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J. Biol. Chem., Vol. 276, Issue 44, 41417-41423, November 2, 2001
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From the Departments of Pediatrics, Medicine, and Cell Biology and
Physiology, Washington University School of Medicine,
St. Louis, Missouri 63110
Received for publication, July 26, 2001, and in revised form, August 27, 2001
Matrilysin (matrix metalloproteinase-7) is
expressed by mucosal epithelia throughout the body and functions in
host defense by activating murine intestinal Despite their specialization to serve distinct functions in
different tissues, mucosal epithelia share common features in their
response to injury and infection. Following injury, epithelial cells
initiate a programmed series of coordinated responses, such as
proliferation, migration, and matrix assembly, to restore the integrity
of the damaged tissue. In addition, by forming a barrier at mucosal
surfaces and by the release of a variety of antimicrobial products,
epithelia provide the first line of innate defense against invading
pathogens. Although seemingly divergent events, the epithelial programs
in response to infection and injury may have co-evolved, particularly
with respect to the genes selectively induced and repressed. After all,
injury provides an opportunity for infection, and infection can lead to
injury. Hence, many of the epithelial products associated with either
of these events may actually be common to both. Matrilysin (matrix
metalloproteinase (MMP)1-7),
a member of the MMP gene family produced primarily by mucosal epithelia, is an example of a protein that is regulated by both infection and injury and serves essential functions in both innate defense and re-epithelialization.
As we demonstrated in an aseptic ex vivo model of airway
damage, matrilysin is prominently expressed by migrating epithelial cells in wounded trachea, and re-epithelialization of the injured airway is markedly delayed in matrilysin-null mice (1). Furthermore, as
discussed below and the subject of this report, the presence of
bacteria, either commensal or pathogenic, is also a potent effector of
matrilysin expression, and we hypothesize that the activity of this
enzyme may be a common and essential component of mucosal defense
mechanisms. For example, intestinal pro- Unlike many MMPs, which are typically expressed or released in response
to injury, disease, or inflammation, matrilysin is expressed by
non-injured, non-inflamed exocrine and mucosal epithelia in most adult
human tissues. For example, matrilysin is produced by the ductal or
glandular epithelia of the skin, salivary glands, pancreas, liver,
breast, intestine, urogenital tract, and other tissues (3-6). We
proposed that exposure to commensal bacteria (and possibly low-level
infection) regulates the widespread expression of matrilysin in healthy
mucosa (7). Indeed, in mice with conventional microflora, matrilysin is
prominently expressed in the Paneth cells of the small intestine, but
it is nearly undetectable in germ-free mice. Expression of this MMP,
however, is fully restored in ex-germ-free mice colonized with just one
species of commensal bacteria (7). In addition, matrilysin expression
in cultured cells and tissues is, depending on its basal levels,
induced or markedly increased by exposure to several strains of
pathogenic Escherichia coli (7). Thus, bacterial exposure
seems to be the physiologic signal that regulates matrilysin expression
in intact epithelia. However, the bacterially derived signal that regulates matrilysin expression is not known.
Lungs of patients with cystic fibrosis (CF) are normal in
utero, but become infected shortly after birth with a number of microorganisms, including Pseudomonas aeruginosa,
Staphylococcus aureus, Haemophilus influenzae,
and Klebsiella pneumoniae. Infection leads to the
development of chronic inflammation, which, in turn, contributes to
further tissue destruction and airway obstruction. Among these
bacteria, P. aeruginosa is an opportunistic microorganism recovered from the airways of most patients with CF and many with impaired host defense. Eventually, >85% of CF patients become colonized with P. aeruginosa (8, 9), an infection that is extremely resistant to eradication (10, 11). The initial step of
bacterial infection, crucial for the development of permanent colonization at later stages, is the adherence of the bacteria to
epithelial cells.
In lungs from patients with CF, matrilysin is prominently expressed by
airway and alveolar epithelial cells (1), and here we assessed if any
of the bacterial species frequently isolated from CF patients mediates
the marked up-regulation of this MMP. Our data demonstrate that
exposure to P. aeruginosa, but not other respiratory
pathogens, is a potent stimulus of matrilysin expression in lung
epithelial cells. A number of bacterial gene products stimulate
pro-inflammatory responses in epithelial cells, and we identified
P. aeruginosa flagellin, the monomeric component of
flagella, as the factor controlling matrilysin expression. Our data
demonstrate that the production of this defense-related metalloproteinase is specifically regulated in airway epithelial cells
by a bacterial product.
Tissue Samples and Cell Culture--
Specimens of recipient
lungs from patients with CF were biopsied at the time of transplant
surgery, and Formalin-fixed, paraffin-embedded samples
(n = 16) were obtained from the Department of Pathology of Washington University (St. Louis, MO) and from Dr. James R. Yankaskas (University of North Carolina). For normal lung, we examined
the tumor-free margins of lung adenocarcinoma tissue (n = 5), recipient lungs of transplant patients with primary pulmonary hypertension (n = 6), and segments of the proximal end
of normal human trachea obtained from donor lungs (n = 7). The human colon adenocarcinoma cell line HT29 and the human lung
carcinoma cell line Calu-3 were obtained from American Type Culture
Collection (Manassas, VA). These cell lines were maintained in RPMI
1640 medium supplemented with 10% fetal bovine serum without antibiotics.
