Surfactant proteins A and D in Eustachian tube epithelium

Reija Paananen1,2, Raija Sormunen1,3, Virpi Glumoff1,2, Martin van Eijk4, and Mikko Hallman1,2

1 Biocenter Oulu, 2 Department of Pediatrics, and 3 Department of Pathology, University of Oulu, FIN-90014 Oulu, Finland; and 4 Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, The Netherlands


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Surfactant protein (SP) A and SP-D are collectins that have roles in host defense. The Eustachian tube (ET) maintains the patency between the upper airways and the middle ear. Dysfunction of local mucosal immunity in ET may predispose infants to recurrent otitis media. We recently described preliminary evidence of the expression of SP-A and SP-D in the ET. Our present aim was to establish the sites of SP-A and SP-D expression within the epithelium of the ET in vivo. With in situ hybridization, electron microscopy, and immunoelectron microscopy, the cells responsible for SP-A and SP-D expression and storage were identified. SP-A expression was localized within the ET epithelium, and the protein was found in the electron-dense granules of microvillar epithelial cells. Being concentrated in the epithelial lining, only a few cells revealed intracellular SP-D, and it was not associated with granules. The SP-A and SP-D immunoreactivities in ET lavage fluid, as shown by Western blot analyses, were similar to those in bronchoalveolar lavage fluid. We propose that there are specialized cells in the ET epithelium expressing and secreting SP-A and SP-D. SP-A and SP-D may be important for antibody-independent protection of the middle ear against infections.

collectin; innate immunity; in situ hybridization; immunoelectron microscopy


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE EUSTACHIAN TUBE (ET) develops embryonically from the first pharyngeal pouch and connects the upper respiratory tract to the middle ear. The ET protects the middle ear from excessive fluctuations of atmospheric pressure, serves as a clearance tract, and may protect the middle ear against the invasion of microbes and other noxious agents from the airways (3). Infections in the middle ear are very common, affecting nearly all children at least once, and ~20% of children have recurrent otitis media (ROM). Dysfunction of the ET is considered to be the principal pathogenetic factor in the susceptibility to ROM, which is largely determined by hereditary factors (9). However, the mechanisms and the genes involved remain unknown. Evidence of the presence of a surface tension-lowering substance in the ET has been found (2). Recently, Paananen et al. (31) described the expression of surfactant protein (SP) A and SP-D in the epithelial lining of the ET. SP-A has also been detected in the middle ear mucosa (8). These data suggest that innate immune responses might play an important role in the protection of the ET against infection.

SP-A and SP-D are hydrophilic collagenous glycoproteins. Together with serum mannose-binding protein, conglutinin, collectin-43, and the recently described human liver collectin CL-L1 (30), they belong to the family of C-type lectins (known as collectins). These proteins consist of a short amino-terminal region, a long collagen-like domain, a neck region, and a carbohydrate recognition domain (CRD). The basic structural unit of each collectin is a trimer based on a collagen-like triple helix. According to the current notion, collectins bind to carbohydrates expressed on the surface of various microorganisms and to specific receptors on phagocytic cells, thus accelerating microbial clearance (6, 16). Individual collectins and their specific structural domains are likely to have particular roles in host defense (12).

SP-A is a 28- to 36-kDa protein expressed primarily by alveolar epithelial cells in the lung. SP-A isolated from bronchoalveolar lavage (BAL) fluid is bound to surfactant aggregates. SP-A improves the SP-B-mediated surface tension-reducing properties of surfactant lipids. However, deletion of the SP-A gene apparently does not affect lung stability (22). SP-A binds to pulmonary type II alveolar cells and immune cells, particularly alveolar macrophages (39b). The proposed functions of SP-A in vitro include enhancement of surface activity (13, 38), maintenance of homeostasis between the extra- and intracellular surfactant pool (7), and involvement in non-antibody-mediated defense against microorganisms (39b). SP-A knockout mice have been shown to have reduced defense against several lung pathogens (24, 25).

