Secretion and gene expression of secretory leukocyte protease inhibitor by human airway submucosal glands

Hiroki Saitoh, Tohru Masuda, Sanae Shimura, Toshiaki Fushimi, and Kunio Shirato

First Department of Internal Medicine, Tohoku University School of Medicine, Sendai 980-8574, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Submucosal glands were isolated within 4 h of death from tracheae and bronchi obtained from autopsied lungs, and the secretory response of secretory leukocyte protease inhibitor (SLPI) was examined with ELISA and a secretory index. Although human neutrophil elastase (HNE) at low concentrations increased SLPI secretion above the control level (i.e., 149% of control level at 10-11 M), HNE at high concentrations significantly decreased it below the control level (i.e., 16% of control level at 10-7 M). The decrease in SLPI concentration was shown to result from the degradation of SLPI by excessive HNE. Methacholine induced significant secretion (i.e., 363% of control level at 10-5 M) that was abolished by both M1 and M3 receptor antagonists. A semiquantitative analysis of SLPI mRNA by RT-PCR and Southern blot showed that compared with the superficial epithelium, submucosal glands had a 30-fold or higher level of SLPI mRNA. Both HNE and methacholine significantly increased the level of SLPI mRNA in submucosal glands in a dose-dependent manner (i.e., 357% of control level at 10-7 M and 175% of control level at 10-5 M, respectively). These findings indicate that human airway submucosal glands can transcribe 30-fold or more SLPI mRNA than the superficial epithelium and that SLPI mRNA transcription and secretion are regulated by both HNE and muscarinic receptors.

neutrophil elastase; muscarinic agonist


    INTRODUCTION
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INTRODUCTION
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AIRWAY INFLAMMATION, present in bronchitis, bronchiectasis, and cystic fibrosis, is characterized by the accumulation of activated neutrophils in the airway mucosa. Azurophilic (intracellular) granule components of neutrophils include neutral serine protease, elastase, and cathepsin G as well as other enzymes such as myeloperoxidase and lysozyme (27). Among the proteases, neutrophil-derived elastase quantitatively represents the major granule component and is well known to be the most potent protease to stimulate airway secretion, accelerate airway inflammation, and damage the airway mucosal tissue (13, 25, 27, 28). Elastase is counterbalanced by two major antiproteases, secretory leukocyte protease inhibitor (SLPI) and alpha 1-antitrypsin (alpha 1-AT) (8). alpha 1-AT is generated in the liver and is present in serum, and SLPI is locally produced in the airways. SLPI is a single nonglycosylated polypeptide chain of 107 amino acids (~11,700 Da) with a boomerang-shaped tertiary structure composed of two domains, with the antiprotease active site for elastase and trypsin residing in the COOH-terminal domain (6).

Expression of the SLPI gene has been extensively examined in superficial epithelial cells from human airways, but SLPI secretion from the superficial epithelium has not yet been clarified (1, 3, 16). Furthermore, in the human lung, immunohistochemical studies (21, 34) have demonstrated that SLPI is predominantly in the serous cells of tracheal and bronchial submucosal glands, and an in situ hybridization experiment (35) has demonstrated that the SLPI gene is in the serous cells of human nasal mucosa. Submucosal glands are abundant in human airways and make a greater contribution to airway secretion than does the superficial epithelium (22), whereas in experimental animals such as mice, rats, and rabbits, the submucosal glands are scarce or absent (10). However, we have limited knowledge as to the production and secretory response of SLPI from human submucosal glands. Although SLPI secretion has been studied in primary cultures of human gland cells (18-20, 32), there have been no gene expression experiments concerning SLPI secretion from human submucosal glands nor have there been comparisons in the gene expression between the superficial epithelium and submucosal glands in human airways. Furthermore, cultured gland cells are known to differ in both structure and function from in vivo submucosal gland cells in human airways (36), and isolated submucosal gland preparations can overcome the limitations of cultured gland cells (20, 23, 26). Hence, we measured the secretory response to agonists from isolated glands by means of ELISA and a secretory index and investigated expression of the SLPI gene with RT-PCR for semiquantification of SLPI mRNA to compare it with that of the superficial epithelium.


