Introduction

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Lung surfactant has been known to be synthesized and secreted by alveolar type II cells and bronchiolar Clara cells, and has been shown to be composed of phospholipids, cholesterol, and lung-specific proteins (1). Four distinct surfactant proteins have been described to date: surfactant proteins A (SP-A), B (SP-B), C (SP-C), and D (SP-D) (1). Among these proteins, the 36-kD glycoprotein SP-A is the most abundant in lung surfactant and, moreover, has been shown to play roles in the defense mechanism through the activity of alveolar macrophages in addition to surfactant function and metabolism (1, 2). The genomic organization of human SP-A was originally determined by White and colleagues (3), and subsequently the human genome has been found to contain two SP-A genes, SP-A1 and SP-A2, that are expressed in alveolar type II cells (4). Meanwhile, lung surfactant materials have been observed in the airway as well as in the alveolar region by morphologic and biochemical analysis of phospholipids in sputum and airway mucus (5, 6). In addition to the speculation that airway surfactant and/or surfactant proteins are derived from the alveolar regions (5), some reports have suggested the local production of surfactant materials by airway walls (7). Both SP-A and SP-B messenger RNA (mRNA) were described in human bronchiolar Clara cells (8, 9), and SP-A mRNA and protein were found in large airways and glands of human fetuses (10, 11). To date, to our best knowledge, there have been no reports showing SP-A localization in submucosal glands of adult human airways. In the present study we investigated the expression of surfactant proteins by the cells in central airways (trachea and bronchi) using both monoclonal antibody and reverse transcription-polymerase chain reaction (RT-PCR) analyses, and found that human airway submucosal glands express the SP-A2 gene and produce SP-A.

	Materials and Methods

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Human trachea and bronchi obtained from autopsied lungs within 4 h of death and from operated lungs of 19 patients (14 men and 5 women, 56 ± 14 yr, mean ± SD) were used for the present study. The diagnoses of the 14 patients who received autopsy were ventricular fibrillation with myocardial infarction (male, 58 yr), congestive heart failure with dilated cardiac myocarditis (male, 45 yr), rupture of abdominal aortic aneurysm (male, 55 yr), polyarteritis nodsa (male, 60 yr), intestinal bleeding with liver cirrhosis (male, 56 yr), acute myelotic leukemia (male, 45 yr), chronic lymphatic leukemia (male, 69 yr), kidney tumor (male, 40 yr), urinary bladder tumor (male, 53 yr), seminoma (male, 38 yr), mediastinal tumor (male, 50 yr), laryngeal carcinoma (female, 62 yr), and lung carcinoma (2 females, 40 and 87 yr). Furthermore, lung tissue samples were obtained from five patients who underwent operations for lung cancer (2 females and 3 males, 65 ± 7 yr). The present study was approved by the Ethics Committee on Human Investigations of Tohoku University School of Medicine (Sendai, Japan).

Isolation of Single Submucosal Glands

Immediately after their removal, the trachea and bronchi were put in cold (4°C) phosphate-buffered saline (PBS) with penicillin (100 IU/ml) and streptomycin (100 µg/ml) and transferred to our laboratory. The external surface of the trachea was cleaned of fat and connective tissues, cut into rings 3 to 4 cm long, and fixed by pins in PBS, posterior (membranous) wall side up. Light from a flexible fiberbronchoscope placed inside the tracheal ring was used to transilluminate the membranous portion of the trachea. The outermost layer and thick smooth muscle layer were carefully removed. The glands could then be easily distinguished from the surrounding connective tissue under a stereoscopic microscope (magnification: ×60-80). Fresh, unstained submucosal gland was isolated using two pairs of tweezers and microscissors. To avoid tissue damage during isolation, care was taken to isolate the gland by picking up some of the connective tissue surrounding the gland (12).

