(Investigative Ophthalmology and Visual Science. 1999;40:3168-3176.)
© 1999
by The Association for Research in Vision and Ophthalmology, Inc.
Evidence for TIMP-1 Protection Against P. aeruginosa–Induced Corneal Ulceration and Perforation
Karen A. Kernacki,
Ronald Barrett and
Linda D. Hazlett
From the Department of Anatomy and Cell Biology, Wayne State University–School of Medicine, Detroit, Michigan.
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Abstract
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PURPOSE. To determine the biological significance of individual endogenous
tissue inhibitors of metalloproteinases (TIMPs) in protection against
tissue destruction using a Pseudomonas
aeruginosa–induced model of corneal ulceration.
METHODS. Corneal TIMP-1, -2, and -3 mRNA levels were compared between young
adult (resistant) and aged (susceptible) mice challenged with P.
aeruginosa. Resistant mice that demonstrated greater amounts of
an individual TIMP were treated with polyclonal antibody (pAb) to that
TIMP. To determine whether TIMP neutralization exacerbated P.
aeruginosa–induced corneal disease, TIMP pAb– and normal
rabbit serum (NRS)- (control) treated mice were examined
macroscopically and histopathologically after infection. Corneal
neutrophil (PMN) myeloperoxidase (MPO) levels also were examined in
these mice.
RESULTS. Greater amounts of TIMP-1 mRNA only were found in corneas of resistant
versus susceptible mice after P. aeruginosa challenge.
Systemic treatment of resistant mice with TIMP-1 pAb resulted in
corneal perforation by 5 to 7 days after infection (PI).
Histopathologic evaluation of corneal tissues from TIMP-1 pAb– versus
NRS-treated mice confirmed that TIMP-1 pAb treatment resulted in
extensive stromal dissolution. This treatment also was associated with
loss of epithelium within the central cornea. Both the histopathology
and PMN MPO enzyme assays also showed an increase in corneal PMN number
following TIMP-1 pAb treatment.
CONCLUSIONS. These studies provide evidence that, after P. aeruginosa
infection, adequate endogenous expression of TIMP-1 in cornea protects
against extensive corneal tissue destruction. The protective effects of
TIMP-1 may be multifactorial. In addition to directly protecting
extracellular matrix components from active matrix metalloproteinases,
TIMP-1 may either directly or indirectly influence recruitment of PMNs
into infected cornea. Finally, TIMP-1 also may affect wound healing and
resurfacing of the corneal epithelium.
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Introduction
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Corneal ulceration is often observed after ocular exposure to
infectious agents such as Pseudomonas aeruginosa or herpes
simplex virus, after chemical or thermal injury to the cornea, or in
association with diseases such as rheumatoid arthritis or vitamin A
deficiency.1
2
3
4
5
6
The condition is characterized by
dissolution of the extracellular matrix (ECM) components of the corneal
stroma, often leading to extensive corneal scarring and perforation.
Despite the nature of the corneal insult, the sequence of events
leading to ulcer formation in the different infectious and
noninfectious conditions are remarkably similar.7
8
9
Previous reports have shown that the presence of a persistent or
recurring epithelial defect precedes stromal
destruction.10
Likewise, animal studies have demonstrated
that loss of basement membrane (BM) underlying the epithelium occurs
after epithelial defect formation, but before stromal involvement,
suggesting that loss of BM constituents may be a regulating step for
initiating stromal degradation.11
12
In addition,
infiltration of neutrophils (PMNs) into affected corneal tissue is
closely associated with corneal stroma dissolution.12
13
14
In ulcerating cornea, destruction of corneal ECM components (BM
constituents and stroma) has been attributed largely to the action of a
family of proteolytic enzymes [matrix metalloproteinases (MMPs)] that
collectively have the ability to degrade virtually all components of
the ECM. These assumptions were based primarily on the ability of
different broad-spectrum MMP inhibitors to block corneal tissue loss or
destruction either in vivo or in vitro.15
16
17
18
19
In contrast,
only a few studies have correlated overall increased expression of one
or more MMP with BM destruction or stromal loss.18
20
MMP activity can be controlled by interaction with tissue inhibitors of
metalloproteinases (TIMPs), the principal natural inhibitors of the
MMPs.21
22
To date, four members of the TIMP family of