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Isolation of single submucosal glands. Human tracheae and bronchi obtained from autopsied lungs 2-4 h after death [11 men and 5 women, 61 ± 4 (SE) yr] were used in the present study. The diagnoses of the 16 patients were dilated cardiomyopathy (male, 48 yr), bleeding of the gastrointestinal tract (male, 24 yr), infectious endocarditis (male, 60 yr), metastatic lung adenocarcinoma (male, 81 yr), pancreatic carcinoma (male, 72 yr), colon cancer (male, 65 yr), mediastinal tumor (male, 50 yr), acute monocytic leukemia (male, 80 yr), renal tumor (male, 40 yr), lung squamous cell carcinoma (female, 87 yr), congestive heart failure with preexisting myocardial infarction (female, 58 yr), chronic bronchitis (female, 73 yr), progressive multifocal leukoencephalopathy (female, 52 yr), lung adenocarcinoma (female, 40 yr), pneumonia with chronic pulmonary emphysema (male, 71 yr), and liver cirrhosis (male, 71 yr). The present study was approved by the Ethics Committee on Human Investigations of Tohoku University School of Medicine (Sendai, Japan).

Immediately after removal, the tracheae and bronchi were put in cold (4°C) phosphate-buffered saline with penicillin (100 IU/ml) and streptomycin (100 µg/ml) and transferred to our laboratory. According to the method previously reported by Shimura et al. (26) and van Seuningen et al. (33), fresh, unstained submucosal glands were isolated with two pairs of tweezers and microscissors under a stereoscopic microscope (×60-80). To avoid tissue damage during the procedure, care was taken to isolate the gland by picking up some of the connective tissue surrounding the gland (26).

In some experiments, we used superficial epithelial layers that were isolated from the tracheae and bronchi with a method similar to that for the isolation of submucosal glands (26, 33). Absence of contamination by submucosal gland cells was confirmed with both histological examination and surfactant protein A cDNA amplification by RT-PCR. A previous experiment by Saitoh et al. (23) has shown that submucosal gland cells can transcribe surfactant protein A mRNA, whereas superficial epithelial cells cannot.

Measurement of SLPI concentration. Isolated submucosal glands were put in a 50-ml conical tube, filled with medium 199 (BioWhittaker, Walkersville, MD) supplemented with penicillin (100 IU/ml) and streptomycin (100 µg/ml), and washed 10 times by gentle shaking. The 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 12-24 h for equilibration. After equilibration and three washes with medium 199, five pieces of isolated submucosal glands were put in plastic dishes (22 mm in diameter) with 2 ml of medium. After a 4-h incubation (period I), the medium was harvested and stored at -80°C until used. The submucosal glands were again washed three times and filled with 2 ml of medium. After 8 or 24 h of incubation with the agonist (period II), the medium was harvested and stored at -80°C until used. The SLPI concentration of the medium was measured with an ELISA kit according to the manufacturer's protocol (R&D Systems, Minneapolis, MN). The secretory response to the agonist is expressed as a percentage of the control level according to the secretory index (9).

Next, we investigated whether SLPI secreted by isolated glands was degraded by excess human neutrophil elastase (HNE). Spent medium containing SLPI secreted by the submucosal glands during equilibration was collected and stored at -80°C until used. Two milliliters of the medium that was diluted from ×0.5 to ×0.125 were incubated with 10-11, 10-9, or 10-7 M HNE or without HNE (control) for 8 h in conditions that were the same as those for the actual culture of the isolated glands (40% O2-5% CO2 at 37°C). The time dependence of SLPI degradation was also investigated. The medium was incubated with 10-7 M HNE for 10 or 30 min or 1, 2, 4, and 8 h. After incubation, SLPI concentrations in the dishes were measured with an ELISA kit (R&D Systems).

