1 Department of Environmental
Health, Acrolein, an unsaturated aldehyde found in smog
and tobacco smoke, can induce airway hyperreactivity, inflammation, and
mucus hypersecretion. To determine whether changes in steady-state
mucin gene expression (Muc2 and
Muc5ac) are associated
with inflammatory cell accumulation and neutrophil elastase activity,
FVB/N mice were exposed to acrolein (3.0 parts/million; 6 h/day, 5 days/wk for 3 wk). The levels of Muc2 and Muc5ac mRNA were determined by RT-PCR, and the presence of Muc5ac protein was detected by immunohistochemistry. Total and differential cell counts were determined from bronchoalveolar lavage (BAL) fluid, and neutrophil elastase activity was measured in the BAL fluid supernatant. Lung Muc5ac mRNA was increased on days 12 and 19, and Muc5ac protein was
detected in mucous granules and on the surface of the epithelium on
day 19. Lung Muc2 mRNA was not
detected at measurable levels in either control or exposed mice.
Acrolein exposure caused a significant and persistent increase in
macrophages and a rapid but transient increase in neutrophils in BAL
fluid. Recoverable neutrophil elastase activity was not significantly
altered at any time after acrolein exposure. To further examine the
role of macrophage accumulation in mucin gene expression, additional strains of mice (including a strain genetically deficient in macrophage metalloelastase) were exposed to acrolein for 3 wk, and Muc5ac mRNA
levels and macrophage accumulation were measured. The magnitude of
macrophage accumulation coincided with increased Muc5ac mRNA levels,
indicating that excessive macrophage accumulation augments acrolein-induced Muc5ac synthesis and secretion after repeated exposure. These findings support a role for chronic monocytic inflammation in the pathogenesis of mucus hypersecretion observed in
chronic bronchitis.
chronic obstructive pulmonary disease; macrophage; metalloelastase; tobacco smoke; air pollution
AIRWAY MUCUS HYPERSECRETION is a common feature in the
pathogenesis of chronic obstructive airway diseases including chronic bronchitis, asthma, and cystic fibrosis. Patients show signs of airway
inflammation, an increased number of mucous cells, and excessive airway
mucus. The major components of mucus are large, heavily glycosylated
proteins (mucins) that provide airway secretions with their
characteristic viscosity, adhesiveness, and elasticity (53). Nine human
mucin genes (MUC1-MUC4,
MUC5ac,
MUC5b, and MUC6-MUC8) have
been identified and partially characterized throughout the
gastrointestinal, reproductive, and respiratory tracts (reviewed in
Ref. 42). Of these, only MUC5ac (43) and MUC5b (55) glycoproteins have
been isolated from human airway secretions.
One mechanism involved in the pathogenesis of mucus hypersecretion is
an increase in the number of mucus-secreting cells throughout the
airway epithelium. This condition can be induced experimentally in
laboratory animals by exposure to respiratory irritants including tobacco smoke (10), O3 (17),
SO2 (30), and acrolein (33). Hypersecretion in rats and mice, with increases in Muc2 mRNA (25) and
Muc5ac mRNA and immunoreactivity (6), has been attributed to the
differentiation of nonsecretory cells to mucus-secreting cells (30).
Similarly, recent in vitro studies demonstrated that differentiation
(altered morphology and increased mucin secretion) is preceded by
increases in MUC2 and MUC5ac mRNA levels in human airway epithelial
cells (15) and by increases in Muc5ac, but not in Muc2, mRNA levels in
rat tracheal epithelial cells (14). Increased Muc5ac, but not Muc2,
mRNA also has been observed to precede mucous cell differentiation in
rats in vivo in response to acrolein exposures (6).
Acrolein (CH2CHCHO) is a potent
irritant found in tobacco smoke [in high concentrations up to 50 parts/million (ppm)], wood smoke, and diesel exhaust and is a
component of photochemical smog (1, 3). After acute acrolein exposure,
inflammatory mediators and neutrophil infiltration increase (31).
