Doxycycline Inhibition of Interleukin-1

in the Corneal Epithelium

Abraham Solomon,
1
Mark Rosenblatt,
1
De-Quan Li,
1
Zuguo Liu,
1
Dagoberto Monroy,
1
Zhonghua Ji,
1
Balakrishna L. Lokeshwar,
2
and Stephen C. Pugfelder
1
PURPOSE. To evaluate the effect of doxycycline on the regulation of interleukin (IL)-1 expression and
activity in human cultured corneal epithelium.
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 g/ml
lipopolysaccharide (LPS), LPS with 10 g/ml doxycycline, or LPS with 0.1 mg/ml methylpred-
nisolone (MP) for 24 hours. The intracellular and supernatant protein amounts of IL-1, the
precursor and mature forms of IL-1, IL-1 receptor antagonist (IL-1 RA), and the intracellular level
of IL-1converting 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-1, IL-1 RA, and ICE.
RESULTS. LPS increased the mRNA and protein amounts of intracellular and released IL-1, mature
IL-1, and IL-1 RA. Doxycycline inhibited the LPS-induced IL-1 increase in the mRNA and protein
amounts in the corneal epithelium and upregulated the expression of the anti-inammatory 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-1 secreted to the conditioned media of HLE was
functionally active in inducing matrix metalloproteinase (MMP)-1 and MMP-3 in cultured corneal
broblasts. Doxycycline signicantly decreased IL-1 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- and
decrease the bioactivity of this major inammatory cytokine. These data may partially explain the
clinically observed anti-inammatory 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 inammatory diseases. (Invest
Ophthalmol Vis Sci. 2000;41:25442557)
K
eratitis sicca, the corneal epithelial disease that devel-
ops in dry eye, is among the most common and prob-
lematic conditions faced by ophthalmologists. In mild
cases, it is associated with symptoms of irritation, redness, and
blurred vision. In the more severe forms, sight-threatening
corneal problems may develop, such as lamentary keratitis,
corneal epithelial erosions, corneal stromal vascularization,
and ulceration. The exact mechanism by which keratitis sicca
develops has not been established. Our group has reported
that inammation may be the primary factor causing this con-
dition.
1
The proinammatory cytokine interleukin (IL)-1 has
been identied as a factor that may play a key role in the
initiation and perpetuation of this inammation. We have ob-
served that as tear clearance from the ocular surface decreases,
the concentrations of both isoforms of the proinammatory
cytokine IL-1, IL-1,
2
and IL-1 (Solomon et al., unpublished
results, 2000), increase in the tear uid. The IL-1 gene family is
a group of potent cytokines that function as major mediators of
inammation and immune response.
3
This family is composed
of three forms: two proinammatory forms, IL-1 and IL-1,
each having a precursor form, and an anti-inammatory form,
IL-1 receptor antagonist (IL-1 RA).
Recent data suggest that the IL-1 cytokines play an impor-
tant role in the regulation of inammation and wound healing
on the ocular surface. IL-1 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
broblasts.
5
Both IL-1 and IL-1 have been found to modulate
matrix metalloproteinase (MMP) expression by corneal stromal
broblasts
6
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 broblasts.
4
These ndings
From the
1
Ocular Surface and Tear Center, Bascom Palmer Eye
Institute, Department of Ophthalmology; and
2
Department of Urology,
University of Miami School of Medicine, Florida.
Supported in part by Public Health Service Research Grant
EY11915 (SCP); Department of Health and Human Services, National
Eye Institute Grant CA61038 (BLL); National Cancer Institute; an un-
restricted Grant from Research to Prevent Blindness; and the Drs.
David and Maureen Smith Ocular Surface and Tear Research Fund.
Submitted for publication November 23, 1999; revised February
23, 2000; accepted March 17, 2000.
Commercial relationships policy: N.
Corresponding author: Stephen C. Pugfelder, Bascom Palmer Eye
Institute, 900 NW 17th Street, Miami, FL 33136.
spugfelder@bpei.med.miami.edu
Investigative Ophthalmology & Visual Science, August 2000, Vol. 41, No. 9
2544 Copyright Association for Research in Vision and Ophthalmology
make IL-1 a prime candidate for inducing ocular surface dis-
ease, especially the chronic subclinical ocular surface inam-
mation of dry eye.
