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1 From the Ocular Surface and Tear Center, Department of Ophthalmology, Bascom Palmer Eye Institute; and the 2 Department of Urology, University of Miami School of Medicine, Florida.
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Abstract |
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METHODS. Unstimulated tear fluid was collected from patients with ocular rosacea and normal control subjects. Levels of MMP-9, MMP-3, and TIMP-1 were determined by enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis. Supernatants from primary human corneal epithelial cultures and human tear fluid were incubated with MMP-3. Cultured epithelial cells and their supernatants were also treated with doxycycline before MMP-3 was added. Gelatin zymography was used to identify activated 82-kDa MMP-9. MMP-9 activity was assessed with a commercial MMP-9 activity assay system.
RESULTS. MMP-9 and TIMP-1 were detected at significantly higher concentrations in rosacea-affected than in normal tear fluids. MMP-3 was detected exclusively in the tear fluid of patients with ocular rosacea who had corneal epithelial disease. Treatment of the supernatant and tear fluid with MMP-3 resulted in two bands with molecular weights of 92 kDa and 82 kDa, representing pro-MMP-9 and activated MMP-9, respectively. Doxycycline added to the conditioned media did not affect activation of MMP-9 by MMP-3. However, 24-hour treatment of corneal epithelial cultures with doxycycline resulted in a lower concentration and activity of MMP-9 in their supernatants.
CONCLUSIONS. MMP-9 and TIMP-1 are produced by the human corneal epithelium and are present in tear fluid. MMP-3 alone is sufficient to activate MMP-9 on the ocular surface. Doxycycline does not directly inhibit this activation by MMP-3, but it decreases MMP-9 activity when added to corneal epithelial cultures.
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concentration and was inversely correlated with
tear fluorescein clearance.2 Ocular rosacea and other dry eye conditions are associated with an increased incidence of recurrent corneal epithelial erosion (RCEE) and sterile corneal stromal ulceration.3 4 RCEE has been reported to occur in up to 12% of patients with ocular rosacea.3 4 We have experienced clinical success in treating patients with RCEE associated with rosacea with doxycycline, a compound with documented anticollagenolytic activity.5 MMP-9 produced by the corneal epithelium has been found to impede re-epithelialization of the cornea after experimental thermal injury in animal models.6 MMP-9 is secreted from cells as a 92-kDa proenzyme.7 Physiological mechanisms exist for suppressing and promoting activation of this enzyme. For example, tissue inhibitors of metalloproteinases (TIMPs) bind the proenzyme and inactivate it.7 In contrast, MMP-3 (stromelysin-1) has been reported to be an efficient activator of MMP-9.8
The purpose of this study was to evaluate human tear fluid from normal subjects and patients with ocular rosacea, with and without corneal epithelial disease, as well as conditioned media from primary human corneal epithelial cultures for the presence of TIMP-1 and MMP-3. We also evaluated the ability of MMP-3 to activate MMP-9 in these fluids and the effect of doxycycline on MMP-3 activation of MMP-9 in a corneal epithelial culture system.
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Materials and Methods |
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Tear fluid samples were collected from two groups of subjects. The first group consisted of patients with the diagnosis of meibomian gland disease associated with rosacea and a Schirmer 1 test score less than 10 mm. The diagnostic criteria for rosacea have been reported.9 The second group consisted of control subjects of similar age and gender distribution with no history of eye disease or ocular surgery, use of eye drops, or symptoms of ocular irritation. For detection of TIMP-1 and pro-MMP-9, tear fluid was collected from 15 patients with ocular rosacea and 8 normal control subjects. For detection of pro-MMP-3, tears were collected from 11 eyes of six patients with ocular rosacea and from 5 eyes of five normal control subjects. In the rosacea group, two eyes had epithelial basement membrane dystrophy, two eyes had RCEEs, and one eye had a nonhealing epithelial defect and sterile stromal thinning. For gelatin zymography to evaluate MMP-3 activation, tear fluid was taken from two normal control subjects who had detectable pro-MMP-9 in their tears.