Animals repeatedly exposed to Several lines of evidence suggest that inflammation and leukocyte
infiltration in response to irritant exposures may contribute to mucous
cell differentiation and mucus hypersecretion. First, neutrophil
elastase induces mucous cell differentiation when instilled into rodent
airways (49) and stimulates mucus secretion from airway gland cells in
tissue culture (50). Second, anti-inflammatory drugs inhibit tobacco
smoke-induced mucous cell differentiation (41) and decrease the
expression of MUC5ac in human airway epithelial cells (27). In
addition, macrophage- and epithelium-derived inflammatory mediators
such as PGE2, 12- and
15-hydroxyeicosatetraenoic acid, tumor necrosis factor- Most previous investigations focused on mucous cell differentiation and
mucin expression in larger rodents and airway cells in culture. Recent
molecular advances and the increasing availability of transgenic and
knockout mice provide unique insights into the role of inflammatory
mediators and pathways controlling the pathogenesis of airway diseases.
The purpose of this study was to extend the previous study by Borchers
et al. (6) in rats by examining the role of inflammatory
cells and mediators in mucin mRNA expression (Muc5ac and Muc2) induced
by acrolein exposures in mice. This was investigated by exposing mice
subchronically to acrolein and measuring lavagable cells, elastase
activity, and airway mucin steady-state mRNA levels. These findings
were compared with immunohistochemical localization of Muc5ac reactive
substances in the airways. Additionally, we sought to determine the
contribution of excess macrophage accumulation to acrolein-induced
Muc5ac mRNA expression. This was examined by measuring macrophage
infiltration and Muc5ac mRNA in different strains of mice including a
strain genetically deficient in macrophage metalloelastase (MME), an
enzyme required for macrophage tissue invasion (48).
Experimental design. To determine
whether acrolein exposure altered airway inflammation, mucus
production, and mucin mRNA expression, FVB/N mice (male, 6-8 wk;
Jackson Laboratories, Bar Harbor, ME) were exposed to 3.0 ppm acrolein
for 6 h/day, 5 days/wk for up to 3 wk. After exposure, bronchoalveolar
lavage (BAL) was performed on half of each exposure group
(n = 3 mice) to determine leukocyte
cell number and composition and elastase activity. In the remainder of
each exposed group (n = 3 mice), the
right inferior lung lobe was ligated and removed for mucin mRNA (Muc2
and Muc5ac) measurements, and the balance of the lung was Formalin
fixed for Muc5ac immunostaining.
To assess the role of macrophage infiltration in mucin production,
C57BL/6J mice, mice genetically deficient in MME
[MME( Acrolein exposure. The mice were
exposed to acrolein as previously described (31). Acrolein vapor was
generated by passing N2 (3-15
ml/min) over a 3-ml reservoir of liquid acrolein (Janssen Chimica, New
Brunswick, NJ). This mixture was diluted with high-efficiency particle-filtered air (400 ml/min) and introduced into a
0.32-m3 stainless steel chamber.
The exposure concentration was analyzed with a method described by
Cohen and Altshuler (9). Briefly, the chamber atmosphere
was sampled with a series of two glass-fritted impingers, each
containing 10 ml of 96% ethanol. A fraction of each sample was mixed
with 50 mM hexylresorcinol (Sigma, St. Louis, MO), 2.1 mM mercury
chloride (Aldrich, Milwaukee, WI), and 29.7 M trichloroacetic acid
(Fisher, Fair Lawn, NJ). Samples and known standards in equal volumes
were heated (65°C for 15 min) and allowed to cool (22°C for 15 min), and the absorbance at 605 nm was measured with a
spectrophotometer (Beckman DU-64).
Tissue preparation. Animals were
killed immediately after exposure by an intraperitoneal injection of
pentobarbital sodium (50 mg/kg of Nembutol; Abbott Laboratories,
Chicago, IL) and severing the posterior abdominal aorta. To obtain lung
tissue for mRNA isolation, the chest cavity was opened, and the right
inferior lobe was clamped, excised, frozen in liquid nitrogen, and
stored at RNA isolation and analysis. Total RNA
was isolated from the frozen tissue by the
guanidinium-phenol-chloroform procedure described by Chomczynski (8).