Bacteria and Other Reagents--
The recombinant E. coli strain AAEC185/pSH2 expresses the complete type 1 pilus operon (type 1+/fimH+) (12)
and was provided by Dr. Scott Hultgren (Washington University). K. pneumoniae strain KPA1 and S. aureus
were provided by Dr. Azzaq Belaaouaj (Washington University). H. influenzae was provided by Dr. Joseph St. Geme III (Washington
University). P. aeruginosa strains 51673 and 39324 (CF
isolates) and strains 10145 and 25619 (laboratory strains) were
obtained from American Type Culture Collection. P. aeruginosa strains PAK and PAK-NP were provided by Dr. Thomas
Ferkol (Washington University). The flagellin mutants PAK-fliC and PAK-NP-fliC and the anti-flagellin
antibody were provided by Dr. Alice Prince (Columbia University, New
York, NY). Bacteria were grown at 37 °C in Luria-Bertani medium
(E. coli and K. pneumoniae), 3% tryptic soy
broth (P. aeruginosa and S. aureus), and
brain/heart infusion (H. influenzae). Gentamycin and
P. aeruginosa (serotype 10) lipopolysaccharide were obtained from Sigma.
Immunohistochemistry and in Situ Hybridization--
Anti-human
matrilysin antiserum was raised in rabbits against a synthetic peptide
corresponding to amino acids 93-108 (13) and affinity-purified as
described (1). Deparaffinized 5-µm sections were processed for
immunohistochemistry using alkaline phosphatase or horseradish
peroxidase as described (1) and were counterstained with Harris
hematoxylin. In vitro transcribed antisense and sense RNA
probes for matrilysin, collagenase-1, stromelysin-1, and 92-kDa
gelatinase were labeled with [ Infection Protocol and Analyses--
Human epithelial cells were
seeded onto six-well plates and grown to ~80% confluency. Inoculi of
bacteria, ranging from 109 to 106
colony-forming units/well, were added to eukaryotic cells in 1 ml of
RPMI 1640 medium and 10% fetal bovine serum and incubated for up to 90 min. Epithelial monolayers were then washed extensively with
phosphate-buffered saline to remove non-adherent bacteria, and the
cultures were further incubated in RPMI 1640 medium and 10% fetal
bovine serum supplemented with 50 µg/ml gentamycin to kill the
remaining extracellular bacteria. At different times post-infection,
total RNA from the cells was prepared with RNAzol (Tel-Text, Inc.,
Friendswood, TX). In experiments performed in the presence of 5%
dialyzed fetal bovine serum, cells were metabolically labeled with
Tran35S-label (ICN, Costa Mesa, CA) for 6 or 24 h, and
conditioned media were collected and analyzed by immunoprecipitation
with specific anti-matrilysin antiserum (13). Northern hybridization
for matrilysin, collagenase-1, MT1-MMP, and GAPDH mRNAs and
gelatin zymography of conditioned media were done as described (7).
Purification of Flagellin--
P. aeruginosa
flagellin was purified as described (16), with some modifications. A
400-ml sample of a 24-h culture of P. aeruginosa ATCC 51673 or 10145 was centrifuged at 10,000 × g for 15 min.
Over a period of 2 h at room temperature, ammonium sulfate was
added to the supernatant to reach 40% saturation. After centrifugation at 20,000 × g for 40 min, the pellets were resuspended
in 8 ml of 50 mM Tris-HCl, pH 9.5, containing 0.5 mM dithiothreitol and dialyzed extensively against the same
buffer. Flagellin was collected by centrifugation at 27,000 × g for 40 min; resuspended in 2 ml of 50 mM
Tris-HCl, pH 7.2, and 0.5 mM dithiothreitol; and analyzed by SDS-polyacrylamide gel electrophoresis on 12% acrylamide gels.
Immunoblotting--
Media samples from the 90-min period of
infection of the epithelial cells were concentrated 28-fold by
lyophilization. Aliquots of concentrated media were separated by 12%
SDS-polyacrylamide gel electrophoresis and transferred by semidry
electrophoretic transfer at 15 V for 20 min to nitrocellulose membranes
(Hybond ECL, Amersham Pharmacia Biotech, Buckinghamshire, United
Kingdom) in 48 mM Tris, 39 mM glycine, 20%
methanol, and 0.0375% SDS. Nonspecific binding sites were blocked by
soaking membranes in 3% nonfat dry milk in Tris-buffered saline at
4 °C overnight. Blots were incubated with a 1:10,000 dilution of
anti-flagellin polyclonal antiserum (17) in blocking buffer for 1 h and washed twice with Tris-buffered saline containing 0.1% Tween 20 for 10 min. Membranes were subsequently incubated with a 1:10,000
dilution of peroxide-linked donkey anti-rabbit IgG (Amersham Pharmacia
Biotech) in blocking buffer for 1 h, washed twice, and developed
with the enhanced chemiluminescence system (Amersham Pharmacia Biotech)
according to the manufacturer's instructions.