Traditional therapies for keratoconjunctivitis have con-
sisted of articial tears and aqueous-conserving therapies, such
as punctal occlusion. Although these therapies transiently im-
prove 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 inammatory environment of the oc-
ular surface would represent a major improvement in the
management of these conditions and would have a major
clinical impact. Consistent with the concept that inammation
is a key feature in the pathophysiology of keratitis sicca is the
nding that both aqueous tear deciency and meibomian gland
disease are effectively treated with the corticosteroid methyl-
prednisolone (MP).
9,10
Unfortunately the long-term use of top-
ical 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-inammatory
therapies that target IL-1 expression in the corneal epithelium.
Systemically administered tetracycline antibiotics have
long been recognized as effective therapies for ocular surface
inammatory diseases. The semisynthetic tetracycline, doxycy-
cline, has been reported to successfully treat the common dry
eye condition acne rosacea,
11
as well as recurrent corneal
erosions
12
and phlyctenular keratoconjunctivitis.
13
We hypoth-
esized that one of the mechanisms of action of doxycycline in
dry eye is the downregulation of the IL-1mediated inamma-
tory 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.
METHODS
Reagents
Dulbeccos modied Eagles medium (DMEM), fetal bovine
serum (FBS), HEPES buffer, F12 (Hams), were from Life Tech-
nologies (Rockville, MD). Tissue culture plates were from Bec-
ton Dickinson (Franklin Lakes, NJ). Cholera toxin subunit A,
epidermal growth factor (EGF), hydrocortisone, LPS (derived
from Serratia marcescens), doxycycline, and MP were from
Sigma (St. Louis, MO). IL- precursor, IL-1converting en-
zyme (ICE), and IL- mature enzyme-linked immunosorbent
assay (ELISA) kits were from Cistron (Pine Brook, NJ); IL-1
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 cov-
ered with a drop of FBS overnight. The explants were cultured
in supplemented hormonal epithelial medium (SHEM) contain-
ing equal amounts of DMEM and Hams F12 medium, supple-
mented with 5% FBS, 0.5% dimethyl sulfoxide, 2 ng/ml EGF, 5
g/ml insulin, 5 g/ml transferrin, 5 ng/ml selenium, 0.5 g/ml
hydrocortisone, 30 ng/ml cholera toxin A, 50 g/ml gentami-
cin, and 1.25 g/ml amphotericin B. Cultures were incubated
at 37C under 95% humidity and 5% CO
2
. The medium was
changed every 2 days. Cultures were maintained for 10 to 14
days until conuence 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 g/ml bacterial
LPS alone or in combination with either 0.1 mg/ml MP or 10
g/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 80C
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 80C 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-1, IL-1 RA, and ICE.
RPA Template Construction
Partial cDNAs for human IL-1, IL-1, IL-1 RA, and glyceralde-
hyde-3-phosphate dehydrogenase (GAPDH) were prepared by
reverse transcriptionpolymerase chain reaction (RT-PCR).
PolyA RNA was isolated from cultured human corneal epi-
thelial cells using oligo-dTcoated beads (Oligotex Direct
mRNA Isolation System; Qiagen, Valencia, CA), according to
the manufacturers instructions. RT was performed using 200
ng mRNA as template and gene-specic primers (see Table 1)
were prepared to human IL-1 (gene accession, X02531), IL-1
(gene accession, X02532), IL-1 RA (gene accession, M63099),
and GAPDH (gene accession, NM 002046), according to the
manufacturers instructions (Superscript II Reverse Transcrip-
tion Kit; Life Technologies). The resultant rst-strand cDNA
was used for PCR (PCR Kit, Life Technologies) using a gene-
specic 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 reamplied using the same upstream primer and a third
gene-specic primer, downstream primer-1. The primers used
in this second amplication 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 Technol-
ogies).
RPA Probes
RPA requires hybridization of sense mRNA to complementary
radiolabeled antisense RNA. Subsequently, double-stranded
IOVS, August 2000, Vol. 41, No. 9 Doxycycline Inhibition of IL-1 in Corneal Epithelium 2545
mRNA-radiolabeled antisense RNA hybrids are treated with
RNase specic for single-stranded RNA. Protected hybrids can
then be resolved and quantied using gel electrophoresis and
autoradiography. Radiolabeled antisense RNA was transcribed
using the a kit (Maxiscript T7; Ambion) and labeling with
[-
32
P]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-1, 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 there-
fore transcribed to yield a specic 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
lm. After transcription, probes were DNase treated to remove
template DNA, and unincorporated nucleotides were removed
using RNA Quick Spin columns (Roche Molecular Biochemi-
cals, Indianapolis, IN). A template set containing DNA tem-
plates for ICE and GAPDH RNA probes, was purchased from
PharMingen (San Diego, CA).
