From the
Surfactant protein A (SP-A) is the most abundant protein
associated with phospholipids in pulmonary surfactant. There are
several lines of evidence that pulmonary and gastrointestinal
epithelium produce closely related surface-active materials, although
the presence of SP-A in gastrointestinal tract has so far not been
reported. Indirect immunofluorescence experiments using different
antibodies raised against rat pulmonary SP-A showed that some jejunal
and colonic but not gastric epithelial cells positively stained for
SP-A. Analysis of the proteins in cell lysates from rat small intestine
and colon studied by Western blot revealed several immunoreactive
bands, including the characteristic triplet of 26-, 32-, and 38-kDa
monomeric proteins, less strongly labeled than in lung cells, and
higher molecular mass forms of 66 and 120 kDa also present in lung
cells. The 66- and 120-kDa bands displayed the expected isoelectric pH
of SP-A after two-dimensional electrophoresis. Alkylation induced
conversion of the 120-kDa form (almost completely) and the 66-kDa form
(partly) into the 26-38-kDa monomeric species. The presence of
SP-A mRNA in rat stomach, small intestine, and colon was then searched
for by conventional cDNA/reverse transcriptase-polymerase chain
reaction. Products of appropriate size (372 base pairs) identical to
that of pulmonary tissue were amplified in small intestine and colon
but not in stomach or in other tissues used as controls. Cloning and
sequencing of rat colon SP-A cDNA revealed the same sequence as the one
reported for rat lung SP-A. Furthermore, analysis of the
transcriptional initiation site of SP-A gene in colon by
anchored-polymerase chain reaction showed that transcription was
initiated at the same site in both colon and lung. These data, which
demonstrate that small intestine and colon express SP-A constitutively
and that this protein is present in some epithelial cells, extend the
concept of intestinal surfactant and underline its close relationships
to pulmonary surfactant.
Rat surfactant-associated protein A (SP-A),
Gastrointestinal mucosae produce and secrete surface-active
phospholipids that share a number of biochemical and physiological
properties with pulmonary surfactant and have been therefore designated
surface-active material
(4, 5, 6) .
Gastrointestinal surfactant has been described as a hydrophobic layer
of surface-active phospholipids between the apical border of epithelial
cells and the luminal contents. Several important properties have been
attributed to this layer, including ability to lubricate the movement
of intraluminal contents (7), to act as a barrier against autodigestion
and ulceration
(6) , or to be a vehicle for secretion of luminal
alkaline phosphatase
(4, 8) .
In alveolar type II
cells, surfactant components are assembled into lamellar bodies, the
intracellular form of storage, before being secreted into alveolar
spaces
(9) . In the stomach, phospholipids are found to be
present in multi-lamellar structures in the air-liquid interface on the
mucosal surface
(10) . In the rat small intestine, Eliakim and
co-workers
(8) described the presence of lamellar bodies
morphologically similar to those in epithelial alveolar cells, in the
enterocytes themselves, in the intercellular spaces, and adjacent to
the apical membrane. Similarly to those in lung epithelium, these
lamellar bodies contain saturated phosphatidylcholine and possess the
ability to lower surface tension
(4, 8) .
Whereas the
presence of pulmonary surfactant proteins SP-B and SP-D has been
reported in intestinal lumen
(11) and SP-D in the
stomach
(39) , SP-A has not yet been identified elsewhere than in
pulmonary tissue. We here report evidence for the expression of
pulmonary surfactant protein A gene in small intestine and colon.
The intestinal expression of SP-A gene
was investigated by immunocytochemistry using antibodies raised against
pulmonary SP-A. Immunoreactivity was clearly observed in a fraction
estimated to be about 15-30% of intestinal and colonic epithelial
cells but not in gastric cells. Staining was either diffuse into
cytoplasm or restricted to brush border ( Fig. 1and
Fig. 2
). Consistent observations have been made by J. N. Freund
and I. Duluc
Immunoreactive forms of pulmonary SP-A are known to display a large
heterogeneity. This is the result of differential glycosylation and
acylation processes occurring at the post-translational level on the
one hand
(26, 27) and of the presence of non-reducible
multimeric forms on the other
(28) . In the present study,
one-dimensional Western blot analysis of jejunal and colonic cells
revealed bands characteristic of SP-A with a pattern similar to that
obtained with pulmonary cells. The amplification procedure that was
used enhanced bands with a molecular weight consistent with multimeric
forms of the protein, while the triplet of molecular masses 26, 32, and
38 kDa, characteristic of monomeric species of pulmonary
SP-A
(1) , appeared faintly. Alkylation of the intestine samples
performed for preventing reassociation of reduced subunits
demonstrated, however, that the immunoreactive band observed in the
120-kDa range corresponds to a reducible multimeric form of SP-A that
has been previously described in pulmonary epithelium (28, 29). The
other immunoreactive band in the range of 66 kDa was less affected by
the alkylation treatment, either in the lung or in the intestine. The
66-kDa band, generally described in Western blots from bronchoalveolar
lavage or purified SP-A, probably represents a non thiol-dependent
dimeric form of the protein that cannot be dissociated by reductive
cleavage. Kuroki et al.(28) demonstrated that neither
alkylation nor reduction plus alkylation completely prevented the
formation of the 66-kDa form in pulmonary tissue. It was further
demonstrated by peptide mapping that this 66-kDa protein was consistent
with a non-disulfide-linked dimer of the 32-38-kDa proteins,
although the nature of the cross-links remained unclear
(30) .