In Vivo Infection and Reverse Transcription-Polymerase Chain
Reaction--
C57BL/6 mice (10-12 weeks old) were anesthetized and
inoculated nasally with 50 µl of sterile saline or a suspension
containing 108 colony-forming units of P. aeruginosa PAK or PAK-fliC. Mice (four/group) were
killed 6 h later, and total lung RNA was isolated. Matrilysin mRNA was detected by reverse transcription-polymerase chain
reaction as described (6) using primers MMat405
(5'-ACTTACCTCGGATCGTAGTG-3') and MMat645 (5'-GTCCAGTACTCATCTTTGTC-3')
and an annealing temperature of 55 °C for 25 cycles. Mouse GAPDH was
amplified in a separate reaction using primers MGapdh205
(5'-ATTCAACGGCACAGTCAAGG-3') and MGapdh859
(5'-GGTCCTCAAGTGTAGCCCAAG-3'). Amplification products were resolved on
8% acrylamide gels and visualized by ethidium bromide staining.
We previously reported that matrilysin is induced in airway
epithelial cells migrating over denuded tissue in an aseptic ex vivo model of tracheal injury (1) and in airway cells of intact trachea infected with pathogenic strains of E. coli (7).
These findings indicate that the expression of matrilysin is regulated by both infection and injury. We also reported that matrilysin is
expressed at high levels by airway epithelial cells at sites of overt
mucosal damage in CF (1).
In a further examination of several CF samples (n = 16), we observed that matrilysin protein was uniformly detected in the intact epithelia of trachea (Fig. 1,
A and B), bronchioles (Fig. 1, E and
G), and alveoli (Fig. 1K). No staining was seen
in samples processed with preimmune IgG (Fig. 1C). In
addition, a moderate to intense signal for matrilysin mRNA
colocalized with cells that had strong staining for the protein (Fig.
1, E and F, G and H), indicating that this MMP was actively expressed in the airway epithelia
of CF lungs. In contrast, matrilysin was typically not seen in the
intact airway epithelia of non-CF lung samples (Fig. 1D),
except in epithelial cells at the edge of wounded mucosa, if
such lesions were present. In several of the non-CF samples (8 out of
18), matrilysin protein was seen in intact epithelium (Fig.
1I); however, signal for the mRNA was not detected, even after extended autoradiographic exposure (Fig. 1J). Intense
staining for matrilysin protein was also seen in intact and wound-edge alveolar type II cells in CF lungs (Fig. 1K), but only a
weak immunostaining signal was detected in alveolar cells bordering sites of denudation in non-CF specimens (Fig. 1L,
arrowheads). A strong signal for matrilysin protein and
mRNA was also seen in the ductal cells of peritracheal and
peribronchial glands in CF samples, but not in non-CF specimens (data
not shown). Signal for collagenase-1 (MMP-1), stromelysin-1 (MMP-3), or
gelatinase-B (MMP-9) mRNA or protein was not detected in the
epithelial layer of CF or non-CF samples (data not shown). Together,
these in vivo observations demonstrate that expression of
matrilysin is potently and invariantly up-regulated in the intact
airway epithelia of CF lungs.
To determine whether infection directly mediates the up-regulation of
matrilysin seen in CF lung tissues, we infected epithelial cells with
various CF pathogens. Inoculi containing 1 × 108
colony-forming units of bacteria, representing a 50:1
bacterial/epithelial cell ratio, were exposed to cells for 90 min. To
eliminate bacteria, monolayers were subsequently washed with
phosphate-buffered saline and treated with gentamycin. Total RNA was
isolated from the cells at 6 h post-infection and analyzed by
Northern blotting. As a positive control for the bacterially mediated
induction of matrilysin expression, we infected cells with a
recombinant E. coli strain engineered to produce type 1 pili
(12), which potently induces matrilysin expression in human mucosal
epithelial cells (7). Infection with a P. aeruginosa strain
isolated from a CF patient (ATCC 51673) markedly stimulated matrilysin
expression in Calu-3 lung carcinoma (30-fold) and HT29 colon carcinoma
(6-fold) epithelial cells (Fig.
2A). P. aeruginosa
was a more potent stimulator of matrilysin expression than E. coli in lung-derived cells (30-fold versus 10-fold,
respectively). In contrast, infection at a comparable multiplicity of
infection (m.o.i.) with S. aureus, H. influenzae, and K. pneumoniae had no effect on matrilysin mRNA
levels (data not shown). The up-regulation of matrilysin mRNA
levels correlated with increased amounts of matrilysin protein, as
detected by immunoprecipitation of the enzyme from the 24-h conditioned
media of infected cells (Fig. 2B).
Regulation of Matrilysin Expression in Airway
Epithelial Cells by Pseudomonas aeruginosa Flagellin*
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-defensins. In normal
adult human lung, matrilysin is expressed at low levels in the airway
epithelium, but is markedly up-regulated in cystic fibrosis (CF).