The purity was estimated by measuring the 260- to 280-nm absorbance
ratio with a spectrophotometer. Purified RNA was stored at
Quantitation of PCR products. PCR
products were quantitated by densitometry measurement as previously
described (6). A solution containing the PCR-amplified DNA (10 µl)
was electrophoresed on a 2% agarose gel containing ethidium bromide
(0.5 µg/ml) in 90 mM Tris phosphate-2 mM EDTA buffer. The DNA bands
in the gel were illuminated with ultraviolet light and photographed
(type 665 black and white film, Polaroid). Band images were scanned and
analyzed by an image-analysis software program (Mocha, Jandel Scientific), and the total intensity (average intensity × total pixels) was measured. All messages examined amplified exponentially (until saturation) according to the amount of target mRNA in the sample. The relationship between mRNA level and band intensity was
determined by a curve-fitting software program (SigmaPlot, Jandel
Scientific) and found to be y = a[1 Immunohistochemistry. Muc5ac protein
was detected with a chicken polyclonal antibody to mouse gastric mucin
(a generous gift from Dr. Samuel Ho, Veterans Affairs Medical Center,
Minneapolis, MN) in paraffin sections (5 µm) of mouse (FVB/N) airways
as previously described (23). This antibody recognizes the peptide
sequence QTSSPNTGKTSTISTT coded by the Muc5ac mRNA found in gastric and lung tissues (47). Muc5ac antigen-antibody complexes were detected with
the Vectastain ABC Peroxidase Elite goat IgG kit (Vector Laboratories,
Burlingame, CA). The enzymatic reaction product was enhanced with
nickel cobalt to give a black precipitate. Endogenous peroxidase was
quenched (15 min at 22°C) with 3%
H2O2
in methanol. The sections were incubated in 2% normal goat serum in
PBS with 0.2% Triton X-100 (blocking solution; 2 h at 22°C) and
incubated (20 h at 4°C) with primary antibody (1:15,000 dilution).
The sections were washed (6 × 5 min) in PBS with 0.2% Triton
X-100 and incubated (30 min at 22°C) with biotinylated goat
anti-chicken antibody (1:2,000 dilution). The sections were treated (15 min at 22°C) with the avidin-biotin blocking kit (Vector
Laboratories) to block endogenous biotin in the tissue. The sections
were incubated with the avidin-biotin-peroxidase complex diluted in
blocking solution (30 min at 22°C), nickel diaminobenzidine in 0.1 M acetate buffer (4 min at 22°C) for development of a colored
reaction product, and Tris-cobalt (4 min at 22°C) and
counterstained with 0.1% nuclear fast red (2 min at 22°C).
PCR cloning. To examine whether Muc2
mRNA levels were altered in acrolein-exposed mice, a partial cDNA
sequence was cloned by PCR with oligonucleotide primers based on rat
Muc2 sequences (38). The protocol was modified by increasing the number
of cycles (n = 40) and lowering the
annealing temperature (Ta = 55°C) to generate a single PCR product at the expected length
(~700 bp). This product was excised from the gel, purified with a DNA purification kit (Wizard, Promega), and reamplified to obtain a
sufficient amount of product for sequence analysis (ABI Prism).
BAL and cell counts. After exposure,
the animals were anesthetized (50 mg/kg of pentobarbital sodium ip) and
exsanguinated. The lungs were then lavaged three times with 1 ml of
Hanks' balanced salt solution (GIBCO BRL). Individual BAL returns were
pooled and stored at 4°C until cell counting and then at
Elastase activity. To determine
whether repeated acrolein exposure caused an increase in recoverable
elastase activity from BAL samples, enzyme activity was assayed
spectrophotometrically with the synthetic substrate
N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Sigma) according to a
modified method of Iwamura et al. (21). The lavage fluid
from each animal was centrifuged (1,000 rpm for 5 min at 24°C;
Marathon 21K/R, Fisher) to pellet the cells, and the supernatant was
removed and stored at Data analysis. Mucin mRNA levels were
determined in duplicate from three or four animals per group and are
presented as means ± SE. Statistical analysis was performed with
the Kruskal-Wallis one-way analysis of variance and Dunn's method for
comparison of groups. Lavage data are also presented as means ± SE
from three to four animals per group. Student's
t-test was used to determine differences from control values. Values with
P Mucin mRNA expression in FVB/N mice.
