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Published ahead of print on May 30, 2003, doi:10.1165/rcmb.2002-0274OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 591-597, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0274OC

Expression and Localization of Lung Surfactant Protein A in Human Tissues

Jens Madsen, Ida Tornøe, Ole Nielsen, Claus Koch, Wolfram Steinhilber and Uffe Holmskov

Department of Immunology and Microbiology, Institute of Medical Biology, University of Southern Denmark, Odense; Department of Pathology, Odense University Hospital, Odense; and Statens Serum Institut, Copenhagen, Denmark; and Altana Pharma, Konstanz, Germany

Address correspondence to: Uffe Holmskov, Department of Immunology and Microbiology, Institute of Medical Biology, University of Southern Denmark, Winsløwparken 21.1, DK-5000 Odense C, Denmark. E-mail: holmskov{at}health.sdu.dk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lung surfactant protein A (SP-A) is a collectin produced by alveolar type II cells and Clara cells. It binds to carbohydrate structures on microorganisms, initiating effector mechanisms of innate immunity and modulating the inflammatory response in the lung. Reverse transcriptase–polymerase chain reaction was performed on a panel of RNAs from human tissues for SP-A mRNA expression. The lung was the main site of synthesis, but transcripts were readily amplified from the trachea, prostate, pancreas, and thymus. Weak expression was observed in the colon and salivary gland. SP-A sequences derived from lung and thymus mRNA revealed the presence of both SP-A1 and SP-A2, whereas only SP-A2 expression was found in the trachea and prostate. Monoclonal antibodies were raised against SP-A and characterized. One of these (HYB 238-4) reacted in Western blotting with both reduced and unreduced SP-A, with N-deglycosylated and collagenase-treated SP-A, and with both recombinant SP-A1 and SP-A2. This antibody was used to demonstrate SP-A in immunohistochemistry of human tissues. Strong SP-A immunoreactivity was seen in alveolar type-II cells, Clara cells, and on and within alveolar macrophages, but no extrapulmonary SP-A immunoreactivity was observed. In contrast to lung surfactant protein D (SP-D), which is generally expressed on mucosal surfaces, SP-A seems to be restricted to the respiratory system.

Abbreviations: bronchoalveolar lavage, BAL • carbohydrate-recognition domain, CRD • mannose-binding lectin, MBL • reverse transcription-polymerase chain reaction, RT-PCR • sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE • lung surfactant protein A, SP-A • lung surfactant protein D, SP-D • Tris-buffered saline, TBS


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lung surfactant protein A (SP-A) is a member of the collectin family, a group of oligomeric proteins in which a collagenous region is connected by an {alpha}-helical neck region to a C-type lectin or carbohydrate-recognition domain (CRD) (1–3). The structural subunit consists of three such polypeptide chains, and the functional protein is made up of six subunits linked by interchain disulfide bridges near the N-terminus (4). Post-translational modifications include complex N-linked glycosylation at Asn187 in the CRD (4).

Two genes encoding SP-A transcripts, SP-A1 and SP-A2, are found in humans (2, 3, 5), and several alleles are known for each gene (6). Transcripts of both genes undergo alternative splicing of 5'-untranslated exons, and the relative expression of different SP-A splice variants differs between individuals (7). Apart from the existence of splice variants, variable cleavage of the signal peptide, which gives rise to an additional cysteine residue in the N-terminal region, contributes to the complexity of the SP-A molecule (4, 8). Pulmonary SP-A may occur as a heterotrimer (4, 9) and the two genes are differentially regulated (10). However, because recombinant SP-A1 can form homotrimers and higher oligomers, these forms may also exist in the lung in vivo (9). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of reduced SP-A indicates a molecular mass of 31–36 kD (11).

SP-A gene knockout mice are more susceptible than wild-type mice to infections with bacterial and viral pathogens, and SP-A deficiency is associated with enhanced inflammation and synthesis of proinflammatory cytokines (12).

