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1 From the Ocular Surface and Tear Center, Bascom Palmer Eye Institute, Department of Ophthalmology; and 2 Department of Urology, University of Miami School of Medicine, Florida.
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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METHODS. Human corneal limbal epithelium (HLE) was cultured from explants
prepared from limbal rings of donor corneas. Primary cultured limbal
epithelial cells were treated with either 10 µg/ml lipopolysaccharide
(LPS), LPS with 10 µg/ml doxycycline, or LPS with 0.1 mg/ml
methylprednisolone (MP) for 24 hours. The intracellular and supernatant
protein amounts of IL-1
, the precursor and mature forms of IL-1ß,
IL-1 receptor antagonist (IL-1 RA), and the intracellular level of
IL-1ß–converting enzyme (ICE) were measured with enzyme-linked
immunosorbent assays (ELISAs). Western blot analysis was performed to
evaluate IL-1 RA protein. mRNA steady state amounts were determined by
RNase protection assay (RPA) for IL-1, IL-1ß, IL-1 RA, and ICE.
RESULTS. LPS increased the mRNA and protein amounts of intracellular and
released IL-1, mature IL-1ß, and IL-1 RA. Doxycycline inhibited
the LPS-induced IL-1ß increase in the mRNA and protein amounts in the
corneal epithelium and upregulated the expression of the
anti-inflammatory IL-1 RA protein. In addition, doxycycline reduced the
steady state level of the cellular ICE protein but did not affect the
level of ICE transcripts. IL-1ß secreted to the conditioned media of
HLE was functionally active in inducing matrix metalloproteinase
(MMP)-1 and MMP-3 in cultured corneal fibroblasts. Doxycycline
significantly decreased IL-1ß bioactivity in the supernatants from
LPS-treated corneal epithelial cultures. These effects were comparable
to those induced by the corticosteroid, MP.
CONCLUSIONS. Doxycycline can suppress the steady state amounts of mRNA and protein of IL-ß and decrease the bioactivity of this major inflammatory cytokine. These data may partially explain the clinically observed anti-inflammatory properties of doxycycline. The observation that doxycycline was equally potent as a corticosteroid, combined with the relative absence of adverse effects, makes it a potent drug for a wide spectrum of ocular surface inflammatory diseases.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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,2
and IL-1ß (Solomon et al., unpublished
results, 2000), increase in the tear fluid. The IL-1 gene family is a
group of potent cytokines that function as major mediators of
inflammation and immune response.3
This family is composed
of three forms: two proinflammatory forms, IL-1 and IL-1ß, each
having a precursor form, and an anti-inflammatory form, IL-1 receptor
antagonist (IL-1 RA).
Recent data suggest that the IL-1 cytokines play an important role in
the regulation of inflammation and wound healing on the ocular surface.
IL-1ß was found in the epithelium, stroma, and endothelium of the
cornea, at the mRNA and protein levels.4
Type 1 receptor
for IL-1 is expressed in stromal fibroblasts.5
Both
IL-1 and IL-1ß have been found to modulate matrix
metalloproteinase (MMP) expression by corneal stromal
fibroblasts6
and their own synthesis in
keratocytes,7
to regulate apoptosis of keratocytes in
response to corneal epithelial wounding,8
and to
upregulate hepatocyte growth factor and keratocyte growth factor in
corneal fibroblasts.4
These findings make IL-1 a prime
candidate for inducing ocular surface disease, especially the chronic
subclinical ocular surface inflammation of dry eye.
Traditional therapies for keratoconjunctivitis have consisted of artificial tears and aqueous-conserving therapies, such as punctal occlusion. Although these therapies transiently improve irritation symptoms, they are often ineffective in treating the severe complications of dry eye, such as recurrent corneal epithelial erosion and corneal stromal ulceration. Therapies targeting the underlying inflammatory environment of the ocular surface would represent a major improvement in the management of these conditions and would have a major clinical impact. Consistent with the concept that inflammation is a key feature in the pathophysiology of keratitis sicca is the finding that both aqueous tear deficiency and meibomian gland disease are effectively treated with the corticosteroid methylprednisolone (MP).9 10 Unfortunately the long-term use of topical corticosteroids is limited by potential sight-threatening side effects, such as glaucoma and cataract. Therefore, there is a clinical need for nontoxic steroid-sparing anti-inflammatory therapies that target IL-1 expression in the corneal epithelium.