Tear Collection
Tear fluid was collected from the inferior tear meniscus, causing
the least irritation possible, using a preweighed polyester wick
(Transorb rods; American Filtrona, Richmond, VA) to obtain the sample
as previously described.10
Wicks were then placed into the
end of a micropipette tip located within a 0.5-ml tube (Eppendorf,
Fremont, CA). The volume of collected tears was determined by
reweighing the tubes containing the rods immediately after tear
collection (model GA110 scale; Ohaus, Florham Park, NJ). The tear
samples were then stored at -80°C until they were used for
enzyme-linked immunosorbent assay (ELISA) or zymography.
Sample Extraction
Immediately before the tears were used for ELISA or zymography,
the samples were maintained at room temperature for 30 minutes. The
tears were then extracted from the saturated wicks by centrifuging at
12,000 rpm for 5 minutes within the pipette tip, after adding a volume
of buffer (ELISA buffer from manufacturer; zymography buffer: 50 mM
Tris/HCl, 0.15 M NaCl, 10 mM CaCl2, 0.005% Brij
35 (23 lauryl ether; Sigma, St. Louis, MO), 0.02% sodium azide
[pH 7.5]). The volume of buffer added to each tube brought the final
volume of tears plus buffer to 20 µl. For example, if the volume of
collected tears was 2 µl, 18 µl of buffer was added. The rods and
pipette were carefully removed, and the tear fluid aspirated.
Corneal Epithelial Cell Culture
Human corneal limbal epithelium was cultured from explants of
human donor corneoscleral rims preserved in modified McCarey–Kaufman
medium that was provided by the Florida Lions Eye Bank. Each
corneoscleral rim was trimmed, the endothelial layer and iris remnants
removed, and the tissue treated with dispase for 15 minutes. Each rim
was dissected into 12 equal parts. Each piece of limbus was placed in a
single well of a six-well plastic culture plate and covered with a drop
of fetal bovine serum overnight. The explants were cultured in medium
containing equal amounts of Dulbecco’s modified Eagle’s medium (DMEM)
and Ham’s F12 medium, enriched with 5% fetal bovine serum, 1% ITS
(insulin, transferrin, selenium), hydrocortisone, epidermal
growth factor, and cholera toxin. After 10 to 14 days of incubation,
the cells were trypsinized and seeded onto 24-well plates at a density
of 105 cells/well. More than 95% of these
passaged cells stained positively for acidic cytokeratins with
monoclonal antibody AE-1 (ICN, Costa Mesa, CA), confirming that they
were epithelial cells. First-passaged epithelial cells were cultured
until confluence and then switched to serum-free medium for 24 hours
before the supernatant was harvested. The viability of these cultured
cells was confirmed with an MTS
{[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenol)-2-(4-sulfophenyl)-2H-tetrazolium]
inner salt} assay (CellTitier 96 Aqueous; Promega, Madison,
WI).
Pro-MMP-3, Pro-MMP-9, and TIMP-1 ELISA
ELISAs for pro-MMP-3, pro MMP-9, and TIMP-1 were performed using
commercial kits (Oncogene, Cambridge, MA). Tear fluid samples were
further diluted in the supplier-provided assay buffer to a final volume
of 50 µl for pro-MMP-3 and 100 µl for pro-MMP-9 and TIMP-1. Human
corneal epithelial cell supernatants were also assayed for pro-MMP-3
and TIMP-1 with a final volume of 200 µl per well. Samples were
incubated for 2 hours at room temperature for 18 hours at 4°C. Assays
were performed in duplicate and completed according to the
manufacturer’s instructions.