Intestinal cells therefore express the native, glycosylated, and
multimeric forms of SP-A, although probably at a lower level when
compared to pulmonary cells. The reason for the relative predominance
of oligomeric forms of SP-A in intestinal cells is not clear. The
possibility that it was only apparent and actually due to the
amplification system cannot be ruled out. Alternatively, different
routes in intracellular processing of the protein in alveolar type II
cells
(31, 32) and in intestinal cells may account for a
different oligomeric pattern in both organs. The finding that the
integrity of disulfide bonds seemed to be necessary for biological
activity of SP-A in inhibition of surfactant-associated phospholipid
secretion
(28) suggests nevertheless the possibility of such
similar structure-associated function in the intestine.
In rat lung,
SP-A gene encodes mRNA containing the entire coding region as well as
untranslated 3`- and 5`-regions
(24, 33) . Analysis of
the intestinal transcripts of SP-A gene detected by PCR demonstrated a
complete identity of nucleotide sequence between lung and intestinal
cDNA. Indeed, we found that the intestinal sequence of SP-A cDNA
completely matched the one obtained by analyzing rat genomic clones
(34). Only minor differences between the nucleotide sequence reported
by Sano for rat lung SP-A cDNA
(24) and the present data were
observed, which were presumably due to typing errors in the previously
published sequence, since both the amino acid deduced sequences were
found to be identical.
Activation of transcription from alternative
promoters present upstream of the same gene is a common mechanism
through which certain genes can be differentially expressed in a
variety of tissues
(35) . We performed anchored PCR in pulmonary
and intestinal extracts to identify the start point of transcription in
these tissues. We found that transcription of the SP-A gene is
initiated at the same site in lung and colon tissues (Fig. 8).
This result confirms the complete homology between both transcripts and
suggests that the same promoter is used in these tissues.
The
presence of immunoreactive SP-A in intestinal brush border indicates
that, similar to the lung, this protein is released in the lumen. Other
secreted proteins have been described as commonly expressed by both
intestine and lung epithelium. Expression of the secreted intestinal
human mucin MUC 2 was recently reported in bronchus and in epithelium
of the small intestine and colon
(36) . The expression of
functional pulmonary proteins in intestinal tissue can be regarded as a
consequence of the common embryological origin shared by both organs,
with lung epithelia being derived from the foregut endoderm of the
embryo. Moreover, during development, both intestine and lung display
an acceleration of maturation after administration of
corticosteroids
(25, 37) . This raises the possibility
that intestinal SP-A gene expression may be regulated by the same
factors that modulate SP-A gene expression in the lung, i.e. glucocorticoids, epidermal growth factor, and cAMP
(25) , an
assumption that future investigations will allow us to test.
An
interesting question raised by the findings reported here is that of
the function of intestinal SP-A. In the lung, the physiological
functions of SP-A are relevant on one hand to surfactant secretion and
structure (formation of tubular myelin and of lipid-rich surface film,
regulation of surfactant secretion and clearance) and on the other hand
to regulation of alveolar macrophage function
(38) .
A first
possible role for intestinal SP-A can be speculated from the known
function of pulmonary SP-A in surfactant organization and secretion.
The extension of the concept of surfactant to the gastrointestinal
tract was made by Hills et al.(5) who discovered that
luminal material extracted from rat gastric mucosa was associated with
typical lamellar bodies and possessed surface-active properties
consistent with its high content in pulmonary-type phospholipids,
principally disaturated phosphatidylcholine. D. H. Alpers'
laboratory
(4, 11) further showed that intestinal tissue
also contained surfactant-like material. This material obtained after
light mucosal scraping of either adult or newborn rat intestine was
also stored intracellularly as lamellar bodies and lowered surface
tension. Its close relationship with lung surfactant was indicated by
the presence of pulmonary proteins SP-B and
SP-D
(4, 11) . The presence of SP-A, however, was not
demonstrated in this material by Western blot in this previous study.
Possibly, this could result from low amounts of SP-A, as suggested by
the necessity of amplifying the detection system in our experiments, or
from rapid degradation of SP-A by luminal proteases. Nevertheless, the
presence of other authentic pulmonary surfactant proteins reported by
these authors is in line with the present data. Our findings that SP-A
was not detected by immunofluorescence and that SP-A gene was not
transcribed in the stomach is also consistent with the recent
observations of Fisher et al.(39) who found abundant
expression of SP-D but not of SP-A, B, and C by gastric mucous
secreting cells.