Systemically administered tetracycline antibiotics have long been recognized as effective therapies for ocular surface inflammatory diseases. The semisynthetic tetracycline, doxycycline, has been reported to successfully treat the common dry eye condition acne rosacea,11 as well as recurrent corneal erosions12 and phlyctenular keratoconjunctivitis.13 We hypothesized that one of the mechanisms of action of doxycycline in dry eye is the downregulation of the IL-1–mediated inflammatory cascade in the corneal epithelium. Therefore, the purpose of this study was to evaluate the effect of doxycycline on the regulation of IL-1 expression and activity in the human corneal epithelium.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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and IL-1 RA ELISA kits were from R&D systems (Minneapolis,
MN); and MMP-1 and MMP-3 ELISA kits were from Oncogene Research
Products of Calbiochem (Cambridge, MA). RNA lysis and RNase protection
kits were from Ambion (Austin, TX). IL-1 RA was from Genzyme
(Cambridge, MA). The BCA protein assay kit was from Pierce (Rockford,
IL).
Culture of Human Corneal Limbal Epithelium
Human corneal limbal epithelium (HLE) was cultured from explants
of human donor corneoscleral rims, provided by the Florida Lions Eye
Bank at the Bascom Palmer Eye Institute. Each corneoscleral rim was
trimmed, the endothelial layer and iris remnants were removed, and the
tissue was treated with dispase for 15 minutes. Each rim was dissected
into 12 equal parts, which were applied to six-well plastic dishes and
covered with a drop of FBS overnight. The explants were cultured in
supplemented hormonal epithelial medium (SHEM) containing equal amounts
of DMEM and Ham’s F12 medium, supplemented with 5% FBS, 0.5%
dimethyl sulfoxide, 2 ng/ml EGF, 5 µg/ml insulin, 5 µg/ml
transferrin, 5 ng/ml selenium, 0.5 µg/ml hydrocortisone, 30 ng/ml
cholera toxin A, 50 µg/ml gentamicin, and 1.25 µg/ml amphotericin
B. Cultures were incubated at 37°C under 95% humidity and 5%
CO2. The medium was changed every 2 days.
Cultures were maintained for 10 to 14 days until confluence and then
switched to the serum-free medium described above, without FBS, for 24
hours before the additions of treatments.
To demonstrate the effect of doxycycline on the corneal epithelium and to compare it with that of a corticosteroid, primary cultures of HLE were treated with 10 µg/ml bacterial LPS alone or in combination with either 0.1 mg/ml MP or 10 µg/ml doxycycline. These treatments were maintained for 24 hours.
After a 24-hour treatment, the culture supernatant was collected from
each well, centrifuged, and stored in -80°C until assayed by ELISA.
Cell lysis solution, containing 50 mM Tris-HCl (pH 7.6), 300 mM NaCl,
and 0.5% Triton X-100, was added to the cells for 3 hours, and the
cellular protein was collected, centrifuged, and stored in -80°C
until assayed by ELISA. In parallel cultures, the cells were subjected
to lysis buffer (Direct Protect; Ambion), and total RNA was isolated
for further assessment by RNase protection assay (RPA). The ELISA and
RPA were targeted at determining the protein and mRNA levels,
respectively, of IL-1, IL-1ß, IL-1 RA, and ICE.
RPA Template Construction
Partial cDNAs for human IL-1, IL-1ß, IL-1 RA, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were prepared by
reverse transcription–polymerase chain reaction (RT-PCR). PolyA+ RNA
was isolated from cultured human corneal epithelial cells using
oligo-dT–coated beads (Oligotex Direct mRNA Isolation System; Qiagen,
Valencia, CA), according to the manufacturer’s instructions. RT was
performed using 200 ng mRNA as template and gene-specific primers (see
Table 1 ) were prepared to human IL-1 (gene accession, X02531),
IL-1ß (gene accession, X02532), IL-1 RA (gene accession, M63099), and
GAPDH (gene accession, NM 002046), according to the manufacturer’s
instructions (Superscript II Reverse Transcription Kit; Life
Technologies). The resultant first-strand cDNA was used for PCR (PCR
Kit, Life Technologies) using a gene-specific upstream primer and the
same downstream primer-2 used for RT. An aliquot of the initial PCR
reaction (except for the GAPDH probe that required only a single round
of PCR) was reamplified using the same upstream primer and a third
gene-specific primer, downstream primer-1. The primers used in this
second amplification were designed with 12 nucleotide additions (four
copies of a trinucleotide repeat containing a single deoxyuracil in
each repeat) at their 5' ends, to facilitate rapid cloning of the
amplimers (pAMP1 System; Life Technologies).
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-32P]CTP (800 Ci/mmol).