inhibitors, TIMP-1, -2, -3, and -4, have been cloned and sequenced from
a number of animal species.23
All the TIMPs inhibit active
MMPs by binding to the MMP active site, forming tight, noncovalent
complexes. In addition, strong interactions between the latent form of
MMP-2 and TIMP-2 and MMP-9 and TIMP-1 have been described. As an
auxiliary means of control, most cells that secrete a MMP also produce
at least one form of TIMP; however, the expression of MMP and inhibitor
are often independently or reciprocally regulated.21
Little has been done to examine the expression and/or participation of
endogenously produced TIMPs in corneal disease. Both TIMP-1, -2, and -3
mRNA and protein have been detected in normal and diseased corneal
tissues.20
24
25
26
In these studies, differences in TIMP
expression or MMP/TIMP ratios were shown in normal versus diseased
human corneas, yet the biological significance of these important data
could not be tested. We recently showed that expression of TIMP-1, -2,
and -3 mRNA were independently regulated in corneal tissue of outbred
mice infected with P. aeruginosa.21
As an
extension of this work, our current studies have compared TIMP-1, -2,
and -3 mRNA expression in mice shown to be either resistant (cornea
heals) or susceptible (cornea perforates) to P. aeruginosa
challenge to determine whether adequate expression of one or more of
the TIMPs protects the cornea of resistant mice from irreversible
tissue destruction. Resistant mice that demonstrated increased
expression of an individual TIMP were injected with a TIMP-specific
polyclonal antibody (pAb) to evaluate the in vivo contribution of the
MMP inhibitor to the resistance phenotype.
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Methods
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Infection of Mice
Young adult (8 weeks) and aged (12 months) BALB/cBy (BALB/c) mice
(Charles River Laboratories, Kingston, NJ) were used for these studies.
Before corneal infection, mice were lightly anesthetized with
isoflurane (Aerrane; Anaquest, Madison, WI) and placed beneath a
stereoscopic microscope at x40 magnification. The central cornea of
the left eye was scarified with three 1-mm incisions using a sterile
26-gauge needle. Random eyes were routinely examined histologically to
ensure that the wounds penetrated only the epithelial basal lamina and
superficial corneal stroma. A bacterial suspension (5 µl) containing
1.0 x 106 colony forming units (CFU) of
P. aeruginosa ATCC strain 19660 (Rockville, MD),
prepared as described previously,28
was applied topically
onto the wounded cornea. Eyes were examined macroscopically 24 hours
after infection (PI) and/or at times described below to ensure that all
mice were similarly infected and to monitor the course of disease in
infected mice, respectively. All animals were treated humanely and in
full compliance with the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research.
Ocular Response to Infection
After P. aeruginosa corneal infection, ocular disease
was graded using the following established scale29
: 0,
clear or slight opacity partially covering the pupil; +1, slight
opacity fully covering the entire anterior segment; +2, dense opacity
partially or fully covering the pupil; +3, dense opacity covering the
entire anterior segment; and +4, corneal perforation. To observe eyes
whose lids were sealed, mice were anesthetized with isoflurane and
sterile phosphate-buffered saline was applied to the lids to permit
their careful partial opening without inducing corneal perforation. A
mean clinical score was calculated for each group of mice to express
disease severity. This was done by summation of the scores for each
group divided by the total number of mice scored at each time point.
For each of the experiments described below [young versus aged and
TIMP-1 pAb– versus normal rabbit serum (NRS)-treated] ocular disease
grades were determined using two different groups of mice
(n = 5 mice/experimental group/experiment) to ensure
reproducibility of the data. Data from a single representative
experiment (young versus aged and TIMP-1 pAb– versus NRS-treated) are
shown in the results.
Quantitation of Corneal TIMP-1, -2, and -3 mRNA Levels
RNase protection assays were used to quantitate corneal levels of
TIMP-1, -2, and -3 mRNA as described previously.27
For
these studies, corneal tissue was collected from young adult and aged
mice before and at 12 hours, 1, 3, and 5 days after corneal challenge
with P. aeruginosa. Immediately after collection, corneas
were flash-frozen in liquid nitrogen and stored at -70°C until
extraction of RNA. Six corneas were pooled for an individual sample.