SP-A is generally recognized as being expressed in alveolar type-II cells, nonciliated (Clara) cells, and submucosal glands of the respiratory airways (13–15), but extrapulmonary expression has also been found in epithelial cells lining the gastrointestinal tract and mammary glands (16–18), in the maxillary sinus, middle ear, and Eustachian tube (19, 20), in mesentery cells (21), and in the prostate gland and thymus (22).

We have previously shown that lung surfactant protein D (SP-D) is generally present on mucosal surfaces and is not restricted to the respiratory system (23). In the present study, the possible extrapulmonary expression of human SP-A was investigated by reverse transcriptase–polymerase chain reaction (RT-PCR) and immunohistochemistry. Expression of SP-A mRNA was clearly demonstrated in certain nonpulmonary tissues like the prostate gland and colon, but translated protein in the form of SP-A immunoreactivity was not found outside the lung.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Buffers and Reagents
Buffers and reagents used were Tris-buffered saline (TBS): 10 mM Tris-HCl buffer, pH 7.4, containing 140 mM NaCl and 0.02% (wt/vol) NaN3; TBS/Tw: TBS containing 0.05% (vol/vol) Tween 20 (polyoxyethylene sorbitan monolaurate; Merck-Eurolab, Darmstadt, Germany); diethanolamine buffer: 10% (vol/vol) diethanolamine-HCl, pH 8.9, containing 0.5 mM MgCl2 and 0.02% (wt/vol) NaN3; sample buffer: 0.1 M Tris-HCl, pH 8.0, containing 1.5% (wt/vol) SDS, 5% (vol/vol) glycerol and 0.2% (wt/vol) bromphenol blue; substrate buffer: 0.1 M Tris-HCl, pH 9.5, containing 100 mM NaCl and 5 mM MgCl2; alkaline phosphatase-conjugated goat anti-rabbit IgG (whole molecule) antibody (Sigma-Aldrich, St. Louis, MO); alkaline phosphatase–conjugated goat anti-mouse IgG (whole molecule) antibody (Sigma-Aldrich). SP-A was purified from lung washings from patients with alveolar proteinosis, as described (4).

RT-PCR
Human total RNA from various tissues (see Figure 1) was purchased from Clontech (Palo Alto, CA). Total RNA from each tissue was in general a pool from different specimens ranging from 1–84 specimens/tissue. RNA from human colon was prepared from five specimens frozen in liquid nitrogen by means of Tri Reagent (Sigma-Aldrich), according to the manufacturer's instructions.



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  		Figure 1. RT-PCR analysis of SP-A-mRNA expression in human tissues. Numbers in parentheses show the number of specimens used per tissue. SP-A and ß-actin transcripts were amplified by PCR with gene-specific primers. The products were analyzed by agarose-gel electrophoresis. For SP-A transcripts, the gel was blotted onto nylon membrane and hybridized with an SP-A probe as described in MATERIALS AND METHODS.

 
First-strand synthesis was performed with the Thermoscript kit (Invitrogen, Carlsbad, CA) from 1 µg of total RNA from each tissue with oligo-dT priming in a volume of 20 µl at 50°C for 50 min. The reaction mixture was then diluted to 100 µl with water and 5 µl used as a template for PCR in a volume of 30 µl containing 200 µM of each dNTP and 1x PCR buffer with 1.5 mM MgCl2 (Invitrogen), 1 U of Taq polymerase (Invitrogen) and 10 pmol of each primer. For SP-A the forward primer HuSP-A forward (5'-GGA AGC CCT GGT ATC CCC GG-3') and the reverse primer HuSP-A reverse (5'-TAA TGG TAT CAA AGT TGA CTG-3') were used. This primer pair has the equivalent location in human SP-A as that used for rat SP-A mRNA by Rubio and coworkers (16) and spans part of the collagenous region and part of the CRD. The primers do not discriminate between SP-A1 and SP-A2 and give rise to a single product of 372 bp. PCR was performed as follows: 1 min at 94°C, 35 cycles of (94°C for 30 s, 57°C for 30 s, and 72°C for 30 s), followed by 72°C for 7 min.