Because CF lungs support a heavy bacterial load, we assessed if
relevant CF pathogens regulate matrilysin expression in human lung
epithelial cells. Indeed, acute infection with Pseudomonas
aeruginosa (but not Staphylococcus aureus,
Haemophilus influenzae, or Klebsiella pneumoniae) induced the expression of matrilysin in Calu-3 lung epithelial cells. Increased matrilysin mRNA levels were detectable at 3 h post-infection and peaked at a 25-fold induction between 6 and 8 h. Both P. aeruginosa CF isolates and laboratory
strains induced matrilysin expression to similar levels. Flagellin, the monomeric precursor of bacterial flagella, was identified as the inductive factor released by P. aeruginosa that regulated
matrilysin expression. In addition, flagellin-null mutants failed to
stimulate matrilysin expression in cultured cells or in lungs infected
in vivo. These data show that P. aeruginosa
(and specifically flagellin) potently stimulates matrilysin expression
in lung epithelial cells and may mediate the overexpression of this
proteinase in CF lungs.
INTRODUCTION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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-defensins, demonstrated
substrates of this MMP, are not activated in matrilysin-null mice,
leading to an impaired ability to battle enteric pathogens (2).
Collectively, our functional data indicate that the catalytic activity
of matrilysin is an essential component of both mucosal defense and repair.
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-35S]UTP as described
(14). Sections were hybridized and washed as described (15). After
autoradiography for 10-21 days, the photographic emulsion was
developed, and the slides were stained with hematoxylin/eosin. In
situ hybridization experiments were done at least twice.
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DISCUSSION
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View larger version (120K):
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Fig. 1.
Up-regulation of matrilysin in cystic
fibrosis. A and B, in tracheal
specimens from CF lungs, strong immunostaining (alkaline phosphatase;
red precipitant) for matrilysin protein was seen in ciliated
cells in the intact epithelium. Staining for matrilysin was often most
intense at the apical edge of the airway epithelium. Mucous-containing
cells did not stain for the protein, and signal was not seen in any
interstitial or inflammatory cells. Shown are tracheal sections from
two different specimens. C, no signal was seen in sections
processed with preimmune serum. Shown is a section serial to that in
B. D, in sections of normal trachea from donor
lungs, signal for matrilysin protein was typically not seen in the
intact mucosa, but was invariably seen in epithelial cells at the edge
of denuded areas. E, in CF samples, strong staining
(peroxidase immunostaining; brown precipitant) for
matrilysin protein was also seen in the intact epithelium of distal
bronchioles. F, in situ hybridization was done on
a section serial to that in E. Under dark-field
illumination, a strong signal for matrilysin mRNA was seen in most
airway epithelial cells. Autoradiographic exposure was for 10 days.
G and H, in this section of CF lung, a collapsed
airway was seen surrounded by a dense inflammatory infiltrate. A
prominent signal for matrilysin protein (G) and mRNA
(H) colocalized to the airway epithelium. Autoradiographic
exposure was for 10 days. I, in lung from a patient with
primary pulmonary hypertension, moderate staining for matrilysin
protein was seen in the bronchiolar epithelium. J, in
situ hybridization for matrilysin mRNA was done on a serial
section. No specific autoradiographic signal was seen, even after an
extended autoradiographic exposure of 21 days. K, in CF
samples, a prominent signal for matrilysin protein was seen in
essentially all alveolar epithelial cells, whether in intact areas or
at the edge of denuded tissue. L, in peripheral lung from
non-CF specimens, weak staining for matrilysin was seen only in
occasional alveoli cells typically at sites with overt epithelial
denudation (arrowheads). Shown is a section of lung from a
patient with primary pulmonary hypertension. With the exception of the
dark-field micrographs, all samples were photographed using Nomarski
optics.
View larger version (49K):
[in a new window]
Fig. 2.
P. aeruginosa-mediated induction
of matrilysin. A, human HT29 (colon) and Calu-3 (lung)
carcinoma epithelial cells were infected for 90 min with type 1 piliated E. coli (Ec) or P. aeruginosa
(Pa; ATCC 51673) at an m.o.i. of 50. Monolayers were then
washed with phosphate-buffered saline and subsequently incubated for
6 h in fresh medium containing antibiotics. Northern blot analysis
was done for matrilysin (MAT) and GAPDH mRNAs.
B, Calu-3 lung epithelial cells were infected with P. aeruginosa 51673 at different m.o.i. values and metabolically
labeled during a 24-h post-infection period. Matrilysin was
immunoprecipitated from the conditioned media with specific antibodies,
and the complexes were resolved on 12% SDS-polyacrylamide gels. The
pro form of the enzyme, which was selectively immunoprecipitated,
migrated at ~28 kDa. Molecular mass standards are shown on the left
in kilodaltons. Autoradiography was done for 72 h. C,
Calu-3 lung epithelial cells were infected for 90 min with P. aeruginosa 51673 at the indicated m.o.i. values. Total RNA was
isolated at 6 h post-infection, and Northern blotting was done
with matrilysin and GAPDH. D, Calu-3 cells were infected for
90 min, and total RNA was prepared at the indicated times
post-infection. Autoradiograms were scanned, and the densitometric
signals for matrilysin mRNA were normalized to those for GAPDH
mRNA. The results are expressed relative to matrilysin levels in
control cells at each time point. E, monolayers of Calu-3
cells were infected for 90 min with P. aeruginosa at an
m.o.i. of 50. P. aeruginosa 51673 and 39324 are strains
isolated from CF patients. P. aeruginosa 10145 and 25619 are
laboratory strains. Total RNA was prepared from the cells at 6 h
post-infection, and Northern analysis was done for matrilysin and GAPDH
mRNA.