Plasmids were digested at a unique BamHI site upstream of
the cloned cDNAs. RNA probes were generated for IL-1, IL-1ß, and
IL-1 RA, and GAPDH. The GAPDH message is a housekeeping gene that is
found to be expressed 10 to 20 times more than the messages for the
cytokines measured in the RPA. The GAPDH probe was therefore
transcribed to yield a specific activity 10 times less than that of the
cytokine probes, to allow simultaneous detection of protected cytokine
probe fragments as well as GAPDH probe fragments, given the range of
sensitivity provided by the x-ray film. After transcription, probes
were DNase treated to remove template DNA, and unincorporated
nucleotides were removed using RNA Quick Spin columns (Roche
Molecular Biochemicals, Indianapolis, IN). A template set containing
DNA templates for ICE and GAPDH RNA probes, was purchased from
PharMingen (San Diego, CA).
RNase Protection Assay for IL-1, IL-1ß, IL-1 RA, and ICE
RNase protection assays were performed (Direct Protect System;
Ambion) as described by the manufacturer. Briefly, cultured human
corneal epithelial cells were resuspended in lysis buffer at
approximately 107 cells/ml. The cell lysis buffer
included concentrated guanidine thiocyanate, which rapidly solubilizes
cells and also rapidly inactivates ribonucleases. Assays were performed
using 50 µl of cell lysate, 105 cpm of each
cytokine probe (specific activity, 5 x 10 5
cpm/µg) and 4 x 104 cpm of the GAPDH
probe (specific activity, 5 x 10 4
cpm/µg) for each sample. Samples were allowed to hybridize overnight
at 37°C. They were then treated for 30 minutes at 37°C with RNase
solution, after which the RNase was inactivated with proteinase K.
Protected RNA fragments were precipitated and separated on a 6%
polyacrylamide urea-Tris-base, boric acid, EDTA (TBE)
sequencing gel.
RPAs for IL-1, IL-1ß, and IL-1 RA were repeated four times on
primary cultures derived from four different donor corneas.
RPAs for ICE were repeated three times on primary cultures from three
donor corneas. Autoradiographs from these gels were scanned and then
analyzed using image analysis software (Gel-Pro; Media Cybernetics,
Silver Spring, MD). The digitized data for each band was plotted, and
the area under the curve for each peak was calculated with
statistical software (GraphPad Prism; GraphPad Software, San Diego,
CA). The value for each cytokine band was divided by the corresponding
value of the GAPDH band in the same lane to calculate the relative mRNA
amount for each gene. Results are shown as means ± SEM of
relative mRNA amounts from three or four experiments.
Immunodetection of IL-1ß Precursor, IL-1ß Mature, IL-1, IL-1
RA, and ICE
The conditioned media and cell lysates of corneal limbal
epithelial cells from four independent primary cultures, derived from
four different donor corneas were collected, centrifuged, and stored at
-80°C until assayed. The concentrations of IL-1ß precursor and
IL-1ß mature in the cell lysates and in the supernatants and of ICE
in cell lysates were measured by ELISAs according to the respective
manufacturer’s protocol. The cellular protein concentration in
cell lysates was measured with the BCA protein assay kit.
Western Blot Analysis for IL-1 RA
To evaluate the expression of IL-1 RA protein in the conditioned
medium and in the cells, we further incubated primary limbal epithelium
with LPS, LPS and MP, LPS and doxycycline, or LPS with MP and
doxycycline, using the same concentrations as described earlier.
Cell lysates and conditioned media containing equal quantities of protein were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using 4% to 15%, 0.75-mm thick polyacrylamide gel (Mini-ready; Bio-Rad, Hercules, CA) at a constant 200 V for 45 minutes, in a mini-protean electrophoresis apparatus (Bio-Rad). A positive control (human recombinant IL-1 RA; R&D) and prestained (7.5–203 kDa) molecular weight protein markers (Bio-Rad) were run simultaneously with the samples. Resolved proteins were transferred to nitrocellulose membranes (BioTrance 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 polyclonal rabbit anti-human IL-1 RA antibody diluted in 1% bovine serum albumin, Tris-buffered saline, and 0.5% Tween 20, the membranes were incubated with IgG-horseradish peroxidase–conjugated goat anti-rabbit (Sigma) diluted 1:80,000 in 1% bovine serum albumin, Tris-buffered saline, and 0.5% Tween 20. Signals were detected with an immunodetection kit (Renaissance Enhanced Chemiluminescence [ECL]; NEN Life Science Products, Boston, MA), and then exposed to x-ray film (Eastman Kodak, Rochester, NY) from 30 seconds to 3 minutes.