Total RNA was extracted from corneal tissues using RNazol B (Tel-Test,
Friendsville, TX) according to the recommendations of the manufacturer.
As described before,27
5 µg of total RNA from each
sample was hybridized overnight at 56°C to 300 pg of the
32P-labeled TIMP-1 and -3 riboprobes, whereas 25
µg of total RNA was used for the TIMP-2 assays. Similarly, various
concentrations of unlabeled sense-strand standard (1.6–50 pg) was
hybridized to the same amount of the respective TIMP-1, -2, or -3
riboprobes. After hybridization, samples were digested with 1000 U of
T1 nuclease (Gibco-BRL, Gaithersburg, MD). Nuclease-protected fragments
were resolved on a 4.5% urea containing sequencing gel. Protected
bands were observed by exposing the dried gel to x-ray film and
quantitated using a MDX Persen Densitometer S II and Image Quant
Densitometric software (Molecular Dynamics, Sunnyvale, CA). This
experiment was performed at least three times using three different
groups of mice. Single values for TIMP-1, -2, or -3 mRNA were obtained
from each of the individual pooled samples. The data from the three
experiments were combined to determine whether statistical differences
in TIMP-1, -2, or -3 expression existed between young and aged mice.
Results are reported as atomoles of TIMP mRNA per microgram of total
RNA (± SEM).
Recombinant TIMP-1 Protein Generation and TIMP-1 pAb Production
Purified murine recombinant TIMP-1 (rTIMP-1) was expressed in
Escherichia coli using the QIAexpress system from Qiagen
(Chatsworth, CA). A cDNA for the entire coding region (minus the signal
sequence) of murine TIMP-1 was generated by polymerase chain reaction
(PCR) using plasmid DNA containing 825 bp of both coding and noncoding
regions of the TIMP-1 gene as the template (plasmid provided by Dylan
Edwards, University of Calgary, Alberta, Canada). PCR primers were
engineered with BamHI (+ strand) and HindIII (-
strand) restriction sites to facilitate ligation into the PQE30
expression vector. At the N terminus of the coding region of the TIMP-1
cDNA, a 6X-His affinity tag coding sequence from the PQE30 vector was
added. The ligation product was transformed into E. coli M15
(pREP). Clones were sequenced using the Sequinase Version 2 kit (US
Biochemicals, Cleveland, OH) to verify insertion of the proper DNA
sequence and in frame orientation within the PQE30 plasmid. Recombinant
protein was generated by inducing bacterial cultures with
isopropylthiogalactoside (IPTG) and purified from bacterial cell
lysates using a resin column (Sepharose CL-6B) to which
nitrilotriacetic acid (NTA) charged with Ni2 +
ions was bound. The Ni2 + ion binds the
6X-His–tagged protein with high affinity and allows purification of
proteins to >95% homogeneity.30
The column purified protein was analyzed on a 12% SDS-polyacrylamide
gel. Gel slices containing the rTIMP-1 protein were excised and used
for the immunization of rabbits. pAb to murine rTIMP-1 was generated by
Great Lakes Biomedical Research (Romeo, MI). The specificity of the
TIMP-1 pAb was determined by Western blot analysis. For these studies,
100 ng of the rTIMP-1 protein or supernatant from a P.
aeruginosa–infected corneal tissue homogenate were diluted in
Laemelli sample buffer containing 2-mercaptoethanol. Proteins in the
individual samples were resolved on 12% SDS-polyacrylamide gels. After
electroblotting onto nitrocellulose, blots were blocked in Blotto
(Tris-buffered saline [TBS] containing 0.5% Tween 20, 3% skim milk
powder, and 2% bovine serum albumin [BSA]). The blocking step was
followed by incubation of an individual blot overnight at 4°C with
the TIMP-1 pAb (1:5000 dilution in TBS with 10% Blotto). The control
blot was incubated similarly in preimmune, NRS. Blots were washed in
TBS–Tween 20 and incubated at room temperature for 2 hours with a
peroxidase-labeled goat anti-rabbit IgG (1 mg/ml) (Amersham, Arlington
Heights, IL). After a final series of TBS–Tween 20 washes, blots were
developed by chemiluminescence as specified by the manufacturer
(Amersham).