Western Blot Analysis
Samples, including purified TIMP-1 (Oncogene) incubated with
purified pro-MMP-9 for 3 hours at 37°C, untreated human corneal
epithelial supernatant, 16x concentrated untreated supernatant,
supernatant incubated with purified TIMP-1 for 3 hours at 37°C, and
supernatant incubated with 0.33 ng/µl human neutrophil elastase
(Sigma) for 3 hours at 37°C, were subjected to sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), using a 4% to
15% gradient, 0.75 mm thick gels (Mini-ready; Bio-Rad, Richmond, CA)
at a constant 200 V for 45 minutes, in an electrophoresis apparatus
(Mini-Protean, Bio-Rad). Prestained (7.5–203 kDa) molecular weight
protein markers (Bio-Rad) were run simultaneously with the samples.
Resolved proteins were transferred to nitrocellulose membranes
(BioTrace NT, Ann Arbor, MI) using a minitank blot apparatus (Bio-Rad).
Membranes were blocked in 3% fat-free milk for 45 minutes. After a
1-hour incubation with the primary antibody (diluted to 1 µg/ml in 50
mM Tris-HCl and 150 mM NaCl [pH 7.4], containing, 1% bovine serum
albumin and 0.5% Tween 20), the membranes were incubated with 0.5
µg/ml IgG-horseradish peroxidase–conjugated goat anti-mouse IgG
(Pierce, Rockford, IL). Specifically bound antibody was detected using
an immunodetection kit (Renaissance Enhanced Chemiluminescence; duPont
NEN, Boston, MA), and then exposed to x-ray film (Eastman Kodak,
Rochester, NY) from 30 seconds to 3 minutes.
Activation of Pro-MMP-9 in Conditioned Media and Tear Fluid
Corneal epithelial culture supernatant was thawed to room
temperature for 1 hour. Ten microliters of the supernatant were added
to each of six tubes (Eppendorf). Different amounts of the catalytic
domain of MMP-3 were added in duplicate: 0.63 ng/µl, 1.5
ng/µl, and 2.3 ng/µl. One tube of each MMP-3 concentration was
incubated at 37°C for 4 hours, and the other tube was incubated at
37°C for 8 hours. Untreated supernatant samples were used as normal
control subjects.
Two tear samples from normal control subjects were incubated with MMP-3 for 4 hours at 37°C. Untreated tear samples from the same individuals were assayed as untreated control samples.
Doxycycline Treatment of Corneal Epithelial Cell Culture and
Conditioned Media
Ten microliters of corneal epithelial supernatant was added to
each of four tubes (Eppendorf). Each of the tubes was incubated for 4
hours at 37°C with the following reagents: 50 µg/ml doxycycline
(Sigma), 50 µg/ml doxycycline + 1.5 ng/µl MMP-3, and 100 µg/ml
doxycycline + 1.5 ng/µl MMP-3.
In addition, cultured human corneal epithelial cells were treated with 10 µg/ml doxycycline for 24 hours at 37°C. Ten microliters of supernatant from these cell cultures was added to each of three tubes (Eppendorf). Two of the tubes were incubated for 4 hours at 37°C with 1.5 ng/µl MMP-3. The third tube was incubated without MMP-3. This set of experiments was performed using both purified MMP-3 catalytic domain and human neutrophil elastase-activated MMP-3 (whole enzyme). These experiments were performed three times. The protein concentration in control and doxycycline-treated cultures was measured with a microprotein assay kit (BCA; Pierce).