IL-1ß Activity Assay
To evaluate the functional activity of IL-1ß secreted
by cultured HLE, we sought to develop a bioassay that is based on the
induction of MMP-1 and MMP-3 in corneal fibroblasts by
IL-1ß.6
IL-1ß has been previously demonstrated to
induce MMP-1 and MMP-3 secretion in human synovial
fibroblasts,14
endometrial stromal cells,15
and fibrochondrocytes.16
We therefore cultured
early-passaged corneal fibroblasts in conditioned media that were
collected from the HLE cultures. The resultant MMP-1 and -3 supernatant
concentrations were measured.
Corneal fibroblasts were cultured as previously described.17 Briefly, the central corneas of donor eye bank eyes were isolated with a 6-mm trephine after removal of the epithelium and the endothelium with a cell scraper. Explant cultures were prepared in the same manner as described earlier for limbal epithelial culture, except that DMEM containing 10% FBS (D-FBS) was used. Cultures were incubated at 37°C under 95% humidity and 5% CO2, and the medium was changed twice a week. Fibroblasts were subcultured with 0.05% trypsin and 0.85 mM EDTA in a calcium-free MEM at 80% to 90% confluence with 1:3 to 1:4 split for three passages.
Third-passage fibroblasts were seeded in six-well tissue culture plates at a density of 2 x 105 cells per well. After 5 days in culture, on confluence, cultures were switched to a serum-free medium containing DMEM supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenium, 50 µg/ml gentamicin, and 1.25 µg/ml amphotericin B (D-ITS). Some cultures were maintained in D-FBS. After a 24-hour incubation in D-ITS, cultures were divided into two groups.
The first group of cultures served as a positive control and were treated directly with human recombinant IL-1ß with one of the following: D-ITS alone, recombinant human (rh)-mature IL-1ß (10 ng/ml), rh-pre-IL-1ß (10 ng/ml), and rh-pre-IL-1ß (10 ng/ml) with matrix MMP-9 (1 µg/ml).
The second group of cultures were treated with conditioned media (CM) derived from HLE cultures that had been treated as described earlier. These treatments included CM from HLE culture treated with medium alone (CM-SHEM), CM from HLE culture treated with LPS (CM-LPS), CM from HLE culture treated with LPS and doxycycline (CM-LPS + doxy), and CM from HLE culture treated with doxycycline (CM-doxy). To exclude the possibility that the drugs contained in the conditioned media (LPS and doxycycline) altered MMP secretion in corneal fibroblasts, two additional treatments were added: SHEM with LPS (10 µg/ml; SHEM-LPS) and SHEM with LPS and doxycycline (10 µg/ml each; SHEM-LPS + doxy).
Cultures were incubated with one of these treatments for 24 hours, and thereafter their supernatants were collected for measurement of MMP-1 and MMP-3 concentrations by ELISAs.
Measurement of ICE Activity
The specific activity of ICE in cell lysates was
measured, as previously described,18
by incubating lysates
with the fluorogenic substrate AcTyr-Val-Ala-Asp-AMC (YVAD-AMC; Upstate
Biotechnology, Lake Placid, NY) in buffer containing 100 mM HEPES, 10%
sucrose, 0.1% NP40, and 10 mM dithiothreitol (pH 7.5) for 1 hour at
37°C. Fluorescence was determined with a fluorometer at 360 nm
(excitation) and 530 nm (emission).
Statistical Analysis
Results are expressed as mean ± SEM of at least three
experiments performed on cultures derived from at least three different
donor corneas, respectively. Statistical analysis was performed using
Student’s t-test or one-way analysis of variance (ANOVA)
where appropriate. P < 0.05 was considered
significant.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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, IL-1ß, and IL-1 RA
were demonstrated at the RNA and protein levels in primary cultures of
human corneal limbal epithelium (Fig. 1A
, first lane; Figs. 2
and 3
5
and 6
). Both precursor and mature forms of IL-1ß were found in
these cultured epithelial cells and their supernatants. Considerably
higher concentrations of the mature IL-1ß were found within the cells
(Figs. 2A
2B) , whereas higher precursor IL-1ß concentrations were
found in the extracellular environment (Figs. 3A
3B)
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The cellular protein concentration of pre-IL-1ß was low, varying between 6.4 ± 6.9 pg/mg protein in cells cultured in medium alone and 23.3 ± 13.6 pg/mg protein in cells stimulated with LPS (Fig. 2A) . The concentration of pre-IL-1ß was not affected by MP or doxycycline.
Considerably higher levels of IL-1ß mature protein were found within the cells (Fig. 2B) . A significant increase of IL-1ß mature was observed after treatment with LPS (from 284.8 ± 50.8 to 550.6 ± 89.1 pg/mg protein; P = 0.035, ANOVA). This LPS-induced elevation was significantly inhibited by the addition of either MP (397.7 ± 38.1 pg/mg protein), or doxycycline (427 ± 47.05 pg/mg protein; P = 0.035, ANOVA).