Immunohistochemical Experiment

Bronchial explants containing cartilage (5 × 5 mm) were fixed with formalin, embedded in paraffin (6 µm thick), and immunohistochemically examined using PE-10, which was a generous gift from Dr. Hosoda at Teijin Bio-Medicine (Tokyo, Japan). PE-10 is a monoclonal antibody against SP-A and its specificity has been confirmed in immunohistochemical studies of lung (10, 13, 14). As controls, alveolar tissues were used for the immunohistochemical experiment. Deparaffinized sections were treated with 5% H₂O₂ in methanol for 30 min at room temperature for the inhibition of endogenous peroxidase, and were washed with running tap water. The sections were incubated with PE 10 at a dilution of 1:100 or 1:500 overnight (16-20 h) and washed with 0.1 M Tris-HCl buffer (pH 7.6). The sections were then incubated with biotinylated horse antimouse immunoglobulin G (IgG) for 30 min at room temperature and washed with Tris-HCl buffer. The sections were further incubated with avidin D-biotinylated horseradish peroxidase solution (Vector Lab, Burlingame, CA) for 30 min at room temperature and washed with Tris-HCl buffer. Peroxidase activity was observed by incubating the sections in diaminobenzidine/imidazole/H₂O₂ medium. After washing with Tris-HCl buffer and distilled water, the sections were counterstained with hematoxylin. Frozen sections (10 µm thick) were also used for the immunohistologic experiment. After the fixation with 10% neutral-buffered formalin (pH 7.4) for 60 min, the sections were processed in the same manner as the formalin-fixed and paraffin-embedded sections.

Measurement of SP-A Concentration

Isolated submucosal glands were put in a 50-ml conical tube filled with Medium 199 (Bio-Whittaker, Walkersville, MD) supplemented with penicillin (100 IU/ml) and streptomycin (100 µg/ml) and washed 10 times by gentle shaking. Submucosal glands were then collected and put in plastic dishes (35 mm in diameter) in a 40% O₂-5% CO₂ humidified incubator at 37°C for 12 to 24 h for the equilibration. After equilibration and three washings with medium 199, about five pieces of isolated submucosal glands were seeded in plastic dishes (22 mm in diameter) with 2 ml media. After 4 h incubation, the media were harvested and stored at 80°C until use. The SP-A concentrations of the media were measured by means of a previously reported method (15). The media (50 µl) were dissolved in 200 µl of buffer solution I (0.6% sodium dodecyl sulfate [SDS]; 2% Triton X-100; 0.01 M PBS, pH 7.2) and 200 µl of peroxidase-labeled PE-10 dissolved in 10 ml of buffer solution II (0.25% skim milk; 0.01 M PBS, pH 7.2) and were mixed thoroughly. SP-A from the bronchoalveolar fluid of alveolar proteinosis patients (250, 125, 62.5, 31.25 ng/ml) was used as the standard material. Plastic beads coated with PC-6 were added to the mixture in the test tube and incubated for 90 min at 37°C. PC-6 is a monoclonal antibody against SP-A and its specificity has been confirmed in an enzyme-linked immunosorbent assay (ELISA) study, as has that of PE-10 (13). The mixture was then removed from the test tube by suction and the beads were washed three times. Then, 400 µl of substrate solution (5 mmol/liter of hydrogen peroxidase and 0.1% phosphate-citrate buffer, pH 4.0) and developer (0.06% tetramethylbenzidine HCl, pH 2.0) were added to the test tube and the tube was incubated for 30 min at 37°C. The reaction was stopped by the addition of 1 ml of reaction stopper (1 N H₂SO₄). The absorbance of each tube was measured at 450 nm by a spectrophotometer. A calibration curve was made by the absorbance of several concentrations of SP-A standard materials.

RT-PCR Analysis of SP-A, SP-B, SP-C, and SP-D mRNA

Isolated submucosal glands (12), cultured tracheal gland cells (16), superficial epithelial tissue peeled from trachea using microforceps, and cultured airway epithelial cells (17) were all used for RT-PCR analysis. As controls, alveolar tissues were obtained by cutting a peripheral portion of the lobe under the pleura with scissors. Total RNA was extracted from each sample within 1 h of the isolation of submucosal glands in ISOGEN (Nippon Gene Co., Ltd., Tokyo, Japan), following the manufacturer's protocol. One microgram of total RNA extracted from the tissue was converted to first-strand cDNA with oligo (dT)_12-18 primer and Moloney murine leukemia virus reverse transcriptase (GIBCO BRL Life Technologies, Inc., Rockville, MD), according to the supplier's instructions. The oligonucleotide primers used in PCR were:

1. For SP-A (primers common to SP-A1 and SP-A2), 5'-GAAGGACGTTTGTGTTGGAA-3' and 5'-TGGATTCCTTGGGACAGCAA-3', and the simultaneously amplified 439-base pair (bp) SP-A1 and SP-A2 cDNA segments;
2. For SP-A1 (primers specific to SP-A1), 5'-CAGGCACGGGAGAGATGGTC-3' and 5'-ATGGAGGCCGACAAGGAGAGCGT-3', and amplified 670-bp SP-A1 cDNA segment;
3. For SP-A2 (primers specific to SP-A2), 5'-CAGGGACGGGAGAGATGGTG-3' and 5'-GGAAACTGAAGGCCAGACAGGAT-3', and amplified 670-bp SP-A2 cDNA segment;
4. For SP-B, 5'-TCATCGACTACTTCCAGAACCAGA-3' and 5'-GCAGATGCCGCCCGCCACCAGAGG-3', and the amplified 371-bp cDNA;
5. For SP-C, 5'-AGCAAAGAGGTCCTGATGGA-3' and 5'-CTAGTGAGAGCCTCAAGACT-3', and the amplified 405-bp cDNA;
6. For SP-D, 5'-TTTGCCAGGAGCTGCAGGGCAA-3' and 5'-AAGTGCTCGCAGACCACAAGACG-3', and the amplified 927-bp cDNA; and
7. For -actin, 5'-ATGGGTCAGAAGGATTCCTAT-3' and 5'-TGACTTAGTTGCGTTACAC-3', and the amplified 1017-bp cDNA.

A total of 0.01 of the cDNA synthesis reaction was combined at a final volume of 20 µl for PCR amplification by using each set of primers and 0.48 units of Tth DNA polymerase (Toyobo, Osaka, Japan) for SP-A, SP-B, SP-C, SP-D, and -actin cDNA segment amplification. The tempertures and cycles for amplification were:

1. SP-A (primers common to SP-A1 and SP-A2): 35 cycles, denaturation at 92°C (1 min), annealing at 54°C (1 min), and elongation at 72°C (3 min);
2. SP-A1 or SP-A2 (primers specific for SP-A1 or SP-A2): 30 or 35 cycles, denaturation at 92°C (1 min), annealing at 60°C (1 min), and elongation at 72°C (3 min);
3. SP-B: 38 cycles, denaturation at 94°C (50 s), annealing at 66°C (1 min), and elongation at 72°C (1.5 min);
4. SP-C: 30 cycles, denaturation at 92°C (1 min), annealing at 54°C (1 min), and elongation at 72°C (3 min);
5. SP-D: 35 cycles, denaturation at 94°C (50 s), annealing at 68°C (1 min), and elongation at 72°C (1.5 min); and
6. -actin: 35 cycles, denaturation at 92°C (1 min), annealing at 54°C (1 min), and elongation at 72°C (3 min).

A total of 6 µl of each PCR product was electrophoresed in a 1% agarose gel containing ethidium bromide.

Restriction Fragment Length Polymorphism and Southern Analysis for Detecting Gene Expression of SP-A1 and SP-A2

For PCR amplification using the SP-A primers (primers common to SP-A1 and SP-A2), 0.5% of the first-strand cDNA synthesis reaction of the isolated submucosal glands or the control alveolar tissue was combined at a final volume of 30 µl with 0.72 units of Tth DNA polymerase for SP-A cDNA segment amplification. PCR was performed for 40 cycles; each cycle consisted of denaturation at 92°C (1 min), annealing at 54°C (1 min), and elongation at 72°C (3 min). The PCR product was put on a Millipore filter (0.05 µm in pore size and 25 mm in diameter; Millipore Co., Bedford, MA), which was floated in sterile water for 1 h to dialyze and remove the PCR buffer (18). After the dialysis, the PCR product was harvested from the filter using a pipette, and 16 µl of the PCR product was digested by 2 µl of the restriction enzyme Apa I and 2 µl of 10× buffer (Toyobo) at 37°C for 8 h. Then, 8 µl of the treated PCR product underwent electrophoresis on 3% agarose gel in Tris-boric acid-ethylenediaminetetraacetic acid (EDTA)- 2Na buffer and was transferred to nylon membranes (Hybond N; Amersham Corp., Arlington Heights, IL).