SP-D is a 43-kDa protein synthesized and secreted by pulmonary alveolar type II cells, nonciliated airway cells, and cells of the gastric mucosa. SP-D expression is also detected in the epithelium of the conducting airways and in the tracheal and bronchial glands of the lower airways (43). Today, SP-D is increasingly recognized as a molecule involved in the host defense system of many organs, and SP-D expression has been found in many mucosal surfaces in human tissues (27). SP-D is nonsedimentable at neutral pH, binding to type II cell apical membranes and alveolar macrophages (42). Deletion of the SP-D gene alters the type II cell morphology, increasing the pool sizes of surfactant (4, 23). Similar to SP-A, SP-D consists of isoforms that differ in apparent molecular mass and isoelectric point (6). SP-D does not bind to the surfactant complex or enhance surface activity. However, it binds to the carbohydrate structures on the surfaces of pathogens, resulting in agglutination of the target (16). Recombinant SP-D inhibited respiratory syncytial virus infectivity both in vitro and in vivo (14). SP-D also stimulates the phagocytosis of Pseudomonas aeruginosa (34).

The mucosa of the fibrocartilaginous portion of the ET consists of ciliated stratified epithelium of the respiratory type, which rests on a basement membrane. The ciliated and mucus cells are concentrated in the lower part of the cartilaginous ET, which contains numerous mucosal folds. The assumed ventilatory function of the tube is accomplished by the pharyngeal muscles, which pull apart the mucous surfaces of the cartilaginous ET (33).

According to the current hypothesis, the local surfactant system of the ET is of major functional significance. Evidence of a surface tension-lowering substance in canines has been shown by ET lavage (ETL) (2). Concentric lamellar bodies have been detected within the epithelial cells and the extracellular spaces of the ET (20). According to preliminary evidence, SP-A and SP-D genes were expressed in the porcine ET epithelium (31). Recently, Paananen et al. (32) also showed SP-B expression within the ET epithelium. The present study establishes the cellular and subcellular sites of SP-A and SP-D expression and the distribution of these proteins within the epithelial lining of the ET.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Porcine ET Preparations and BAL

The pharyngeal opening of the ET was prepared, and the cartilaginous part was dissected. Tissue samples from the porcine ET were recovered from its pharyngeal, middle, and distal segments, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight, and embedded in paraffin. Four-micrometer cross sections were mounted on SuperFrost Plus (Menzel-Gläser, Braunschweig, Germany) slides and used for in situ hybridization studies. For protein preparations, ETL was performed with 1× PBS as described previously (31). BAL was performed as a control. Briefly, the trachea was cannulated, and the airways were filled with 0.9% saline at a pressure of 30 cmH2O. The fluid was collected from the airways with gentle suction. The procedure was repeated three times, and the obtained lavage yields were combined.

Antibodies

The polyclonal sheep anti-SP-A antibody was a kind gift from Prof. S. Hawgood (University of California, San Francisco, CA). The antiserum was purified with a HiTrap protein A column (Pharmacia, Uppsala, Sweden). The porcine anti-SP-D polyclonal antibody used was raised in rabbit against purified porcine SP-D. The IgG fraction was obtained and affinity purified with an immobilized porcine SP-D-Sepharose column. The rat polyclonal antibody against SP-D was a kind gift from Prof. E. Crouch (Barnes-Jewish Hospital, St. Louis, MO).

Immunohistochemistry

Cross sections of the ET were immunostained with the purified sheep anti-SP-A antibody and the pig anti-SP-D antibody as previously described (31). For SP-A staining, 3-amino-9-ethylcarbazole complex was used as the chromogen.

Electron Microscopy

For electron microscopy, tissue pieces from the porcine ET were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. They were then postfixed in 1% osmium tetraoxide in phosphate buffer, dehydrated in acetone, and embedded in Epon LX 112 (Ladd Research Industries, Burlington, VT). Thin sections were cut with a Reichert Ultracut E microtome and examined under a Philips CM 100 transmission electron microscope with an acceleration voltage of 80 kV.