Another possible role for the intestinal SP-A stems
to the known antimicrobial properties of pulmonary SP-A. SP-A belongs
to a family of animal lectin-like proteins encoded by different genes
that share a characteristic carbohydrate-recognition
domain
(40) . It has been stressed for instance that SP-A and
mannose-binding proteins (MBPs) possess strong homology in their
overall organization into collagenous and ligand-binding domains and in
their amino acid sequences
(41, 42) . Each of these
proteins consists of polypeptides that are linked by disulfide bonds
that immediately precede the collagenous domains at their NH
Other proteins of the
C-type lectin family, the pancreatitis-associated protein III and the
Reg protein (the pancreatic stone protein) have been found in either
intestinal mucosa
(16, 46) or in the human colon (47).
These proteins were present in the cryptic proliferative compartment of
the small intestine
(46) and in the undifferentiated (but not in
the differentiated) cells of the two human colon carcinoma cell lines
Caco-2 and HT-29
(48) . These proteins associated with a
proliferative/undifferentiated state are generally believed to play a
role in the immune defense system of the intestine. This has already
been demonstrated for the pancreatitis-associated protein, which
induces the aggregation of several bacterial strains
(49) .
Altogether, this suggests that SP-A may belong to a family of
structurally related proteins with antibacterial functions.
On the
basis of their surface-active properties, surfactant-like materials
have now been reported in a wide variety of tissues including inner ear
duct
(50) , oesophagus
(51) , biliary duct
(52) ,
human Caco-2 cells
(53) , gastric
(5, 6) , and
intestinal mucosa
(11, 49) . In view of the presence of
surfactant-associated proteins in specific segments of the
gastrointestinal tract, pointed out by the compositional similarities
shared by different surfactants, there is a need to redefine and extend
the concept of surfactant.
We are very grateful to Dr. J. A. Whitsett
(Cincinnati, OH) for kindly providing anti-rat SP-A antibody and to Dr.
P. Mangeat (CNRS, Montpellier, France) for the gift of monoclonal
gastric anti-H
(
)
an octadecameric sialoglycoprotein (monomer,
M
= 26,000-38,000), binds strongly to
surfactant glycerophospholipids and acts in a calcium-dependent manner
to promote the transformation of the secreted lamellar bodies to
tubular myelin within the pulmonary alveolus
(1) . SP-A is also
believed to play an important role in mediating surfactant
reutilization through its binding to specific receptors on the type II
pneumocytes
(2) and to be implicated in alveolar defense
mechanisms through opsonization of microorganisms and promotion of
phagocytosis
(3) . Cloned SP-A cDNA predicts a peptide, which
after glycosylation is 32-38 kDa. The mature peptide is organized
into distinct amino-terminal collagenous intermediary
phospholipid-binding and carboxyl-terminal lectin-like domains.
Preparation of Tissues and Cell
Isolation
The presence of SP-A protein was studied on
epithelial cells isolated from stomach, small and large intestine, and
lung of adult male Wistar rats (200-250 g, Charles River,
France). Pulmonary epithelial cells were prepared according to Richards
et al.(12) . Gastric cells were obtained as previously
described
(13) . Intestinal cells were isolated from jejunal and
colonic mucosae according to Weiser's procedure
(14) with
minor modifications. The upper half of rat small intestine and colon
was flushed free of content with cold phosphate-buffered saline (PBS)
(150 mM NaCl, 30 mM KCl, 10 mM
NaHPO
, 15 mM
KH
PO
), pH 6, everted, and washed twice in the
same solution. The everted segments were filled with PBS and incubated
under agitation in a water bath at 37 °C in 10 ml of a dispersing
solution containing 1% bovine serum albumin, 10 mM glucose,
2.5 mM glutamine, 1.5 mM EDTA, 0.5 mM
dithiothreitol, 0.01% trypsin inhibitor in PBS, pH 6. Cells were
collected by centrifugation (900
g, 3 min). Incubation
media from two successive 15-min periods were pelleted and washed twice
in PBS, pH 7. Collected cells were used for indirect immunofluorescence
and for immunoblotting studies.
Antibodies
Two different anti-rat SP-A
antibodies were used for indirect immunofluorescence and immunoblotting
studies: a polyclonal antibody raised in guinea pig against SP-A
isolated from rat lung lavage and prepared in our laboratory
(15) and a polyclonal anti-SP-A antibody raised in guinea pig,
kindly provided by Dr. J. A. Whitsett (Children's Hospital
Medical Center, Cincinnati, OH). Goat anti-guinea pig antibody coupled
to peroxidase (Sigma, L'Isle d'Abeau, France) or
biotinylated goat anti-guinea pig antibody (Vector, France) was used as
the second antibody. Texas Red streptavidin and streptavidin peroxidase
were both purchased from Amersham (Les Ulis, France). Fluorescein
isothiocyanate-phalloidin (Sigma), a specific marker of F-actin and a
monoclonal antibody raised in mouse directed against rat gastric
H,K
-ATPase (kindly provided by Dr. P.
