First Department of Internal Medicine, Tohoku University School of Medicine, Sendai 980-8574, Japan
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ABSTRACT |
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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 1011 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
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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
1-antitrypsin (
1-AT) (8).
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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METHODS |
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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).
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 -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
-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 · min1 · ml
1 of the
[
-32P]ATP-labeled SLPI and
-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--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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RESULTS |
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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 (1011 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.
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
1-AT resulted in an apparent
inhibition of the decrease in the SLPI secretion by HNE (Fig. 1),
whereas
1-AT itself did not alter the SLPI secretion as
described in a previous report (28).
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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 107 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
1-AT did not alter
the SLPI concentration in the spent medium in a dose- or time-dependent
manner.
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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
105 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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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
-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
-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
-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
-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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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
107 M (Fig. 5). The
increase in the mRNA level with HNE was inhibited by pretreatment with
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
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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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 105 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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DISCUSSION |
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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 -actin cDNAs were amplified by RT-PCR and the SLPI
cDNA level was normalized by
-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
-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 × 109 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
(106 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 (105 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.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Brent Bell for reading the manuscript.
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FOOTNOTES |
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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.
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