The supernatant concentrations of IL-1ß precursor protein showed an increase from 13.24 ± 6.31 pg/mg protein in untreated cells to 42.17 ± 13.9 pg/mg protein in LPS-treated cells (Fig. 3A) . This change was not significant. The addition of either MP or doxycycline caused a small, nonsignificant decrease in the supernatant concentration of pre-IL-ß. In contrast, the mature form of IL-ß in culture supernatant significantly increased after LPS treatment (from 3.47 ± 1.42 to 11.29 ± 3.96 pg/mg protein), and decreased to baseline when either MP (4.71 ± 1.41pg/mg protein) or doxycycline (2.9 ± 0.61 pg/mg protein) was added to LPS-treated cultures (P = 0.026, ANOVA; Fig. 3B ). The IL-1ß mature to precursor ratio decreased after the addition of either MP or doxycycline, indicating an inhibitory effect of these two drugs on the activation of IL-1ß (Fig. 3C) .
Functional Activity of IL-1ß Secreted from Cultured Corneal
Epithelium
Mature IL-1ß, but not precursor IL-1ß, significantly increased
MMP-1 and MMP-3 secretion by corneal fibroblasts compared with the
media control (D-ITS), showing that this is a feasible method for
determining IL-1ß bioactivity (Figs. 4A
4C
). Preincubation of precursor IL-1ß with MMP-9, an MMP that is
known to process precursor IL-1ß to the mature biologically active
form,19
resulted in MMP-1 and MMP-3 levels comparable to
those observed with mature IL-ß. MMP-1 and -3 were detected in
corneal fibroblast cultures treated with HLE-conditioned media (Figs. 4B
4D)
. Significantly increased production of these MMPs was observed
when the LPS-treated HLE-conditioned media were added. Treatment of the
fibroblast cultures with conditioned media from LPS +
doxycycline–treated HLE abrogated this LPS-induced increase (Figs. 4B
4D)
. In contrast, direct treatment of the corneal fibroblasts with LPS
resulted in negligible protein concentrations, excluding a direct
effect of drug that remained in the HLE-conditioned media.
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by Endotoxin and MP showed upregulation after stimulation with
LPS. This increase was partially reversed when either MP or doxycycline
was added to LPS (Fig. 1B)
. The changes in the mRNA amounts for IL-1
were not statistically significant, although they followed a trend
similar to that of IL-1ß.
The intracellular IL-1 protein concentration significantly increased
from 525 ± 151 to 1134 ± 155 pg/mg protein
(P < 0.001, ANOVA) in LPS-treated cultures and
remained high when either MP or doxycycline was added to the culture
(Fig. 5A
). A similar trend was observed in the culture supernatants (Fig. 6A ). LPS increased IL-1 concentration from 3.4 ± 1.2 to
10.7 ± 2.7 pg/mg protein (P = 0.055), but neither
MP nor doxycycline affected the concentration of this cytokine. The
cellular concentration of IL-1 protein in corneal epithelial cells
was 10-fold higher than the concentration released into the culture
medium.
Effect of Doxycycline on IL-1 RA
IL-1 receptor antagonist was upregulated by LPS alone or with
either MP or doxycycline at the mRNA and protein levels. LPS induced a
3.5-fold increase of mRNA expression (P = 0.017), which
was partially inhibited when MP was added but was unchanged when
doxycycline was added to LPS (Fig. 1B)
.
No differences were noted between the four culture groups in the concentrations of the intracellular IL-1 RA protein (Fig. 5B) . However, the concentrations of IL-1 RA in the cultures supernatants demonstrated a significant increase when either MP or doxycycline was added to LPS (862 ± 222, 2730 ± 333, and 2230 ± 229 pg/mg protein for control, LPS + MP, and LPS + doxycycline, respectively (P < 0.001; Fig. 6B ).
Western blot analysis for IL-1 RA demonstrated a marked increase in the bands for culture supernatants when doxycycline was added to LPS. The combination of doxycycline and MP added to LPS demonstrated the highest IL-1 RA protein expression (Fig. 7 , top). The expression of the intracellular IL-1 RA was not changed by any of the treatments, when compared with medium alone (Fig. 7 , bottom). These data correspond with the ELISA data, showing significant changes in secreted IL-1 RA, whereas the intracellular expression was stable (Figs. 5B 6B) .
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ratio markedly
decreased after the addition of LPS alone, but addition of either of
these two drugs to LPS returned the ratio to that observed in the
control cultures (Fig. 6C)
.