Acrolein exposure increased Muc5ac steady-state mRNA levels in a
time-dependent manner (Fig. 1). No change
occurred through the first week of exposure. However, after 2 wk of
exposure, Muc5ac levels were greater than the control level (~5-fold
increase on day 12) and increased
further after 3 wk (~10-fold increase on day
19). In contrast, no measurable Muc2 mRNA was
detected by RT-PCR in control or exposed animals (Fig.
2). This was determined after 40 cycles of
amplification with primers that generate a PCR product from mouse small
intestine (an organ with high Muc2 expression) and rat trachea RNA
samples. To confirm that the primers were amplifying Muc2 sequences in mouse tissue, the PCR product from the mouse small intestine was purified and sequenced. This sequence was determined to be 90% identical to rat Muc2. The Muc2 nucleotide sequence has been submitted to GenBank with accession number AF080584.
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
4.0 ppm acrolein develop
histological changes, including epithelial damage, mucus
hypersecretion, and bronchiolitis marked by excessive macrophage
accumulation in the airways, characteristic of obstructive airway
diseases (11, 13).
, and
macrophage-derived mucus secretagogue (MMS-68) can increase mucus
secretion and mucus gel thickness (12, 32, 34, 51).
METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
/
)] and littermate control mice
[MME(+/+)] were exposed to 3.0 ppm acrolein for 6 h/day, 5 days/wk for 3 wk. MME(
/
) mice were selected because macrophages
in these mice lack the capacity to invade the lung after tobacco smoke
exposure (18). C57BL/6J mice were used for comparison because the
knockout mice were generated on this genetic background. After
exposure, the lungs were either lavaged
(n = 4 mice/group) or frozen
(n = 4 mice/group) for mRNA isolation
and Muc5ac and
-actin mRNA quantitation. Control mice (n = 4/group) were exposed for 3 wk to
high-efficiency particle-filtered air.
70°C. To obtain tissue for histological analysis,
a cannula was inserted in the middle of the trachea, and the lung was
instilled with 10% phosphate-buffered Formalin (30 cmH2O for 1 min; Fisher). The
trachea was ligated, and the inflated lung was immersed in fixative for
24 h (7). The fixed tissues were dissected after 24 h, and midlobe
sections of the left lung were washed with phosphate-buffered saline
(PBS), dehydrated through graded ethanol solutions (30-70%), and
processed into paraffin blocks (Hypercenter XP, Shandon).
70°C until amplified by reverse transcription-polymerase chain reaction (RT-PCR). For RT-PCR, primers were generated from published sequences of mouse Muc5ac (47), rat Muc2 (38), and murine
-actin (56). Primer sequences were as follows: Muc5ac 3'
primer, 5'-AGT CTC TCT CCG CTC CTC TCA AT-3' and Muc5ac
5' primer, 5'-CAG CCG AGA GGA GGG TTT GAT CT-3'; Muc2
3' primer, 5'-CAC ACA GCG ACC TTT CTC AT-3' and Muc2
5' primer, 5'-ACC CTC CTC CTA CCA CAT TG-3'; and
-actin 3' primer, 5'-CAG GAT GGC GTG AGG GAG
AGC-3' and
-actin 5' primer, 5'-AAG GTG TGA TGG
TGG GAA TGG-3'. Transcripts were reverse transcribed in a 10-µl
mixture of 250 ng of total RNA with 25 U of Superscript II reverse
transcriptase (GIBCO BRL, Life Technologies, Grand Island, NY) in
buffer containing 10 mM dithiothreitol, 1 mM deoxynucleotide
triphosphates (Promega, Madison, WI), 10 U of RNAsin (Promega), and 0.2 µM 3' oligonucleotide primer. First-strand synthesis consisted
of primer annealing (25°C for 10 min) and template extension
(42°C for 45 min; Biometra thermocycler, Tampa, FL). Newly
synthesized cDNA was amplified by PCR in 50 µl with 0.75 U of
Taq polymerase (GIBCO BRL) in
Taq buffer (GIBCO BRL) containing 1.5 mM MgCl2 and 0.2 µM 5'
primer. The amplification conditions consisted of a 20-s period at
94°C and a 30-s period at the annealing temperature
(Ta) specific for each primer
pair followed by 30 s plus a 1-s period/cycle for n number of cycles (Muc5ac,
Ta = 56.8°C,
n = 30; Muc2,
Ta = 55°C, n = 40;
-actin,
Ta = 58°C,
n = 18). The specificity of each PCR product was confirmed by dye-terminator sequencing with an ABI Prism
cycle-sequencing kit (Perkin-Elmer, Foster City, CA).
