Abstract
purpose. To investigate the expression and regulation of membrane-type matrix
metalloproteinases (MT-MMPs) 4, 5, and 6 in the mouse corneas infected
with Pseudomonas aeruginosa.
methods. C57BL/6J mice were intracorneally infected with P.
aeruginosa. The expression of MT4-, MT5-, and MT6-MMP was
detected at both the mRNA and protein levels by RT-PCR and immunoblot
analysis. Immunohistochemical staining was performed to localize the
expression of MT4- and MT5-MMP in the mouse corneas.
results. Expression of MT4- and MT5-MMP was detected in the normal (uninfected)
cornea by RT-PCR and immunoblot analysis. When infected with P.
aeruginosa, the corneas showed significant induction of each
MT-MMP. Localization of MT4- and MT5-MMP revealed that the expression
of MT5-MMP was restricted to the epithelial tissue in the normal
cornea, whereas the induced expression of MT4- and MT5-MMP was
predominantly in the substantia propria, which contained most of the
infiltrating cells. MT6-MMP expression was not detected in the
uninfected cornea but was upregulated in the infected corneas.
conclusions. Expression of MT4-, MT5-, and MT6-MMP was induced in corneas infected
with P. aeruginosa. Immunohistochemistry showed
predominant immunoreactivity of MT4- and MT5-MMP in the substantia
propria. Previous histologic studies have revealed different patterns
of inflammatory cell infiltration with an increased number of
polymorphonuclear neutrophils (PMNs) during the early stage of
inflammation and increased macrophages during the late stage. These
results indicate a good correlation between the overexpression of the
MT-MMPs in the infected corneas and the inflammatory response—that is,
leukocyte infiltration—indicating that inflammatory cells such as
macrophages and PMNs may play a role in the upregulation of MT-MMPs
during corneal infection, which in turn can cause the destruction of
corneal tissue.
Recently, studies in our laboratory showed that
membrane-type matrix metalloproteinases (MT-MMPs) 1, 2, and 3 were all
upregulated in mouse corneas infected with
P. aeruginosa and
the peak of the induction correlated to the inflammatory events,
suggesting the important role of MT-MMPs in corneal infection and
destruction.
1
MMPs are a group of structurally related and zinc-dependent enzymes
that play a crucial role in physiologic and pathologic processes, such
as embryonic development, endometrial cycling, wound healing,
rheumatoid arthritis, tumor invasion, and metastasis.
2 MMPs have been classified into five broad categories based on their
substrate specificity and domain organization. They include
collagenases (MMP-1, -8, -13, and -18), stromelysins (MMP-3, -10, and
-11), gelatinases (MMP-2 and -9), MT-MMPs (MMP-14, -15, -16, -17, -24,
and -25), and other MMPs (MMP-7, -12, -19, -20, -21/22, and
-26).
3 4 5 6 7 Since the first member was identified by Sato et
al. in 1994,
8 the MT-MMP subgroup has grown rapidly and
now has six members named after the transmembrane domains that anchor
the molecules on the plasma membrane.
3 5 6 MT-MMPs are
important in turnover of the extracellular matrix (ECM), because of
their dual functions. Similar to other MMPs, they directly cleave ECM
proteins, including type I and III collagens, fibronectin, and laminin.
In addition, they activate other MMPs such as pro-MMP-2 (gelatinase A)
and pro-MMP-13 (collagenase 3) and form an enzymatic cascade for
regulation of ECM degradation.
9 10
Although classified in the same subgroup, MT4-, MT5-, and MT6-MMP are
different from the other three MT-MMPs in some respects. For example,
MT4-MMP has the least sequence identity with other MT-MMPs, except for
MT6-MMP.
11 12 Both MT4-MMP and MT6-MMP are predominantly
expressed in leukocytes, and both are
glycosylphosphatidylinositol-anchored MMPs.
12 13 14 15 In
addition, MT4-MMP also shows TNF-α convertase activity and can
potentially process pro-TNF-α to the 17-kDa form.
11 These characteristics of MT4-MMP and MT6-MMP suggest that they play a
role in inflammation. However, the normal functions of MT4-, MT5-, and
MT6-MMP remain unknown. Their expression and regulation in the cornea
have not been reported.