RT-PCR analysis of SLPI mRNA. Isolated submucosal glands and superficial epithelial tissue were used for RT-PCR analysis. Total RNA was extracted from each sample within 1 h of isolation of the submucosal glands and superficial epithelial tissue or after a 36-h incubation without any stimulation from ISOGEN (Nippon Gene, 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 primers and Moloney murine leukemia virus reverse transcriptase (GIBCO BRL, Life Technologies, Rockville, MD) according to the supplier's instructions. The oligonucleotide primers used in the PCR were 5'-ATGAAGTCCAGCGGCCTCTT-3' and 5'-ATGGCAGGAATCAAGCTTTC-3' for SLPI and the amplified 408-bp SLPI cDNA segments and 5'-ATGGGTCAGAAGGATTCCTAT-3' and 5'-GTGTAACGCAACTAAGTCA-3' for beta -actin and the amplified 1,017-bp cDNA. One one-hundredth of the cDNA synthesis reaction was combined in a final volume of 20 µl for PCR amplification by using each set of primers and 0.48 U of Tth DNA polymerase (Toyobo, Osaka, Japan) for SLPI and beta -actin cDNA segment amplification. The conditions for amplification were denaturation at 92°C (1 min), annealing at 54°C (1 min), and elongation at 72°C (3 min).

Southern blot analysis. Six microliters of the PCR-amplified product underwent electrophoresis on 3% agarose gels in Tris-boric acid-EDTA disodium buffer and were transferred to nylon membranes (Hybond N, Amersham, Arlington Heights, IL). Hybridization was performed overnight in a solution with a final concentration of 5× saline-sodium phosphate-EDTA buffer, 5× Denhardt's solution, 0.5% sodium dodecyl sulfate (SDS), 20 µg/ml of denatured salmon sperm DNA, and 5 × 105 counts · min-1 · ml-1 of the [gamma -32P]ATP-labeled SLPI and beta -actin oligonucleotide probes 5'-CTTCAAGTCACGCTTGCACT-3' and 5'-GATGACCCAGATCATGTTTG-3', respectively. SLPI-specific probes were designed according to the sequences previously reported (30). These probes were labeled with T4 polynucleotide kinase (New England Biolabs) according to the manufacturer's recommendation. After hybridization, the nylon membranes were washed once in 2× sodium chloride-sodium citrate (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 50°C for 15 min. The imaging plate of a bio-image analyzer (BAS 2000, Fuji Photo Film, Tokyo, Japan) was exposed to the membrane. The scanned image produced by the imaging plate was analyzed by the bio-image analyzer. The amplified cDNA levels were calculated from the radioactivity of the hybridization signals.

Reagents. Acetyl-beta -methylcholine chloride [methacholine (MCh)], medium 199, and HNE were purchased from Wako Pure Chemicals (Osaka, Japan), BioWhittaker (Walkersville, MD), and Elastin Products, (Owensville, MO), respectively. 11{[2-(Diethylamino)methyl-1-piperidinyl]acetyl}-5,11-dihydro-6H-pyreido-(2,3-b)(1,4)-benzo-diazepin-6-one (AF-DX116) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) were generous gifts from Boehringer-Ingelheim (Ingelheim, Germany) and Dr. R. Barlow (University of Bristol, Bristol, UK), respectively. All other reagents for the present experiments were from Sigma (St. Louis, MO).

Statistical analysis. Data are means ± SE. For multiple mean comparisons, analysis of variance and Duncan's multiple range test were used. For mean comparison, two-tailed paired or unpaired Student's t-test was used, and the Cochran-Cox t-test was used when Bartlett's test for uniformity of variance showed it to be nonuniform. P < 0.05 was considered significant.


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No significant differences in SLPI secretion in response to HNE, MCh, or dexamethasone (Dex) were observed between the results from 8 h of stimulation and those from 24 h of stimulation (24-h duration of period II). Therefore, we describe the results from the 8-h stimulation except where stated otherwise.