TIMP-1 pAb Treatment
A group of five young adult BALB/c mice were injected
intraperitoneally with 0.2 ml serum containing the TIMP-1 pAb at 1 and
3 days before and at 1 and 3 days after corneal P.
aeruginosa challenge. Control young adult BALB/c mice
(n = 5) were similarly treated with 0.2 ml NRS. Each of
the pAb treatment experiments were performed in duplicate to ensure
reproducibility of the data. Data for representative experiments are
presented in the results.
Histopathology
For histopathologic analysis, whole eyes were enucleated from
three mice from each experimental group (TIMP-1 pAb– versus
NRS-treated) at 3 days PI. Enucleated eyes were immersed immediately in
PBS, rinsed, and placed in a fixative containing 1% osmium tetroxide,
2.5% glutaraldehyde, and 0.2 M Sorenson’s phosphate buffer (pH 7.4),
(1:1:1) at 4°C for a total of 3 hours. Eyes were transferred into
fresh fixative after 1.5 hours. Eyes were then dehydrated in graded
ethanols and embedded in Epon-araldite as described
previously.28
Thick sections (1.5 µm) were cut, stained
with a modified Richardson’s stain, and observed. Representative
sections were photographed with a Zeiss Axiophot photomicroscope
equipped with bright field optics using Ilford pan F film (Moberley,
Cheshire, UK).
Quantitation of PMN in Corneal Tissues
A myeloperoxidase (MPO) assay was used to quantitate the number of
PMN infiltrating the cornea after infection.31
32
At 3 and
5 days PI, individual corneas (n = 3/group/time) were
collected from TIMP-1 pAb– and NRS-treated mice. Corneas were excised
at the limbus with a sterile razor blade, and noncorneal tissue was
removed by dissection. After collection, individual corneas were
immersed in 1.0 ml of 50 mM phosphate buffer (pH 6.0) containing 0.5%
hexadecyltrimethylammonium bromide. Samples were sonicated for 10
seconds on ice, freeze-thawed three times, sonicated a second time, and
centrifuged at 14,000g for 10 minutes to remove cellular
debris. An aliquot of the supernatant (0.1 ml) was added to 2.9 ml of
the 50 mM phosphate buffer containing o-dianisidine
dihydrochloride (16.7 mg/100 ml) and hydrogen peroxide (0.0005%). The
change in absorbency at 460 nm was monitored continuously for 5 minutes
using a Genesis 2 spectrophotometer (Spectronics, Rochester, NY). The
slope of the line was determined for individual samples and used to
calculate units of MPO in the individual corneas. One unit of MPO
activity is defined as that degrading 1 µmol of
peroxide/min.32
Results are reported as units of MPO per
cornea. This experiment was performed in duplicate; representative data
are shown in the results section.
Statistical Analysis
An unpaired, two-tailed Student’s t-test was used to
determine statistical significance for the mean clinical score data,
RNase protection, and MPO assays. Mean differences were considered
significant at the confidence level of P 
0.05.
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Results
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Infection in Young Adult and Aged BALB/c Mice
After ocular challenge with P. aeruginosa, the response
to ocular disease was graded in young adult and aged BALB/c mice to
determine whether inbred mice respond to corneal infection in a manner
similar to young adult and aged outbred mice. Figure 1
shows the calculated mean clinical scores from 24 hours to 7 days PI,
as described previously for aged Swiss–ICR mice,33
the
central cornea of inbred aged mice was not completely obscured by
corneal opacity by 24 hours PI. In these mice, a dense ring of opacity
was observed at the limbus, whereas the central cornea typically
displayed faint opacity indicative of a delayed infiltration of PMN
into that area. Although this response is not defined within the
conventional grading scale described by Hazlett et al.,29
aged mice were assigned a grade of +1 because of the faint opacity
within the central cornea. Young adult mice displayed faint opacity
across the entire anterior segment (+1) at 1 day PI. Although young
adult mice did not progress past a +2 ocular disease grade throughout
the experiment, all the aged mice showed +4 ocular disease grades
(corneal perforation) by 7 days PI. Significant differences between
young adult and aged mice were found from 3 to 7 days PI
(P = 0.006, 0.011, and 0.001 at 3, 5, and 7 days PI,
respectively).