Immunoelectron Microscopy

Immunoelectron microscopy was performed as described by Sormunen et al. (37) with a slightly modified procedure. Fresh porcine ET and lung tissues were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 1 h. Small tissue pieces were immersed in 2.3 M sucrose and frozen in liquid nitrogen. Thin cryosections were cut with a Leica Ultracut UCT microtome. For immunolabeling, the sections were first incubated in 5% BSA with 0.1% cold-water fish skin gelatin (Aurion, Wageningen, The Netherlands). Antibodies and gold conjugate were diluted in 0.1% BSA-c (10% solution of acetylated BSA with Kathon CG as a preservative; Aurion) in PBS, and all washes were performed in 0.1% BSA-c in PBS. The sections were then incubated with antibodies raised against SP-A and SP-D for 45 min. After being washed, the sections were treated with a protein A-gold complex (size 10 nm) prepared according to the method described by Slot and Geuze (36) for 30 min. The control samples were prepared by performing the same labeling procedure in the absence of the primary antibody. The sections were embedded in methyl cellulose and examined as described in Electron Microscopy.

In Situ Hybridization

Preparation of the probes. In situ hybridization was carried out essentially according to the instructions supplied by Boehringer Mannheim (Mannheim, Germany). Briefly, a 365-bp CRD fragment of the SP-A cDNA sequence from the porcine lung and a 313-bp CRD fragment from the SP-D cDNA from the porcine lung were subcloned into pBluescript (Stratagene, La Jolla, CA) and pGEM-T Easy (Promega, Madison, WI) vectors. The plasmids were linearized. Antisense and sense SP-A and SP-D UTP-digoxigenin (Dig)-labeled riboprobes were synthesized with 19 U of T7 RNA polymerase (Promega), 20 U of T3 RNA polymerase (Promega), or 20 U of Sp6 RNA polymerase (Promega). The transcription reaction contained 1.2 µg of linearized plasmid, 1× transcription buffer (Promega), 10 mM dithiothreitol, 1× Dig reaction mix (Boehringer Mannheim), and RNase inhibitor (Pharmacia) for 2 h at 37°C. After DNase I digestion, the probes were purified with lithium chloride-ethanol precipitation and detected on agarose gel. The labeling was detected with a Dig detection kit (Boehringer Mannheim).

Hybridization protocol. Four-micrometer paraffin sections were treated with proteinase K (5 µg/ml) at 37°C for 30 min and postfixed in 4% paraformaldehyde in PBS. The sections were acetylated with 0.25% acetic anhydride in triethanolamine buffer, washed with 4× SSC, and covered with 200 µl of a prehybridization mix [50% (vol/vol) formamide, 4× SSC, and 40 µg/ml of single-strand DNA] for 2 h at 58°C followed by 200 µl of a hybridization mixture (40% formamide, 10% dextran sulfate, 1× Denhardt's solution, 4× SSC, 10 mM dithiothreitol, 40 µg/ml of yeast tRNA, and 40 µg/ml of denatured salmon sperm DNA) containing 300 ng/ml of the Dig-labeled riboprobe. A GelBond film (FMC BioProducts, Rockland, ME) was applied. Hybridization was allowed to occur at 58°C for 42 h. After hybridization, the unbound probe was removed from the sections by treatment with RNase A followed by high-stringency washes. The hybridized probe was detected by incubating the sections with the anti-Dig antibody conjugated with alkaline phosphatase. The color reaction took place overnight with nitro blue tetrazolium/ 5-bromo-4-chloro-3-indolyl phosphate (Boehringer Mannheim) in Tris · HCl, pH 9.5, with 5 mM levamisole. The reaction was stopped with 10 mM Tris · HCl buffer, pH 8, containing 1 mM EDTA, and the sections were counterstained with 0.02% fast green FCF (Sigma). As a positive control, sections from the porcine lung were hybridized with the same riboprobes. Negative controls consisted of the same tissue sections hybridized in an identical fashion with the Dig-labeled sense riboprobe. As another negative control, tissue sections hybridized with the antisense riboprobe were subjected to a color reaction without anti-Dig antibody incubation.