Effect of Doxycycline on ICE
The mRNA transcripts of ICE were demonstrated in nontreated limbal
epithelial cells (Fig. 8)
, but no changes were demonstrated among the different treatments. The
active p20 subunit of ICE protein was demonstrated by ELISA in limbal
epithelium cell lysates (Fig. 9)
. Its concentration increased significantly after stimulation with LPS
(from 6.4 ± 1.1 to 8.5 ± 0.6 pg/µg protein;
P = 0.011, ANOVA). There was no significant change in
the cellular concentration of ICE protein in the MP-treated group.
Doxycycline, however, significantly reduced the intracellular protein
concentration when compared with LPS alone (6.3 ± 1.8 pg/µg
protein, P = 0.011, ANOVA).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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, IL-1ß, and IL-1 RA) were
expressed at low levels. A strong inflammatory stimulation with LPS
upregulated all three forms. When MP was added to LPS, mRNA expression
of all three forms was inhibited, with IL-1 RA inhibited to a lesser
degree. When doxycycline was added as the anti-inflammatory agent, only
the level of IL-1ß RNA was decreased. Both doxycycline and the
corticosteroid decreased the concentration of mature IL-1ß protein
and increased the concentration of the anti-inflammatory IL-1 RA
protein in the supernatants of limbal epithelial cultures. Two main mechanisms have been identified for activating precursor IL-ß into its mature form. The first involves ICE, an intracellular cysteine protease that is highly specific for the cleavage of IL-1ß.18 ICE has been reported to be upregulated by immunologic stimuli such as LPS and granulocyte-macrophage colony-stimulating factor (GM-CSF).22 To the best of our knowledge, this is the first demonstration of ICE expression by the corneal epithelium. We demonstrated ICE at the mRNA level and found that this gene is not regulated by either MP or doxycycline. Others have reported that LPS induced only a moderate increase of ICE activity in monocytes and that transcriptional induction of the ICE gene does not play a role in LPS-induced ICE activation.23 Immunodetection of the active p20 fragment of ICE by ELISA in this study demonstrated the protein in nontreated cells, which was increased by LPS, moderately decreased by MP, and significantly decreased by doxycycline. It therefore appears that doxycycline may decrease IL-1ß at the protein level through direct inhibition of ICE.
A second mechanism for IL-1ß activation involves the MMPs. MMP-9, -2, and -3 can process precursor IL-ß into its active form.19 Activated forms of these MMPs are found in the tear fluid of patients with delayed tear clearance.2 This suggests that these enzymes may activate precursor IL-1ß in the extracellular environment as found in our conditioned media. Among these various MMPs, MMP-9 was observed to produce the most stable biologically active IL-1ß. It is possible that doxycycline inhibits the conversion of precursor IL-1ß produced by the corneal epithelium, into its mature form, by inhibiting proteolytic cleavage by MMP-9. MMP-9 is the principal gelatinase produced by the human corneal epithelium.6 We have observed a more than 70% decrease in MMP-9 activity in doxycycline-treated corneal epithelial cultures.24 This finding is consistent with reports of doxycycline’s decreasing MMP-9 activity in nonocular tissues. For example, doxycycline was shown to inhibit MMP-1 and MMP-9 activities in inflamed gingival tissue of adult patients with periodontitis.25 Therefore, another possible mechanism of inhibition of IL-1ß activation by doxycycline may be MMP-9–mediated.
Within cells, the majority of IL-1ß was in the mature form, whereas in the supernatant, the precursor form was higher. LPS stimulation increased the concentration of mature IL-1ß. Consistent with this finding was an increase in IL-1 bioactivity in the supernatants of these stimulated cultures. This finding is similar to the normal human tear fluid, in which we have detected predominantly precursor IL-1ß, with very little mature form. The release of IL-1ß has been reported to occur during apoptosis or cell death.26 If the presence of IL-1ß in the culture supernatant were due to release from dying cells, we would expect to find mainly mature IL-1ß. Therefore, it is possible that the corneal epithelium in our culture systems, as well as in vivo, releases precursor IL-1ß into the environment, where it is susceptible to proteolytic cleavage and activation. Indeed, treatment of our cultures with either MP or doxycycline appeared to inhibit activation of precursor IL-1ß in the supernatant of LPS-activated cells.