RNase Protection Assay for IL-1, IL-1, IL-1 RA,
and ICE
RNase protection assays were performed (Direct Protect Sys-
tem; Ambion) as described by the manufacturer. Briey, cul-
tured human corneal epithelial cells were resuspended in lysis
buffer at approximately 10
7
cells/ml. The cell lysis buffer in-
cluded concentrated guanidine thiocyanate, which rapidly
solubilizes cells and also rapidly inactivates ribonucleases. As-
says were performed using 50 l of cell lysate, 10
5
cpm of each
cytokine probe (specic activity, 5 10
5
cpm/g) and 4
10
4
cpm of the GAPDH probe (specic activity, 5 10
4
cpm/g) for each sample. Samples were allowed to hybridize
overnight at 37C. They were then treated for 30 minutes at
37C with RNase solution, after which the RNase was inacti-
vated with proteinase K. Protected RNA fragments were pre-
cipitated and separated on a 6% polyacrylamide urea-Tris-base,
boric acid, EDTA (TBE) sequencing gel.
RPAs for IL-1, IL-1, 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 anal-
ysis 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-1 Precursor, IL-1
Mature, IL-1, IL-1 RA, and ICE
The conditioned media and cell lysates of corneal limbal epi-
thelial cells from four independent primary cultures, derived
from four different donor corneas were collected, centrifuged,
and stored at 80C until assayed. The concentrations of IL-1
precursor and IL-1 mature in the cell lysates and in the
supernatants and of ICE in cell lysates were measured by
ELISAs according to the respective manufacturers 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 condi-
tioned 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 concentra-
tions as described earlier.
Cell lysates and conditioned media containing equal quan-
tities 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 (7.5203
kDa) molecular weight protein markers (Bio-Rad) were run
simultaneously with the samples. Resolved proteins were trans-
ferred 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 peroxidaseconjugated goat anti-rabbit (Sigma) di-
luted 1:80,000 in 1% bovine serum albumin, Tris-buffered sa-
line, and 0.5% Tween 20. Signals were detected with an
immunodetection kit (Renaissance Enhanced Chemilumines-
cence [ECL]; NEN Life Science Products, Boston, MA), and
TABLE 1. Primers Used in RNAse Protection Assay
cDNA Size (bp) Upstream Primer (53) Downstream Primer-1 (53) Downstream Primer-2 (53)
IL-1 407 CAA GGA GAG CAT GGT GGT
AGT AGC AAC CAA CG
GCA CTG GTT GGT CTT CAT
CTT GGG C
TAG TGC CGT GAG TTT CCC
AGA AGA AGA GGA GG
IL-1 348 GCT ACG AAT CTC CGA CCA
CCA CTA CAG C
CCT TGT ACA AAG GAC ATG
GAG AAC ACC
CTT ATC ATC TTT CAA CAC
GCA GGA CAG G
IL-1 RA 308 CCA TTC AGA GAC GAT CTG
CCG ACC
GCT TGT CCT GCT TTC TGT
TCT CGC
CTG TCT GAG CGG ATG AAG
GCG AAG C
GAPDH 188 GAC ATC AAG AAG GTG GTG
AAG CAG GC
CCA AAT TCG TTG TCA TAC
CAG GAA ATG AGC
2546 Solomon et al. IOVS, August 2000, Vol. 41, No. 9
then exposed to x-ray lm (Eastman Kodak, Rochester, NY)
from 30 seconds to 3 minutes.
IL-1 Activity Assay
To evaluate the functional activity of IL-1 secreted by cultured
HLE, we sought to develop a bioassay that is based on the
induction of MMP-1 and MMP-3 in corneal broblasts by IL-1.
6
IL-1 has been previously demonstrated to induce MMP-1 and
MMP-3 secretion in human synovial broblasts,
14
endometrial
stromal cells,
15
and brochondrocytes.
