©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Pulmonary Surfactant Protein A (SP-A) Is Expressed by Epithelial Cells of Small and Large Intestine (*)

Sandrine Rubio (1), Thierry Lacaze-Masmonteil (2) (3), Bernadette Chailley-Heu (1), Axel Kahn (3), Jacques R. Bourbon (1), Robert Ducroc (1)(§)

From the (1) From INSERM U.319, Université Paris 7, 2 Place Jussieu, 75251 Paris, Cedex 05, (2) Service de Pédiatrie et de Réanimation Néonatale, Hôpital Antoine Béclère, Clamart, and (3) INSERM U.129, Institut Cochin de Généitique Moléculaire, 24 rue du Fg St-Jacques, 75014 Paris, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Rat surfactant-associated protein A (SP-A),() 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.

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.


MATERIALS AND METHODS

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 KHPO), 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 NHCl 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) .

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() with staining of intestinal epithelium in situ with other SP-A antibodies.

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 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) .

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.


FOOTNOTES

*
This research was funded in part by the Comité National de Lutte Contre les Maladies Respiratoires et la Tuberculose (Grant 92 MR 2) and by Mutuelle Générale de l'Education Nationale (Grant 704 022). These data were presented in part at the American Gastroenterological Association meeting in New Orleans, LA, May 15-18, 1994. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 331-44-27-63-28; Fax: 331-43-25-67-89.

The abbreviations used are: SP-A, surfactant protein A; PBS, phosphate-buffered saline; IEF, isoelectric focusing; RT-PCR, reverse transcriptase-polymerase chain reaction; bp, base pair(s); MBP, mannose-binding proteins.

J. N. Freund, personal communication.


ACKNOWLEDGEMENTS

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,K-ATPase antibody. Thanks are due to C. Ilic for skillful technical assistance.


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