Secretory response to human HNE. SLPI secretion from human submucosal glands in response to HNE is shown in Fig. 1. Although HNE at low concentrations (10-11 to 10-9 M) induced an increase above the control level (i.e., 149% of control level at 10-11 M), HNE at high concentrations (10-8 to 10-7 M) induced a significant decrease in the SLPI secretion below the control level, reaching a response of 16% of control level at 10-7 M. alpha 1-AT (2 × 10-6 M) was mixed, incubated with 10-7 M HNE for 15 min at room temperature, and used for period II as described in a previous report (1). The inactivation of HNE by pretreatment with alpha 1-AT resulted in an apparent inhibition of the decrease in the SLPI secretion by HNE (Fig. 1), whereas alpha 1-AT itself did not alter the SLPI secretion as described in a previous report (28).


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Fig. 1.   Secretory leukocyte protease inhibitor (SLPI) secretion from isolated submucosal glands in response to human neutrophil elastase (HNE) stimulation for 8 h. Values are means ± SE of 3-12 experiments; nos. in parentheses, no. of experiments. HNE produced an increase in SLPI secretion above control level (i.e., 149% of control level at 10-11 M) at 10-11 to 10-9 M, whereas it induced a significant decrease below control level (i.e., 16% of control level at 10-7 M) at high concentrations (10-8 to 10-7 M). Pretreatment with alpha 1-antitrypsin (alpha 1-AT; 2 × 10-6 M) inhibited the decrease with 10-7 M HNE. star  P < 0.05 vs. control. star star P < 0.01 vs. control. star star star P < 0.001 vs. control. §§§ P < 0.001 vs. 10-7 M HNE alone.

Degradation of secreted SLPI by HNE. The SLPI concentration in the spent medium from the equilibration decreased after incubation with HNE as shown in Fig. 2. The SLPI concentration in the control medium after 8 h of incubation was 160 ng/ml and that of the medium after incubation with HNE decreased in a dose-dependent manner, reaching 59 ng/ml at 10-7 M (37% of the control level). The SLPI concentration in the medium with serial dilutions also decreased after incubation with HNE in a dose-dependent manner, reaching 10-16% of control level at 10-7 M. Furthermore, the decrease in SLPI concentration with HNE was time dependent. For example, the concentrations after incubation with 10-7 M HNE for 10 and 30 min and 1, 2, 4, and 8 h were 36, 33, 30, 24, 21, and 19% of control level (97 ng/ml), respectively. In contrast, 10-5 M MCh, 10-5 M Dex, or 2 × 10-6 M alpha 1-AT did not alter the SLPI concentration in the spent medium in a dose- or time-dependent manner.


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Fig. 2.   Degradation of SLPI secreted from isolated submucosal glands by HNE. The medium from isolated submucosal glands incubated for 24 h (which contained SLPI) was collected, diluted to serial concentrations (×1, ×0.5, ×0.25, and ×0.125) and incubated with and without (control) HNE for 8 h. Solid bars, control; hatched bars, 10-11 M HNE; crosshatched bars, 10-9 M HNE; open bars, 10-7 M HNE. The SLPI concentration was decreased in a dose-dependent manner, reaching 10-37% of the serial dilution of the control sample at 10-7 M HNE.

Secretory response to MCh. A significant increase in the SLPI secretion in response to MCh was observed in a dose-dependent manner, reaching 363% of control level at 10-5 M (Fig. 3) The increase with MCh was abolished by treatment with atropine that was added 15 min before the beginning of period II as shown in Fig. 3. Furthermore, we examined the effect of the M1 receptor antagonist pirenzepine, the M2 receptor antagonist AF-DX116, and the M3 receptor antagonist 4-DAMP methiodide on SLPI secretion from isolated submucosal glands, adding them 15 min before the beginning of period II. Both pirenzepine and 4-DAMP abolished the increase with MCh, whereas AF-DX116 did not alter it, as shown in Fig. 3.


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Fig. 3.   SLPI secretion from isolated submucosal glands in response to methacholine (MCh) for 8 h of stimulation. Values are means ± SE of 3-12 experiments; nos. in parentheses, no. of experiments. MCh induced a significant SLPI secretion in a dose-dependent manner (i.e., 363% of control level at 10-5 M). The increase with MCh was abolished by atropine (ATRO), pirenzepine (PZ), and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), whereas AF-DX116 did not alter it. star  P < 0.05 vs. control. star star P < 0.01 vs. control. §§ P < 0.01 vs. 10-5 M MCh alone.