Reaction products were analyzed by agarose gel electrophoresis followed by alkaline (0.4 M NaOH) upward capillary transfer for 3 h onto a nylon membrane (Hybond N+; Pharmacia Amersham Biotech, Freiburg, Germany). All hybridization and washing procedures were performed as described by Madsen and colleagues (23). A PCR-amplified fragment from the lung was sequenced and used as probe, with the incorporation of [{alpha}-32P]dATP by means of hexamer random priming and an oligo labeling kit (Amersham Pharmacia Biotech). The amount of RNA used in the RT-PCR was normalized with reference to human ß-actin as described by Grønlund and coworkers (24), except that first-strand synthesis was performed with oligo-dT priming.

One microliter of the PCR product from the trachea, thymus, and prostate was used as template for a second round of PCR, performed as described above, to obtain enough material for sequence verification (Figure 2).



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  		Figure 2. Sequence data of the re-amplified fragments from thymus, lung, trachea, and prostate. Nucleotide numbering for SP-A1 and SP-A2 is shown on top. Differences between SP-A1 and SP-A2 are marked in red. Nucleotides that are different in SP-A1 and SP-A2 are marked by arrows.

 
Production of Monoclonal Antibodies against SP-A
CF1 x BALB/c female mice were immunized by intraperitoneal injections with 10 µg purified SP-A adsorbed onto 1 mg aluminum hydroxide gel (Alhydrogel, Superfoss, Denmark) in 0.25 ml 0.1 M sodium phosphate buffer, pH 7.4. The injections were repeated at intervals of 14 d. Mice with a high antibody titer against SP-A, as determined by direct enzyme-linked immunosorbent assay on SP-A–coated microtiter plates, were selected for fusion of spleen cells. Fusions and screening of hybridoma clones were performed as described previously (23). All the selected monoclonal antibodies were of the IgG1 subclass and their specificity was tested by Western blotting.

SDS-PAGE and Western Blotting
The source of SP-A for SDS-PAGE and Western blotting was derived from a fraction of bronchoalveolar lavage (BAL) fluid from patients with alveolar proteinosis that has been separated by Mono Q ion-exchange chromatography and previously used for purification of the SP-D–binding molecule gp-340 (25). For SDS-PAGE, samples contained ~ 500 ng of separated BAL protein and ~ 50 ng of recombinant SP-A1 or SP-A2. The samples were reduced by heating to 100°C for 1 min in sample buffer containing 60 mM dithiothreitol, and were then alkylated by the addition of iodoacetamide to a final concentration of 90 mM. Unreduced samples were treated in the same way, except that dithiothreitol was omitted.

SDS-PAGE and Western blotting for Figure 3 were performed in the PhastSystem (Amersham Pharmacia Biotech). Proteins were separated on 8–25% polyacrylamide gradient gels with the discontinuous buffer system and blotted onto polyvinylidene difluoride membrane (Immobilon P; Millipore, Bedford, MA). SDS-PAGE and Western blotting for Figure 4 were performed with 4–20% polyacrylamide gradient gels using the discontinuous buffer system and blotted onto polyvinylidene difluoride membrane (Immobilon P; Millipore). The membranes were incubated with the selected monoclonal mouse antibodies (anti-human SP-A antibody: HYB 238–4; anti-human SP-D antibody: HYB 245–1; anti-human mannose-binding lectin (MBL) antibody: HYB 131–1) diluted 1:1,000 (stock concentration 1 mg/ml) followed by alkaline phosphatase–coupled goat anti-mouse IgG (1 µg/ml) in TBS/Tw containing a final concentration of 0.5 M NaCl. The membranes were washed and developed with 0.1 mg/ml nitro blue tetrazolium (N-6876; Sigma-Aldrich) and 0.17 mg/ml potassium 5-bromo-4-chloro-3-indolylphosphate (B-6149; Sigma-Aldrich) in substrate buffer until sufficient color development was obtained. Visualization of markers and total amount of protein on the membrane was performed by incubating the membrane in gold-solution (2 mM AuCl2-solution, 0.1% Tween 20, and 10 mM trisodium citrate, pH 3.2) overnight at room temperature. The membrane was afterward washed twice in water.