For PCR amplification by using primers specific to SP-A1 or SP-A2, 1% of the first-strand cDNA synthesis reaction was combined at a final volume of 20 µl with 0.48 units of Tth DNA polymerase for SP-A cDNA segment amplification. PCR was performed for 30 or 35 cycles; each cycle consisted of denaturation at 92°C (1 min), annealing at 60°C (1 min), and elongation at 72°C (3 min). A total of 6 µl of the treated PCR products using primers specific to SP-A1 or SP-A2 underwent electrophoresis on the 3% agarose gel and were transferred to the different nylon membranes because of the same base pairs. All other maneuvers were the same as those using the SP-A primers.

Hybridization was performed overnight in a solution with a final concentration of 5× sodium sulfate phosphate EDTA buffer (SSPE), 5× Denhardt's solution, 0.5% SDS, 20 µg/ml denatured salmon sperm DNA, and 5 × 10⁵ cpm/ ml of the [-³²P]adenosine triphosphate-labeled SP-A1 oligonucleotide probe or the SP-A2 oligonucleotide probe. Southern blot analysis was performed by competition hybridization with allele-specific oligonucleotide probes; the ³²P-labeled SP-A1 probe (5'-CACCTGGAGAAATGCCAT-3') with a 10-fold amount of the unlabeled SP-A2 probe or the ³²P-labeled SP-A2 probe (5'-CGCCTGGAGAAACACCAT-3') with a 10-fold amount of the unlabeled SP-A1 probe were simultaneously added to the buffer for hybridization to increase the specificity in the hybridization of SP-A1 or SP-A2. SP-A1- and SP-A2-specific probes, designed according to the sequences previously reported (12), were labeled with T4 polynucleotide kinase (Toyobo) according to the manufacturer's recommendation. After hybridization, the nylon membranes were washed once in 2× sodium citrate-sodium chloride (SSC)/0.1% SDS at room temperature for 5 min; three times in 1× SSC/0.1% SDS at room temperature for 10 min; and once in 1× SSC/ 0.1% SDS at 53°C for 15 min. Autoradiography was performed by exposing X-ray film (Fuji Photo Film Co., Tokyo, Japan) to the washed membranes.

Northern Analysis for Measuring SP-A mRNA Level

Ten micrograms of total RNA extracted from alveolar tissue and 10 µg of total RNA from submucosal glands were electrophoresed on a 1% agarose gel containing formaldehyde and transferred to nylon membranes (Hybond N; Amersham). The 439-bp DNA fragment (a mixture of SP-A1 and SP-A2 cDNA fragment) amplified by RT-PCR from lung tissue as described previously was used as the probe. The amplified cDNA fragment was purified by using QIA quick gel extraction kit (QIAGEN, Inc., Chatsworth, CA) according to the manufacturer's protocol. The cDNA fragments were labeled with [-³²P]dCTP using the Multiprime DNA labeling system (Amersham). Hybridization was carried out overnight in a solution with a final concentration of 5× SSPE, 5× Denhardt's solution, 0.5% SDS, 20 µg/ml denatured salmon sperm DNA, and 5 × 10⁵ cpm/ml of mixture of SP-A1 and SP-A2 cDNA probe. After hybridization, membranes were washed once in 2× SSC/0.1% SDS at room temperature for 1 min, once in 2× SSC/0.1% SDS at room temperature for 10 min, twice in 1× SSC/0.1% SDS at room temperature for 10 min, and once in 1× SSC/0.1% SDS at 53°C for 15 min. An imaging plate for a bio-image analyzer (BAS 2000; Fuji Photo Film Co.) was exposed to the membrane. The scanned image produced by the imaging plate was analyzed by the bio- image analyzer. The mRNA levels were calculated on the basis of the radioactivity of the hybridization signals.

