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From the Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Abstract
PURPOSE. Interleukin (IL)-1
and IL-6 levels in the cornea are greatly elevated during the
early stages after an alkali burn in mice. The authors investigated the
effect of synthetic inhibitor of matrix metalloproteinase (SIMP) on the
expression of inflammatory cytokines in alkali-burned murine corneas
and evaluated the clinical appearance of the eyes.
METHODS. After 0.5N NaOH-alkali burns to 400 corneas of ICR mice, 200 received
400 µg/ml of SIMP topically 4 times a day while 200 corneas were
similarly treated with vehicle only. At days 4, 7 and 14 after injury,
each cornea was assigned a clinical score for corneal opacity, corneal
epithelial defect, hyphema and cataract. Extracts of injured corneas in
each group were then assayed for cytokine production using ELISA
systems for IL-1, IL-1ß, IL-6 and tumor necrosis factor-
(TNF-).
RESULTS. The levels of IL-1, IL-1ß and IL-6 were significantly lower in the
SIMP-treated group than in the vehicle-treated group 7 days after the
burn. However, levels of these cytokines were similar in the SIMP and
non-SIMP groups at days 4 and 14. Levels of TNF- did not differ
between both groups at any postinjury time. In the SIMP-treated
corneas, there was less opacification and hyphema formation and
epithelial regeneration was faster.
CONCLUSIONS. Topical application of SIMP in alkali-burned murine corneas reduced the
expression of IL-1, IL-1ß, and IL-6 and lessened the severity of
the injury.
The matrix metalloproteinase (MMP) family of enzymes are found in cornea.1 2 3 4 5 An increased release of MMPs after an alkali burn is thought to be responsible for the destruction of the alkali-burned cornea,6 7 8 9 a condition that is typically uncontrollable. Previous reports have revealed that matrix metalloproteinase inhibitors (MMPI), such as a thiol peptide,10 tissue inhibitor of matrix metalloproteinase (TIMP),11 and synthetic inhibitor of matrix metalloproteinase (SIMP)12 13 are effective in the treatment of alkali injuries. These reports demonstrated that MMPI-treated eyes have less inflammatory cell infiltration and less destruction of corneal collagen compared to vehicle-treated control eyes. However, the mechanisms of effect of MMPIs have not been fully clarified.
Our recent research has demonstrated clearly that interleukin (IL)-1
and IL-6 are highly expressed in the cornea during the early stages
after an alkali burn and may play an essential role in
polymorphonuclear leukocyte (PMN) infiltration or associated corneal
damage after the burn.14
15
To more fully understand the
mechanisms of MMPI action, we investigated the effect of MMPI on
cytokine expression in the alkali-burned murine cornea.14
Materials and Methods
Synthetic Inhibitor of Matrix Metalloproteinase
We used SIMP developed by Galardy and
associates.12
16
This is an extremely potent inhibitor of
fibroblast collagenase, gelatinase, and stromelysin and is also known
as Galardin or GM6001. The compound is a dipeptide analogue with the
structure N-[2(R)-2-(hydroxamido
carbonylmethyl)-4-methylpentanoyl]-L-tryptophane
methylamide. For topical application, SIMP was dissolved at a
concentration of 400 µg/ml in
N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic
acid (HEPES) buffer containing 2% dimethyl sulfoxide (pH 7.4). SIMP
was gifted from Sankyo Co., Ltd. (Tokyo, Japan). For use as the control
vehicle (pH 7.4), HEPES buffer containing 2% dimethyl sulfoxide was
prepared. The solutions were stored at 4°C until use.
Animals
Seven-week-old female ICR mice were used in this experiment. The
animals were housed in plastic cages in a room with a 12-h light/12-h
dark cycle. They were free from ocular disease. All mice were treated
in compliance with the guidelines of the ARVO Resolution on the Use of
Animals in Ophthalmic and Vision Research, and the experimental
protocol was approved by the Committee for Animal Research, Kyoto
Prefectural University of Medicine.