Tetracyclines have been used widely to treat several localized
inflammatory diseases, such as chronic acne, periodontitis, and the
dermatologic and ocular manifestations of
rosacea.11
27
28
29
One study27
reported
dramatic improvement in the symptoms and signs of ocular rosacea in 111
patients treated with oral tetracycline or doxycycline. The exact
mechanisms of doxycycline-induced suppression of the corneal epithelium
IL-1ß system are not clear. Tetracycline was found to inhibit the
LPS-induced IL-1ß secretion, but not the mRNA accumulation in human
monocytes, suggesting a posttranscriptional block in cytokine
production.30
In addition, tetracycline blocked
LPS-stimulated tumor necrosis factor (TNF)- secretion, by inducing
retention of membrane-associated TNF- in monocyte cell membranes,
thus preventing this cytokine’s release into the culture
media.31
Because no transport mechanism has been
identified for the IL-1ß protein, this mechanism of inhibition is
unlikely.
The functional activity of IL-1ß generated from our limbal epithelial cultures was tested on corneal fibroblasts, measuring their MMP-1 and -3 secretion. This bioassay provides an in vitro system that closely resembles the in vivo situation, wherein epithelial cells of the ocular surface produce inflammatory cytokines that regulate MMP secretion by corneal and conjunctival fibroblasts.6 Using MMP-1 and -3 secretion as a measure of IL-1ß activity in conditioned media from HLE cultures, we found significantly lower concentrations of these matrix-degrading enzymes when conditioned media from LPS + doxycycline-treated cultures were added. This result may be either IL-1ß–mediated or a result of a direct effect of doxycycline on these enzymes.
Intervention in one of the IL-1ß regulatory mechanisms may provide important strategies for the treatment of various inflammatory processes on the ocular surface. The increased tear fluid concentrations of IL-1ß in dry eye conditions may result from release of proinflammatory cytokines, especially IL-1ß, from stressed or damaged ocular surface epithelium into the tear film. A low volume of tear fluid and its delayed clearance from the ocular surface may further increase the concentration of IL-1ß. This in turn may induce recruitment and activation of ocular surface inflammatory cells. Increased activity of proteolytic enzymes (such as MMP-9) in the tear fluid of patients with delayed tear clearance may also process precursor-IL-1ß to its mature form, further increasing the concentration of IL-1ß in the tear fluid. This inflammatory cascade could be responsible for the signs and symptoms of ocular irritation so commonly encountered in dry eye. Pharmacologic intervention may be capable of breaking this vicious inflammatory cycle. We have demonstrated that doxycycline has the potential to reduce the protein levels of cellular and extracellular mature IL-1ß at a level comparable with that of a potent corticosteroid. We therefore conclude that based on our in vitro model, the efficacy of doxycycline for treatment of keratitis sicca may be due to inhibition of the IL-1ß system.
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Footnotes |
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Submitted for publication November 23, 1999; revised February 23, 2000; accepted March 17, 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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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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concentration and fluorescein clearance in ocular rosacea Invest Ophthalmol Vis Sci 40,2506-2512This article has been cited by other articles:
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  V A Smith, D Khan-Lim, L Anderson, S D Cook, and A D Dick Does orally administered doxycycline reach the tear film? Br. J. Ophthalmol., June 1, 2008; 92(6): 856 - 859. [Abstract] [Full Text] [PDF]
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 C.-C. Sun, J.-H. Su Pang, C.-Y. Cheng, H.-F. Cheng, Y.-S. Lee, W.-C. Ku, C.-H. Hsiao, J.-K. Chen, and C.-M. Yang Interleukin-1 Receptor Antagonist (IL-1RA) Prevents Apoptosis in Ex Vivo Expansion of Human Limbal Epithelial Cells Cultivated on Human Amniotic Membrane Stem Cells, September 1, 2006; 24(9): 2130 - 2139. [Abstract] [Full Text] [PDF]
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 D. N. Meli, R. S. Coimbra, D. G. Erhart, G. Loquet, C. L. Bellac, M. G. Tauber, U. Neumann, and S. L. Leib Doxycycline Reduces Mortality and Injury to the Brain and Cochlea in Experimental Pneumococcal Meningitis Infect. Immun., July 1, 2006; 74(7): 3890 - 3896. [Abstract] [Full Text] [PDF]