16
We therefore cul-
tured early-passaged corneal broblasts in conditioned media
that were collected from the HLE cultures. The resultant
MMP-1 and -3 supernatant concentrations were measured.
Corneal broblasts were cultured as previously de-
scribed.
17
Briey, the central corneas of donor eye bank eyes
were isolated with a 6-mm trephine after removal of the epi-
FIGURE 1. (A) RNase protection as-
say of RNA extracted from primary
cultured human corneal epithelial
cells. Cells were treated with either
10 g/ml bacterial LPS alone or in
combination with either 0.1 mg/ml
MP (LPS MP) or 10 g/ml doxycy-
cline (LPS Doxy). (B) mRNA
amounts for IL-1, IL-1, and IL-1 RA
corrected for the different amounts
of GAPDH. Data are the mean SEM
of four different experiments on pri-
mary cultures from four different do-
nor corneas. A signicant increase in
the mRNA amounts of IL-1 was ob-
served after treatment with LPS, with
a subsequent signicant decrease to
the control level when either MP
or doxycycline was added to LPS
(*P 0.037, ANOVA). Similar nonsig-
nicant changes occurred in the
IL-1 mRNA expression. The mRNA
amounts of IL-1 RA were signicantly
increased by LPS (**P 0.017,
ANOVA) but were not markedly
changed with the addition of MP or
doxycycline.
IOVS, August 2000, Vol. 41, No. 9 Doxycycline Inhibition of IL-1 in Corneal Epithelium 2547
thelium and the endothelium with a cell scraper. Explant
cultures were prepared in the same manner as described ear-
lier for limbal epithelial culture, except that DMEM contain-
ing 10% FBS (D-FBS) was used. Cultures were incubated at
37C under 95% humidity and 5% CO
2
, 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% conuence with 1:3 to 1:4 split for three
passages.
Third-passage broblasts were seeded in six-well tissue
culture plates at a density of 2 10
5
cells per well. After 5 days
in culture, on conuence, cultures were switched to a serum-
free medium containing DMEM supplemented with 5 g/ml
insulin, 5 g/ml transferrin, 5 ng/ml selenium, 50 g/ml gen-
tamicin, and 1.25 g/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 rst group of cultures served as a positive control and
were treated directly with human recombinant IL-1 with one
of the following: D-ITS alone, recombinant human (rh)-mature
IL-1 (10 ng/ml), rh-pre-IL-1 (10 ng/ml), and rh-pre-IL-1 (10
ng/ml) with matrix MMP-9 (1 g/ml).
The second group of cultures were treated with condi-
tioned 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 secre-
tion in corneal broblasts, two additional treatments were
added: SHEM with LPS (10 g/ml; SHEM-LPS) and SHEM with
LPS and doxycycline (10 g/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.
FIGURE 2. Cellular protein amounts
of the precursor (A) and the ma-
ture (B) forms of IL-1 measured
by ELISA in cell lysates of HLE col-
lected after 24 hours in serum-free
medium and stimulated with ei-
ther bacterial LPS, LPS MP, or
LPS doxycycline (LPS Doxy).
Protein amounts are expressed in
picograms per milligram of total
cellular protein assayed in corre-
sponding cellular lysates. Data are
the mean SEM from four inde-
pendent experiments performed
in HLE cultures from four different
donor corneas. LPS induced a sig-
nicant increase in the intracellu-
lar concentration of the mature
IL-1 protein, with subsequent de-
crease when MP or doxycycline
was added (*P 0.035, ANOVA).
2548 Solomon et al. IOVS, August 2000, Vol. 41, No. 9
Measurement of ICE Activity
The specic activity of ICE in cell lysates was measured, as
previously described,
18
by incubating lysates with the u-
orogenic substrate AcTyr-Val-Ala-Asp-AMC (YVAD-AMC; Up-
state Biotechnology, Lake Placid, NY) in buffer containing
100 mM HEPES, 10% sucrose, 0.1% NP40, and 10 mM dithio-
threitol (pH 7.5) for 1 hour at 37C. Fluorescence was
determined with a uorometer at 360 nm (excitation) and
530 nm (emission).