Gelatin Zymography and MMP-9 Activity
Gelatinase activity in the corneal epithelial supernatant and tear
fluid was measured by gelatin zymography. Culture-conditioned media (10
µl) and diluted tear samples were incubated with SDS gel sample
buffer for 30 minutes at room temperature and electrophoresed on a 10%
SDS-polyacrylamide gel containing gelatin (1 mg/ml). After
electrophoresis, the proteins were renatured by washing the gel twice
in 0.25% Triton X-100 (20 minutes per wash) followed by an 18-hour
incubation at 37°C in the digestion buffer (50 mM Tris-HCl [pH 7.4]
containing 0.15 M NaCl, 10 mM CaCl2, 2 µM
ZnSO4, 1 mM phenylmethylsulfonyl fluoride,
0.005% Brij35, and 0.02% sodium azide). After incubation, the gel was
briefly rinsed in distilled water and stained with 0.25% Coomassie
brilliant blue R250 in 40% isopropanol solution for 2 hours. The gel
was destained with 7% acetic acid. Gelatinase activity in the gel was
visible as a clear area on a blue background, indicating where the
gelatin had been digested. The molecular weights of the gelatinases in
the samples were determined from protein markers (Bio-Rad) and purified
92-kDa pro-MMP-9 that were run in separate lanes on the gel. Gels were
then photographed (Polaroid, Cambridge, MA). For experiments comparing
band densities, the photographs were scanned (Scan Jet 4C;
Hewlett–Packard, Palo Alto, CA). Relative band densities in the
digitized images were determined by computer with a gel analysis
software program (Gel-Pro Analyzer; Media Cybernetics, Silver Springs,
MD).
MMP-9 activity in culture conditioned media (100 µl) was assessed with an MMP-9 activity assay system (Biotrak; Pharmacia Biotech, Piscataway, NJ).
Statistical Analysis
The t-test was used to compare statistical levels of
MMP-9 and TIMP-1 concentrations in human tear fluid samples, MMP-9
concentrations in untreated and doxycycline-treated cultures, and
protein concentrations in culture supernatants.
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2
9
and
IL-1ß14
in the tear fluid of patients with ocular
rosacea. Furthermore, we have recently reported an increased level of
MMP-9 (both latent and activated forms) in tear fluid of eyes with
ocular rosacea and RCEEs.2
Barro et al.15
noted significantly elevated levels of MMP-9, both latent and active
forms, in the tear fluid of patients with corneal transplant failure
compared with those with successful grafts. Although both latent-MMP-9 and TIMP-1 concentrations were elevated in tear fluids obtained from patients with rosacea compared with normal control subjects, we found no significant difference in the TIMP-1-to-pro-MMP-9 ratio between the two groups. It has been previously reported that in some systems, devoid of other MMP secretion, a ratio of TIMP-1 to MMP-9 less than 1 may promote the activation of MMP-9 by other proteinases, such as MMP-3.16 In the tear fluid of patients with rosacea, we found the TIMP-1-to-MMP-9 ratio to be more than 1, and yet we had detected the 82k-Da activated form of MMP-9 in 50% of tear samples obtained from patients with ocular rosacea in earlier research.2
The presence of active MMP-9 in the tear fluid of patients with ocular rosacea in our previous study,2 despite the high levels of TIMP-1 detected in our present study, suggests an alternate mechanism of activation of this enzyme in the ocular surface milieu. One such mechanism could be a concomitant increase in the levels of both MMP-3 and elastase. The latter protease is known to degrade TIMP-1 and activate MMP-3.17 Indeed, the concentration of MMP-3 in the tear fluid of patients with rosacea was also elevated, compared with that in normal volunteers, in whom this enzyme was undetectable (<3.75 ng/ml) in our assay. Consistent with this proposed mechanism is our finding that MMP-3 was undetectable in culture supernatants of corneal limbal epithelial cells where only pro-MMP-9 (92 kDa) activity was detected. These observations, together with our finding that TIMP-1 was degraded by neutrophil elastase, suggests the possibility that MMP-9 is activated by MMP-3 on the ocular surface of patients with rosacea. These enzymes may be secreted by the neutrophils and macrophages that have been reported to infiltrate the ocular surface in rosacea,18 or other conditions associated with delayed tear clearance.19 Although we did not attempt to measure elastase activity in our tear samples simultaneously (because of low sample volume), its accumulation in conditions of delayed tear clearance (e.g., during sleep) has been reported. Inflammatory leukocytes were observed to be the likely source of this elastase.19
Human neutrophil elastase has been reported to be capable of degrading TIMP-1.17 We obtained a similar result, in that the immunoreactive epitope on TIMP-1 (essentially the intact TIMP-1 molecule) produced by the corneal epithelium was degraded by neutrophil elastase. Neutrophil elastase has also been reported to be the principal caseinolytic protease in tear fluid.19 Elevated activity of this enzyme has been observed in the closed-eye state,19 a condition during which most RCEEs occur.20 Once freed from TIMP-1, pro-MMP-9 is amenable to activation by MMP-3. Thus, it is possible that degradation of TIMP-1, by neutrophil elastase in the closed eye, leads to increased MMP-9 activity, dissolution of the corneal epithelium basement membrane, and epithelial detachment.