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  		Figure 3. SDS-PAGE–Western blots of bronchoalveolar lavage (BAL) developed with the monoclonal antibody HYB 238-4 or stained with colloidal gold (A, lanes 3 and 4). (A) Lane 1: reduced BAL proteins; lane 2: unreduced BAL proteins; lane 3: reduced BAL proteins stained with colloidal gold; lane 4: unreduced BAL proteins stained with colloidal gold. (B) All samples were reduced. Lane 1: BAL proteins; lane 2: N-deglycosylated BAL proteins; lane 3: collagenase-treated BAL proteins; lane 4: recombinant SP-A1; lane 5: recombinant SP-A2.

 


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  		Figure 4. SDS-PAGE–Western blots of purified human SP-A, SP-D, and MBL developed with the monoclonal antibodies HYB 238-4 (anti–SP-A); HYB 245 (anti–SP-D), and HYB 131-1 (anti-MBL). Lane 1: purified human SP-A; lane 2: purified human SP-D; lane 3: purified human MBL.

 
Deglycosylation and Collagenase Treatment of SP-A
Fractionated BAL protein (~ 500 ng) was N-deglycosylated by enzymatic digestion with N-glycosidase F for 18 h at 37°C by means of the PGNase F kit (New England Biolabs, Beverly, MA).

Fractionated BAL protein (~ 500 ng) was also incubated for 24 h at 37°C with collagenase (0.25 U per 1–10 µg of protein) from Clostridium histolyticum (C-0773; Sigma-Aldrich) in 25 mM Tris-HCl buffer, pH 7.4, containing 10 mM CaCl2, or in the same buffer containing 10 mM EDTA instead of CaCl2 as a control.

All samples were analyzed by SDS-PAGE in the reduced state followed by Western blotting with HYB 238–4 as described above.

Expression of Recombinant Human SP-A1 and SP-A2
SP-A1 and SP-2 were expressed in Chinese hamster ovary cells and purified as described by Voss and colleagues (9). Briefly, the recombinant proteins were purified from the culture supernatant of stably transfected cells by affinity chromatography on a mannose-agarose column (Sigma-Aldrich). The isolated material was more than 90% pure as estimated by SDS-PAGE of ~ 100-ng samples (9).

Immunohistochemistry
Sections (4 µm thick) were cut from noncommercial paraffin-embedded blocks of normal human tissue fixed in neutral buffered 4% formaldehyde. Sections were mounted on ChemMate Capillary Gap Slides (Dako, Glostrup, Denmark), dried at 60°C, deparaffined, and rehydrated. Antigen retrieval was optimized with heat-induced epitope retrieval using three different buffers: (i) 10 mM sodium citrate, pH 6; (ii) 10 mM Tris containing 0.5 mM ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid, pH 9; and (iii) Target Retrieval Solution (Dako). After optimization antigen retrieval was performed by microwave heating in Target Retrieval Solution (Dako). Three Tissue Tek containers (Miles, Elkhart, IN), each with 24 slides in 250 ml buffer, were placed on the edge of a turntable inside the microwave oven. Slides were heated for 11 min at full power (900W), then for 15 min at 400W, and then left in the buffer for a further 15 min. Any endogenous biotin was then blocked with a biotin-blocking system (Dako). The slides were incubated with the monoclonal mouse anti-human SP-A antibody (HYB 238–4) diluted 1:3,000 (stock concentration 1 mg/ml) or the monoclonal mouse anti-human SP-D antibody HYB 245–1 diluted 1:800 (stock concentration 1 mg/ml) for 25 min at room temperature. Immunostaining was performed with the ChemMate horseradish peroxidase/3,3'-diaminobenzidine tetrahydrochloride (HRP/DAB) detection kit (K5001; Dako) using automated equipment (TechMate 1000; Dako), and was followed by brief nuclear counterstaining in Mayer's hematoxylin. Cover slips were mounted with AquaTex (Merck Eurolab).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of SP-A in Human Tissues Analyzed by RT-PCR
A fragment of 372 bp extending from the collagen region into the CRD of human SP-A was amplified by RT-PCR applied to total RNA from 20 different human tissues (Figure 1). Expression of SP-A mRNA was most pronounced in the lung, but was also clearly seen in the trachea, prostate gland, and pancreas. Low expression was observed in thymus, colon, and salivary gland, whereas no expression was detected in the stomach, small intestine, liver, mammary gland, kidney, uterus, placenta, testis, spleen, skeletal muscle, adrenal gland, brain, and heart. All samples were normalized with respect to ß-actin. The amplified fragments from the lung, trachea, thymus, and prostate were isolated and sequenced. Analysis of the sequence data showed that both SP-A1 and SP-A2 genes were expressed in human lung and thymus, whereas only SP-A2 was expressed in the trachea and prostate gland (Figure 2).