Western Blot Analyses of SP-A and SP-D

Porcine ETL and BAL fluids were used for the analyses. After removal of the cells by centrifugation (500 g for 10 minutes at 4°C), the samples were centrifuged for 2 h at 21,000 g at 4°C to collect the sedimentable lipid-protein complexes. SP-D was analyzed in the supernatant fraction and SP-A in the sedimentable aggregate fraction. The supernatant obtained was concentrated with an Ultrafree-4 centrifugal filter unit with a Biomax-10K NMWL membrane (Millipore, Bedford, MA). Before Western blot analyses, the samples were analyzed for the total protein content with the Bradford assay kit (Bio-Rad, Hercules, CA) with BSA as a standard. The ET supernatant samples containing 50 µg of protein and 20 µg of protein from BAL samples as well as the ET aggregate samples containing 20 µg of protein and 2 µg of protein from the BAL samples were solubilized in Laemmli buffer containing 10% (vol/vol) 2-mercaptoethanol were boiled for 5 min and applied to a 12% SDS-PAGE gel. After electrophoresis, the proteins were transferred onto a nitrocellulose membrane with a blotting apparatus (Bio-Rad). After being blocked overnight in a mixture containing 5% nonfat milk and 0.05% Tween 20 in Tris-buffered saline, the nitrocellulose sheets were incubated with rabbit anti-sheep SP-A serum diluted 1:15,000 or with porcine SP-D antibody diluted 1:5,000. A secondary anti-rabbit IgG antibody conjugated with horseradish peroxidase was visualized with a chemiluminescent detection system (ECL Plus Western Blotting Analysis System, Amersham).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Morphology and Localization of SP-A and SP-D

The porcine ET is a curved tube. The dorsal part is closely sheltered by cartilage, and the lower segment has an elongated lumen with numerous inferior mucosal folds (Fig. 1A). The ET epithelium consists of stratified epithelium that morphologically resembles the lower airway epithelium. The ET epithelium could be described as columnar ciliated epithelium that also contains cuboidal epithelial cells. Goblet cells, ciliated cells, and cells with microvilli are present as well, and a few distinct macrophages can be detected. The apical areas of the cells with microvilli contained electron-dense secretory granules (Fig. 2).


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Fig. 1.   Light microscopy of paraffin-embedded sections of Eustachian tube (ET). A: hematoxylin and eosin-stained cross section from the middle segment of the cartilaginous ET. Right, floor part with mucosal folds; left, roof part inside the cartilage. Box in A: part of epithelium magnified in B and C. B: immunostaining of ET epithelium with polyclonal anti-sheep surfactant protein (SP) A antibody. C: immunostaining of ET epithelium with polyclonal anti-pig SP-D antibody. Bar in C is also for B.



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Fig. 2.   Electron micrograph of an ET epithelial cell with microvilli and apparent secretory granules (arrows).

Both immunostaining and expression were studied throughout the circumference of the epithelium in the middle segment of ET. The pharyngeal and more distal parts of the ET were similarly studied. SP-A immunostaining was detected in the tubal floor where the mucosal folds exist. The distribution of SP-A expression was similar as studied by in situ hybridization. Both protein and mRNA could be detected in the epithelium of the tubal floor both pharyngeally and distally. Light-microscopic studies showed SP-A staining to be concentrated close to the mucosal folds. Intense SP-A staining was detected in the apparent secretory cells apically (Fig. 1B). The gobletlike cells also showed immunopositivity for SP-A. Similar to SP-A, SP-D immunostaining was concentrated in the mucosal folds. SP-D additionally showed light diffuse staining in the ET epithelium throughout the circumference. This was evident in both the pharyngeal and distal parts of the ET. However, some cells showed more intense staining both apically and basally (Fig. 1C). The apical staining for SP-D was stronger and more diffuse than that for SP-A, for which the positivity was specifically located in the apical cells. Some cells in the middle of the epithelium showed immunopositivity for both SP-A and SP-D. Lung sections served as positive controls in which the positive staining for both SP-A and SP-D could be localized in the alveolar type II cells (data not shown).

Immunoelectron microscopy confirmed the light-microscopic findings. SP-A was localized in the microvillar epithelial cells, more specifically, in the electron-dense material of apically located granules (Fig. 3A). Furthermore, lamellar structures were occasionally detected in the granules of gobletlike cells, and this material showed positive labeling for SP-A (Fig. 3C). More diffuse labeling was detected in the dark long microvillar cells (data not shown). Traces of SP-A label could also be seen in the ET lumen attached to lipid material or to the microvilli of the apical cells (Fig. 3A). The few macrophages found within the epithelial layer contained SP-A (data not shown). This labeling is in accordance with the light-microscopic findings.