Mangeat, CNRS, Montpellier, France), was used to assess the quality of
intestinal and gastric preparations in indirect immunofluorescence.
Anti-mouse-fluorescein isothiocyanate was purchased from
Diagnostic-Pasteur (Institut Pasteur, France). All antibodies were
diluted in 1% (mass/volume) bovine serum albumin in PBS and used at
optimal concentrations determined after serial dilutions.
Indirect Immunofluorescence
Two different
preparations were run for dispersed cells. The first one consisted in a
1-h fixation with 3% paraformaldehyde in PBS (v/v), washing in PBS,
snap-freezing, and 5-µm sectioning with aid of a cryostat (CM 3000,
Leica). The second one consisted of dropping whole cells on the glass
bottom of observation chambers by centrifugation at 600 g for 1 h, followed by fixation for 1 h with paraformaldehyde and
cautious washing in PBS. Both preparations were treated for 15 min in
50 mM NH
Cl to avoid endogenous fluorescence and
then incubated for 45 min with 1% bovine serum albumin in PBS and for 1
h with the anti-SP-A antibodies. After washing twice in PBS, 0.01%
Tween 20 (PBS-T) and once in PBS, biotinylated goat anti-guinea pig IgG
antibody was applied for 1 h. Preparations were washed again in PBS-T
and PBS, and Texas Red streptavidin was applied for 1 h. Controls were
incubated only with Texas Red streptavidin or with non-immune sera.
Samples were mounted in PBS-glycerol and observed with a Nikon
microscope equipped with UV epi-illumination.
Samples for Electrophoresis
Pulmonary
surfactant fraction
(16) and isolated pulmonary epithelial cells
were used as SP-A-containing controls. Cells isolated from jejunum,
colon, and lung were lysed by sonication for 30 s, and aliquot
fractions were taken to estimate protein content by the method of
Bradford
(17) . Samples were then either boiled in Laemmli
reducing buffer
(18) containing 10% -mercaptoethanol and
immediately electrophoresed or, in some experiments, further processed
for delipidation by chloroform-methanol extraction as described by
Bligh and Dyer
(19) , solubilized, and boiled for 5 min in 0.5 ml
of 0.1 M Tris-HCl (pH 8.2), 1% SDS, 1%
-mercaptoethanol
before they were proceeded to SDS-polyacrylamide gel electrophoresis.
In some instances, reduced sulfhydryl bonds were alkylated by addition
of 0.15 ml of 1 M iodoacetamide in 0.1 M Tris-HCl (pH
8.2). Following incubation at 37 °C for 40 min, proteins were
boiled in Laemmli reducing buffer and subjected to electrophoresis.
One- and Two-dimensional
Electrophoresis
One-dimensional gel electrophoresis was
performed through 12% SDS-polyacrylamide slab gels according to Laemmli
(18) using the Mini-Protean II apparatus from Bio-Rad.
Two-dimensional gel electrophoresis was performed as described by
O'Farrell
(20) using only delipidated samples of pulmonary
and intestinal isolated cells. Surfactant fraction isolated from rat
lung was treated the same way as other samples. Before electrophoresis,
samples were diluted in a buffer containing 9.5 M urea, 2%
Nonidet P-40, 2% ampholines, and 100 mM dithiothreitol to
charge 20 µg of proteins per sample. The isoelectric focusing (IEF)
dimension was established using pH 3-10 ampholines (Pharmacia,
France) in a tube gel (1 75 mm). IEF was conducted at 500 V for
10 min and then at 750 V for 3 h 30 min. The IEF gels were then
extruded, equilibrated for 10 min in Laemmli reducing buffer, and
electrophoresed in the second dimension using a 12% SDS-polyacrylamide
slab gel as above. Gels were either silver stained according to the
method of Morrissey
(21) or transferred on membrane and
immunoblotted.
Protein Blotting and
Immunostaining
Electrophoresed samples were electroblotted
to a nitrocellulose membrane with a semidry blotting apparatus
(Pharmacia). After blocking in 5% (mass/volume) non-fat milk in
Tris-buffered saline containing 0.05% Tween 20 (TBS-T), the
nitrocellulose sheets were incubated overnight at 4 °C with guinea
pig antibodies raised against SP-A. Non-immune serum activity was also
controlled. After washing three times in TBS-T, the membrane was
incubated for 1 h at room temperature either with a goat anti-guinea
pig antibody coupled to peroxidase or with a biotinylated goat
anti-guinea pig IgG antibody. In the latter instance, after washings in
TBS-T, blots were exposed to streptavidin peroxidase for 1 h. After
extensive washings with TBS-T, blots were developed in both instances
with a chemiluminescent detection system (ECL Western blotting,
Amersham).