exp(
bx)] + c, where a is the amplitude of the exponential,
b is the rate constant, and
c is the zero intercept. For each
RT-PCR, a serial dilution (1,000-32 ng) of total mouse lung mRNA
was amplified and included on each gel to obtain an internally
consistent reference curve. All samples were analyzed in the linear
portion of the curve. The relative amount of mRNA was determined by
comparing the total intensity of each sample against the standard
curve. Mucin mRNA levels are expressed as the multiple of
increase after normalization to
-actin.
70°C until assayed for elastase activity. Total cell counts
were determined with a hemocytometer. Differential cell counts (>300
cells) were performed on Diff-Quik-stained (Baxter Diagnostics, McGaw
Park, IL) cytospin slides (Cytospin3, Shandon Scientific) from 250 µl of lavage fluid.
70°C until assayed for elastase
activity. Briefly, 100 µl of lavage supernatant or human leukocyte
elastase standard (0.1 ng/ml to 10.0 µg/ml; Elastin Products,
Owensville, MO) and 100 µl of 0.2 mM substrate in Tris buffer (0.1 M
Tris, 0.5 M NaCl, and 0.01% NaN3,
pH 7.5) were incubated at 25°C for 30 min. The reaction was
terminated by the addition of 100 µl of acetic acid, and elastase activity was quantitated by measuring the release of
p-nitroanilide (absorbance at 410 nm)
with a 96-well plate reader (Bio-Tek EL309, Burlington, VT).
0.05 were considered significant.
RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
[in a new window]
Fig. 1.
Time course of Muc5ac mRNA expression in acrolein-exposed mice.
Top: densitometry values of Muc5ac
mRNA levels in FVB/N mice exposed to acrolein [3.0 parts/million
(ppm), 6 h/day, 5 days/wk]. Whole lung mRNA was isolated, and
Muc5ac levels were amplified by RT-PCR. Data were normalized to
-actin levels and are expressed as ratio of levels in exposed to
control mice (Exposed/Control). Values are means ± SE;
n = 3 animals/day.
* Significantly different from control mice,
P
0.05. Bottom: duplicate determinations of
Muc5ac RT-PCR products from control (samples
1-3) and
week 3 exposed
(samples
4-6) mouse
lungs. Amplified DNA was separated by electrophoresis on a 2% agarose
gel containing ethidium bromide, illuminated with ultraviolet light,
and photographed. Photographic images were scanned and quantified by
image-analysis software, and total intensity (average intensity × total pixels) was measured. Each pair is a sample obtained from an
individual mouse.
View larger version (34K):
[in a new window]
Fig. 2.
Muc2 mRNA expression in mouse and rat tissues. Total RNA was reverse
transcribed and amplified with Muc2 primers to generate an ~700-bp
product (nos. on left).
Lane 1: 100-bp DNA ladder;
lane 2: 1,000 ng of control mouse lung
RNA; lane 3: 1,000 ng of
day 19 acrolein-exposed FVB/N mouse
lung RNA; lane 4: 500 ng of
day 19 acrolein-exposed mouse lung
RNA; lane 5: 50 ng of mouse small
intestine RNA; lane 6: 250 ng of
control rat trachea RNA. Photograph is negative image of an ethidium
bromide-stained 2% agarose gel. Data are representative of results
obtained from 3-5 separate experiments.
Airway morphology and Muc5ac
immunostaining. Morphological changes, including
thickening of the basement membrane and a shift from cuboidal
epithelium to a more columnar surface epithelium in the conducting
airways, occurred in FVB/N mice exposed to acrolein (3.0 ppm, 3 wk;
Fig. 3, C
and D). Alcian blue
positive-staining cells were absent throughout the entire exposure
period (data not shown). Muc5ac immunoreactivity accompanied the
acrolein-induced increase in Muc5ac steady-state mRNA levels. Airway
Muc5ac staining was absent in control, day
1, and day 5 animals
but was observed in the airways of mice exposed for 12 or 19 days (Fig.