Corneal Alkali Burn
An alkali burn was created as described previously.14
Briefly, 2 µl of 0.5N NaOH was instilled to 400 corneas of ICR mice,
with no subsequent eye-washing; 60 unburned corneas were used to
confirm normal cytokine levels.
Treatment of Mouse Alkali Burns with SIMP
After alkali injuries to 400 corneas, 200 were randomly assigned
for treatment with vehicle containing 400 µg/ml of SIMP, whereas the
other 200 controls were assigned for treatment with vehicle only.
Treatment was started 2 hours after the burn, and one drop of either
SIMP solution or vehicle was applied topically four times a day, every
3.5 hours from 8 AM to 10 PM. This treatment regimen was continued for
14 days after the alkali burn.
At 4, 7, and 14 days after the burn, animals were sacrificed by severing the spinal cord. After the eyes were extracted, external examinations of each eye were performed by two independent examiners who were unaware of whether the animal had received SIMP or vehicle. Eyes were assessed for the presence of corneal opacity, corneal epithelial defects, hyphema, and cataracts. Each cornea was assigned a clinical score according to the area of corneal opacity using the after classification: 0, no opacity; 1, opacity covers less than one third of the corneal surface; 2, opacity covers more than one third and less than two thirds of the corneal surface; and 3, opacity covers more than two thirds of the corneal surface. The corneal epithelial defects were assigned a clinical score as follows: 0, no defects or superficial puctate keratitis; 1, epithelial defects cover less than one third of the corneal surface; 2, epithelial defects cover more than one third and less than two thirds of the corneal surface; 3, epithelial defects cover more than two thirds of the corneal surface. The presence of hyphema or cataract was classified as absent or present: 0, no hyphema; 1, existence of hyphema; 0, no cataract; 1, existence of cataract.
Histologic analyses also were performed for six SIMP-treated and six vehicle-treated eyes 14 days after the burn. These eyes were fixed with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS; pH 7.4) and then dehydrated with 20% sucrose in 0.1 M PBS. Specimens were embedded in O.C.T. compounds (Miles Inc., Elkhart, IN), after which 8-µm cryostat sections were cut, air-dried, and stained with hematoxylin-eosin.
Cytokine Quantitation
To examine changes in cytokine expression with topical application
of SIMP, the concentrations of IL-1, IL-1ß, IL-6, and TNF- were
measured in both SIMP- and vehicle-treated corneas 4, 7, and 14 days
after the burn, using enzyme-linked immunosorbant assay (ELISA) systems
for IL-1, IL-1ß (Genzyme Co., Cambridge, MA), IL-6 (Endogen, Inc.,
Cambridge, MA), and TNF- (Genzyme Co.). Uninjured corneas were used
to confirm normal cytokine levels. At each time point, the eyes were
extracted, and the supernatants of corneal lysates were prepared as
described previously.14
17
Briefly, excised corneas 3 mm
in diameter were frozen in liquid nitrogen and then smashed and
homogenized in PBS at a ratio of 100 µl per cornea. The supernatants
were collected by centrifugation at 1500g for 10 minutes and
stored at -80°C until use. Each test sample comprised 6 to 7 whole
corneas, with 9 to 12 samples (60–80 corneas) used at each time point
in both groups.
Statistics
Clinical scores were compared for significance using the
Mann–Whitney U test. ELISA values were compared using
Student’s t-test.
Results
Treatment of Mouse Alkali Burn with SIMP
Corneal opacification was graded as moderate to severe 4
days after an alkali burn in SIMP- and non-SIMP–treated eyes.
Thereafter, it gradually receded until 14 days after the burn in both
groups. The score for corneal opacification was significantly lower at
postinjury days 7 and 14 in the SIMP-treated group than that in
controls (Fig. 1
A). The score for corneal epithelial defects was significantly lower
than controls at day 7 in the SIMP-treated group. At postinjury day 14,
the corneal epithelium had almost healed in both groups (Fig. 1B)
. In
the vehicle-only group, hyphema was observed in 22% (average score,
0.22) of eyes 4 days after injury, in 49% (average score, 0.49) of
eyes 7 days after injury, and in 43% (average score, 0.43) of eyes 14
days after injury. In contrast, in the SIMP-treated group, hyphema was
observed in 28% (average score, 0.28) of eyes at day 7. This was
significantly lower than in the control group (Fig. 1C) . The scores for
cataract were lower in the SIMP-treated group than in controls at all
time points, but these were not statistically significant (Fig. 1D)
.