In the present study, we explored expression of newly identified MT4-,
MT5-, and MT6-MMP in the corneas of naïve C57BL/6J mice
infected with P. aeruginosa. RT-PCR, immunoblot analysis,
and immunohistochemical staining revealed the significant induction of
MT4- and MT5-MMP expression at both the mRNA and protein levels in
naïve mice over an 8-day period. Similar results were obtained
for MT6-MMP gene expression using only RT-PCR due to the absence of
available antibody to mouse MT6-MMP.
Stock cultures of P. aeruginosa 19660 (ATCC,
Rockville, MD) were stored at 4°C on tryptose agar slants (Difco
Laboratories, Detroit, MI) and were used for the inoculation of 50 to
75 ml of broth medium containing 5% peptone (Difco Laboratories) and
0.25% trypticase soy broth (BBL Microbiology Systems, Cockeysville,
MD). Strain 19660 is hemolytic and lecithinolytic and produces exotoxin
A, alkaline protease, and elastase under appropriate culture
conditions. Cultures were grown on a rotary shaker at 37°C for 16 to
18 hours, centrifuged at 8000 rpm at 4°C for 10 minutes, washed with
normal saline (Travenol Laboratories, Cambridge, MA), and diluted to a
concentration of 2 × 1010 colony-forming
units per milliliter. A standard curve was developed to relate viable
counts to optical density at 440 nm.
All animals were treated in accordance with the ARVO Statement
for the Use of Animals in Ophthalmic and Vision Research. Age-matched
C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME), each weighing 18
to 22 g, were infected at 14 weeks of age. Before infection, they
were lightly anesthetized with ether and placed beneath a stereoscopic
microscope. The corneal surface was then gently incised with three 1-mm
incisions using a sterile 26-gauge needle, taking care not to penetrate
the anterior chamber or to damage the sclera. A bacterial suspension (5μ
l) containing 108 colony-forming units was
topically delivered onto the wounded cornea using a micropipette with a
sterile disposable tip. Mice were examined 24 hours later to verify
infection. Controls consisted of scratched and unscratched mice that
were uninfected.
At selected time points after infection, mice were killed and corneas
were excised. Individual samples consisted of 12 pooled corneas per
time point for RT-PCR and immunoblot analysis. Single corneas from
different time points were used for immunohistochemical staining and
histologic study. Immediately after isolation, corneas were rinsed in
sterile saline and then processed for the purpose of the different
assays. Control mice were treated similarly.
Total RNA was extracted from the harvested corneas with
extraction reagent (TRIzol; Gibco, Grand Island, NY) according to the
manufacturer’s instruction. The total RNA was dissolved in water
treated with diethylpyrocarbonate (DEPC), and the concentration was
measured by a spectrophotometer (UV-1601; Shimadzu, Kyoto, Japan). The
samples were treated with DNase I (0.2 U/μl; Ambion, Austin, TX) to
remove possible DNA contamination. All the reagents needed for RT-PCR
were purchased from Perkin Elmer (Norwalk, CT) except Taq DNA polymerase and the dNTP mixture, which were purchased from Gibco.
RT-PCR was performed as previously described.
1 Briefly, RT
was performed in 0.65-ml RNase-free tubes under optimized conditions in
a DNA thermal cycler (model 480; Perkin Elmer). Equal amounts of total
RNA (500 ng) from each sample were used for this reaction. The whole
product of RT in each tube was amplified by PCR. Cycle parameters were
generally a 1-minute melting step at 95°C, a 1-minute annealing step
at 55°C, and a 2-minute extension step at 72°C. Thirty cycles were
selected for amplification of all target genes, based on results of the
experiments that tested the linear range of amplification with
different cycles. The following specific primers for MT4-, MT5-, and
MT6-MMP were designed and prepared according to the available
information for these specific genes (GenBank, provided in the public
domain by the National Center for Biotechnology information, Bethesda,
MD, and available at http://www.ncbi.nlm.nih.gov/genbank). Because the
mRNA sequence for mouse MT6-MMP has not been reported, the primers for
human MT6-MMP were prepared and used, assuming there is high homology
between human and mouse MT6-MMP. The primers of mouse MT4-MMP (5′-TGG
TCT GAT GGT GCA TCC TA-3′ and 5′-TGC AGG AAC ATA CTG CCA GA-3′)
amplified a 205-bp product (gene sequence 1550-1754); the primers of
mouse MT5-MMP (5′-GTG ACA GCT CCC CAT TTG AT-3′ and 5′-TAG AGT GCT CCA
AGC CCA GT-3′) amplified a 192-bp product (682-873); and the primers of
human MT6-MMP (5′-ATG GCC TGC AGC AAC TCT AT-3′ and 5′-AGG GGC CTT TGA
AGA AGA AA-3′) amplified a ∼200-bp product. There were two negative
controls: one without reverse transcriptase and the other one without
specific primers. The housekeeping gene 18S RNA (Ambion) was also
amplified and used as an internal control for the comparison of all
time point target genes. Finally, the amplified genes were resolved by
1% agarose gels and revealed by ethidium bromide staining. RT-PCR for
each MT-MMP was repeated at least twice.