3). Muc5ac staining was localized to the apical surface of the airway
epithelium and cytoplasmic granules of airway epithelial cells.
|
Airway inflammation in FVB/N mice.
Acrolein exposure resulted in a biphasic response in inflammatory cell
infiltration in FVB/N mice (Fig. 4). The
first phase (day 1) was marked by a
decrease in the number of macrophages compared with the number of
control cells (2.5 ± 0.7 × 105 vs. 0.9 ± 0.4 × 105 cells). In addition, the
percentage of neutrophils increased (0.6 ± 0.1 vs. 6.7 ± 1.7%
of total cells recovered by lavage). The increase in neutrophils was
accompanied by a small, but not significant, increase in free elastase
activity compared with that in control cells.
|
The second phase consisted of an increase in macrophage and neutrophil
levels equivalent to the control level. By day
5, total macrophage cell counts increased significantly
over preexposure control counts and peaked at day
19 (6.1 ± 0.3 × 105 and 9.7 ± 1.2 × 105 cells, respectively; Fig. 4,
top). The increase in neutrophil percentage observed on
day 1 had decreased by
day 5 and remained low (<1%) until
the end of the experiment (Fig. 4,
middle). Elastase activity was
unchanged at all times measured (Fig. 4,
bottom). In additional tests with
C57BL/6J, MME(+/+), and MME(/
) mice, neutrophil levels
measured on the last day of a 3-wk exposure were also low (<1%) and
comparable to control values.
Strain differences in inflammation and Muc5ac mRNA
induction. Repeated exposure to acrolein caused a
significant increase in lavagable macrophages in C57BL/6J and MME(+/+)
mice but not in MME(/
) mice (Fig.
5, top).
C57BL6/J mice responded similarly to FVB/N mice (approximately a
fourfold increase) with a greater than fourfold increase in macrophages
after 3 wk of exposure (1.5 ± 0.2 × 105 vs. 7.4 ± 1.0 × 105 cells). MME(+/+) mice
exhibited a twofold increase (2.5 ± 0.5 × 105 vs. 5.5 ± 1.0 × 105 cells), and
MME(
/
) mice showed no change in lavagable macrophages.
|
The increase in Muc5ac steady-state mRNA levels in C57BL/6J mice after
acrolein exposure was similar to that of FVB/N mice (Fig. 5,
bottom). However, this response
was graduated in the same pattern as that for macrophages, with FVB/N
mice having the greatest increase in Muc5ac mRNA, followed by C57BL/6J,
MME(+/+), and MME(/
) mice. Increases in Muc5ac mRNA
coincided with an increased accumulation of macrophages in the lung
(Fig. 5).
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DISCUSSION |
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The present study demonstrates that acrolein exposure increases Muc5ac mRNA and immunoreactive product in the mouse airway epithelium. The increase in Muc5ac expression was delayed, developing over 2-3 wk of exposure. This response was associated with an increase in macrophages recovered by BAL. The magnitude of macrophage cell increase was correlated with an increase in Muc5ac mRNA levels.
Previously, MUC5ac has been found to be a major component in human airway secretion (20), and MUC5ac mRNA is distributed throughout the submucosal glands and surface epithelium (2). Induction of MUC5ac mRNA levels has been demonstrated by several investigators (6, 14, 16, 54) to coincide with airway mucous cell differentiation in mouse, rat, and human tissues. In our study, repeated exposure to 3.0 ppm acrolein caused a time-dependent increase in Muc5ac, but not in Muc2, steady-state mRNA levels. Muc5ac mRNA levels were elevated on days 12 and 19 of exposure (Fig. 1). Mucus hypersecretion has been previously reported in rats exposed to acrolein (13, 33), and this has subsequently been demonstrated to involve the induction of Muc5ac mRNA and immunoreactive protein (6). We also found Muc5ac synthesis (mRNA and immunoreactivity) to increase in mice after acrolein exposure (Figs. 1 and 3). Although the immunoreactivity with Muc5ac is positive, glycosyltransferase activities were not examined. Therefore, our observation does not rule out potential cross-reactivity with mucous carbohydrate structures, possibly induced by acrolein exposures independent of changes in Muc5ac backbone content.