Comparison of SLPI mRNA levels between the superficial epithelium and submucosal glands. The SLPI cDNA segment was amplified from both isolated submucosal glands and superficial epithelial layers. For comparison of the SLPI mRNA level in these two tissues, semiquantitative analysis of SLPI mRNA by RT-PCR was performed to amplify the cDNA segments of the SLPI and beta -actin genes. To assess the level of SLPI cDNA semiquantitatively, we first determined the number of PCR cycles at which the saturation of amplification started as previously reported by Fushimi et al. (7). The cDNA template that showed the thickest band after 19 PCR cycles was used. Different amounts of the same cDNA template were amplified for 13-25 cycles by using SLPI-specific primers. Each PCR product was subjected to Southern blot hybridization with a 32P-labeled SLPI oligonucleotide probe. There was a linear relationship between the level of the amplified SLPI cDNA fragment and the amount of the template at 13 and 19 cycles but not at >22 cycles. Similarly, there was a linear relationship between the level of the amplified beta -actin cDNA fragment and the amount of the template at 20-26 cycles but not at 29 cycles. Two independent experiments were performed with the same results. Therefore, we amplified the SLPI and beta -actin cDNA fragments for 16 and 23 cycles, respectively, during which the target cDNA was linearly amplified. These PCR products were transferred to nylon membranes after electrophoresis and Southern blot were performed. Radioactivity of the Southern blot was calculated, and the amplified SLPI cDNA level was normalized by the amplified beta -actin cDNA level. The semiquantitative procedure showed that the SLPI mRNA level of submucosal gland cells was 30-fold higher than that of the superficial epithelial cells in the comparison of 1 µg of total RNA that was converted to first-strand cDNA (Fig. 4). No significant differences between samples within 1 h of isolation (n = 5 experiments) and those after 36 h of incubation (n = 10 experiments) were observed, and the latter is shown in Fig. 4. Furthermore, no significant detectable amount of SLPI protein was found in the medium sample from a 36-h culture of the superficial epithelial layer.


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Fig. 4.   Semiquantitative analysis of SLPI mRNA of human tracheal submucosal glands and superficial epithelium. Total RNA was extracted from isolated submucosal glands and superficial epithelium layers of the same individuals and converted to first-strand cDNA. SLPI mRNA transcription was assessed by semiquantitative analysis with RT-PCR and Southern blot and was normalized to the radioactivity of amplified beta -actin cDNA (SLPI/beta -actin). The SLPI mRNA transcription level of the submucosal gland was 30.4-fold of the epithelium. Nos. in parentheses, no. of experiments. star star star P < 0.001 vs. human tracheal epithelium.

Gene expression in response to HNE and MCh. To evaluate the transcription of SLPI mRNA from isolated submucosal glands in response to HNE and MCh, we performed semiquantitative analysis as described in Comparison of SLPI mRNA levels between the superficial epithelium and submucosal glands and compared the results with those of untreated control samples. In response to HNE, the SLPI mRNA level was significantly increased in a dose-dependent manner, reaching 357% of control level at 10-7 M (Fig. 5). The increase in the mRNA level with HNE was inhibited by pretreatment with alpha 1-AT, and the mRNA level was not significantly different from that in control samples as shown in Fig. 5. SLPI transcription was not influenced by alpha 1-AT alone. Furthermore, SLPI mRNA significantly increased after stimulation with MCh (i.e., 175% of control level at 10-5 M; Fig. 6).


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Fig. 5.   Effect of HNE on SLPI mRNA transcription of isolated submucosal glands assessed by semiquantitative analysis with RT-PCR and Southern blot. A: example of Southern blot of cDNA segment amplified with primers specific to SLPI and beta -actin. Southern blot was performed with 32P-labeled SLPI- and beta -actin-specific oligonucleotide probes. HNE enhanced the transcription of SLPI mRNA in dose-dependent manner. B: after 8 h of incubation with HNE, the radioactivity of amplified SLPI cDNA was normalized by that of amplified beta -actin cDNA. Values are means ± SE; nos. in parentheses, no. of experiments. HNE stimulation increased the SLPI mRNA transcript level in a dose-dependent manner, reaching 357% of control level at 10-7 M, which was significantly inhibited by pretreatment with alpha 1-AT (2 × 10-6 M). star  P < 0.05 vs. control. §§ P < 0.01 vs. 10-7 M HNE alone.