Characterization of the Monoclonal Antibody
The specificity of the monoclonal SP-A antibody used for immunohistochemistry was analyzed by Western blotting of fractionated BAL separated on SDS-PAGE in the reduced and unreduced state (Figure 3). The antibody recognized specific bands of 33 and 60 kD in the reduced state, with a very faint band at 110 kD (Figure 3A, lane 1). Bands were observed in the stacking gel in the unreduced state (Figure 3A, lane 2). Gold staining was used to visualize the amount of protein applied to the gel (Figure 3A, lanes 3 and 4). The antibody recognized SP-A both after N-deglycosylation (Figure 3B, lane 2) or after treatment with collagenase (Figure 3B, lane 3). In addition, the antibody recognized both recombinant SP-A1 and SP-A2 in the reduced state (Figure 3B, lanes 4 and 5). Putative cross-reactivity of the monoclonal anti–SP-A antibody with human SP-D and MBL was tested by SDS-PAGE and Western blotting analysis of purified human SP-A, SP-D, and MBL (Figure 4). The antibody recognized only purified human SP-A and did not react with purified human SP-D or MBL either in the nonreduced or reduced state. The anti-human SP-D antibody (HYB 245-1) recognized primarily a band with the molecular weight of ~ 43 kD in the reduced state and a band of ~ 160 kD in the nonreduced state. The anti-human MBL antibody reacted primarily with a band with the molecular weight of ~ 32 kD in the reduced state and with a ladder of different bands in the nonreduced state (Figure 4). The anti-MBL antibody revealed trace amounts of MBL in the SP-D preparation.

Immunohistochemistry
Strong immunoreactivity was observed in alveolar type II cells, in nonciliated bronchial cells (Clara cells), and in a subset of alveolar macrophages in the lung (Figures 5A–5D). The alveolar macrophages showed a distinct granular staining, some granules being located within the cytoplasm, others close to the plasma membrane (Figures 5D–5E). A few of the submucosal glands stained positive for SP-A (Figure 5F). No staining was observed in the lung or submucosal glands when HYB 238-4 was replaced with an antibody directed against ovalbumine (HYB 99-1) of the same subclass as HYB 238-4 (Figure 5G).



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  		Figure 5. Immunohistochemical localization of SP-A in normal lung tissue using the monoclonal antibody HYB 238-4 and counterstained with Mayer's hematoxylin. Original magnification: A, lung section x50; B, type II cells x400; C, Clara cells x400; D, alveolar macrophages in the lung; E, alveolar macrophages in the bronchia; F, submucosal glands; G, control staining with HYB 99-1 x400.

 
A panel of different extrapulmonary tissues including pancreas, small and large intestines, thymus, prostate gland, and cells lining the peritoneal cavity were stained for SP-A. No SP-A immunoreactivity was seen in extra pulmonary tissues (Figures 6A–6F) even when the concentration of primary antibody used was increased 10-fold (not shown). In contrast, mesentery cells (Figure 6G) and an array of different epithelial cells showed strong SP-D immunoreactivity (23). All investigated tissues were negative when using HYB 99-1 or when no primary antibody was used (not shown).