FIGURE 3. Supernatant protein
amounts of the precursor (A) and
the mature (B) forms of IL-1 mea-
sured by ELISA in conditioned media
of HLE. Treatment conditions, ex-
pression of data, and number of ex-
periments are as described in Figure
2. LPS induced a signicant increase
in the extracellular concentration of
the mature IL-1 protein, with sub-
sequent decrease when MP or doxy-
cycline was added. (*P 0.026,
ANOVA). The IL-1 mature-to-pre-
cursor ratio of protein concentra-
tions (C) was markedly reduced after
the addition of either MP or doxycy-
cline.
IOVS, August 2000, Vol. 41, No. 9 Doxycycline Inhibition of IL-1 in Corneal Epithelium 2549
Statistical Analysis
Results are expressed as mean SEM of at least three experi-
ments performed on cultures derived from at least three different
donor corneas, respectively. Statistical analysis was performed
using Students t-test or one-way analysis of variance (ANOVA)
where appropriate. P 0.05 was considered signicant.
RESULTS
Expression of the IL-1 Genes in the Human
Corneal Epithelium
All three genes of the IL-1 family: IL-1, IL-1, and IL-1 RA were
demonstrated at the RNA and protein levels in primary cultures
of human corneal limbal epithelium (Fig. 1A, rst lane; Figs. 2
and 3, 5 and 6). Both precursor and mature forms of IL-1 were
found in these cultured epithelial cells and their supernatants.
Considerably higher concentrations of the mature IL-1 were
found within the cells (Figs. 2A, 2B), whereas higher precursor
IL-1 concentrations were found in the extracellular environ-
ment (Figs. 3A, 3B).
Regulation of IL-1 by Endotoxin, Doxycycline,
and MP
The IL-1 mRNA was expressed at very low levels in the
nonactivated corneal epithelium. However, when stimulated
with LPS, it was signicantly upregulated (sevenfold; P
0.037) when compared with its baseline level (Figs. 1A, 1B).
FIGURE 4. Functional activity of IL-1 measured by MMP-1 and -3 secretion from corneal broblasts treated with conditioned media from HLE. (A)
MMP-1 secretion from corneal broblasts treated with serum-free medium (D-ITS), rh-mature IL-1 (mIL-1), rh-precursor IL-1 (pIL-1), and
precursor IL-1 treated with MMP-9 (pIL-1 MMP-9). Both mIL-1 and pIL-1 MMP-9 signicantly increased MMP-1 protein expression, when
compared with D-ITS alone (*P 0.03, t-test). (B) MMP-1 secretion from corneal broblasts treated with conditioned media (CM) from HLE cultures
that had been treated with SHEM alone (CM-SHEM), LPS (CM-LPS), LPS doxycycline (CM-LPS doxy), or doxycycline alone (CM-doxy). Corneal
broblasts were also directly treated with medium containing LPS (SHEM-LPS) or LPS doxycycline (SHEM-LPS doxy). A signicant increase in
MMP-1 secretion was noted with CM-LPS compared with CM-SHEM, indicating that the IL-1 protein from HLE cultures was functionally active
(*P 0.01, t-test). (C) MMP-3 secretion from corneal broblasts treated as in (A). Both mIL-1 and pIL-1 MMP-9 signicantly increased MMP-3
protein expression, when compared with D-ITS alone (*P 0.005, **P 0.003, respectively). (D) MMP-3 secretion from corneal broblasts treated
as in (B). Whereas CM-LPS signicantly increased MMP-3 secretion compared with CM-SHEM (*P 0.04), CM-doxy decreased MMP-3 secretion
(**P 0.01), both indicative of the activity of IL-1 protein derived from the HLE cultures. A signicant decrease of MMP-3 after direct treatment
with SHEM-LPS doxy (***P 0.03) is a result of the direct effect of doxycycline on MMP-3. Data are expressed as treatment means SEM from
three independent experiments derived from conditioned media from three HLE cultures.
2550 Solomon et al. IOVS, August 2000, Vol. 41, No. 9
The addition of either MP or doxycycline to the culture signif-
icantly inhibited the LPS-induced upregulation (P 0.037) and
reversed IL-1 expression almost to its basal level.
The cellular protein concentration of pre-IL-1 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 stimu-
lated with LPS (Fig. 2A). The concentration of pre-IL-1 was
not affected by MP or doxycycline.