Sequencing of SP-A PCR Product

The amplified 439-bp SP-A PCR product from alveolar tissue (a mixture of SP-A1 and SP-A2 cDNA) was purified by using the QIA quick gel extraction kit (QIAGEN, Inc.) according to the manufacturer's protocol. Sequencing was performed by a dye terminator cycle sequencing method using an ABI PRIZM 310 Genetic Analyzer (Perkin-Elmer, Foster City, CA).

	Results

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The immunohistochemical experiment using PE-10 revealed the localization of SP-A over serous cells of airway submucosal glands in both paraffin and frozen sections, as shown in Figure 1A. Mucous cells of airway submucosal glands or airway superficial epithelial cells were not stained with PE-10 (Figures 1A and 1B). Although the degree of the immunoperoxidase staining over serous cells was different among the samples from the five patients, differences between operated (n = 3) and autopsied (n = 2) lungs were not clear. Control sections incubated with a nonspecific mouse IgG did not show any significant immunoperoxidase staining. Alveolar type II cells in the control alveolar tissue were more strongly stained than the serous cells in glands (Figure 1C). Furthermore, although the lumen of almost all bronchi was free from cells or substances, substances positive for PE-10 attached to the detached cells and exudates were observed in the lumen of a few airways from the autopsied lungs (Figure 1B). These findings indicate that SP-A is localized in serous cells of airway submucosal glands and secreted into the lumen of the airways.

View larger version (170K): [in this window] [in a new window]   View larger version (160K): [in this window] [in a new window]   View larger version (141K): [in this window] [in a new window]   Figure 1.   Light micrographs of paraffin section of human airway and alveolar tissues obtained from autopsied lungs. (A) A positive immunoperoxidase stain was observed over serous cells but not over mucous cells of submucosal gland. A monoclonal antibody against human lung SP-A PE-10 was used at a dilution of 1:500. Bar = 100 µm. (B) Superficial epithelium that was not positively stained. Arrow shows positively stained substances in the bronchial lumen. PE-10 was used at a dilution of 1:500. Bar = 100 µm. (C) Alveolar type II cells in the control alveolar tissue that were more strongly stained than serous cells of airway submucosal glands. PE-10 was at a dilution of 1:500. Bar = 100 µm.

A significant concentration of SP-A (1.2 ± 0.08 ng/ml/h, SEM, n = 40) was detected in the culture medium of isolated submucosal glands by ELISA, indicating that submucosal glands of airways can secrete SP-A.

The SP-A cDNA segment was amplified from isolated submucosal glands (n = 8), whereas cultured gland cells did not show a clear band of SP-A cDNA (n = 5), as shown in Figure 2. None of the SP-B, SP-C, or SP-D cDNA segments were amplified from isolated submucosal gland (n = 8) or cultured gland (n = 5) cells, although all of these cDNAs were amplified from the control alveolar tissue (n = 6). None of the SP-A, SP-B, SP-C, or SP-D cDNA segments were amplified from airway superficial epithelial cells (n = 5) or cultured airway epithelial cells (n = 5) (Figure 3). These findings indicate that human airway submucosal glands transcribe the SP-A gene alone and not the SP-B, SP-C, or SP-D gene.

View larger version (61K): [in this window] [in a new window]   Figure 2.   RT-PCR analysis of SP-A gene. Isolated submucosal glands showed a clear band of SP-A cDNA (lane 2), whereas human cultured gland cells did not (lane 3). Furthermore, the cDNA segment was not amplified from superficial epithelial cells from autopsied tissue (lane 4) or cultured epithelial cells (lane 5). Lane 1 and M represent the control alveolar tissue and DNA marker, respectively.

View larger version (36K): [in this window] [in a new window]   Figure 3.   RT-PCR analysis of SP-A, SP-B, SP-C, and SP-D genes in the control alveolar tissue (A), isolated airway submucosal gland (G), and airway superficial epithelium (E) obtained from operated and autopsied lungs. SP-A cDNA segments with an expected size of 439 bp were amplified by PCR for 35 cycles from isolated submucosal glands as the control alveolar tissue. In contrast, airway epithelial cells failed to show the SP-A gene expression. SP-B (371 bp), SP-C (405 bp), and SP-D (927 bp) cDNA segments were all amplified from the control alveolar tissue but not from isolated submucosal glands or airway superficial epithelium. M represents DNA marker.