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Fig. 6.   Semiquantitative analysis with RT-PCR and Southern blot of SLPI mRNA of human tracheal submucosal glands with MCh and dexamethasone (Dex) stimulation. Values are means ± SE; nos. in parentheses, no. of experiments. MCh and Dex stimulation for 8 h induced significant increases of 175 and 277% of control level in SLPI mRNA transcription level, respectively. star star P < 0.01 vs. control.

Dex effect on gene expression and secretion. Because corticosteroids are known to induce an increase in the SLPI mRNA transcription level in airway epithelial cell lines (2), we investigated the effects of Dex on both SLPI protein secretion and mRNA transcription in submucosal glands. Although Dex stimulation induced a significant increase in the SLPI mRNA level (i.e., 277% of control level at 10-5 M) as shown in Fig. 6, Dex itself (8- or 24-h incubation, 10-9 to 10-5 M) did not stimulate SLPI secretion from submucosal glands (Fig. 7). However, Dex significantly augmented the MCh-induced SLPI secretion from isolated glands as shown in Fig. 7.


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Fig. 7.   SLPI secretion from human tracheal submucosal glands in response to Dex stimulation for 8 h. Values are means ± SE; nos. in parentheses, no. of experiments. Although Dex itself did not alter SLPI secretion, it significantly augmented SLPI secretion with MCh. star star P < 0.01 vs. control. §§§ P < 0.001 vs. 10-5 M MCh alone.


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Because human tissue from autopsied lungs was used for the present experiments, it was possible that postmortem changes, including ischemia and lytic processes, might have affected the experimental results, producing large variations in the data in addition to differences in cell numbers and populations in the isolated submucosal glands. To minimize the variations, we used airways removed 2-4 h after death for the present experiments. Furthermore, all data are expressed as a percentage of the corresponding control values, and a secretory index (9) was used for the analysis of the SLPI concentration.

Previous histochemical analyses have shown the localization of SLPI protein in the serous cells of submucosal glands and faint and/or no positive staining of superficial epithelial cells (1, 4, 21, 34, 35). In an in situ hybridization study with SLPI-specific oligonucleotide probes, SLPI mRNA was found in the serous cells of submucosal glands but not in surface epithelial cells of bronchial mucosa from normal and emphysema patients (33). For quantification of SLPI mRNA, we performed a semiquantitative analysis with RT-PCR because total RNA obtained from approximately five pieces of submucosal glands in a culture dish was <10 µg and therefore too small for Northern blot analysis. The present experiments, in which both SLPI and beta -actin cDNAs were amplified by RT-PCR and the SLPI cDNA level was normalized by beta -actin cDNA, revealed a 30-fold or higher level of SLPI mRNA in human airway submucosal gland cells than in superficial epithelial cells. Taken together with these findings, it seems likely that submucosal glands play a major role in SLPI production and secretion as well as in mucus secretion in human airways.