Mouse Muc2 mRNA was not detectable in the lung but was readily detected with sensitive RT-PCR methods in the intestine of mice (Fig. 2). This suggests that Muc2 may be less important than Muc5ac in the early pathogenesis of airway mucus hypersecretion. Similarly, Muc2 mRNA was undetectable in differentiated rat tracheal epithelial cells in vitro nor did Muc2 mRNA increase in rat lungs after acrolein exposure (6). In rats, Muc2 mRNA was found in control animals, whereas in mice, Muc2 was undetectable even after acrolein exposure. Although Muc2 mRNA is present in human airway epithelium (24), Muc2 protein has not been detected in human respiratory secretions by biochemical or immunological methods (20, 55).
Muc5ac immunoreactivity was observed in small cytoplasmic granules of mucous cells, on apical surface epithelium, and in the airway lumen in mice exposed for 12 or 19 days (Fig. 3). No staining was observed in unexposed mice or mice exposed up to 1 wk. This suggests that epithelial cells acquire the capacity to secrete mucus during the repeated exposures. Characteristic large, apically oriented granules observed by carbohydrate staining (Alcian blue, pH 2.5) in rats after toxicant inhalation (6, 10, 17) were not observed in this study. The reasons for this difference remain to be determined but may involve species differences in both airway epithelial cell composition and inhalation dosimetry.
Cell differentiation could involve a shift from serous cells to mucous cells. In rats, time-dependent decreases in serous cells coincide with increases in mucous cells as evident by a shift in carbohydrate staining and electron densities of secretory granules (30). In the intrapulmonary airways of pathogen-free rats and mice, mucous cells are rare or absent. The rat airway epithelium consists primarily of basal and ciliated cells, with as much as 20% serous cells (26), whereas in the mouse, the primary cell types are the ciliated and Clara cells (39). In contrast to the rat, serous cells in pathogen-free mice account for <1% of the epithelium. Thus we speculate that because mice have fewer initial serous cells, few true mucous cells can develop over a short 3-wk exposure.
Differences in the histological development of mucous cells between rats and mice may also be due to differences in the amount of acrolein deposited along the airways. More acrolein may reach the intrapulmonary airways of rats compared with that in mice. Rats and mice both decrease their respiratory rate in response to acrolein exposure (4, 52). However, mice decrease their respiratory rate by 50% at a concentration six times less than that in rats (1 vs. 6 ppm). In addition, the capacity to absorb aldehydes in the upper respiratory tract (nasal passages) is greater in the mouse than in the rat (35). The differences in breathing pattern and nasal absorption could act together to diminish regional deposition of acrolein in the airways of the lower respiratory tract of the mouse.
The mechanism involved in the induction of Muc5ac message and protein is not known but is likely to involve processes mediated by the excessive accumulation of leukocytes in the airways. A significant and persistent increase in pulmonary macrophages was associated with an augmentation of Muc5ac induction. In FVB/N mice, for example, the macrophage population doubled by day 5 and quadrupled by day 19 (Fig. 4, top). Histological studies conducted in rats repeatedly exposed to acrolein also describe an increase in macrophages associated with mucus hypersecretion (29, 33). In mice exposed for 4 days to acrolein and carbon black, however, no increase in lavagable macrophages was observed (22). Monocyte inflammation has also been reported after acute O3 and SO2 exposures (45, 57) and in SO2-induced bronchitis in dogs (46). Furthermore, smokers with chronic bronchitis exhibit three- to fourfold increases in lung macrophages (5, 44).
Details regarding a causal relationship between monocytic inflammation
and increased mucin synthesis are limited. Mullen et al. (36, 37) noted
that the severity of mucus hypersecretion in patients with chronic
bronchitis was related to monocyte inflammation. This relationship is
supported by our data demonstrating that the magnitude of Muc5ac
expression is influenced by macrophage accumulation in the different
mouse strains tested (Fig. 5). FVB/N and C57BL/6 mice exhibited
comparable increases in macrophage accumulation and showed the greatest
increases in Muc5ac mRNA expression, whereas MME(+/+) mice exhibited a
smaller increase in both macrophages and Muc5ac levels. Most
strikingly, MME(/
) mice failed to accumulate additional
macrophages and subsequently showed the smallest increase in Muc5ac expression.