Considerably higher levels of IL-1 mature protein were
found within the cells (Fig. 2B). A signicant increase of IL-1
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 signicantly inhibited by the addi-
tion 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-1 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 signicant. The addition of
either MP or doxycycline caused a small, nonsignicant de-
crease in the supernatant concentration of pre-IL-. In contrast,
the mature form of IL- in culture supernatant signicantly
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-1 mature to precursor ratio de-
creased after the addition of either MP or doxycycline, indicat-
ing an inhibitory effect of these two drugs on the activation of
IL-1 (Fig. 3C).
FIGURE 5. Cellular protein amounts of
IL-1 (A) and IL-1 RA (B) measured by
ELISA in cell lysates of HLE. Treatment,
stimulants, expression of data, and
number of experiments are as de-
scribed in Figure 2. LPS, LPS MP, and
LPS doxy induced a signicant in-
crease in the intracellular concentra-
tion of the mature IL-1 protein, when
compared with control. (*P 0.001,
ANOVA).
IOVS, August 2000, Vol. 41, No. 9 Doxycycline Inhibition of IL-1 in Corneal Epithelium 2551
FIGURE 6. Supernatant protein amounts
of IL-1 (A) and IL-1 RA (B) measured
by ELISA in conditioned media of HLE.
Treatment, stimulants, expression of
data, and number of experiments are
as described in Figure 2. LPS, LPS
MP, and LPS doxy induced a border-
line signicant increase in the extracel-
lular concentration of the mature IL-1
protein, when compared with control
(*P 0.055, ANOVA). The IL-1 RA
protein secretion was signicantly in-
creased by both LPS Mp and LPS
doxy (**P 0.001, ANOVA). The IL-1
RA to IL-1 ratio of protein concentra-
tions (C) was markedly reduced after
LPS treatment but returned to the con-
trol value after the addition of either
MP or doxycycline.
2552 Solomon et al. IOVS, August 2000, Vol. 41, No. 9
Functional Activity of IL-1 Secreted from
Cultured Corneal Epithelium
Mature IL-1, but not precursor IL-1, signicantly increased
MMP-1 and MMP-3 secretion by corneal broblasts compared
with the media control (D-ITS), showing that this is a feasible
method for determining IL-1 bioactivity (Figs. 4A, 4C). Prein-
cubation of precursor IL-1 with MMP-9, an MMP that is
known to process precursor IL-1 to the mature biologically
active form,
19
resulted in MMP-1 and MMP-3 levels comparable
to those observed with mature IL-. MMP-1 and -3 were de-
tected in corneal broblast cultures treated with HLE-condi-
tioned media (Figs. 4B, 4D). Signicantly increased production
of these MMPs was observed when the LPS-treated HLE-condi-
tioned media were added. Treatment of the broblast cultures
with conditioned media from LPS doxycyclinetreated HLE
abrogated this LPS-induced increase (Figs. 4B, 4D). In contrast,
direct treatment of the corneal broblasts with LPS resulted in
negligible protein concentrations, excluding a direct effect of
drug that remained in the HLE-conditioned media.
Regulation of IL-1 by Endotoxin and MP
The mRNA of IL-1 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 signicant, al-
though they followed a trend similar to that of IL-1.
The intracellular IL-1 protein concentration signicantly
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 signicant in-
crease 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 doxycy-
cline 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 signicant changes in secreted
IL-1 RA, whereas the intracellular expression was stable (Figs.
5B, 6B).
At the supernatant level, the IL-1 RA-to-IL-1 ratio mark-
edly 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 demon-
strated 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 signicantly
after stimulation with LPS (from 6.4 1.1 to 8.5 0.6 pg/g
protein; P 0.011, ANOVA). There was no signicant change
in the cellular concentration of ICE protein in the MP-treated
group. Doxycycline, however, signicantly reduced the intra-
cellular protein concentration when compared with LPS alone
(6.3 1.8 pg/g protein, P 0.011, ANOVA).
Although the active part of ICE (the p20 isoform) was
readily measured by ELISA, we could not demonstrate ICE
activity in our samples using the ICE-specic uorogenic sub-
strate (YVAD-AMC). A possible explanation is that this assay,
which was previously described for detecting ICE in mono-
cytes and macrophages
20
and THP-1 myelomonocytic cells,
21
did not have sufcient sensitivity to detect this enzymes activ-
ity in HLE.