Reverse Transcriptase-Polymerase Chain Reaction
(RT-PCR)
We used the RT-PCR to amplify the SP-A and
-actin mRNAs. This technique identified the presence or absence of
these mRNAs but did not quantify the transcripts. Total RNAs were
extracted from stomach, small intestine, colon, and lung in 5
M guanidium thiocyanate and purified through a cesium chloride
gradient according to the method of Chomczynski and Sacchi
(22) .
2-µg RNAs were reverse transcribed by the random hexanucleotide
priming method using Moloney murine leukemia virus reverse
transcriptase (Life Technologies, Inc.). One-fourth of the cDNA
products was used in the amplification reaction with oligonucleotide
primers specific for SP-A and
-actin gene message. Amplification
cycles were performed in a DNA thermal cycler (Perkin-Elmer Corp.),
each cycle consisting of incubations for 30 s at 94 °C, 30 s at 55
°C, and 60 s at 72 °C in 10 mM Tris-HCl, pH 8.3,
containing 3.5 mM MgCl
, 50 mM KCl, 250
µM dNTP, and 2.5 units of Taq DNA polymerase
(Boehringer Mannheim) in the presence of each sequence-specific SP-A
and
-actin primers. For SP-A, the 5`-primer was located in the rat
pulmonary SP-A third exon (5`-GGAAGCCCTGGGATCCCTGG-3`), and the
3`-primer was complementary of part of the rat pulmonary SP-A fifth
exon (5`-TAATGGTATCAAAGTTGACTG-3`). An aliquot fraction of the
amplified products was then submitted to electrophoresis through a 8%
(mass/volume) polyacrylamide gel, blotted onto nylon filter, and
hybridized with a probing oligonucleotide located in the rat pulmonary
third exon (5`-CCTGGTGCACCTGGAGA-3`) to confirm the specificity of the
amplified bands. All oligonucleotides used in this study were purchased
from Genset.
Cloning and DNA Sequencing of the Amplified
Intestinal cDNA
The amplification of the cDNA using the
polymerase chain reaction was performed for 38 cycles as described
above with minor changes. The 5`-primer 2 located in the second exon
(5`-CAGAAGCCACTGGGGATA-3`) and the 3`-primer 26
(5`-CAGTGTGAGGGTTCATCT-3`) located after the stop codon were used to
amplify the total coding part of the pulmonary cDNA. Polymerase chain
reaction products were then purified, cloned by blunt-end ligation into
a Bluescript vector (Stratagene), and further sequenced by the
sequenase dideoxy chain termination method (sequenase, U. S.
Biochemical Corp.).
Anchored Polymerase Chain
Reaction
Extension of oligonucleotide 33
(5`-CAGTGTGAGGGTTCATCT-3`) complementary to a fragment of the fourth
exon was carried out with reverse transcriptase for synthesis of cDNA.
The excess free nucleotide was removed, and the cDNA was 3`-tailed
using deoxynucleotidyl terminal transferase (Boehringer) under
conditions designed to add less than 20 dGMP residues
(23) . cDNA
amplification was carried out with the nested reverse oligonucleotide 3
(in third exon, 5`-GTGTCCACGTTCTCCAGG-3`) and the 5`-anchored primers.
These primers for the poly(dG) end were a mixture of the large primer
5`-GCATGCGCGCGGCCGCGGAGGCCCCCCCCCCCCCC-3` and the short primer
5`-GCATGCGCGCGGCCGCGGAGCC-3` in a ratio of 1:9
(23) .
Amplification was performed for 35 cycles under the previously
described conditions. A second amplification reaction was performed for
25 cycles using 2 µl of the first amplification products. We used
for this amplification the short 5`-anchored primer and oligonucleotide
22 in the second exon (5`-TCAAGAAGAGGGTGAAGGC-3`). Then, part of the
polymerase chain reaction product was submitted to electrophoresis
through a 1% (mass/volume) agarose gel, blotted onto nylon filter, and
hybridized with the 5`-labeled oligonucleotide 8
(5`-GCTGCAGGCTCTGTATGTGG-3`).
RESULTS
Analysis of SP-A in Intestinal Cells
In
a first step, the presence of SP-A in gastric, jejunal, and colonic
cells was searched for by indirect immunofluorescence study using two
types of cell preparations: frozen sections of pelleted isolated cells
(Fig. 1) and freshly isolated entire cells directly dropped and
fixed on a glass substratum (Fig. 2) to control the possible
diffusion of the antigen. The quality control of cell isolation was
assessed by staining gastric cells with
H,K
-ATPase antibody that labeled only
parietal cells (Fig. 1b) and by staining intestinal
cells with phalloidin that labeled actin filaments of the apical brush
borders (Fig. 1f). Two different polyclonal anti-rat
pulmonary SP-A antibodies of distinct origin were used. As shown in
Fig. 1
, a clear labeling of frozen epithelial cells dispersed
from jejunum (Fig. 1, c and d) or colon
(Fig. 1e) was observed both with our antibody directed
against rat SP-A (Fig. 1, c and e) and with the
one provided by Dr. Whitsett (Fig. 1d). Staining was
found at the apical part of the cells or diffuse into cell cytoplasm.