SP-A1 and SP-A2 cDNA segments, which were simultaneously amplified by the common oligonucleotide PCR primers, were digested by Apa I and electrophoresed. Electrophoresis of the digested SP-A cDNA segment amplified from alveolar tissue revealed two bands, 439 and about 220 bp (Figure 4). According to the previously described SP-A1 and SP-A2 cDNA sequence (4), however, the amplified 439-bp SP-A1 cDNA segment was expected to be digested to 217, 120, and 102 bp by Apa I, whereas the SP-A2 cDNA segment was not expected to be digested, as shown in Figure 5. Contrary to our expectation, no fragments of the 102- (or 120-)bp SP-A1 cDNA segments were identified. These findings suggest that the SP-A1 gene does not contain the two cleavage sites by Apa I but only one site in this area, and that the band of about 220 bp revealed by agarose gel electrophoresis contains two DNA fragments of 217 and 222 bp. In contrast to the control alveolar tissue, the amplified SP-A cDNA segment of the isolated submucosal gland was not digested by Apa I, as shown in Figure 4.

View larger version (53K): [in this window] [in a new window]   Figure 4.   SP-A cDNA segments from isolated submucosal gland (gland) and the control alveolar tissue (alveolar tissue) that were amplified using SP-A primers (common to SP-A1 and AP-A2) for 40 cycles, digested by Apa I, and electrophoresed. A and P represent PCR products treated with restriction enzyme Apa I and PCR products without the treatment, respectively. n = 1-3 experiments. Amplified and digested SP-A cDNA segment of the control alveolar tissue revealed two bands of 439 and about 220 bp. On the other hand, the amplified SP-A cDNA segment of isolated submucosal gland was not digested by Apa I, showing only one DNA band of 439 bp.

View larger version (14K): [in this window] [in a new window]   Figure 5.   According to the SP-A1 and SP-A2 sequences previously described (4), common PCR primers were designed in exon II and exon V to amplify both SP-A1 and SP-A2 cDNA fragments simultaneously. The figure shows expected cleavage sites by the restriction enzyme Apa I located in the amplified SP-A cDNA segment. The SP-A1 cDNA segment is expected to be digested into three fragments of 102, 120, and 217 bp by Apa I, whereas SP-A2 is not expected to have an Apa I site in this area. The specific oligonucleotide probes of SP-A1 and SP-A2 for Southern blot are also shown.

The electrophoresed and amplified cDNA segment was transferred to the nylon membrane and then hybridized with ³²P-labeled SP-A1- or SP-A2-specific oligonucleotide probes. By the SP-A1 probe, a digested SP-A1 cDNA segment of 222 bp and an undigested SP-A1 cDNA fragment of 439 bp were detected in the control alveolar tissue but no band was detected in the isolated submucosal glands (Figure 6). The DNA band of 439 bp was expected to be an undigested SP-A1 cDNA fragment because of the incomplete digestion by Apa I. Some SP-A1 and SP-A2 cDNA fragments might have formed a heteroduplex cDNA in the last PCR cycle that had not been completely digested. The SP-A1 probe did not detect a DNA band of 120 bp, confirming the speculation that SP-A1 lacks the expected first cleavage site by Apa I, as shown in Figure 5. Using the SP-A2 specific probe, only the 439-bp SP-A2 cDNA fragment was detected in both alveolar tissue and isolated submucosal glands (Figure 6). The Apa I-digestion experiment was performed using samples from eight patients (six autopsied and two operated lungs), and all samples showed similar findings as described previously, without significant variation among patients.