Figure 2
shows the typical appearance of corneas 14 days after injury. Although
SIMP-treated corneas (Figs. 2A
2C)
contained few inflammatory cells
and little tissue destruction, control corneas treated with vehicle
only (Figs. 2B
2D)
disclosed massive inflammatory cell infiltration
and severe stromal destruction.
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levels were greatly increased
4 and 7 days after the burn (its value was approximately 8.5 times the
baseline value). Although the expression of IL-1 4 days after injury
was not different between SIMP- and vehicle-treated corneas, the level
of IL-1 in the SIMP-treated group at day 7 was 32.5 ± 5.5
pg/cornea (mean ± SEM), significantly lower than in
vehicle-treated corneas (45.2 ± 2.7 pg/cornea) (Fig. 3)
. At day 14 after the burn, the levels of IL-1 had returned almost
to normal levels in both groups. The level of IL-1ß at day 7 also was
significantly lower in the SIMP-treated group (5.6 ± 0.4
pg/cornea) than in controls (8.0 ± 1.0 pg/cornea) (Fig. 4)
. At days 4 and 14 after injury, the levels of IL-6 did not differ
between the two groups; however, the concentration of IL-6 in the
SIMP-treated group at day 7 was 14.1 ± 2.7 pg/cornea,
significantly lower than in controls (29.3 ± 6.6 pg/cornea) (Fig. 5)
. Levels of TNF- were elevated approximately 1.2 times the baseline
value in both the SIMP- and vehicle-treated corneas at day 7; however,
the levels did not differ between the two groups (Fig. 6)
.
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Alkali injury of the cornea is characterized by the massive
infiltration of PMNs into the stroma and the severe destruction of
corneal collagens. In our recent experiment, among the 10 cytokines
examined, IL-1 and IL-6 were found to be induced strongly in
alkali-burned corneas, the peak production of IL-1 being 3 days
after injury and the peak production of IL-6 being 7 days after
injury.14
15
IL-1 and IL-6 can lead to infiltration by
PMNs, after which these cells release superoxide, prostaglandins,
lysosomal enzymes, or MMPs, which may cause corneal stromal melting.
IL-1 also induces MMPs in cultured corneal stromal cells18
and is known to induce corneal epithelial and stromal cells to express
IL-6 and IL-8.19
20
21
In addition, a recent report has
demonstrated clearly that IL-1 induces MMPs and IL-8 in corneal
fibroblasts extracted from wounds.22
In view of this,
inflammatory cytokines upregulated after an alkali burn are considered
to be responsible for cell infiltration and collagen destruction.
The present study has demonstrated that the topical application of SIMP
significantly reduces the expression of IL-1, IL-1ß, and IL-6 in
alkali-burned mouse corneas 7 days after the injury. It is noteworthy
that at 4 days after injury, the levels of IL-1, IL-1ß, and IL-6
were the same in SIMP- and vehicle-treated corneas, implying that the
reduction of these cytokines that are seen 7 days after injury probably
is a result of the indirect action of the SIMP. One kind of SIMP (thiol
peptide) recently has been shown to have an inhibitory effect on the
chemotaxis of PMNs.23
However, on the basis of our data
demonstrating that IL-1 and IL-6 after an alkali burn were mainly
expressed in regenerating epithelium as opposed to infiltrating
cells,14
we consider that the reduction of
inflammatory cytokines by the SIMP used in these experiments is
not the result of the inhibitory effect on the chemotaxis of PMNs.
Previous work has shown that the levels of IL-1 and IL-6 after
an alkali burn correlate well with the concentration of alkali
solution.14
15
In view of this, it is likely that the
expression of inflammatory cytokines in regenerating
epithelium14
correlates with the severity of the
destruction of corneal collagens. We speculate that degraded corneal
collagens induce regenerating epithelium to express IL-1, IL-1ß,
and IL-6 and that the suppression of stromal degradation by SIMP
results in less expression of cytokines in alkali-burned cornea.