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  		Figure 6. Normal human tissues processed for SP-A (A–F) or SP-D (G) immunohistochemistry with the monoclonal antibodies HYB 238-4 for SP-A and HYB 245-1 for SP-D. The tissues were counterstained with Mayer's hematoxylin. Original magnification: A, pancreas x400; B, thymus x200; C, small intestine x100; D, colon x100; E, prostate x100; F and G, peritoneal wall with mesothelial cells x200.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present article describes the expression of SP-A in the human lung and in nineteen extrapulmonary tissues, as determined by RT-PCR and immunohistochemistry with a monoclonal antibody. SP-A was clearly demonstrated in pulmonary epithelial cells, and although SP-A mRNA was demonstrated in extrapulmonary tissues, no protein expression was found in the corresponding tissues by immunohistochemistry.

It has long been established that alveolar type II cells and nonciliated bronchial cells (Clara cells) in the lung are the major site of synthesis of SP-A (13). Transcripts of both SP-A1 and SP-A2 are expressed in lungs of humans and the genes are differentially regulated (10, 26). The ratio of expression of the two genes varies between human individuals and depends on the specific SP-A alleles present (4, 5, 9, 27, 28).

Northern blotting, RT-PCR, and immunohistochemistry have previously been used to detect SP-A in selected extrapulmonary tissues from different species. Both SP-A1 and SP-A2 transcripts have been detected in the human small and large intestine (17), whereas the SP-A2 gene was predominantly expressed in the trachea (14, 15). SP-A mRNA has also been detected in the thymus, prostate gland, and brain (22, 29). Only one SP-A gene is known in the rat and has been shown to be transcribed in mesentery cells (21) and in the small and large intestine (16) as well as in the lung. In the present study, SP-A mRNA was found to be strongly expressed in the human lung and trachea, as well as in extrapulmonary organs such as the prostate and pancreas. Weak expression was found in thymus, salivary gland, and colon. Sequencing of RT-PCR products from the lung and thymus showed expression of both SP-A1 and SP-A2 mRNA, whereas SP-A2 mRNA was clearly predominant in the prostate gland and trachea. This is in accordance with recent reports showing that SP-A2 expression predominates in the trachea with only a very low level of SP-A1 expression (14, 15).

The selected monoclonal antibody HYB 238-4 raised against SP-A recognized bands of 33 kD and 60 kD in reduced BAL proteins. This is in accordance with previous findings for SP-A and is presumed to correspond to forms of one and two polypeptide chains, respectively (4, 11). Unreduced BAL proteins gave HYB 238–4-reactive bands in the stacking gel. When analyzed by size chromatography, SP-A from BAL fluid from patients with pulmonary alveolar proteinosis emerges in two fractions, one near the void volume of the column (30). This fraction contains multimerized large aggregates of SP-A and is probably responsible for the immunoreactivity observed in the stacking gel when BAL proteins are electrophoresed in the unreduced state. Furthermore, the antibody did not cross-react with purified human SP-D or SP-A.

It has previously been reported that both monoclonal and polyclonal antibodies raised against purified human SP-A cross-reacted with carbohydrate epitopes present on blood-group antigen A–positive erythrocytes (31). Furthermore, both monoclonal and polyclonal antibodies raised against blood-group antigen A cross-reacted with SP-A (31). The reactivity of SP-A with these antibodies disappeared after N-glycosidase treatment of the SP-A (31). HYB 238-4 reacted equally well with SP-A before and after treatment of SP-A with N-glycosidase F, indicating that this antibody reacts with a peptide epitope and not with the asparagine-linked carbohydrate moiety of SP-A. The antibody also reacted with collagenase-treated SP-A, showing that the epitope is located in the neck-CRD region of the protein.