No staining was found in isolated gastric cells
(Fig. 1a). Controls using non-immune serum or secondary
antibody alone were not stained (not shown). Similar results were found
with freshly isolated entire cells using our anti-SP-A antibody as
shown for jejunal cells in Fig. 2, a and b.
Figure 1:
Immunodetection of SP-A in cells
dispersed from rat stomach (a), jejunum (c and
d), or colon (e). Cells were fixed in
paraformaldehyde (3% in PBS) and cryo-sectioned (5 µm), then
subjected to 3-step indirect immunofluorescence. Cells were incubated
with an antibody raised in guinea pig against rat SP-A, prepared in our
laboratory (a, c, and e), or with an
antibody provided by Dr. J. A. Whitsett (d). Staining was
found both in jejunal and colonic but not in gastric cells. In
c, d, and e, epithelial cells were labeled
either at their apical part (arrows) or in cytoplasm
(arrowhead). b, labeling of the gastric
H,K
-ATPase; f, labeling of
the brush border of colonic cells by phalloidin
(arrow).
Figure 2:
Indirect immunofluorescent staining of
unsectioned cells isolated from rat jejunum (a and
b). The whole cells fixed in paraformaldehyde (3% in PBS) were
incubated with the antibody prepared in our laboratory against rat
SP-A. Note the presence of SP-A immunoreactivity at the apex of cells
(arrows).
Characterization of SP-A protein was then performed by Western blot
analysis from one-dimensional gel electrophoresis under reducing
conditions, comparing colonic samples with pulmonary samples. First,
the classical immunolabeling in two steps was used
(Fig. 3A). In isolated pulmonary cells as well as in
surfactant fraction, the characteristic SP-A triplet of 26, 32, and 38
kDa corresponding to the non-glycosylated and glycosylated monomers (1)
was strongly labeled (Fig. 3A, b and
c, respectively). In pulmonary cell sample, a 66-kDa band was
also present (Fig. 3A, b). By contrast, no band
was labeled in colonic samples (Fig. 3A, a). In
a second step, the biotin-streptavidin system was used
(Fig. 3B). With this technique, pulmonary surfactant
fraction appeared similarly labeled as after the classical two-step
technique (Fig. 3B, c). Pulmonary cell sample
was, however, differently labeled; the characteristic monomeric triplet
was faintly labeled whereas two bands in the range of 66 and 120 kDa
were strongly labeled (Fig. 3B, b). In colonic
cells, bands were this time labeled with a pattern identical to that in
pulmonary cells (Fig. 3B, a). The same results
were observed using the two anti-SP-A antibodies (not shown). No bands
were labeled with non-immune serum (not shown).
Figure 3:
Western blot analysis of SP-A in rat colon
and lung epithelial cells and in pulmonary surfactant fraction after
one-dimensional 12% SDS-polyacrylamide gel electrophoresis in reducing
conditions. 80 µg of protein were run for intestinal and pulmonary
cells and 5 µg for pulmonary surfactant fraction. Immunodetection
was made with the anti-SP-A antibody provided by Dr. J. A. Whitsett;
the same results were obtained with the antibody raised in the lab (not
shown). Exposure time was 3 min. PanelA, ECL system
without amplification; lanea, colonic cells;
laneb, lung cells; lanec,
surfactant fraction. The monomeric forms of SP-A (26, 32, and 38 kDa)
were strongly labeled in surfactant fraction and lung cells; a higher
molecular weight protein was faintly labeled in the fraction and more
markedly in lung cells. Colonic cell sample was not labeled. PanelB, ECL system after amplification with biotin
streptavidin; lanea, colonic cells; laneb, lung cells; lanec, surfactant
fraction; laned, molecular weight markers run in
parallel. Pattern of surfactant fraction was the same as without
amplification. In both colonic and lung cell samples, monomeric forms
of SP-A were faintly labeled, while bands around 66 and 120 kDa were
strongly labeled.
To better
characterize the high molecular forms, two-dimensional electrophoresis
was then performed. In Fig. 4, the silver staining and the
immunoreactive pattern of proteins isolated from pulmonary
(Fig. 4, a and b) or colonic (Fig. 4,
c and d) epithelial cells is reported. Among the
numerous spots revealed (Fig. 4, a and c), only
three spots were labeled with the anti-SP-A antibody in pulmonary
(Fig. 4b) and colonic (Fig. 4d)
epithelial cells. These immunoreactive spots observed at 66 and 120 kDa
were colocalized in both tissues at the acidic pI appropriate to SP-A.