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Figure 1. Ocular disease response in young adult and aged BALB/c mice. Ocular
disease grades were averaged in young adult and aged mice at individual
times after infection (n = 5 mice/experimental
group). Results are reported as mean clinical score ± SEM.
(P > 0.05 at 1 day PI and P =
0.006, 0.011, and 0.001 at 3, 5, and 7 days PI, respectively.)
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TIMP mRNA Levels during P. aeruginosa Corneal Infection
Corneal TIMP mRNA levels were quantitated in resistant (young
adult) and susceptible (aged) mice before and after P.
aeruginosa challenge to determine whether differences in the
regulation of one or more of the TIMPs could account, in part, for the
disparate response to infection. TIMP-1, -2, and -3 mRNA levels were
measured in corneal tissues collected before and at 12 hours, 1, 3, and
5 days PI. The data for three individual experiments were combined and
are shown in Figures 2
3
4
. While TIMP-1 mRNA transcripts were not found in uninfected corneal
tissues from either young adult or aged mice, TIMP-1 mRNA was detected
in both experimental groups by 12 hours PI (Fig. 2)
. Peak TIMP-1 mRNA
expression was detected in both experimental groups at 1 day PI. TIMP-1
mRNA levels were significantly greater in corneas from young adult
versus aged mice from 1 to 5 days PI (P = 0.001, 0.01, and
0.041 at 1, 3 and 5 days PI, respectively). Alternatively, low levels
of TIMP-2 message were found in both young adult and aged mice before
and after corneal challenge (Fig. 3)
. No significant differences in
TIMP-2 expression were detected between young adult and aged mice at
any of the times tested. Constitutive TIMP-3 mRNA expression was
detected in both experimental groups of mice (Fig. 4)
. Corneal
challenge with P. aeruginosa showed a slight decrease in
TIMP-3 levels from 12 hours to 5 days PI. However, similar to the
TIMP-2 data, no significant differences in corneal TIMP-3 mRNA levels
were found between young adult and aged mice.
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Figure 2. TIMP-1 mRNA levels in P. aeruginosa–infected corneas.
TIMP-1 mRNA levels were determined in pooled corneal samples
(n = 6 corneas/sample) collected from young adult
and aged BALB/c mice before and after corneal challenge with P.
aeruginosa. The results from three separate experiments
containing a single pooled sample per time point were combined. Results
are reported as atomoles of TIMP-1 mRNA per microgram of total RNA (±
SEM). (P > 0.05 before and at 12 hours PI and
P = 0.001, 0.010, and 0.041 at 1, 3, and 5 days PI,
respectively.)
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Figure 3. TIMP-2 mRNA levels in P. aeruginosa–infected corneas.
TIMP-2 mRNA levels were determined in pooled corneal samples
(n = 6 corneas/sample) collected from young adult
and aged BALB/c mice before and after corneal challenge with P.
aeruginosa. The results from three separate experiments
containing a single pooled sample per time point were combined. Results
are reported as atomoles of TIMP-2 mRNA per microgram of total RNA (±
SEM). (P > 0.05 at all time points tested.)
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Figure 4. TIMP-3 mRNA levels in P. aeruginosa–infected corneas.
TIMP-3 mRNA levels were determined in pooled corneal samples
(n = 6 corneas/sample) collected from young adult
and aged BALB/c mice before and after corneal challenge with P.
aeruginosa. The results from three separate experiments
containing a single pooled sample per time point were combined. Results
are reported as atomoles of TIMP-3 mRNA per microgram of total RNA (±
SEM). (P > 0.05 at all time points tested.)
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Generation of rTIMP-1 Protein and TIMP-1 pAb
As notable differences in only TIMP-1 expression were found
between young adult versus aged BALB/c mice, we next focused our
attention on determining if these differences were biologically
relevant. To do this, murine rTIMP-1 protein was generated using a
bacterial protein expression and purification system (see Materials and
Methods). The rTIMP-1 protein was used to generate a murine
TIMP-1–specific pAb. Figure 5
shows bacterial cell lysates (IPTG induced and uninduced) analyzed by
SDS-PAGE. A 21-kDa protein, corresponding to the molecular weight of
nonglycosylated murine TIMP-1,34
was detected only in
lysates from bacterial cultures induced with IPTG. After affinity
column purification of rTIMP-1, a single 21-kDa band was observed (Fig. 5)
. pAb generated against the recombinant protein reacted specifically
with the 21-kDa rTIMP-1. This pAb also reacted specifically with a
28-kDa protein in P. aeruginosa–infected corneal
homogenates (Fig. 6)
. The predicted molecular weight of glycosylated murine TIMP-1 is
approximately 28 kDa.34
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Figure 5. SDS-polyacrylamide gel showing expression and purification of rTIMP-1
from E. coli cell lysates. (Lane 1)
lysate from uninduced E. coli culture; (lane
2) lysate from IPTG induced E. coli culture;
(lane 3) affinity column purified rTIMP-1 protein.
