillation of
bSP-A. Few gold grains were seen within intracellular compartments. The
overlying extracellular surfactant had the greatest density of
immunolabeling, mostly connected with vesicular forms (arrowheads). N,
nucleus. LB, lamellar body. Bar, 0.5 µm.
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At the 2-h period, some electron-microscopic images (Fig.
4) revealed a few gold particles
associated with the apical plasma membrane as well as single gold
grains within small cytoplasmic vesicles. Other type II cell structures
such as multivesicular bodies were not immunoreactive. LBs were more
heavily labeled than any other organelle (Fig.
5); their average labeling density significantly exceeded the labeling density of cytoplasm and nucleus at
all time periods. The LBs were significantly more densely labeled at
2 h compared with the density of their labeling at the earlier time periods (P < 0.05). Table 2 and Fig.
6 summarize the time-dependent differences in gold grain density of type II cell compartments.

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Fig. 4.
Apical portion of a type II cell 2 h after
intratracheal instillation of bSP-A. Arrows, immunoreactive sites
overlying the apical plasma membrane; arrowheads, small electron-lucent
cytoplasmic vesicles. LBs also are immunolabeled. In Figs. 3-5,
preservation of the LB is a compromise between retaining ultrastructure
and maintaining antigenicity. Bar, 0.5 µm.
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Fig. 5.
A type II cell from an animal 2 h after
intratracheal instillation of bSP-A. After immunogold reaction, LBs
were labeled with gold. Single gold particles could be seen within the
other intracellular compartments. * Portions of LBs shown enlarged
in the inset. Arrowheads, gold particles projected over the
LB content. Bars, 0.5 µm.
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Fig. 6.
Average gold grain density within intracellular
compartments of type II cells at 3 different time points after
intratracheal instillation of bSP-A. * Significant difference in LB
labeling at 2 h compared with 7 and 30 min.
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Clara cells.
Clara cell profiles were studied from bronchioles that contained
recognizable instilled dose and stained positive for bSP-A. At all
three time periods, Clara cell nuclear and cytoplasmic compartments had
lower average labeling densities compared with Clara cell secretory
granules and mitochondria. Clara cell secretory granules were not more
densely labeled than Clara cell mitochondria (Fig.
7). No significant changes in
labeling of Clara cell cytoplasm, nucleus, secretory granules, or
mitochondria were found within the 2-h time period. Table 3 and Fig.
8 show the average gold grain density
data of Clara cell compartments obtained at different time periods
after instillation of bSP-A.

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Fig. 7.
Clara cell profile from an animal 2 h after
intratracheal instillation of bSP-A. Gold particles labeled surfactant
lipid forms (arrows), which overlaid the luminal part of the cell.
Clara cell secretory granules (arrowheads) possessed few gold particles
as did the other intracellular compartments. Bar, 0.5 µm.
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Fig. 8.
Average gold grain density within intracellular
compartments of Clara cells at 3 different time points after
intratracheal instillation of bSP-A. No significant time-dependent
changes in gold grain density was found for any intracellular
compartment of Clara cells.
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We counted gold grains over five to six ciliated cell profiles from a
single animal at each time point. The labeling density at each time
point was 0.2-0.3 for ciliated cell cytoplasm, 0.2-0.3 over
the nucleus, and 0.15-0.4 over the mitochondria. These mean values
are similar to those of comparable compartments in Clara and type II
cells, but no statistical comparisons are possible.
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DISCUSSION |
In this study, we used human SP-A bound to biotin and a polyclonal
antibody (Calbiochem) against biotin to reveal the immunoreactive sites
that presumably corresponded to those extra- and intracellular compartments that contained the instilled bSP-A. Postembedding immunogold staining is an assay performed on sections of embedded tissue, and the immunolabeling is restricted to the surface of the
sections, resulting in a relatively low signal density because only
those binding sites exposed can interact with the specific antibodies.
We believe that the observed signal distribution closely reflects the
true protein distribution based on antibody control sites and the lack
of nonspecific background labeling on the resin. Lung tissue cells were
found to have low levels of immunoreactive endogenous biotin, a vitamin
that serves as a coenzyme of certain enzymes. Therefore, antibody
binding to endogenous biotin during immunocytochemistry did not appear
to be a problem (12).
Extracellular surfactant compartments.
Extracellular surfactant forms have a very similar phospholipid
composition to each other, and TM has been formed from pure lipids plus
purified SP-A and SP-B (20). It is not clear how lipid
molecules and endogenous surfactant proteins interact in vivo to
achieve the different patterns of three-dimensional organization in
MFs, TM, vesicles, or monolayers or where exactly exogenously added
SP-A might be localized. It is unknown to what extent lipid molecules
might interfere with antigen-antibody interactions and therefore might
have an impact on our morphological demonstration of the antigens.
Walker et al. (21) detected endogenous SP-A in frozen
sections of rat lung with a polyclonal antibody. They found that LBs had a low level of immunostaining for endogenous SP-A, whereas TM was
heavily labeled. Haller et al. (7) also described
high-density immunogold labeling of TM for endogenous SP-A compared
with the labeling over LBs. They reported enhancement of SP-A labeling over the peripheral membranes of multilamellar structures during presumptive TM formation. Inhibition of the antigen-antibody reaction in the tightly spaced lipid-rich environment of the LBs may have accounted for some of this differential immunostaining.