Figure 4:
Silver staining (left side) and
immunoblot analysis (ECL system with amplification, right
side) of proteins isolated from pulmonary (a and
b) and colonic (c and d) epithelial cells
and run in two-dimensional electrophoresis. 20 µg of delipidated
proteins were separated by IEF in the horizontal plane (pH range,
3.0-10.0) followed by electrophoresis in a 12% polyacrylamide gel
in the vertical plane. Blots were labeled with Whitsett's
anti-SP-A antibody. Note that multimeric forms of SP-A protein with the
molecular masses observed after SDS-polyacrylamide gel electrophoresis
(i.e. 66 and 120 kDa, arrowhead) and with appropriate
pI (acidic pH is on the rightside) are colocalized
in both colonic and pulmonary epithelial
cells.
To further explore the identity of the 66- and 120-kDa bands as SP-A
multimers, the reduced sulfhydryl bonds of proteins extracted from
isolated cells were alkylated for preventing the possible reassociation
of monomeric forms. Samples submitted either to delipidation or to
delipidation plus alkylation were electrophoresed in one dimension and
immunoblotted. Whereas delipidated samples, from pulmonary as well as
from colonic cells (Fig. 5, a and c), exhibited
the same pattern as non-delipidated samples (Fig. 3B,
a and b) with stronger labeling of 66- and 120-kDa
forms, in delipidated and alkylated samples, the immunoreactive 120-kDa
band faded away, while the bands of 26, 32, and 38 kDa were reinforced
(Fig. 5, b and d). The 66-kDa immunoreactive
band was apparently less affected by alkylation treatment, either in
lung or in the intestine, since it decreased slightly in intensity but
did not disappear. Two-dimensional electrophoresis was then applied to
an alkylated colonic epithelial cell sample run in parallel with a
pulmonary surfactant fraction sample. The silver staining and
immunoreactive pattern are depicted in Fig. 6, a and
b, and Fig. 6, c and d, respectively.
The monomeric species of SP-A were again observed after alkylation
treatment (Fig. 6d). These forms, less intensively
labeled than the multimeric forms, colocalized with the characteristic
triplet labeled in pulmonary surfactant fraction
(Fig. 6b). Altogether, these results demonstrate that
the 120-kDa immunoreactive band present in both pulmonary and colonic
epithelia is a multimeric form of SP-A, the reassociation of which can
be prevented by alkylation. The same results were observed with jejunal
cell preparation (not shown).
Figure 5:
One-dimensional Western blot analysis of
SP-A after delipidation and alkylation treatments. Approximately 50
µg of proteins isolated from colonic (lanesa and
b) or from lung (lanesc and d)
epithelial cells were separated by 12% SDS-polyacrylamide gel
electrophoresis in reducing conditions and immunoblotted with
Whitsett's anti-SP-A antibody. ECL system with amplification was
used; exposure time was 30 s. Only the multimeric forms at 66 and 120
kDa were strongly labeled in delipidated samples isolated from colon
(lanea) or from lung (lanec).
After alkylation by contrast, this exposure time was sufficient for
revealing bands corresponding to the characteristic triplet of SP-A
monomers in colonic cells (laneb) as well as in
pulmonary cells (laned), while labeling of the
120-kDa immunoreactive band was decreased markedly in both
tissues.
Figure 6:
Silver staining (left side) and
immunoblot analysis (ECL system with amplification, right
side) of proteins isolated from pulmonary surfactant fraction
(a and b) and of alkylated proteins from colonic
epithelial cells (c and d) submitted to
two-dimensional electrophoresis. 20 µg of proteins were deposited
and separated by IEF in the horizontal plane (pH range,
3.0-10.0), followed by electrophoresis in a 12% polyacrylamide
gel in the vertical plane. Blots were labeled using Whitsett's
anti-SP-A antibody. In the pulmonary surfactant fraction, the antibody
labeled only the triplet of monomeric forms of SP-A (b) at the
appropriate acidic pI (acidic pH is on the right). In
alkylated proteins from colonic cells (d), several bands not
revealed without alkylation colocalized with the characteristic
pulmonary monomeric forms.
Analysis of SP-A mRNA
The presence of
SP-A transcripts in the gastrointestinal tract was studied by RT-PCR in
different tissues using the lung as a positive control organ and
-actin cDNA amplification as an internal control. Specific SP-A
cDNAs synthesized from total RNAs were selectively amplified with the
two oligonucleotides described under ``Materials and
Methods.'' As expected, Southern blot analysis of the amplified
cDNA revealed in the lung a signal of 372 bp (Fig. 7, lanee). After 23 amplification cycles, similar signals were
observed in duodenum, jejunal, and colon tissues (Fig. 7,
lanesb-d); these signals could be detected
sooner, i.e. after 17 cycles (not shown). No positive signal
was detected in stomach (Fig. 7, lanea),
kidney, muscle, and brain (not shown) even after 32 amplification
cycles, although
-actin cDNA was amplified in these tissues.