FIGURE 7. Western blot analysis of
IL-1 RA in conditioned media (top)
and in cell lysates (bottom) of HLE
cells cultured in medium alone (Con-
trol), or treated with LPS, LPS MP,
LPS doxycycline (LPS doxy), or
the combination of LPS with MP and
doxycycline (LPS MP doxy).
The positive control is rh-IL-1 RA.
Samples were taken from the same
cultures used for the ELISA analyses
shown in Figures 5B and 6B. The data
are representative of four experi-
ments.
IOVS, August 2000, Vol. 41, No. 9 Doxycycline Inhibition of IL-1 in Corneal Epithelium 2553
DISCUSSION
This study demonstrates the effects of doxycycline on the
expression patterns of the IL-1 gene family in the human limbal
epithelium in response to a strong inammatory stimulus. The
results of our study demonstrate a novel inhibitory effect of
doxycycline on the expression of the inammatory cytokine
IL-1, with a concomitant upregulation of the anti-inamma-
tory IL-1 RA (Table 2). We further report on the expression of
ICE (caspase-1) in the corneal epithelium, at both the mRNA
and protein levels, and demonstrate an inhibitory effect of
doxycycline on that enzyme as well. These effects are compa-
rable to those of the corticosteroid MP. Our results imply that
some of the clinically proven benets of tetracycline com-
pounds (tetracycline, doxycycline, and minocycline) in treat-
ing the ocular surface disease of dry eye may be mediated
through their regulatory effects on the synthesis, processing,
or release of IL-1.
FIGURE 8. RPA of primary cultured
human corneal epithelial cells. Treat-
ment conditions are described in Fig-
ure 1. (B) mRNA amounts for ICE
corrected for the different amounts
of GAPDH. Data are the mean SEM
of three experiments on primary cul-
tures from three donor corneas. No
signicant changes were noted be-
tween the different treatments.
2554 Solomon et al. IOVS, August 2000, Vol. 41, No. 9
We have demonstrated that in nonstimulated corneal lim-
bal epithelial cells, all three forms of IL-1 (IL-1, IL-1, and IL-1
RA) were expressed at low levels. A strong inammatory stim-
ulation with LPS upregulated all three forms. When MP was
added to LPS, mRNA expression of all three forms was inhib-
ited, with IL-1 RA inhibited to a lesser degree. When doxycy-
cline was added as the anti-inammatory agent, only the level
of IL-1 RNA was decreased. Both doxycycline and the corti-
costeroid decreased the concentration of mature IL-1 protein
and increased the concentration of the anti-inammatory IL-1
RA protein in the supernatants of limbal epithelial cultures.
Two main mechanisms have been identied for activating
precursor IL- into its mature form. The rst involves ICE, an
intracellular cysteine protease that is highly specic for the
cleavage of IL-1.
18
ICE has been reported to be upregulated
by immunologic stimuli such as LPS and granulocyte-macro-
phage colony-stimulating factor (GM-CSF).
22
To the best of our
knowledge, this is the rst 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 tran-
scriptional 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, mod-
erately decreased by MP, and signicantly decreased by doxy-
cycline. It therefore appears that doxycycline may decrease
IL-1 at the protein level through direct inhibition of ICE.
A second mechanism for IL-1 activation involves the
MMPs. MMP-9, -2, and -3 can process precursor IL- into its
active form.
19
Activated forms of these MMPs are found in the
tear uid of patients with delayed tear clearance.
2
This suggests
that these enzymes may activate precursor IL-1 in the extra-
cellular environment as found in our conditioned media.
Among these various MMPs, MMP-9 was observed to produce
the most stable biologically active IL-1. It is possible that
doxycycline inhibits the conversion of precursor IL-1 pro-
duced by the corneal epithelium, into its mature form, by
inhibiting proteolytic cleavage by MMP-9. MMP-9 is the princi-
pal 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 nding
is consistent with reports of doxycyclines decreasing MMP-9
activity in nonocular tissues. For example, doxycycline was
shown to inhibit MMP-1 and MMP-9 activities in inamed
gingival tissue of adult patients with periodontitis.
25
Therefore,
another possible mechanism of inhibition of IL-1 activation by
doxycycline may be MMP-9mediated.
Within cells, the majority of IL-1 was in the mature form,
whereas in the supernatant, the precursor form was higher.
LPS stimulation increased the concentration of mature IL-1.