To confirm the specificity of the primers, the amplified DNA sequences
were subjected to restriction enzyme digestion. The 205-bp PCR product
was treated with SmaI (Gibco), which recognizes the sequence
CCC∧GGG. The 192-bp product was treated with MboI (Gibco),
which recognizes the sequence ∧GATC. Each of them was incubated with
respective enzyme (5 U) for 90 minutes, with different reaction buffer
and at different temperature, according to the manufacturer’s
instruction. The results were revealed by 3% agarose gel
electrophoresis.
The ∼200-bp PCR product was purified with a kit (MinElute; Qiagen,
Valencia, CA) according to the manufacturer’s instruction. The
concentration of the purified DNA was measured by the spectrophotometer
at 260 nm. The purified DNA at a concentration of 10 ng/μl and human
MT6-MMP primers at a concentration of 1 pM/μl were then prepared. The
DNA samples were analyzed in the DNA Sequencing Facility at the Center
for Molecular Medicine and Genetics (Wayne State University, Detroit,
MI).
Cornea samples were immediately frozen with liquid nitrogen
after isolation from mice. To enrich the extracts with membrane-bound
proteins, TBS-CM buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1 mM
CaCl2, and 1.5% Triton X-114) was used. Samples
were homogenized with TBS-CM buffer on ice and then centrifuged at
12,000 rpm at 4°C for 2 minutes. The supernatants were collected and
incubated at 37°C for 3 minutes, followed by centrifugation at 12,000
rpm for 2 minutes. The detergent phase, which contained enriched
membrane-bound proteins, was saved for immunoblot analysis. The
concentrations of the total protein were measured with a bicinchoninic
acid (BCA) protein assay. Equal amounts of individual samples (5 μg)
were mixed with 5 μl of 4× sample loading buffer (0.125 M Tris-HCl[
pH 6.8], 4% SDS, 40% glycerol, and 0.02% bromphenol blue)
containing β-mercaptoethanol and boiled for 5 minutes. The samples
and a prestained molecular weight marker (Bio-Rad, Cambridge, MA) were
electrophoresed on 12% SDS gels and subsequently transferred to
nitrocellulose membranes. The membranes were blocked for 30 minutes in
blocking reagent (Blotto; Santa Cruz Biotechnology, Santa Cruz, CA;
TBS, containing 0.5% Tween 20, 3% nonfat milk, and 2% bovine serum
albumin) and then incubated with a polyclonal anti-human MT4-MMP
(AB854,1 μg/ml; Chemicon, Temecula, CA) and a polyclonal anti-human
MT5-MMP antibody (AB924, 0.2 μg/ml; R&D, Minneapolis, MN),
respectively, on a rocker at room temperature for 2 hours. The species
reactivity for MT5-MMP is positive to both human and mouse according to
the information from R&D. In addition, alignment of mouse MT4- and
MT5-MMP protein sequences with that of human revealed 82.0% and 92.6%
of homology, respectively. Samples treated with nonimmune (normal)
rabbit IgG or without primary antibody treatment were processed as the
negative control. Afterward, the blot was incubated with anti-rabbit
secondary antibody conjugated with horseradish peroxidase (0.5 μg/ml;
Roche Molecular Biochemicals, Indianapolis, IN) at room temperature for
1 hour. Finally, the blot was developed by a chemiluminescence kit
(Amersham, Arlington Heights, IL), and MT4- and MT5-MMP were visualized
as dark bands with corresponding molecular weights. Immunoblot analysis
of each MT-MMP was repeated at least twice.