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Fig. 3.   Localization of SP-A and SP-D in ET epithelial cells and alveolar type II epithelial cells. A: immunoelectron micrograph showing SP-A localization in apparent secretory granules of microvillar ET epithelial cell and in ET lumen (arrows). B: SP-D immunolabeling of amorphous airspace material from the ET lumen (arrows). C: gobletlike ET epithelial cell showing SP-A gold label in secretory granules of lamellar structures (arrows). D: microvillar ET epithelial cell showing SP-D gold label diffusely in the cytoplasm (arrows). E: alveolar type II epithelial cell showing SP-A gold label in lamellar bodies and at the periphery of the lamellar bodies (arrows). F: SP-D immunolabeling of amorphous airspace exudate of alveoli and lamellar bodies of alveolar type II epithelial cell (arrows). The absence of clear lamellae in lamellar bodies is due to the cryoprocessing method.

The most prominent labeling with SP-D was seen in the apical plasma membrane and in the amorphous material lying on the epithelial cells (Fig. 3B). Few epithelial cells with microvilli showed diffuse labeling with the SP-D antibody (Fig. 3D). In these cells, secretory granules had no detectable immunoreactivity. Some macrophages were also labeled (data not shown). This labeling supports our light-microscopic findings. In the alveolar type II epithelial cells, the SP-A label was concentrated in small vesicles apically (data not shown), in lamellar bodies, and in structures resembling the endoplasmic reticulum (Fig. 3E). Traces of SP-D label were observed in the lamellar bodies, and large quantities were observed in the alveolar amorphous exudate (Fig. 3F). The absence of clear lamellar bodies in alveolar type II cells is caused by the fixation and processing for cryosections, described earlier by Voorhout et al. (40).

In Situ Hybridization

To identify the cellular sites of SP-A and SP-D mRNA expression in the porcine ET, a series of in situ hybridization studies with sections of porcine ET was performed. With specific RNA probes for SP-A and SP-D, intense labeling with the antisense probes was seen in some cells in the floor part of the epithelial cell lining of the ET. SP-A mRNA was mainly detected basally and in the cells in the middle of the epithelium, whereas only light staining could be seen more apically. Similar to SP-A, strong SP-D labeling was also detected basally and in the middle of the epithelium. Unlike SP-A, SP-D mRNA was additionally detected in the apical cells. The signals with the SP-A and SP-D antisense probes are shown in Fig. 4, A and B. There was no labeling in the sections hybridized with either a sense SP-A (Fig. 4E) or SP-D (Fig. 4F) probe nor in the control samples not treated with the anti-Dig antibody (data not shown). The expression patterns were similar in the pharyngeal and more distal parts of the ET, concentrating close to the mucosal folds in the floor of the tube lumen. The roof part of the ET did not show any expression for SP-A or SP-D (data not shown). The labeling specificity was tested with lung tissue as a positive control. Intense staining was detected in the alveolar type II epithelial cells with the SP-A (Fig. 4C) and SP-D (Fig. 4D) antisense probes.


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Fig. 4.   Light micrographs showing the localization of SP-A and SP-D mRNAs in ET epithelium and alveoli. A: in situ hybridization with antisense SP-A probe. B: in situ hybridization with antisense SP-D probe. C: in situ hybridization of porcine lung section with antisense SP-A probe showing SP-A mRNA in type II alveolar epithelial cells. D: in situ hybridization of porcine lung section with antisense SP-D probe showing SP-D mRNA in type II alveolar epithelial cells. E: negative control with sense SP-A probe. F: negative control with sense SP-D probe. A-F are the same magnification. Fast green FCF was used for counterstaining.

Western Blot Analyses of ETL and BAL Fluids

We found the characteristic monomeric SP-A protein triplet of 30-38 kDa in ETL fluid (Fig. 5A). The immunoreactivity of ETL fluid was similar to that detected in BAL fluid. The antibody directed against sheep pulmonary SP-A also labeled the 28-kDa protein in the lipid aggregate fraction of the ETL and BAL fluids.