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 T. J. Kaitu'u, J. Shen, J. Zhang, N. B. Morison, and L. A. Salamonsen Matrix Metalloproteinases in Endometrial Breakdown and Repair: Functional Significance in a Mouse Model Biol Reprod, October 1, 2005; 73(4): 672 - 680. [Abstract] [Full Text] [PDF]
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 Y. Shoshani, J. Pe'er, V. Doviner, J. Frucht-Pery, and A. Solomon Increased Expression of Inflammatory Cytokines and Matrix Metalloproteinases in Pseudophakic Corneal Edema Invest. Ophthalmol. Vis. Sci., June 1, 2005; 46(6): 1940 - 1947. [Abstract] [Full Text] [PDF]
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D.-Q. Li, Z. Chen, X. J. Song, L. Luo, and S. C. Pflugfelder Stimulation of Matrix Metalloproteinases by Hyperosmolarity via a JNK Pathway in Human Corneal Epithelial Cells Invest. Ophthalmol. Vis. Sci., December 1, 2004; 45(12): 4302 - 4311. [Abstract] [Full Text] [PDF]
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 M. Ueta, T. Nochi, M.-H. Jang, E. J. Park, O. Igarashi, A. Hino, S. Kawasaki, T. Shikina, T. Hiroi, S. Kinoshita, et al. Intracellularly Expressed TLR2s and TLR4s Contribution to an Immunosilent Environment at the Ocular Mucosal Epithelium J. Immunol., September 1, 2004; 173(5): 3337 - 3347. [Abstract] [Full Text] [PDF]
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V A Smith and S D Cook Doxycycline--a role in ocular surface repair Br. J. Ophthalmol., May 1, 2004; 88(5): 619 - 625. [Abstract] [Full Text] [PDF]
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P. M. Stuart, F. Pan, X. Yin, Z. Haskova, S. Plambeck, and T. A. Ferguson Effect of Metalloprotease Inhibitors on Corneal Allograft Survival Invest. Ophthalmol. Vis. Sci., April 1, 2004; 45(4): 1169 - 1173. [Abstract] [Full Text] [PDF]
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 T. Krakauer and M. Buckley Doxycycline Is Anti-Inflammatory and Inhibits Staphylococcal Exotoxin-Induced Cytokines and Chemokines Antimicrob. Agents Chemother., November 1, 2003; 47(11): 3630 - 3633. [Abstract] [Full Text] [PDF]
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D. Q. Li, T. Y. Shang, H.-S. Kim, A. Solomon, B. L. Lokeshwar, and S. C. Pflugfelder Regulated Expression of Collagenases MMP-1, -8, and -13 and Stromelysins MMP-3, -10, and -11 by Human Corneal Epithelial Cells Invest. Ophthalmol. Vis. Sci., July 1, 2003; 44(7): 2928 - 2936. [Abstract] [Full Text] [PDF]
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 B. Axisa, I. M. Loftus, A. R. Naylor, S. Goodall, L. Jones, P. R.F. Bell, M. M. Thompson, and C. Napoli Prospective, Randomized, Double-Blind Trial Investigating the Effect of Doxycycline on Matrix Metalloproteinase Expression Within Atherosclerotic Carotid Plaques * Editorial Comment Stroke, December 1, 2002; 33(12): 2858 - 2864. [Abstract] [Full Text] [PDF]
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M. Mohri, P. S. Reinach, A. Kanayama, M. Shimizu, J. Moskovitz, T. Hisatsune, and Y. Miyamoto Suppression of the TNF{alpha}-Induced Increase in IL-1{alpha} Expression by Hypochlorite in Human Corneal Epithelial Cells Invest. Ophthalmol. Vis. Sci., October 1, 2002; 43(10): 3190 - 3195. [Abstract] [Full Text] [PDF]
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V. B. Mahajan, C. Wei, and P. J. McDonnell III Microarray Analysis of Corneal Fibroblast Gene Expression after Interleukin-1 Treatment Invest. Ophthalmol. Vis. Sci., July 1, 2002; 43(7): 2143 - 2151. [Abstract] [Full Text] [PDF]
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 C.-H. Chang, Y. Huang, A. C. Issekutz, M. Griffith, K.-H. Lin, and R. Anderson Interleukin-1{alpha} Released from Epithelial Cells after Adenovirus Type 37 Infection Activates Intercellular Adhesion Molecule 1 Expression on Human Vascular Endothelial Cells J. Virol., January 1, 2002; 76(1): 427 - 431. [Abstract] [Full Text] [PDF]
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A. Solomon, D. Dursun, Z. Liu, Y. Xie, A. Macri, and S. C. Pflugfelder Pro- and Anti-inflammatory Forms of Interleukin-1 in the Tear Fluid and Conjunctiva of Patients with Dry-Eye Disease Invest. Ophthalmol. Vis. Sci., September 1, 2001; 42(10): 2283 - 2292. [Abstract] [Full Text] [PDF]
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A. Solomon, M. Rosenblatt, D. Monroy, Z. Ji, S. C Pflugfelder, and S. C G Tseng Suppression of interleukin 1{alpha} and interleukin 1{beta} in human limbal epithelial cells cultured on the amniotic membrane stromal matrix Br. J. Ophthalmol., April 1, 2001; 85(4): 444 - 449. [Abstract] [Full Text]
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