The corneas, harvested on days 0 (uninfected) and 6 after
infection (peak inflammatory response), were fixed with 10% buffered
formalin overnight, dehydrated with increased concentrations of ethanol
and 100% xylene, infiltrated with paraffin overnight, and embedded in
fresh paraffin. The tissues were sectioned (4 μm) to prepare slides,
and the slides were deparaffinized, rehydrated, and treated with
proteinase K (Sigma, St. Louis, MO). Normal horse serum and 3%
hydrogen peroxide were applied separately to reduce nonspecific
staining and to remove endogenous peroxidase. The slides were treated
with specific primary antibodies recognizing mouse MT4- and MT5-MMP
(the same as used for immunoblot analysis) in a humidified chamber at
room temperature for 2 hours, followed by subsequent treatment with a
biotinylated anti-rabbit secondary antibody and the ABC reagents
(Vectastain; Vector Laboratories, Burlingame, CA). Positive staining
was exhibited by diaminobenzidine (DAB; Vector) treatment appearing as
brown granules.
Semiquantitative RT-PCR was performed to determine whether normal
mouse corneas contain mRNA for MT4-, MT5-, and MT6-MMP and whether the
MT-MMP expression is modulated by corneal infection. The corneas were
harvested on days 0 (control), 1 through 5, and 8 after infection and
total RNA was prepared. As shown in
Figure 1 , the gene expression for MT4- and MT5-MMP was found in normal
(uninfected) mouse corneas, whereas MT6-MMP was not detectable by human
MT6-MMP primers in uninfected corneas. When mice were infected with
P. aeruginosa, the MT4-MMP mRNA expression in the cornea
remained the same on the first 4 days and was then induced on day 5,
remaining high through day 8. The MT5-MMP mRNA expression in the cornea
infected with
P. aeruginosa did not change for the first few
days and then showed slight reduction until day 8, when significant
induction of the MT5-MMP mRNA expression was observed. In contrast,
RT-PCR results showed a ∼200-bp mRNA segment that was amplified by
human MT6-MMP primers and peaked at day 4. DNA sequencing and alignment
with human MT6-MMP revealed 46.6% of gene homology of this DNA segment
when compared with human MT6-MMP (data not shown). In addition, there
was no significant similarity between the amplified DNA and other known
genes.
To verify the amplified MT-MMPs, the amplified DNA sequences were
subjected to restriction enzyme digestion. The amplified MT4-MMP
product was cleaved by SmaI at the site between base pairs
78 and 79, which generated two smaller pieces (78-bp and 127-bp bands).
The amplified MT5-MMP product was hydrolyzed by MboI at the
site between bp 64 and 65, which generated two bands of 64 bp and 128
bp (data not shown).
There was no difference in any MT-MMP mRNA expression between the
scratched corneas and the unscratched corneas on day 0, indicating that
corneal abrasion did not affect the mRNA expression (data not shown).
In addition, negative control samples (those not treated with reverse
transcriptase or amplified without specific primers) did not exhibit
any MT-MMP expression (data not shown).
To confirm MT-MMP expression at the protein level and to compare MT-MMP
expression in the corneas at different time points during the
infection, immunoblot analysis of corneal extracts was performed. As
shown in
Figure 2 , MT4-MMP with a molecular weight of ∼68 kDa was detected in the
uninfected mouse corneas (day 0 sample), which had a molecular weight
similar to human MT4-MMP identified in brain
extracts.
13 In corneas infected with
P.
aeruginosa, the expression of MT4-MMP was gradually induced and
remained at a high level on day 8. MT5-MMP expression in corneas was
different from that of MT4-MMP. MT5-MMP with a molecular weight of∼
39 kDa, probably the soluble species, was found in normal
corneas.
5 The expression was reduced from day 1 through
day 5 after infection. There was a significant induction of MT5-MMP
expression on day 7 after infection. These results were consistent with
the gene expression of MT5-MMP during the infection.
MT-MMP expression at the protein level in the scratched corneas was
comparable to that of the unscratched corneas on day 0. No signals were
detected in any negative control samples (data not shown).
To localize MT-MMP expression in the mouse corneas,
immunohistochemical staining was performed using the corneal samples of
normal and infected mice. As shown in
Figure 3 , no positive staining of MT4-MMP was found in the normal cornea,
although immunoblot analysis showed a weak band of MT4-MMP on day 0
(normal cornea), perhaps because there is much less sensitivity in
immunohistochemical staining than in immunoblot analysis. On day 6,
MT4-MMP expression was localized in both epithelial tissue (weak
positive staining) and substantia propria (strong positive staining).
MT5-MMP expression was observed in corneas of normal mice, mainly
localized in the epithelium. On the sixth day after infection, the
distribution of MT5-MMP was not restricted to the epithelium but was
also detected in the substantia propria.