The present semiquantitative RT-PCR analysis in which SLPI cDNA or beta -actin cDNA was further normalized by untreated control samples showed an upregulation of SLPI mRNA transcription in human airway submucosal glands with HNE in a dose-dependent manner. Similarly, neutrophil elastase at high concentrations is reported to increase the SLPI transcription level in a human airway epithelial cell line (1, 24). In contrast to the upregulation of the mRNA level with HNE, the SLPI concentration in cultures from isolated submucosal glands decreased to 16% of control level in response to HNE at high concentrations (10-8 to 10-7 M) in the present experiment, although HNE at low concentrations (10-11 to 10-9 M) induced a small increase (~50% increase above the control level) in SLPI secretion. A cytotoxic effect of HNE on gland cells seems unlikely because no significant lactate dehydrogenase release was observed in the present as well as in a previous report (25), and upregulation of the mRNA level was induced by high concentrations of HNE. Furthermore, many previous experiments (13, 25, 28) have revealed that mucus, glycoconjugates, or macromolecule secretion from airway explants and submucosal glands in various species including human is significantly stimulated by HNE or neutrophil-derived elastase in a dose-dependent manner, reaching a maximum response 10-fold larger than that with other agonists and with a threshold concentration much lower than that of other agonists. Therefore, it is likely that SLPI secreted from isolated submucosal glands was degraded by excess HNE. The present experiment confirmed the decrease in SLPI concentration in both dose- and time-dependent manners when the spent medium from isolated glands was incubated with HNE.

However, it is possible that a monoclonal antibody of ELISA can recognize some proteolytic products or bound forms of SLPI. Although we have no direct evidence, taken together with the present findings, we speculate that excess HNE degrades the site of SLPI that the antibody can recognize. There have been some reports supporting this idea. Recombinant SLPI is known to be fragmented into two proteins when the molar ratio of elastase to SLPI is excessive (2.0 and 4.0) (17). It has been revealed that proteolytic enzymes from saliva break SLPI into four fragments (10.0-, 7.0-, 5.5-, and 3.0-kDa proteins) and that the site of SLPI (Leu72-Met73-Leu74), which is the location of the elastase inhibitory region (6, 31), is cleaved (17). Another experiment has shown that recombinant SLPI is cleaved at Cys18-Leu19 and then at Met73-Leu74 by Pseudomonas aeruginosa elastase and loses the inhibitory activity to the elastase (the molar ratio of elastase to SLPI is 2,000) (29). In the present degradation experiment, the elastase-to-SLPI molar ratio was calculated to be 25 because 4.0 × 10-9 M SLPI was incubated with 10-7 M HNE. It is possible that incubation with an excessively high concentration of HNE might have resulted in the disappearance of the active elastase-inhibitory center of SLPI. Furthermore, the monoclonal antibody for the present experiments was shown to have the ability to neutralize the bioactivity of recombinant human SLPI (11), suggesting that the epitope of SLPI for these antibodies is the site of the active inhibitory center.

Because it is impossible to compare SLPI secretion with the SLPI mRNA transcription level, it is uncertain whether SLPI mRNA transcriptional upregulation by HNE results from the direct action of HNE or from negative feedback after SLPI secretion in submucosal glands. It is possible that SLPI secretion from submucosal glands consists of two processes, synthesis and exocytosis, as seen in the mucin secretion from human airway submucosal glands by elastase stimulation (25). Recently, Maizieres et al. (14) reported that a very short stimulation (up to 10 min) with HNE (10-6 M) produced a rapid exocytosis of SLPI to 370% of control level and that the influx of extracellular Ca2+ and intracellular Ca2+ concentration ([Ca2+]i) oscillations regulate the rapid exocytotic responses in cultured human gland cells. Although Kikuchi et al. (12) have reported that the two nuclear binding proteins SLPI-B1 and SLPI-B2 bind to a promoter region of the SLPI gene in the type II pneumocyte cell line, the regulation of these nuclear binding proteins by various agonists including HNE remains unclear. Thus the mechanism of SLPI mRNA transcriptional regulation by elastase stimulation in airway submucosal gland cells remains to be studied.