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Figure 6. TIMP-1 Western blot analysis. Purified rTIMP-1 protein (100 ng)
(lane 1) and supernatant from P.
aeruginosa infected corneal homogenates (lane 2)
were analyzed by Western blot analysis using the TIMP-1 pAb as the
primary Ab. Lane 3 shows the same corneal tissue
supernatant incubated with NRS as the primary Ab.
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TIMP-1 pAb Treatment
Because resistant young adult mice expressed greater amounts of
TIMP-1 mRNA, we next tested whether systemic injection of the TIMP-1
pAb would exacerbate P. aeruginosa–induced corneal disease.
For these studies, TIMP-1 pAb or NRS were administered to young adult
mice at 1 and 3 days before and at 1 and 3 days after P.
aeruginosa corneal challenge. The ocular response to infection was
examined from 1 to 7 days PI. Mean clinical scores for the TIMP-1 pAb–
and NRS-treated mice were calculated and are shown in Figure 7
. Both experimental groups initially (1 day PI) displayed similar ocular
disease grades. Although mice given NRS did not progress past a +2
ocular disease grade over the time period examined, corneal perforation
was evident by 5 to 7 days PI in mice treated with the TIMP-1 pAb.
Significant differences between TIMP-1 pAb– and NRS-treated mice were
observed between 3 and 7 days PI (P = 0.0039, 0.008, and
0.0001 at 3, 5, and 7 days PI, respectively). Figure 8
shows slitlamp photomicrographs of representative eyes photographed at
3 days PI. By 3 days PI, mice treated with TIMP-1 pAb showed dense
corneal opacity covering the entire anterior segment of the eye (Fig. 8A)
. In addition, all mice treated with TIMP-1 pAb showed limbal blood
vessel ingrowth into the peripheral cornea and extensively swollen eye
lids. In contrast, mice treated with NRS showed dense corneal opacity
covering only the central cornea (Fig. 8B) . Neither blood vessel
ingrowth nor lid swelling was apparent in NRS-treated mice at this or
any other time after infection.
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Figure 7. Ocular disease response in TIMP-1 pAb– and NRS-treated mice. Young
adult BALB/c mice were treated with either TIMP-1 pAb or NRS before and
after P. aeruginosa corneal challenge
(n = 5 mice/experimental group). Ocular disease
grades were averaged at individual times after infection. Results are
reported as mean clinical score ± SEM. (P >
0.05 at 1 day PI and P = 0.0039, 0.008, and 0.0001
at 3, 5 and 7, days PI, respectively.)
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Figure 8. Slitlamp photomicrographs of P. aeruginosa–infected
mouse eyes from TIMP-1 pAb– and NRS-treated mice. At 3 days PI,
representative eyes from TIMP-1 pAb– and NRS-treated mice were
photographed using a slit-lamp (x25 magnification). (A) eye
from TIMP-1 pAb–treated mouse; (B) eye from NRS-treated
mouse.