View larger version (99K): [in this window] [in a new window]   Figure 6.   Southern blot of amplified cDNA segment using SP-A primers (common to SP-A1 and SP-A2). Southern blot was performed using 32P-labeled SP-A1- and SP-A2-specific oligonucleotide probes. Using the SP-A1 probe, 439 bp in the alveolar tissue and 222 bp of the cDNA segment were detected when digested by Apa I, whereas the amplified cDNA segment from isolated submucosal gland was not detected (upper panel). Using the SP-A2 probe, a 439-bp cDNA segment from both the alveolar tissue and the isolated submucosal gland was detected (lower panel). Symbols are the same as in Figure 4.

Next, an alternative experiment was performed to demonstrate the presence of SP-A1 or SP-A2 transcript. The SP-A1 or SP-A2 cDNA segment was independently amplified by PCR primers specific for SP-A1 or SP-A2, and then hybridized with ³²P-labeled SP-A1- or SP-A2-specific oligonucleotide probes. As shown in Figures 7 and 8, in submucosal glands (n = 3) the SP-A2 cDNA segment alone was amplified, whereas both SP-A1 and SP-A2 cDNA segments were amplified in control alveolar tissue (n = 3). Furthermore, neither the SP-A1 nor SP-A2 cDNA segment was amplified in isolated superficial epithelium (n = 3) (Figures 7 and 8). Taken with the results of the Apa I-digestion experiment, these findings confirmed that airway submucosal glands express the SP-A2 gene alone, in contrast to the alveolar type II cells which express both SP-A1 and SP-A2 genes.

View larger version (57K): [in this window] [in a new window]   Figure 7.   SP-A cDNA segments from the control alveolar tissue (alveolar tissue), isolated submucosal gland (gland), and superficial epithelium (epithelium), amplified using primers specific to SP-A1 (A1) or SP-A2 (A2) for 30 (alveolar tissue) or 35 (isolated glands and epithelium) cycles and electrophoresed. Using primers specific to SP-A1, a band showing 670 bp was observed in alveolar tissue but not in isolated glands. Using primers specific to SP-A2, a band showing 670 bp was observed in both alveolar tissue and isolated glands. Superficial epithelium did not show any bands using primers specific to SP-A1 or SP-A2. M represents DNA marker.

View larger version (33K): [in this window] [in a new window]   Figure 8.   Southern blot of cDNA segment amplified using primers specific to SP-A1 or SP-A2. Using the SP-A1 probe (SP-A1), a 670-bp cDNA was detected in the alveolar tissue but not in the isolated glands. Using the SP-A2 probe (SP-A2), a 670-bp cDNA segment from both the alveolar tissue and the isolated submucosal gland was detected. Symbols are the same as in Figure 7.

To examine the sequence of the area around the expected Apa I site, we amplified the 439-bp SP-A cDNA from the alveolar tissue (a mixture of SP-A1 and SP-A2 cDNA fragments) and performed direct sequencing. The result showed a single sequence, which was the same as that reported for the SP-A2 gene (4). It was assumed from these findings that SP-A1 (¹²⁰⁵CTGGGCCC¹²¹²) has the same sequence as SP-A2 (¹¹³³CCTGGCCC¹¹⁴⁰) in this area, which was not digested with Apa I.

We performed Northern blot analysis to compare the SP-A mRNA level of isolated submucosal gland with that of alveolar tissue. Amplified cDNA fragments of SP-A from alveolar tissue by PCR (a mixture of SP-A1 and SP-A2) were used as the SP-A mRNA probe. In the electrophoresis of total RNA with ethidium bromide stain, the bands of 28 S and 18 S ribosomal RNA (rRNA) of alveolar tissue were more abundant than those of isolated glands, despite the fact that the amount of total RNA was same (10 µg). Quantification of the SP-A mRNA level of the submucosal gland by calculating the radioactivity revealed one-eighteenth of the amount in alveolar tissue.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the present study using a monoclonal antibody, we found a significantly positive signal for SP-A on the serous cells of submucosal glands from human adult airways, although it was weaker than that of alveolar type II cells. SP-A protein was also detected in the culture medium of the isolated glands. However, the concentration of SP-A estimated by ELISA using the same antibody was lower than that of alveolar tissue (19). Both SP-A1 and SP-A2 genes contain the nucleotide sequence corresponding to the peptides (4) that react with the monoclonal antibody PE 10 (20, 21), suggesting that PE-10 is able to detect both SP-A1 and SP-A2 equally. Therefore, it seems unlikely that the difference in SP-A1 and SP-A2 expression from the alveolar tissue contributes to the lower value of SP-A detected in ELISA and immunohistochemistry of the submucosal glands. By Northern blot analysis, the signal of SP-A mRNA in isolated submucosal glands was found to be one-eighteenth of that in alveolar tissue. Although the SP-A mRNA level was normalized by the amount of total RNA (10 µg) in the present study, if the amplified cDNA level of SP-A had been normalized by 28 S rRNA or -actin mRNA the difference in the SP-A mRNA level between the glands and alveolar tissue might not be so large. A large variation in the SP-A mRNA level, reported in adult surgical lung specimens, might also have contributed to the difference (22). In the present experiment using cultured tracheal gland cells, we failed to detect a clear SP-A expression by RT-PCR, suggesting that it requires further differentiation of gland cells (17).