To date, different secretory responses of SLPI in cultured human tracheal gland cells have been reported despite the use of the same experimental conditions (agonist, concentration, and stimulation time). Tournier et al. (32) reported that phenylephrine, carbachol, and isoproterenol all induced an increase in SLPI secretion, whereas Merten et al. (20) reported that none of them altered SLPI secretion. Furthermore, Merten and colleagues (18, 19) found that acetylcholine upregulated SLPI secretion and norepinephrine downregulated it. It is well known that the differentiation of gland cells to serous and/or mucous cells strongly depends on the culture conditions (36). Furthermore, it is possible that various receptor expressions and functions vary among cultured cells (8). The isolated submucosal gland preparation used for the present study has the advantage of overcoming these limitations of cultured gland cells. Cholinergic muscarinic receptor stimulation is well known to potently induce human airway submucosal gland secretion (9). In the present experiment, SLPI secretion was stimulated by MCh in a dose-dependent manner that was abolished by atropine, pirenzepine, and 4-DAMP. Autoradiographically, the coexistence of M1 and M3 receptors over human airway submucosal glands has been demonstrated by Mak and Barnes (15). Therefore, it is possible that both M1 and M3 receptors are involved in the SLPI secretion from human airway submucosal glands. Meanwhile, 4-DAMP has been shown to have an antagonistic action on M1 receptors to a degree similar to its action on M3 receptors (5). Furthermore, electrolyte or fluid secretion from airway submucosal glands in which serous cells play a main role is suggested to be coupled mainly with M1 receptor stimulation (37). Therefore, there still remains the possibility that SLPI secretion from human airway submucosal glands is regulated by the activation of mainly M1 receptors.

The SLPI mRNA transcription level increased to 1.8-fold of the control level after MCh (10-5 M) stimulation, and this increase seems to be too small to correspond to the increase (5.2-fold of the control level) in SLPI protein secretion. SLPI secretion is reported to reach a peak within 10-30 min after stimulation with acetylcholine (18). It is possible that the increase in SLPI mRNA level with MCh involves a mechanism different from that of the rapid secretion (or exocytosis, degranulation) through a rise in [Ca2+]i and may include a negative feedback mechanism. Although the regulation of SLPI secretion or mRNA is not fully understood, it is possible that diverse mechanisms are responsible.

An increase in the SLPI transcription level with Dex was also observed in human airway submucosal glands in the present study as well as in a previous report with human epithelial cell lines (2). In contrast to our expectation, SLPI secretion did not increase after treatment with Dex for 8-24 h. The 5'-flanking region of the SLPI gene has a consensus sequence of glucocorticoid response elements and several consensus sequences of activator protein (AP)-1 that are related to transcriptional regulation by corticosteroids (2, 6). Maruyama et al. (16) reported that the transcriptional upregulation of the SLPI gene by phorbol 12-myristate 13-acetate stimulation was not related to the consensus sequence of AP-1, which is the putative phorbol 12-myristate 13-acetate response element (-812, -575, and -470 bp). Therefore, regulation of SLPI mRNA transcription by glucocorticoid stimulation may be related to glucocorticoid response elements and not the putative consensus sequences of AP-1 that are on the 5'-flanking region of the SLPI gene (16). Although we have no definite explanations for the discrepancy between SLPI secretion and mRNA level in response to Dex, the synthesis of SLPI after upregulation of SLPI mRNA transcription may be increased, resulting in an increased amount of intracellular SLPI. The degranulation or exocytosis of SLPI seems to require a [Ca2+]i rise as suggested by a previous report (18) and appears to be regulated through a mechanism different from that of SLPI mRNA. In fact, in the present experiment, Dex significantly augmented the MCh-induced SLPI secretion from the airway submucosal glands.

In conclusion, the present study showed that 1) human airway submucosal glands can transcribe 30-fold or more SLPI mRNA than does the superficial epithelium, 2) SLPI mRNA transcription and secretion by submucosal glands are regulated by both HNE and muscarinic receptors, and 3) the possibility that SLPI secreted by submucosal glands was degraded by excessive HNE. These findings are important not only for understanding inflammation in diseased airways, including those in chronic bronchitis patients, but also for the development of new therapies.


    ACKNOWLEDGEMENTS

We gratefully acknowledge Brent Bell for reading the manuscript.


    FOOTNOTES

This study was partially supported by Scientific Grant 0101304 from the Ministry of Education, Science, and Culture of Japan.

Address for reprint requests and other correspondence: K. Shirato, First Dept. of Internal Medicine, Tohoku Univ. School of Medicine, Aoba-ku, Sendai 980-8574, Japan (E-mail: shirato{at}int1.med.tohoku.ac.jp).

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 29 February 2000; accepted in final form 22 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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