We infected Calu-3 lung cells for 90 min with increasing m.o.i. values of P. aeruginosa and assessed the response at different times post-infection. Even with an initial inoculum of 1 × 107 bacteria (representing a 5:1 bacteria/epithelial cell ratio), matrilysin mRNA levels were elevated 5-fold at 6 h post-infection (Fig. 2C). Up-regulation of matrilysin expression was observed as early as 3 h post-infection and peaked at 6 h; the levels of matrilysin mRNA remained elevated at 72 h post-infection (Fig. 2D). A similar time course of bacterially mediated induction of matrilysin mRNA levels was seen in HT29 colon epithelial cells (data not shown).
We assessed the response of lung epithelial cells to infection with a variety of P. aeruginosa strains to determine whether the regulation of matrilysin is affected by the pathogenicity of the bacteria. Infection with either P. aeruginosa CF isolates (ATCC 51673 and 39324) or with laboratory strains (ATCC 10145 and 25619) markedly stimulated matrilysin mRNA levels (Fig. 2E). Similarly, matrilysin expression was also induced in HT29 colon epithelial cells infected with these strains (data not shown). Of the other MMPs we examined, we did not detect expression of collagenase-1 or any change in the levels of MT1-MMP mRNA (see Fig. 4E). In addition, as assessed by substrate zymography, infection with P. aeruginosa did not affect the levels of gelatinases A (MMP-2) and B (MMP-9) (data not shown).
To assess the role of bacterial adherence, we examined the effect of
type IV pili, which mediate the adherence of P. aeruginosa to eukaryotic cells (18). Calu-3 cells were infected with a wild-type
strain (PAK) or with an isogenic mutant lacking the PilA subunit
(PAK-NP). This mutant does not produce pili and hence adheres poorly to
eukaryotic cells compared with the parental strain (17). However,
matrilysin expression was stimulated to similar levels in cells
infected with either strain (Fig.
3A). Because adherence of
P. aeruginosa was not critical for matrilysin expression, we
searched for preformed soluble bacterial factors that mediate induction
of this MMP. Preparations of lipopolysaccharide (LPS) from P. aeruginosa failed to induce matrilysin in Calu-3 lung cells (Fig.
3B). In addition, the presence of gentamycin during the
90-min infection period did not prevent induction (Fig. 3B),
and matrilysin expression was also induced by infection with heat-killed bacteria (data not shown). Thus, bacteria do not need to be
metabolically active to influence matrilysin expression.
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We next investigated the effect of proteins released by P. aeruginosa on matrilysin expression in lung epithelial cells.
Proteins in the supernatants of 1- and 2-day cultures were concentrated by ammonium sulfate precipitation and analyzed by SDS-polyacrylamide gel electrophoresis. The pattern of proteins secreted by the bacterial cells was overtly different from the pattern seen in cell lysates (Fig.
4A). Treatment of Calu-3 cells
with concentrated supernatants from either 1- or 2-day cultures
resulted in similar induction of matrilysin (data not shown). Boiling
the supernatants for 20 min did not prevent matrilysin induction, but
proteinase K treatment obliterated the inductive effect (data not
shown). Sequencing of the most prominent band (migrating at 55 kDa)
identified this protein as flagellin, the monomeric component of
bacterial flagella (19). Flagellin was easily purified to homogeneity
from 1-day culture supernatants of P. aeruginosa 51673 (CF
isolate) and 10145 (laboratory strain) by 40% ammonium sulfate
precipitation (Fig. 4, A and B). As demonstrated
by immunoblotting (Fig. 4C) and by Coomassie Blue staining
(data not shown), flagellin was secreted by the bacteria in a time- and
dose-dependent manner during the infection period.
Treatment of Calu-3 cells with 0.2 µg/ml purified flagellin for
6 h resulted in a 12-fold induction of matrilysin mRNA levels
(Fig. 4D). Based on the purification yield and the comparison of band staining intensities by Western blotting and protein
staining, 0.2 µg/ml flagellin was roughly equivalent to direct
infection at an m.o.i. of 200. Furthermore, the induction of matrilysin
obtained with purified flagellin was comparable to that seen with
intact bacteria (Fig. 3; see Fig. 5 below), suggesting that flagellin
is the major bacterial factor regulating expression of MMP. As we found
for infection with P. aeruginosa, flagellin did not affect
expression of MT1-MMP, collagenase-1 (Fig. 4E), and
gelatinases A (MMP-2) and B (MMP-9) (data not shown).