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Fig. 5.   Western blot analyses of Eustachian tube lavage (ETL) and bronchoalveolar lavage (BAL) fluids. A: immunostaining of ETL and BAL fluids with anti-sheep SP-A antibody. B: immunostaining of nonsedimentable fractions of ETL and BAL fluids with anti-porcine SP-D antibody. Nos. at left, protein molecular mass standards. The samples were boiled under reducing conditions with 10% beta -mercaptoethanol, and the proteins were separated by 12% SDS-PAGE.

The antibody against porcine SP-D revealed intense labeling of a 48-kDa protein in the nonsedimentable fractions of both the ETL and BAL fluids (Fig. 5B). The same filters were also immunostained with the rabbit anti-rat SP-D antibody. This antibody revealed similar immunoreactivity, although the intensity was weaker.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, the expression and localization of SP-A and SP-D within the epithelial cells and in the epithelial lining fluid of the ET were defined. With an in situ hybridization technique, immunohistochemistry, electron microscopy and immunoelectron microscopy, the cells expressing SP-A and SP-D in the ET were identified. The SP-A mRNA label was evident in several epithelial cells in the microvillar epithelium of the tubal floor. SP-A mRNA-producing cells seem to be more abundant than cells expressing SP-D mRNA. The present results complement recent RT-PCR and Northern hybridization data by Paananen et al. (31) on the expression of SP-A and SP-D in the porcine ET. In addition to alveolar type II cells and Clara cells (41), SP-A and SP-D expression has been previously detected in the epithelium of the conducting airways and in the tracheal and bronchial glands of the lower airways (21, 43). The porcine ET epithelium could be classified as a modified transitional respiratory epithelium where several cell types, including ciliated cells, goblet cells, nonciliated cells with secretory granules, nonciliated cells without secretory granules, intermediate cells, and basal cells, are present. It has been proposed that goblet, intermediary, and dark granulated cells may be different phenotypes of the same secretory cell (17). Basal cells are considered to be undifferentiated cells that become one of the epithelial cell types as they undergo differentiation (26). The localization of SP-A in both goblet and granulated cells is consistent with this concept.

The tubal floor, where ciliated cells are prominent, is involved in mucociliary transport (33). The expression of both SP-A and SP-D was detected near these mucosal folds. With immunohistochemistry, SP-D showed light diffuse staining apically throughout the whole circumference of the ET lumen, indicating the secretion of SP-D. On the basis of electron microscopy, only a few cells with the cytoplasmic SP-D immunolabel were found. Consistent with the light-microscopic findings, SP-D was detected in the ET lumen, on the surface of apical cells, and attached to the plasma membrane. Similar labeling of amorphous airspace material in the lung has been previously reported by Crouch et al. (5). The pattern of immunogold labeling suggests that a bulk of SP-D was secreted rather than associated with intracellular organelles involved in storage or degradation. In contrast to the SP-D findings, SP-A was detected intracellularly. SP-A immunoreactivity was identified in the electron-dense granules of the microvillar epithelial cells. In the gobletlike cells, some apical granules contained lamellar structures, which were positively labeled for SP-A. A recent study (32) revealed both immunolabeling and expression of SP-B in ET epithelium. Similar to SP-B, SP-A was detected in the apical secretory granules of microvillar cells. In contrast, the granules of the gobletlike cells containing SP-A were free of SP-B labeling. Although the microvillar epithelial cells contained both SP-A and SP-D, they were distinctly different from alveolar type II epithelial cells. Besides the morphological differences, in ET cells, the intensity of SP-A gold labeling was distinctly higher than that of SP-B, suggesting more intense expression and secretion of SP-A. The lack of clear lamellar bodies in alveolar type II epithelial cells in the present study may be explained by the processing method for cryosectioning, which does not include osmium tetroxide, the lipid fixation agent in conventional electron microscopy. To obtain better lipid preservation, we also tried a freeze-substitution technique (40), but the immunolabeling intensity was weaker and the cellular morphology was less well preserved.