However, we cannot completely exclude the possibility that SIMP
may prevent the activation or inhibit the expression of these cytokines by a mechanism(s) independent of stromal collagens.
Previous reports have demonstrated that MMPI prevents the ulceration of rabbit corneas after an alkali burn.11 12 13 In this study, using a mouse alkali-burn model, we confirmed the beneficial effect of SIMP on corneal opacification, epithelial regeneration, and hyphema formation after the burn, although the effect is not immediate. Recently, Saika and associates24 demonstrated that TIMPs enhance the spreading of the corneal epithelium ex vivo and the proliferation of corneal epithelial cells in vitro. Our data indicated that MMPIs also promote epithelial regeneration in vivo. Interestingly, we found that the incidence of hyphema formation was significantly lower in SIMP-treated eyes than it was in non-SIMP treated eyes 7 days after an alkali burn. Because trabecular meshwork cells and ciliary muscle cells express MMPs,25 26 our results suggest that hyphema formation in alkali-burned eyes may be the result of the destruction of trabecular meshwork, iris, and ciliary body by MMPs. Hyphema complications are clinical features of some cases of alkali-burned eyes, and it is worth considering the possibility that MMPI may be useful in preventing the occurrence of hyphema after alkali injury.
Although IL-1, IL-1ß, and IL-6 are cytokines lacking a
transmembrane domain precursor, TNF- contains a transmembrane
domain, and membrane-anchored pro–TNF- is proteolytically processed
to the mature TNF-.27
Several MMPs are responsible for
the processing of mature TNF-,27
a processing that
MMPIs can inhibit.28
29
We found that the protein level of
TNF- after our alkali burn did not differ between the SIMP-treated
and control groups. This may be because both precursor and mature
TNF- were contained in the supernatants of each sample, and both
were detected by ELISA systems. Alternatively, it could be because the
upregulation of TNF- after the burn is only slight, making it
impossible to detect the effect of SIMP on TNF- expression. We
predict that in conditions in which the expression or the processing of
TNF- is excessively upregulated, MMPI may have an inhibitory effect
on TNF- expression. Interestingly, Fas ligand, a major inducer of
apoptosis, belongs to the TNF family and is prevented from shedding by
MMPI.30
We predict that further mechanisms may exist in
the action of MMPI that need to be established.
The anticollagenase activity of MMPI has been considered to be the main mechanism by which it protects alkali-burned eyes from becoming ulcerated; however, the inhibitory effect of MMPI on the expression of inflammatory cytokines after the burn also may be responsible for the reduced damage seen in MMPI-treated eyes.
Acknowledgements
The authors thank Andrew J. Quantock for helpful comments and critical reading of the manuscript.
Footnotes
Supported in part by a grant in-aid for scientific research (09877336, 08457467, 09771459) from the Japanese Ministry of Education, Culture and Science; a research grant from the Japanese Ministry of Health and Welfare; and the intramural research fund of the Kyoto Prefectural University of Medicine.
Submitted for publication January 7, 1999; revised April 6, 1999; accepted May 5, 1999.
Proprietary interest category: N.
Corresponding author: Chie Sotozono, Department of Ophthalmology, Kyoto Prefectural University of Medicine, Hirokoji, Kawaramachi, Kamigyoku, Kyoto 602-0841, Japan. E-mail: csotozon@eyeeye.ophth.kpu-m.ac.jp
References
by human corneal epithelial cells Nippon Ganka Gakkai Zasshi 95,728-732[Medline][Order article via Infotrieve]
autocrine feedback loop Invest Ophthalmol Vis Sci 38,1367-1379 precursor by metalloproteinases Nature 370,555-557[Medline][Order article via Infotrieve]
processing by a metalloproteinase inhibitor Nature 370,558-561[Medline][Order article via Infotrieve]
(TNF ) and abrogates endotoxin-induced lethality Shock 7,427-431[Medline][Order article via Infotrieve]
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