Consistent with this nding was an increase in IL-1 bioactivity
in the supernatants of these stimulated cultures. This nding is
similar to the normal human tear uid, in which we have
detected predominantly precursor IL-1, with very little ma-
ture form. The release of IL-1 has been reported to occur
during apoptosis or cell death.
26
If the presence of IL-1 in the
culture supernatant were due to release from dying cells, we
would expect to nd mainly mature IL-1. Therefore, it is
possible that the corneal epithelium in our culture systems, as
well as in vivo, releases precursor IL-1 into the environment,
where it is susceptible to proteolytic cleavage and activation.
Indeed, treatment of our cultures with either MP or doxycy-
cline appeared to inhibit activation of precursor IL-1 in the
supernatant of LPS-activated cells.
Tetracyclines have been used widely to treat several local-
ized inammatory diseases, such as chronic acne, periodonti-
FIGURE 9. Protein amounts of intracellular ICE measured by ELISA in
cell lysates of HLE. Treatment, stimulants, and expression of data are
described in Figure 2. ICE protein amounts were signicantly elevated
after stimulation with LPS (*P 0.011, ANOVA), slightly reduced after
the addition of MP, and returned to the nonstimulated level after the
addition of doxycycline. Data are from ve independent experiments
performed on HLE cultures from ve donor corneas.
TABLE 2. Signicant Changes of mRNA and Protein Expression of the IL-1 System by Different Treatments in HLE Cultures
Treatment
IL-1 Mature IL-1 IL-1 RA ICE
mRNA Protein mRNA Protein mRNA Protein mRNA Protein
Intracellular
LPS (compared with control) 1 1 1 1 1
LPS MP (compared with LPS) 2 2
LPS doxy (compared with LPS) 2 2 2
Supernatant
LPS (compared with control) 1 1
LPS MP (compared with LPS) 2 1
LPS doxy (compared with LPS) 2 1
1Signicant increase; 2signicant decrease.
IOVS, August 2000, Vol. 41, No. 9 Doxycycline Inhibition of IL-1 in Corneal Epithelium 2555
tis, and the dermatologic and ocular manifestations of rosa-
cea.
11,2729
One study
27
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-1 system are not clear. Tetracycline was found to inhibit
the LPS-induced IL-1 secretion, but not the mRNA accumula-
tion in human monocytes, suggesting a posttranscriptional
block in cytokine production.
30
In addition, tetracycline
blocked LPS-stimulated tumor necrosis factor (TNF)- secre-
tion, by inducing retention of membrane-associated TNF- in
monocyte cell membranes, thus preventing this cytokines
release into the culture media.
31
Because no transport mecha-
nism has been identied for the IL-1 protein, this mechanism
of inhibition is unlikely.
The functional activity of IL-1 generated from our limbal
epithelial cultures was tested on corneal broblasts, 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 inammatory
cytokines that regulate MMP secretion by corneal and conjunc-
tival broblasts.
6
Using MMP-1 and -3 secretion as a measure of
IL-1 activity in conditioned media from HLE cultures, we
found signicantly lower concentrations of these matrix-de-
grading enzymes when conditioned media from LPS doxy-
cycline-treated cultures were added. This result may be either
IL-1mediated or a result of a direct effect of doxycycline on
these enzymes.
Intervention in one of the IL-1 regulatory mechanisms
may provide important strategies for the treatment of various
inammatory processes on the ocular surface. The increased
tear uid concentrations of IL-1 in dry eye conditions may
result from release of proinammatory cytokines, especially
IL-1, from stressed or damaged ocular surface epithelium into
the tear lm. A low volume of tear uid and its delayed
clearance from the ocular surface may further increase the
concentration of IL-1. This in turn may induce recruitment
and activation of ocular surface inammatory cells. Increased
activity of proteolytic enzymes (such as MMP-9) in the tear
uid of patients with delayed tear clearance may also process
precursor-IL-1 to its mature form, further increasing the con-
centration of IL-1 in the tear uid. This inammatory 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 inamma-
tory cycle. We have demonstrated that doxycycline has the
potential to reduce the protein levels of cellular and extracel-
lular mature IL-1 at a level comparable with that of a potent
corticosteroid. We therefore conclude that based on our in
vitro model, the efcacy of doxycycline for treatment of kera-
titis sicca may be due to inhibition of the IL-1 system.
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