The differences observed between FVB/N, C57BL/6, and MME(+/+) mice in
macrophage accumulation are not surprising because strain differences
are known to exist for other inflammatory events like O3-induced neutrophil recruitment
in mice (28). Interestingly, even in the absence of monocytic
inflammation [MME(/
)], a small but
significant increase in Muc5ac expression was observed (Fig. 5). This
suggests that acrolein stimulated resident cells (epithelial cells or
macrophage) and suggests that acrolein can directly increase mucin
production. Whether MME has a role in the development of mucus
hypersecretion by direct effects on the surface epithelium remains to
be determined.
The neutrophil percentage increased in the lavage fluid of mice after day 1 of acrolein exposure but made up <1% of the total cells by day 5 (Fig. 4, middle). This peak neutrophilic response is accompanied by a small and insignificant increase in free elastase activity (Fig. 4, bottom). Although neutrophil elastase can induce mucous cell differentiation when instilled intratracheally, the amount required for this response far exceeds the amount measured after acrolein exposure. In previous elastase instillation experiments, >100 µg/25 g body wt was the threshold dose at which mucous cell differentiation occurs within 21 days (49). In our experiment, elastase levels of 0.9-1.2 µg/25 g body wt were recovered from the airways of exposed mice (Fig. 4, bottom). This suggests that neutrophil-derived elastase levels were too low to produce mucus hypersecretion. However, these data are limited in that BAL mainly samples the alveolar compartment. Higher elastase concentrations may occur focally along the airways at sites where neutrophils may accumulate. Matrix-bound elastase or elastase present at the basal surface of epithelial cells released by migrating neutrophils may be difficult to sample by BAL. Nonetheless, because little change in elastase activity was noted and the amount required for mucous cell conversion was ~100-fold greater, it seems unlikely that neutrophil influx is responsible for the induction of Muc5ac expression.
The specific mechanisms and mediators involved in mucous cell differentiation are unknown. Activated macrophages can release mediators, including cytokines, arachadonic acid metabolites, platelet-activating factor, reactive oxygen species, and MMS-68, that increase mucus secretion (12, 19, 34, 40, 51). These studies are short term (hours) and performed on cells that already have the capacity to store and secrete mucus and, therefore, may not accurately reflect the complex changes that must occur over weeks to produce mucous cell differentiation after repeated irritant exposures.
In summary, acrolein exposure induced the expression of Muc5ac mRNA levels and Muc5ac antibody staining in mouse airway epithelium that was preceded and accompanied by an increase in BAL macrophages. Furthermore, the increase in Muc5ac mRNA levels was significantly reduced in transgenic mice lacking macrophage accumulation. These findings suggest that acrolein can induce mucin synthesis by direct (chemical irritation) and indirect (monocyte-mediated) mechanisms. The latter provides a role in excessive macrophage accumulation in the augmentation of mucin synthesis and secretion in chronic obstructive pulmonary disease. Future studies on the mechanisms of Muc5ac induction in vivo and the potential role of macrophage-derived mediators could improve our understanding of the pathogenesis of airway mucus hypersecretion in obstructive pulmonary diseases.
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ACKNOWLEDGEMENTS |
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We thank Dr. Samuel Ho for the generous gift of the Muc5ac antibody and Sherri Profitt for excellent technical assistance.
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FOOTNOTES |
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This study was supported by the National Institute of Environmental Health Sciences Grants R01-ES-06562, R01-ES-06677, and P30-ES-06096 and National Heart, Lung, and Blood Institute Grant R01-HL-58275.
M. Borchers is a recipient of a University of Cincinnati (Ohio) Graduate Assistantship, and this work is in partial fulfillment of the requirements for the PhD degree at the University of Cincinnati.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. Leikauf, Dept. of Environmental Health, Univ. of Cincinnati, PO Box 670056, Cincinnati, OH 45267-0056 (E-mail: Leikaugd{at}ucmail.uc.edu).
Received 26 August 1998; accepted in final form 28 April 1999.
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