Our results for exogenously added bSP-A immunostaining of TM were
different. The exogenous bSP-A was present in the extracellular surfactant compartments at all three times we studied. This protein was
found associated with lipid-rich vesicular components almost immediately after the instillation and was detectable throughout the
period studied. Nevertheless, incorporation of bSP-A into TM was very
modest at the early time period and was still low 2 h after the instillation.
There are several possible explanations for our findings of the
distribution and time course of bSP-A labeling of alveolar surfactant
compartments. The human alveolar proteinosis source of the SP-A and our
processing of it for biotinylation could affect its distribution,
although we think that unlikely because the type II cells incorporated
bSP-A into LBs in a time-dependent manner as expected of native SP-A
(6, 9, 16, 25,
28). Our method of biotinylation of SP-A was identical to
that of Ryan et al. (16). Those authors found bSP-A
inhibited phospholipid secretion from isolated (rat) type II cells and
that specific bSP-A binding to type II cells was present.
We do not know whether TM structures inhibited penetration of the
antibody and, therefore, whether the availability of the antigen to the
antibody was the same in the lattices as it was over other lipid-rich
components. TM is a pool of highly organized lipid and protein
molecules usually showing boundary lipid lamellae facing other
extracellular surfactant forms nearby (15,
24). A transitional zone apparently exists only between
MFs and TM. The tubes of TM that are contiguous to MFs in those pools
could be closed at both their ends, possibly inhibiting access to
exogenous protein (11). Lattices of TM may be saturated
with native SP-A, and thus few sites for binding of exogenous SP-A
might be available.
Labeling of the hypophase could represent an estimate of the amount of
lipid-free SP-A (or lipid-poor SP-A) that might be important for
regulatory functions of the protein. Our estimate of ~2% of the
total bSP-A accounted for in the hypophase is consistent with the
estimate of free SP-A made by Baritussio et al. (3).
Type II cells.
Prior reports by several investigators (9,
16) have indicated that type II cells recycle surfactant
lipid and SP-A. Young et al. (28) previously used
125I-labeled human alveolar proteinosis SP-A and found a
time-dependent uptake of the radiolabel from the alveolar space into
rat lung type II cell LBs. As a positive control for the present use of bSP-A, we analyzed type II cells and found the expected time-dependent concentration of bSP-A into the LBs. Our electron micrographs showed
exogenous SP-A possibly attached to the apical plasma membrane of type
II cells and subsequently internalized, but we could not determine
whether the transporting organelles were coated pits or coated vesicles
because our processing of lung tissue for immunoelectron microscopy
resulted in poor preservation of specialized membrane structural
detail. Our immunolabeling data were consistent with trafficking of
SP-A from the apical plasma membrane via cytoplasmic vacuoles to the
LBs of type II cells. Quantitative analysis demonstrated a significant
increase in antigen density within LBs after 2 h compared with
that at the earlier periods.
Clara cells.
Clara cells have been the object of extensive studies, but their role
in surfactant biology is not yet defined. The Clara cell of rat lung is
a site of production of SP-A mRNA and protein, but little is known
about the processing of the protein by Clara cells (21).
In particular, a possible recycling of SP-A by Clara cells has not been
previously directly addressed.
The cytoplasm and nuclei of Clara cells in this study showed about the
same intensity of immunolabeling as the analogous compartments of type
II cells, and both were unchanged during the 120-min period studied.
The mitochondria of Clara cells contained more immunoreactive biotin
labeling than the mitochondria in the type II cells. Kuhn (12) showed that immunoreactive biotin stores in the
mitochondria of Clara cells were uniquely greater than the
immunoreactive biotin in the mitochondria of type II or ciliated cells.
Other organelles of Clara cells did not have high biotin levels
according to Kuhn. Clara cell granules showed no time-dependent
increase in immunolabeling for bSP-A. We expected that a recycling of
exogenous SP-A would be signified by a time-dependent increase in
labeled SP-A within those cellular compartments participating in
recycling. Because Clara cell granules were maximally labeled by 7 min
(the earliest we were able to instill the bSP-A and then perfusion fix
the lungs), it is possible that a very fast recycling process would be
missed if the recycling were complete in a few minutes. Likewise, our longest measurements at 2 h would not detect a recycling process that reached a steady state over an even longer period.
In conclusion, our immunoelectron-microscopic observation of exogenous
bSP-A instilled into adult rat lung suggests that specific surfactant
lipid forms as well as two different epithelial cells uniquely
incorporate this protein during the 2 h after instillation.
There was a strong compartmentalization of bSP-A extracellularly.
Lipid-rich vesicular components had the greater portion of
immunoreactive protein, whereas TM and hypophase were relatively poorly immunolabeled.
Although both type II and Clara cells synthesize and apparently secrete
SP-A, they do not internalize SP-A in the same way. The uptake of bSP-A
by type II cells was confirmed, but Clara cells did not demonstrate a
time-dependent uptake. We interpret these results as evidence against
recycling of SP-A by the Clara cell, at least within the 7-min to 2-h
time frame of our experiments.
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ACKNOWLEDGEMENTS |
We were assisted in the statistical analysis of the data by Dr.
Steven Grambow (Durham Veterans Affairs Medical Center, Durham, NC). We
are grateful to Dr. C. A. Piantadosi for providing the alveolar
proteinosis lavage fluid samples.
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FOOTNOTES |
This work was supported by Veterans Affairs Merit Review and National
Heart, Lung, and Blood Institute Grants HL-32188 and HL-30923.
Address for reprint requests and other correspondence: J. Savov, Durham VAMC (151), 508 Fulton St., Durham,
NC 27705 (E-mail: jsavov{at}acpub.duke.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
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