As stated before, MMP-3 was not produced by cultured human corneal epithelium. Similar findings were reported by Fini and Girard1 in rabbit corneal epithelial cultures. This metalloproteinase was also not detected in normal unstimulated human tear fluid. In contrast, MMP-3 was detected in tear fluid from subjects with delayed tear clearance, with the highest concentrations in the tear fluid obtained from patients with either RCEE or frank corneal stromal ulceration. These findings suggest that the pathologic alterations of the ocular surface that accompany delayed tear clearance may also contribute to the increased production of MMP-3 by cells on the ocular surface, such as infiltrating leukocytes. Delayed tear clearance may also lead to increased retention of this enzyme in the tear fluid and to an accumulation of plasmin, another known activator of MMP-9.21
A 4-hour incubation in vitro with the catalytic domain of MMP-3 was found to completely convert 92-kDa pro-MMP-9 present in both tear fluids and corneal epithelial supernatants to its active 82-kDa form. This finding suggests that MMP-3 serves as an activator of pro-MMP-9 in human tear fluid in vivo.
MMP-9 has been implicated as a causative factor in delayed corneal surface wound healing and re-epithelialization.6 A similar role for MMP-9 in rosacea and RCEE would have significant clinical implications. Activation of MMP-9 may be one of the key pathophysiological events in the corneal ulcers that develop in ocular surface disease. Some or many events in this cascade may be mediated by inflammatory cytokines. For example, we have found that tear fluid from patients with rosacea has greater concentrations of the 17-kDa mature form of IL-1ß and lower concentrations of the biologically inactive 31-kDa precursor form than tear fluid from normal eyes.14 Activation of this important inflammatory cytokine from its precursor to mature form may be an important step in the inflammatory cascade. The mechanism of extracellular conversion of pro-IL-1ß to its biologically active mature form is not completely understood22 ; however, activated MMP-9 has been reported to be a key enzyme responsible for this conversion in the extracellular environment.23 We are currently investigating the mechanisms of conversion of pro-IL-1ß to its mature form on the ocular surface. Mature IL-ß is a potent inflammatory factor that causes and promotes inflammation and neural activation.24
We report here for the first time, the effect of and the potential use of doxycycline on the activity of pro-MMP-9 released by human corneal epithelium, as well as its enzymatic activation by MMP-3. We found that doxycycline at a nontoxic concentration markedly decreased the activity of pro-MMP-9. However, doxycycline did not affect the activation of pro-MMP-9 by MMP-3, a finding consistent with a previously reported study that doxycycline does not affect MMP-3 activity.25 Our finding could explain the observed clinical efficacy of doxycycline in the treatment of RCEE and the prophylaxis of its recurrence.
Ocular rosacea and RCEE are serious eye conditions that cause discomfort and can threaten vision. An effective mechanism to inhibit MMP-9 activity in vivo, possibly by using a nontoxic inhibitor of MMPs, such as doxycycline, has the potential to bring measurable relief to patients who have these conditions.
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Acknowledgements |
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Footnotes |
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Submitted for publication August 6, 1999; revised December 22, 1999; accepted January 5, 2000.
Commercial relationships policy: N
Corresponding author: Stephen C. Pflugfelder, Bascom Palmer Eye Institute, 900 NW 17th Street, Miami, FL 33136. spflugfelder{at}bpei.med.miami.edu
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