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Corneas from TIMP-1 pAb– or NRS-treated mice also were examined
histopathologically at 3 days PI and marked differences were noted
between the two experimental groups (Fig. 9)
. TIMP-1 pAb–treated mice exhibited centrally thinned corneas
(approximately 1/2 of normal), denuded epithelium in the central
cornea and numerous free bacteria and intact PMNs throughout the stroma
(Figs. 9A 9B)
. PMNs filled the anterior chamber and were associated
with the surface of the ocular lens. In the cornea, Descemet’s
membrane and the endothelium were denuded from limbus to limbus. In the
peripheral cornea, epithelium was present. PMN were frequently seen as
well as a few mononuclear cells. In control (NRS-treated) mice, the
central cornea was not thinned or degraded (stromal collagen intact) as
extensively and epithelium was intact centrally (Figs. 9C
9D) . PMN
were observed in the superficial half of the stroma and free bacteria
in the stroma were also plentiful. Descemet’s membrane and the
endothelium were intact and few PMN were present in the anterior
chamber. In the peripheral cornea, the epithelium was intact, and PMNs
were observed in the superficial stroma. Lastly, as treatment of mice
with TIMP-1 pAb was qualitatively associated with a greater number of
PMN in infected corneal tissue, we used an established MPO
assay32
to quantitate PMN number in infected corneal
tissue collected from TIMP-1 pAb– and NRS-treated mice. These data are
presented in Figure 10
. Significantly greater amounts of MPO activity were detected in corneas
from TIMP-1 pAb– versus NRS-treated mice at both of the times tested
(P = 0.006 and 0.005 at 3 and 5 days PI, respectively).
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Figure 9. Light-microscopic corneal histopathology of TIMP-1 pAb– (A,
B) or NRS- (C, D) treated mice at 3
days PI. Arrow in (A) and (C)
denotes region of central cornea. (B) and (D) are
a higher magnification of the central cornea. Bars, 100 µm.
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Figure 10. Corneal MPO activity in TIMP-1 pAb– and NRS-treated mice. Three
individual corneas were collected from TIMP-1 pAb– and NRS-treated
mice at 3 and 5 days PI and analyzed for PMN MPO activity. Results are
reported as units MPO/cornea (± SEM). (P = 0.006 and
0.005 at 3 and 5 days PI, respectively.)
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Discussion
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During normal wound healing processes, it has been suggested that
an appropriate balance exists between ECM degradation and deposition,
such that the overall outcome is maintenance of integrity and function
of the affected tissue. Alternatively, in pathologic states, this
balance is thought to be altered in a manner that promotes progressive
ECM degradation or extensive deposition of fibrotic
tissue.21
In ulcerative corneal disease, there is evidence
to suggest that alteration of the ratio of MMPs:TIMPs plays a role in
progressive stromal degradation. In this regard, topical application of
either recombinant TIMP or synthetic MMP inhibitors prevented or
delayed corneal ulceration in various disease
models.15
16
17
18
19
In another study, Riley et
al.20
demonstrated increased MMP-1 and decreased TIMP-1
immunostaining in ulcerating versus normal human corneas. Likewise,
Kenney et al.25
described an increase in the ratio of
MMP-2/TIMP-1 in corneas of keratoconus patients. Although suggesting a
general role for MMPs and TIMPs in these diseases, neither study was
able to directly examine the participation of individual endogenously
produced TIMPs in protection against corneal destruction. Therefore, to
address this problem, we used a resistant (cornea heals) versus
susceptible (cornea perforates) model of P.
aeruginosa–induced corneal disease to identify those TIMPs that
are involved in corneal wound healing. In this model, young adult (8
weeks) BALB/c mice restore corneal clarity within 2 weeks after ocular
P. aeruginosa challenge, whereas aged mice undergo corneal
perforation within 7 days PI (Fig. 1)
.
When TIMP-1, -2, and -3 mRNA levels were examined in young adult and
aged inbred mice before and after P. aeruginosa corneal
challenge, we found that all three of the TIMPs were independently
regulated in corneal tissue (
Figs. 2-4
). These data, using a
well-defined inbred mouse model system, confirm and extend our previous
results using outbred young adult Swiss-ICR mice.27
Based
on promotor regions of the TIMP genes and previous gene induction
studies, these results are not suprising.35
In the current
studies, TIMP-1 mRNA was not expressed in uninfected corneas of either
experimental group under the conditions tested. However, after corneal
challenge, significantly greater amounts of TIMP-1 were detected in
corneas of young adult and aged mice from 1 to 5 days PI. Ascroft et
al.36
recently reported data that complements our current
studies. Their work suggested that the decreased TIMP-1 mRNA and
protein expression in cutaneous wounds from aged versus young
individuals was associated with the reported impaired wound healing in
the aged.