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To corroborate further that flagellin is the bacterial factor
responsible for P. aeruginosa-mediated induction of
matrilysin, we infected Calu-3 cells with mutants of fliC,
the gene that encodes flagellin (16). Exposure for 90 min to the mutant
strains PAK-fliC and PAK-NP-fliC, in which an
interrupted copy of the gene has replaced the wild-type fliC
sequence (17), did not affect matrilysin mRNA levels, whereas
infection with the wild-type strain PAK and the pilin mutant PAK-NP
resulted in a 10-fold induction (Fig. 5A). In addition, we prepared
crude supernatants (i.e. 40% ammonium sulfate-precipitated
proteins from the 2-day culture supernatants) from the wild-type PAK
strain and the isogenic flagellin PAK-fliC mutant. The
protein patterns from these supernatants differed only in the presence
or absence of flagellin (Fig. 5B). Treatment of Calu-3 cells
with PAK (but not PAK-fliC) supernatants up-regulated matrilysin expression by 8-fold (Fig. 5C), further
identifying flagellin as the major factor that promotes matrilysin
expression.
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Finally, to determine whether flagellin is relevant to the induction of
matrilysin expression in vivo, we infected mice intranasally with wild-type P. aeruginosa (PAK) or with the
PAK-fliC mutant. Mice were killed 6 h post-infection,
and matrilysin mRNA expression was examined in lung tissues.
Reverse transcription-polymerase chain reaction showed that matrilysin
mRNA levels increased in PAK-infected animals compared with the
levels seen in control mice (Fig. 6). In
contrast, infection with the mutant PAK-fliC strain had no
detectable effect on matrilysin mRNA levels.
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We have demonstrated that matrilysin is prominently and invariantly expressed in the airway and respiratory epithelia of lungs from patients with CF and that exposure of lung epithelial cells to P. aeruginosa, a relevant CF pathogen, markedly stimulates the expression of this MMP. In contrast, S. aureus, H. influenzae, and K. pneumoniae, other pathogens found in CF lungs, did not influence matrilysin expression. The increase in matrilysin mRNA levels was rapid and detectable at low m.o.i., suggesting that epithelial cells readily respond to bacterial exposure by up-regulating this MMP. Furthermore, the specificity of the response to P. aeruginosa suggests that this bacterium selectively triggers signaling pathways that control matrilysin expression in epithelial cells. Because most adult CF patients are permanently colonized with one or more P. aeruginosa strains, these findings may explain the pattern of increased expression of matrilysin we observed in CF lung tissues (Fig. 1).
The pathogenesis of the airway infection in CF follows a pattern in which initial colonization by non-mucoid variants of P. aeruginosa, often combined with more transient infections with H. influenzae, K. pneumoniae, and S. aureus, almost invariably results in chronic colonization by mucoid variants of P. aeruginosa (10). Our results show that both CF isolates and non-CF strains of P. aeruginosa induce the expression of matrilysin to comparable levels, suggesting that the up-regulation of matrilysin could occur early in the establishment of the disease. Indeed, we found that a short infection led to rapid and sustained expression. In addition, the marked sensitivity of lung epithelial cells to P. aeruginosa exposure indicates that even low levels of initial colonization by the bacterium could turn on the expression of the enzyme. However, it is currently unclear whether matrilysin is initially engaged in tissue repair and innate defense mechanisms or whether the enzyme contributes to tissue damage from the onset of the infection, further facilitating the adherence of P. aeruginosa to injured tissue (20).
The adherence of P. aeruginosa to lung epithelial cells depends on the expression of type IV pili (21), and mutants of the pilin subunit gene pilA induce a diminished inflammatory response (22). Our data demonstrate that the lack of PilA did not affect matrilysin induction by P. aeruginosa. Therefore, we hypothesized that a preformed soluble bacterial factor was responsible for the induction of matrilysin expression. Indeed, we found that a factor was present in the supernatant of overnight bacterial cultures and that it was heat-stable and sensitive to proteinase degradation. We identified flagellin, the monomeric component of bacterial flagella (16, 23), as the secreted protein that induced matrilysin expression. Similarly, flagellin from Salmonella, enteroaggregative E. coli, and Pseudomonas species induces a program of epithelial cell activation and is a potent pro-inflammatory mediator in intestinal and lung epithelial cells (17, 24-27). Although adherence may not be directly critical for induction of matrilysin, binding of bacteria to tissue could potentiate the host response signal by increasing the pericellular bacterial density and hence the concentration of flagellin at the epithelial cell surface.
Previous reports suggested that bacterial soluble virulence factors can
induce MMP activation or expression (28, 29); however, to our
knowledge, this is the first example of flagellin up-regulating the
expression of an MMP. In this regard, P. aeruginosa LPS had
no significant effect on matrilysin expression, corroborating our
previous finding that the factor responsible for the E. coli-mediated up-regulation of matrilysin in colon epithelial
cells is not LPS-related (7). LPS can induce the expression of genes
encoding innate defense molecules in airway epithelial cells, such as
the mucin muc2 (30). However, human BD-2, an
inducible 
-defensin, does not respond strongly to LPS in a variety
of epithelial cells, but is up-regulated by bacteria, interleukin-1,
and other pro-inflammatory mediators (31-33). As we reported (7),
matrilysin is induced in epithelial cells treated with
interleukin-1, tumor necrosis factor-, and interleukin-6, albeit
to a much lesser extent than in infected cells. Thus, matrilysin and
human BD-2 may share common regulatory mechanisms and respond to
similar stimuli. Indeed, expression of human BD-2 in human colon
epithelial cells is induced by Salmonella enteritidis
flagellin (34). Insight into the response of respiratory epithelial
cells to airway pathogens has recently been provided by microarray
analysis of genes differentially expressed after infection with
P. aeruginosa and Bordetella pertussis,
illustrating the potent pro-inflammatory response mounted by epithelial
cells upon interaction with bacteria (35, 36). Therefore, our data include matrilysin in the panoply of genes induced early and strongly by bacterial exposure. (Incidentally, matrilysin cDNA is not
included in the arrays used in these studies).