Human SP-A is encoded by two different genes, SP-A1 and SP-A2, whose divergence of nucleotide sequences was detected in all introns and exons; the highest divergence was about 13% in the upstream region, intron I, and the noncoding portion of exon V, and other sequences of all exons and introns have a much lower divergence (4). A macromolecular structural comparison of recombinant SP-A derived from two human coding sequences (SP-A1 and SP-A2) has been reported (23). McCormick and Mendelson reported that 65% of the SP-A mRNA was derived from the SP-A2 gene, whereas only 35% was from SP-A1 gene in human adult peripheral lung tissue (24). Meanwhile, it is also reported that only SP-A1 transcripts were detected in lung tissue from a 28-wk gestation neonate and that in midgestation fetal lung, 65% of the SP-A mRNA was found to be SP-A1 and 35% was SP-A2 (24). The present study revealed that the SP-A2 gene alone was expressed in human adult airway submucosal glands. The lack of SP-A1 gene expression in the gland cells was observed in all patients examined in the present study, although there have been reports of wide variation in the relative abundance of SP-A1 and SP-A2 gene expression in peripheral lungs (24).

SP-A is known to have a role in the host defense with alveolar macrophage interaction and to enhance the phagocytosis of a number of microbial species, including viruses and bacteria (25). One explanation for the function is based on the macromolecular structure of SP-A, which is similar to the subcomponent C1q of the first component of complement C1 (30). In addition, Tenner and associates reported that, like C1q, SP-A can enhance FcR- and CR1-mediated phagocytosis by macrophages (25). The SP-A2 gene is known to be far more inducible than that encoding SP-A1 in the lung tissue (24). SP-A responds to acute inflammation in the same manner as does mannose-binding protein, with increases in mRNA and protein production (31). Macrophages are found not only in the peripheral airways and alveoli but also in the fluid layer and walls of large bronchi (32). Furthermore, SP-A is known to contribute to the surfactant property of phospholipids and the formation of tubular myelin, and natural surfactant with SP-A has better effects than an artificial surfactant without SP-A in the surfactant therapy (33). Because surfactant is known to have an important function in maintaining mucociliary clearance in the airways (34), it can be postulated that SP-A facilitates the mucociliary clearance through the enhanced surfactant property and/ or the assembly of the mucociliary layer (35). Taken with the localization of SP-A in larger airway, we speculate that SP-A produced through the SP-A2 gene expression by airway submucosal glands is secreted into the airway lumen and plays a role in the host defense of airways. Morphometric analysis has shown that the surface area from the trachea to the segmental bronchi is 1:500 to 1:5,000 or less than that of the alveolar space (36). Therefore, it might be that the SP-A secreted by airway submucosal glands is enough to function as a host-defense substance even though the SP-A gene expression was much smaller than that of the alveolar tissue.

In this study we showed that human airway submucosal gland cells can transcribe the SP-A2 gene and produce SP-A protein in a manner different from those of peripheral airway and alveoli. Although further studies are neededincluding those on the regulation of SP-A protein secretion and gene expression in airway submucosal glandsit is likely that SP-A produced from airway submucosal glands plays a role in the host defense of airways.