Because differences in only TIMP-1 mRNA levels were detected between
resistant and susceptible mice, the remainder of this study focused on
determining if systemic neutralization of TIMP-1 protein exacerbated
corneal disease pathology in resistant mice and on the initial
characterization of the effects of this treatment. Gipson et
al.37
recently used a similar TIMP neutralization approach
and found increased influx of PMN into lung tissue and intensification
of lung injury after TIMP-2 pAb versus preimmune serum treatment. As
predicted, treatment of resistant mice in the present study with the
TIMP-1 pAb converted these mice to the susceptible phenotype. Corneal
perforation was evident in TIMP-1 pAb– versus NRS-treated mice by 5 to
7 days PI, similar to that observed with susceptible aged mice (Figs. 1
7) .
Histopathologic evaluation of corneas from TIMP-1 and NRS-treated mice
corroborated the macroscopic findings (Fig. 9)
. By 3 days PI, the
epithelium in TIMP-1 pAb–treated mice was centrally denuded and the
stromal layer was thinned to approximately 1/2 that of normal.
Alternatively, the epithelium in NRS-treated mice was present from
limbus to limbus and the stroma was not degraded as extensively. These
data are in accordance with previous studies that showed a loss or
defect in regeneration of the epithelium preceded stromal collagen
dissolution.10
In addition to inhibition of BM degrading
MMP activity, it has also been suggested that endogenous TIMPs may
influence healing of the corneal epithelium by enhancing spreading and
proliferation of the epithelial cells.38
In the experiments described herein, both the histopathology and PMN
MPO assays demonstrated a significantly increased number of PMNs in
corneas of mice treated with the TIMP-1 pAb (Figs. 6 7)
. The presence
of a large number of PMNs also has been associated with stromal
collagen degradation in various corneal ulcerative
models.12
13
14
30
Accordingly, Schultz et
al.19
demonstrated reduction of PMN influx into
alkali-burned corneas and ultimately prevention of corneal ulceration
after treatment with a synthetic MMP inhibitor. The mechanism by which
TIMP-1 prevents corneal PMN influx after P. aeruginosa
challenge remains unknown. However, based on the data reported herein
as well as in previous studies, various possibilities may be considered
for future testing. In this regard, PMNs, on activation, have the
ability to release MMP-8 (PMN interstitial collagenase) and MMP-9
(gelatinase B) from secondary granules.39
PMN collagenase
can directly degrade stromal collagen and generate collagen peptide
fragments that are chemotactic for PMN.40
Inhibition of
MMP-8 activity by TIMP-1 may therefore help to control this cyclic
response. Likewise, it has been suggested that PMNs use MMP-9 to
traverse endothelial BM and extravasate into inflamed
tissues.41
Finally, TIMP-1 may reduce PMN infiltration
into the cornea by blocking the shedding of the L-selectin adhesion
molecule present on activated PMN. It has been suggested that release
of L-selectin from PMNs after the initial attachment to the endothelium
facilitates entry into subendothelial tissues. Shedding of the
L-selectin molecule was shown to be inhibited by synthetic MMP
inhibitors.42
Furthermore, a study by Pfister et
al.43
showed that a synthetic MMP inhibitor directly
affected PMN chemotaxis.
In summary, we have investigated the role of TIMP-1, -2, and -3 in a
P. aeruginosa-induced model of ulcerative corneal disease.
Because differences in the expression of only TIMP-1 could be detected
between resistant and susceptible mice, we focused our study on this
TIMP. These studies, using a systemic TIMP-1 pAb treatment protocol,
are the first to show that expression of adequate endogenous levels of
TIMP-1 in cornea after P. aeruginosa challenge is associated
with protection against extensive stromal destruction and corneal
perforation. The data also strongly suggest that TIMP-1 may be
protective in several phases of the ulcerative process, including
epithelial resurfacing, BM and/or stromal ECM loss, and PMN
infiltration.
|
Footnotes
|
|---|
Supported by the National Institutes of Health, National Eye Institute
Grants R01 EY02986 and P30 EY04068 and by the Fight for Sight
Fellowship PD97013.
Submitted for publication May 14, 1999; revised July 21, 1999; accepted August 6, 1999.
Commercial relationships policy: N.
Corresponding author: Karen A. Kernacki, Department of Anatomy/Cell
Biology, Wayne State University, 540 East Canfield, Detroit, MI
48201. E-mail: kkernack{at}med.wayne.edu
|
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Top
Abstract
Introduction