Finally, it is tempting to speculate that matrilysin and other innate
defense molecules, such as mucins and defensins, are part of a general
response of epithelial cells to bacterial exposure and that these
molecules play pivotal roles in host defense. However, high levels of
production or activity of these molecules can be detrimental and could
be major contributors to the processes of tissue destruction and airway
obstruction that eventually kill CF patients. For example, a high level
of activity of matrilysin, an MMP capable of acting on a variety of
extracellular matrix molecules, could result in the exaggerated
degradation of any of these substrates and alterations in tissue
architecture. Similarly, an excessive production of mucins causes
clogging of the CF airways due to the accumulation of material that
cannot be efficiently removed by the mucociliary clearance mechanism.
Finally, the concentration of defensins in CF sputum/fluid could be so
high as to be cytotoxic to airway epithelial cells (37-39). In sum, we
provide evidence that flagellin, a potent virulence factor, strongly
up-regulates the expression of matrilysin in lung epithelial cells and
that this host enzyme is an early marker of bacterially induced
inflammation. Work in progress aims to elucidate the role of matrilysin
activity in the relationship between injury and repair in the
pathogenesis of cystic fibrosis.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Drs. Scott Hultgren, Azzaq Belaaouaj, Joseph St. Geme III, and Thomas Ferkol for providing bacterial strains; Dr. Alice Prince for providing bacterial strains and anti-flagellin antibody; and Dr. James R. Yankaskas for providing sections from human tissue samples. We also thank Dr. Ulpu Saarialho-Kere and Jill Roby for the immunohistochemical and in situ hybridization analyses and Dr. Robert Mecham and Thomas Broekelmann for protein sequencing.
| |
FOOTNOTES |
|---|
* This work was supported by National Institutes of Health Grants HL54619 and HL29594 (to W. C. P.) and DE14040 (to C. L. W.).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.
To whom correspondence should be addressed: Dept. of Pediatrics,
Campus Box 8208, Washington University, 660 S. Euclid Ave., St. Louis,
MO 63110. Tel.: 314-286-2862; Fax: 314-286-2894; E-mail: parks_w@kids.wustl.edu.
Published, JBC Papers in Press, August 29, 2001, DOI 10.1074/jbc.M107121200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: MMP, matrix metalloproteinase; CF, cystic fibrosis; MT1, membrane type 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; m.o.i., multiplicity of infection; LPS, lipopolysaccharide.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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| 1. | Dunsmore, S. E., Saarialho-Kere, U. K., Roby, J. D., Wilson, C. L., Matrisian, L. M., Welgus, H. G., and Parks, W. C. (1998) J. Clin. Invest. 102, 1321-1331 |
| 2. | Wilson, C. L., Ouellette, A. J., Satchell, D. P., Ayabe, T., López-Boado, Y. S., Stratman, J. L., Hultgren, S. J., Matrisian, L. M., and Parks, W. C. (1999) Science 286, 113-117 |
| 3. | Wilson, M. J., Garcia, B., Woodson, M., and Sinha, A. A. (1992) Biol. Reprod. 47, 683-691 |
| 4. | Rodgers, W. H., Matrisian, L. M., Giudice, L. C., Dsupin, B., Cannon, P., Svitek, C., Gorstein, F., and Osteen, K. G. (1994) J. Clin. Invest. 94, 946-953 |
| 5. | Saarialho-Kere, U. K., Crouch, E. C., and Parks, W. C. (1995) J. Invest. Dermatol. 105, 190-196 |
| 6. | Wilson, C. L., Heppner, K. J., Rudolph, L. A., and Matrisian, L. M. (1995) Mol. Biol. Cell 6, 851-869 |
| 7. | López-Boado, Y. S., Wilson, C. L., Hooper, L. V., Gordon, J. I., Hultgren, S. J., and Parks, W. C. (2000) J. Cell Biol. 148, 1305-1315 |
| 8. | Pier, G. B., Grout, M., Zaidi, T. S., and Goldberg, J. B. (1996) Am. J. Respir. Crit. Care Med. 154, S175-S182 |
| 9. | Bals, R., Weiner, D. J., and Wilson, J. M. (1999) J. Clin. Invest. 103, 303-307 |
| 10. | Govan, J. R., and Deretic, V. (1996) Microbiol. Rev. 60, 539-574 |