In ETL fluid, SP-A appeared as a typical protein triplet of 30- to 38-kDa bands, which is characteristic of a reduced alveolar SP-A monomer. Both ETL and BAL fluids also had a 28-kDa band, which is the size of unglycosylated SP-A. Glycosylated SP-A is thought to be an immunologically more active form of SP-A (39c). The differences in the polymerization of SP-A could lead to a different binding capacity to microbial components and an altered biological function (15).

The nonsedimentable fraction of ETL fluid showed SP-D immunoreactivity similar to that of BAL fluid. The 48-kDa SP-D was prominent. It has been suggested that SP-D is processed differently in bronchiolar cells compared with type II cells (28). According to a recent study by van Eijk et al. (39a), porcine SP-D has three structural differences compared with other species. One of these is an extra cysteine in the collagenous region. This could lead to a different oligomerization and a more stable heteromer. The other differences are the three-amino acid insertion and a potential extra N-glycosylation site in the CRD. Therefore, there may be size differences between porcine SP-D (48 kDa) and that of the other species studied so far (43 kDa).

According to previous evidence (2, 3, 20), the epithelial lining of the ET contains surface-active material, which possibly protects the epithelial lining and facilitates the muscle-driven opening of the ET during swallowing. Our present results suggest that there are specialized cells in the ET epithelium that express SP-A and SP-D and that these proteins are secreted into the ET lumen. Whether SP-A is involved in the alleged surface tension-reducing function of ET surfactant remains unknown. Immunoelectron microscopy revealed no evidence of tubular myelin, which is a typical form of extracellular alveolar surfactant. The ET protects the middle ear by preventing unwanted nasopharyngeal foreign materials from entering the middle ear. The protective function is dependent on the length of the tube, the radius of its lumen, and the compliance of its walls (3). We propose that the local defense system in the ET plays an important role in preventing middle ear infections. Because the ET is the route for pathogens from the upper airway into the middle ear, the dysfunction of the local mucosal immunity in the ET may predispose infants to ROM. The floor part of the ET lumen and its mucociliary blanket are postulated to be responsible for the clearance of secretions from the middle ear into the nasopharynx (3). Because SP-A and SP-D mRNA expression is detected close to the mucosal folds in the floor part of the ET lumen, these collectins are likely to participate in the local host defense. In the lung, the postulated main role for SP-A and SP-D is to interact directly with carbohydrates on the surface of microbial pathogens and with the surface receptors of inflammatory cells, thereby initiating a variety of effector mechanisms involved in host defense. Similar to the lower airways, SP-A and SP-D in the mucosal surface of the ET could prevent the spreading of specific infections from the upper airways to the middle ear. The long list of foreign substances that have been reported to bind to the lung collectins also includes Streptococcus pneumoniae and Hemophilus influenzae (11, 29, 39), which, apart from causing respiratory infections, are the major bacteria causing middle ear infections. Respiratory syncytial virus and influenza viruses, i.e., the respiratory viruses that play an important role in the disruption of normal ET function, are also on the list of pathogens with which SP-A and SP-D interact (1, 14, 39). An infection of the upper respiratory tract initiates a cascade of events that finally leads to the development of acute otitis media as a complication. The ET is the route for pathogens from the upper airways to the middle ear, and the lack of protective agents in the ET mucosa may predispose infants to ROM. Recently, it was shown that specific SP-A haplotypes differ between children susceptible to ROM (35). However, the specific functions of SP-A and SP-D in the ET lumen remain to be defined. We propose that the collectins expressed in the ET epithelium are involved in the clearance of pathogens and may act as immunomodulators.


    ACKNOWLEDGEMENTS

We thank Maarit Hännikäinen, Elsi Jokelainen, and Sirpa Kellokumpu for excellent technical assistance and Drs. Vesa Anttila, Jussi Rimpiläinen, and Matti Pokela and the Pouttu Food Company (Kannus, Finland) for supplying the research material.


    FOOTNOTES

This research was supported by grants from Biocenter Oulu (Oulu, Finland) and the Academy of Finland.

Address for reprint requests and other correspondence: R. Paananen, Dept. of Pediatrics and Biocenter Oulu, Univ. of Oulu, FIN-90014 Oulu, Finland (E-mail: rpaanane{at}cc.oulu.fi).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 27 February 2001; accepted in final form 24 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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