Corneal damage caused by
P. aeruginosa infection starts
with inflammatory reactions such as edema, leukocyte infiltration, and
angiogenesis. With continuation of the inflammation, corneal ulceration
may occur, and corneal perforation may eventually take place because of
the stromal dissolution.
16 17 18 One of the possible
mechanisms responsible for corneal destruction is that both host and
bacterial proteases contribute to the degradation of Bowman’s membrane
and the ECM, which leads to severe corneal damage. Evidence for this
hypothesis is based on the observations that purified proteases are
able to cleave ECM proteins and that bacterial proteases can cause
keratitis by activating host proteases.
19 20 In addition,
the two gelatinases MMP-2 and -9 have been identified in corneal
tissues and shown to play a role in corneal ulceration and tissue
remodeling in a noninfectious disease model.
21 22 23
Although a number of studies have begun to reveal the effects of some
MMPs on corneal tissues under physiological and pathologic conditions,
little is known about the expression and regulation of MT-MMPs in the
cornea. Our recent work on MT1-, MT2-, and MT3-MMP showed that these
three MT-MMPs are all present in the normal mouse cornea and that there
is a significant induction of each of them in the corneas of
naïve (unimmunized) mice intracorneally infected with
P.
aeruginosa, whereas the induction in the infected corneas of
immunized mice reaches a much lower level and has a shorter
duration.
1
In the present study, we further investigated the expression and
regulation of another three recently identified MT-MMPs—MT4-, MT5-,
and MT6-MMP, in the mouse corneas infected with
P.
aeruginosa. MT4- and MT5-MMP were identified in the normal
(uninfected) mouse cornea at both the mRNA and protein levels, whereas
MT6-MMP was not detectable by RT-PCR. Similar to MT1-, MT2-, and
MT3-MMP, expression of MT4-, MT5-, and MT6-MMP also showed a
significant induction in corneas when mice were intracorneally infected
with
P. aeruginosa. However, the induction pattern was
different from that previously observed (MT1-, MT2-, and MT3-MMP).
MT4-MMP expression in the infected corneas did not change dramatically
until day 5 after infection. It was reported that MT4-MMP was mainly
expressed by leukocytes, especially by monocytes and
macrophages.
11 Indeed, our data showed strong positive
staining of MT4-MMP in the cornea on day 6 after infection. Moreover,
our previous histologic study also showed increased macrophage
infiltration of the corneas during days 6 and 9 after
infection.
1 Therefore, the infiltrating macrophages seemed
responsible for the induction of MT4-MMP expression in the cornea
during the late stage of the inflammation. Although MT5-MMP was
primarily identified in human brain tumor,
24 basal
expression of MT5-MMP was also found in our corneal studies, primarily
localized in the epithelial tissue.
Downregulation of MT5-MMP during the early stage of the infection (days
2–4) possibly resulted from injury of epithelial cells, because they
seem to be the only source of MT5-MMP in the normal cornea, according
to our studies of immunohistochemical staining. Later induction of
MT5-MMP was probably also due to the infiltration of macrophages into
the infected cornea. MT6-MMP, also called leukolysin,
12 was not detected in the normal cornea by RT-PCR, but with infection,
there was a significant induction of MT6-MMP expression in corneas. It
was reported that MT6-MMP was predominantly expressed in
leukocytes.
6 12 Our data regarding MT6-MMP induction
correlate well with our previous findings on corneal inflammation as
quantified by bacterial numbers, PMNs measured by myeloperoxidase,
arachidonic acid metabolites, and cytokines.
25 26 27 In
addition, previous histologic studies showed that PMNs peak at day 6
after infection and then diminish gradually, which has good correlation
with the MT6-MMP induction pattern.
1 Thus, induced
expression of MT6-MMP in the cornea may be produced by PMNs.
As members of the MMP family, MT4- and MT5-MMP hydrolyze a variety of
ECM components, such as fibronectin, gelatin, fibrinogen, fibrin, and
proteoglycans.
11 28 The induced enzymatic activity during
the inflammation may cause degradation of the Bowman membranes and ECM
components, leading to severe corneal damage. Similar to other MT-MMPs,
MT4-, MT5-, and MT6-MMP are also capable of activating
pro-MMP-2.
5 6 29 Therefore, the induction of these MT-MMPs
in the mouse corneas infected with
P. aeruginosa may also be
responsible for corneal damage caused by other proteases that are
activated by these MT-MMPs.
The authors thank Julianna M. Berk for technical assistance.