| 11. | Singh, P. K., Schaefer, A. L., Parsek, M. R., Moninger, T. O., Welsh, M. J., and Greenberg, E. P. (2000) Nature 407, 762-764 |
| 12. | Langermann, S., Palaszynski, S., Barnhart, M., Auguste, G., Pinkner, J. S., Burlein, J., Barren, P., Koenig, S., Leath, S., Jones, C. H., and Hultgren, S. J. (1997) Science 276, 607-611 |
| 13. | Busiek, D. F., Ross, F. P., McDonnell, S., Murphy, G., Matrisian, L. M., and Welgus, H. G. (1992) J. Biol. Chem. 267, 9087-9092 |
| 14. | Saarialho-Kere, U. K., Kovacs, S. O., Pentland, A. P., Olerud, J., Welgus, H. G., and Parks, W. C. (1993) J. Clin. Invest. 92, 2858-2866 |
| 15. | Prosser, I. W., Stenmark, K. R., Suthar, M., Crouch, E. C., Mecham, R. P., and Parks, W. C. (1989) Am. J. Pathol. 135, 1073-1088 |
| 16. | Brimer, C. D., and Montie, T. C. (1998) J. Bacteriol. 180, 3209-3217 |
| 17. | Feldman, M., Bryan, R., Rajan, S., Scheffler, L., Brunnert, S., Tang, H., and Prince, A. (1998) Infect. Immun. 66, 43-51 |
| 18. | Comolli, J. C., Waite, L. L., Mostov, K. E., and Engel, J. N. (1999) Infect. Immun. 67, 3207-3214 |
| 19. | Totten, P. A., and Lory, S. (1990) J. Bacteriol. 172, 7188-7199 |
| 20. | de Bentzmann, S., Plotkowski, C., and Puchelle, E. (1996) Am. J. Respir. Crit. Care Med. 154, S155-S162 |
| 21. | Strom, M. S., and Lory, S. (1993) Annu. Rev. Microbiol. 47, 565-596 |
| 22. | Tang, H., Kays, M., and Prince, A. (1995) Infect. Immun. 63, 1278-1285 |
| 23. | Spangenberg, C., Heuer, T., Burger, C., and Tummler, B. (1996) FEBS Lett. 396, 213-217 |
| 24. | DiMango, E., Zar, H. J., Bryan, R., and Prince, A. (1995) J. Clin. Invest. 96, 2204-2210 |
| 25. | Steiner, T. S., Nataro, J. P., Poteet-Smith, C. E., Smith, J. A., and Guerrant, R. L. (2000) J. Clin. Invest. 105, 1769-1777 |
| 26. | Gewirtz, A. T., Simon, P. O., Schmitt, C. K., Taylor, L. J., Hagedorn, C. H., O'Brien, A. D., Neish, A. S., and Madara, J. L. (2001) J. Clin. Invest. 107, 99-109 |
| 27. | Eaves-Pyles, T., Murthy, K., Liaudet, L., Virag, L., Ross, G., Garcia-Soriano, F., Szabo, C., and Salzman, A. L. (2001) J. Immunol. 166, 1248-1260 |
| 28. | de Bentzmann, S., Polette, M., Zahm, J. M., Hinnrasky, J., Kileztky, C., Bajolet, O., Klossek, J. M., Filloux, A., Lazdunski, A., and Puchelle, E. (2000) Lab. Invest. 80, 209-219 |
| 29. | Firth, J. D., Putnins, E. E., Larjava, H., and Uitto, V. J. (1997) Infect. Immun. 65, 4931-4936 |
| 30. | Li, J. D., Dohrman, A. F., Gallup, M., Miyata, S., Gum, J. R., Kim, Y. S., Nadel, J. A., Prince, A., and Basbaum, C. B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 967-972 |
| 31. | Becker, M. N., Diamond, G., Verghese, M. W., and Randell, S. H. (2000) J. Biol. Chem. 275, 29731-29736 |
| 32. | Harder, J., Meyer-Hoffert, U., Teran, L. M., Schwichtenberg, L., Bartels, J., Maune, S., and Schroder, J. M. (2000) Am. J. Respir. Cell Mol. Biol. 22, 714-721 |
| 33. | O'Neil, D. A., Porter, E. M., Elewaut, D., Anderson, G. M., Eckmann, L., Ganz, T., and Kagnoff, M. F. (1999) J. Immunol. 163, 6718-6724 |
| 34. | Ogushi, K., Wada, A., Niidome, T., Mori, N., Oishi, K., Nagatake, T., Takahashi, A., Asakura, H., Makino, S., Hojo, H., Nakahara, Y., Ohsaki, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Moss, J., and Hirayama, T. (2001) J. Biol. Chem. 276, 30521-30526 |
| 35. | Ichikawa, J. K., Norris, A., Bangera, M. G., Geiss, G. K., van't Wout, A. B., Bumgarner, R. E., and Lory, S. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 9659-9664 |
| 36. | Belcher, C. E., Drenkow, J., Kehoe, B., Gingeras, T. R., McNamara, N., Lemjabbar, H., Basbaum, C., and Relman, D. A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 13847-13852 |
| 37. | Zhang, H., Porro, G., Orzech, N., Mullen, B., Liu, M., and Slutsky, A. S. (2001) Am. J. Physiol. Lung Cell. Mol. Physiol. 280, L947-L954 |
| 38. | Lichtenstein, A., Ganz, T., Selsted, M. E., and Lehrer, R. I. (1986) Blood 68, 1407-1410 |
| 39. | Soong, L. B., Ganz, T., Ellison, A., and Caughey, G. H. (1997) Inflamm. Res. 46, 98-102 |
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