SP-A1 and SP-A2 are 96% identical at the protein level and, apart from an Asp to Glu substitution at position 247, all differences between SP-A1 and SP-A2 are found in the collagenous region. As HYB 238–4 reacts with collagenase-treated SP-A, it was therefore likely to react with both SP-A1 and SP-A2. This was confirmed by Western blotting of recombinant SP-A1 and SP-A2, which showed that the monoclonal antibody did not differentiate between the two gene products. The recombinant SP-A molecules differed slightly in mobility from native SP-A because of their different glycosylation in the Chinese hamster ovary cells (9). The monoclonal antibody HYB 238-4 therefore recognizes a peptide epitope located in the neck-CRD region that is shared by SP-A1 and SP-A2.

Immunohistochemistry with this antibody confirmed the presence of SP-A immunoreactivity in alveolar type II cells and bronchial nonciliated epithelial cells (Clara cells). In most of the alveolar macrophages the staining appeared to be present in the phagolysosome compartment, but some macrophages showed a distinct granular staining of the cell membrane. The presence of SP-A in alveolar macrophages does not appear to be due to an expression of SP-A but rather to direct uptake of SP-A or uptake associated with phagocytosis of microbial material (32, 33).

No SP-A immunoreactivity was detected in any extrapulmonary tissues, neither in those showing high expression of SP-A mRNA, such as the prostate gland and thymus, nor in those showing low mRNA expression, such as the large intestine and salivary gland. This contrasts with previous reports in which extrapulmonary SP-A immunoreactivity was reported in epithelial cells of small and large human and rat intestines by immunohistochemistry with polyclonal antibodies (16, 17). SP-A immunoreactivity with polyclonal antibodies has also been detected in the middle ear of rabbits and pigs (19, 20).

The expression of SP-A mRNA in the small and large intestine is very low in comparison with that in the lung (17), and the possibility cannot be excluded that the level of protein expression in the intestine may be too low to be detected by our immunohistochemical technique. However, as blood-group antigens are known to be present throughout the gastrointestinal tract, it is possible that the strong signal seen by Lin and coworkers (17) in the small and large intestine is due to cross-reaction of the polyclonal antibody with blood group antigens.

There have been several reports of SP-A mRNA expression in the prostate (17, 22, 29), and SP-A immunoreactivity has been detected in seminal fluid by SDS-PAGE and Western blotting (34). We found high levels of SP-A2 mRNA levels in the prostate. SDS-PAGE and Western blotting of three samples of seminal fluid from different donors did not give bands corresponding to SP-A (not shown). No SP-A immunoreactivity was seen in the prostate from six different individuals by SP-A immunohistochemistry. This is surprising because the antibody has been shown to react with recombinant SP-A2. Apart from HYB 238-4 we have used three additionally monoclonal antibodies raised against SP-A (HYB 238-1 to HYB 238-3) for immunohistochemical localization of SP-A. None of the antibodies showed staining in any extrapulmonary tissues examined, including the prostatic gland (not shown).

Apart from difference in sensitivity between the RT-PCR technique and the immunohistochemistry, the discrepancy between the two observations is difficult to explain.

Recently, Hawgood and coworkers found extrapulmonary expression of both SP-A and SP-D mRNA in the mouse. Whereas mouse SP-D protein was localized in an array of extrapulmonary tissues by immunohistochemistry, SP-A protein seemed to be restricted to the lung using this method (35). These results parallel our obtained data on the localization in humans of SP-A in this article and previously for SP-D (23).

We conclude that although SP-A mRNA can be demonstrated in extrapulmonary tissues, the expression of SP-A at the protein level seems to be restricted to the respiratory system. This contrasts with the expression of SP-D, which seems to have a wide distribution on mucosal surfaces.


   Acknowledgments
 
This work was supported by the Benzon Foundation, the Danish Medical Research Council, the Novo-Nordic Foundation, Fonden til Lægevidenskabens Fremme, and the EU contract number QLK2 CT 2000 0035.

Received in original form November 26, 2002

Received in final form May 20, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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