Figure 7:
Southern blot autoradiogram of
RT-PCR-amplified SP-A cDNAs. 2 µg of total RNAs extracted from
stomach (lanea), duodenum (laneb), jejunum (lanec), colon (laned), and lung (lanee) were used for
cDNA synthesis. The specific primers for SP-A and -actin gene
message were used for amplification as described under ``Materials
and Methods.'' After electrophoresis and blotting, hybridization
was performed with 5`-labeled oligonucleotides. SP-A mRNA was amplified
in lung and in various intestinal segments but not in stomach, despite
a much larger number of amplification
cycles.
To
determine the sequence of the amplified product in the colon,
complementary DNA of SP-A was amplified in colon with the primer pair 2
and 26, which allowed the amplification of the total coding part of the
pulmonary SP-A cDNA to occur. This amplified cDNA was then cloned into
the blunted EcoRV site of Bluescript. Two positive clones were
sequenced and compared with a genomic clone. No difference was found
between these clones, suggesting that intestinal and pulmonary cDNA
sequences were completely matched. Only some minor changes with the
nucleotide sequence reported by Sano et al.(24) were
found. These differences were present in genomic and intestinal cDNA
clones as well (not shown).
Determination of Initiation Site of Transcription by
Anchored PCR
To determine the transcriptional initiation
site in intestinal epithelium, complementary poly(G)-tailed cDNA was
synthesized from total RNA extracted from rat colonic cells and lung
and kidney homogenates. Oligonucleotide 33 (fourth exon) was used for
cDNA synthesis. The 5`-anchored priming oligonucleotides previously
described
(23) and reverse oligonucleotide 3 were designed for
the first amplification of the tailed cDNA. Then, part of this first
amplified product was submitted to a second reaction of amplification
using the short 5`-anchored priming oligonucleotide and reverse
oligonucleotide 22 (second exon). The expected size of this new
amplified fragment is about 143 bp (35 bp corresponding to the
5`-35-oligonucleotide anchor, the 48 bp of the first full exon and the
first 60 bp of the second exon spanning from the splicing junction to
the 5`-extremity of reverse primer 22). After the second amplification,
Southern blot analysis of the amplified cDNA revealed in colon and in
lung a signal of about 150 bp, the intensity of which increased with
the number of cycles (Fig. 8). No signal was detected after 25
cycles of amplification of tailed cDNA synthesized from kidney RNA,
used as negative control. Hybridization of these fragments with
oligonucleotide 8 revealing identical signals indicates that the
transcriptional initiation site for the SP-A gene is the same in the
lung and in the colon.
Figure 8:
Southern blot autoradiogram of the
anchored polymerase chain reaction products. Total RNAs extracted from
adult rat colonic cells (C), lung (L), or kidney
(K) homogenates were used for cDNA synthesis. After
electrophoresis and blotting, hybridization was performed with
5`-labeled oligonucleotide 8. Oligonucleotide 33 was used for cDNA
synthesis. Oligonucleotide 22 and the 5`-anchor primers (see
``Materials and Methods'') were designed for amplification.
Amplified products display the same size in colon and lung, indicating
an identical initiation site.
DISCUSSION
The present study establishes that SP-A, the major
hydrophilic pulmonary surfactant-associated protein, is constitutively
expressed by epithelial cells of both small and large intestine of the
rat. This is the first report of an ectopic expression of SP-A gene
thus far considered as lung specific. It was not thought to be normally
expressed, even at low level, by tissues other than pulmonary
epithelium, more specifically alveolar type II cells and non-ciliated
bronchiolar Clara cell, making SP-A a specific marker of lung
epithelial cells
(25) .
(
)
with staining of intestinal
epithelium in situ with other SP-A antibodies.
terminus, whereas the COOH-terminal domain of both is lectin-like
and carbohydrate binding. In addition to this parallel in overall
organization, SP-A and MBP sequences can be aligned with only three
gaps to show 30% identity in sequence. Virtually all invariant residues
in the carbohydrate-recognition domain of MBPs are found in
SP-A
(41) . Another protein containing a collagen-like domain,
the complement protein C1q, which binds in a glycosylated-dependent
manner on the Fc domain of immunoglobulins
(43) , also presents a
close homology to SP-A
(44) and MBPs
(41) . It has been
speculated that these proteins may be functionally related
(40) .
It could therefore be assumed that SP-A might participate in a
primitive form of immune response in intestine epithelium as has also
been shown to be the case in the
lung
(3, 44, 45) .
,K
-ATPase antibody.
Thanks are due to C. Ilic for skillful technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.