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. Pflugfelder1
`
`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 mg/ml
`lipopolysaccharide (LPS), LPS with 10 mg/ml doxycycline, or LPS with 0.1 mg/ml methylpred-
`nisolone (MP) for 24 hours. The intracellular and supernatant protein amounts of IL-1a, the
`precursor and mature forms of IL-1b, IL-1 receptor antagonist (IL-1 RA), and the intracellular level
`of IL-1b– 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-1a, IL-1b, IL-1 RA, and ICE.
`RESULTS. LPS increased the mRNA and protein amounts of intracellular and released IL-1a, mature
`IL-1b, and IL-1 RA. Doxycycline inhibited the LPS-induced IL-1b 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-1b 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-1b 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-b 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. (Invest
`Ophthalmol Vis Sci. 2000;41:2544 –2557)
`
`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 filamentary 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 inflammation may be the primary factor causing this con-
`
`From the 1Ocular Surface and Tear Center, Bascom Palmer Eye
`Institute, Department of Ophthalmology; and 2Department 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. Pflugfelder, Bascom Palmer Eye
`Institute, 900 NW 17th Street, Miami, FL 33136.
`spflugfelder@bpei.med.miami.edu
`
`2544
`
`dition.1 The proinflammatory cytokine interleukin (IL)-1 has
`been identified as a factor that may play a key role in the
`initiation and perpetuation of this inflammation. We have ob-
`served that as tear clearance from the ocular surface decreases,
`the concentrations of both isoforms of the proinflammatory
`cytokine IL-1, IL-1a,2 and IL-1b (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-1a and IL-1b,
`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 impor-
`tant role in the regulation of inflammation and wound healing
`on the ocular surface. IL-1b 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-1aand IL-1bhave 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
`
`Investigative Ophthalmology & Visual Science, August 2000, Vol. 41, No. 9
`Copyright © Association for Research in Vision and Ophthalmology
`
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`IOVS, August 2000, Vol. 41, No. 9
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`Doxycycline Inhibition of IL-1 in Corneal Epithelium 2545
`
`make IL-1 a prime candidate for inducing ocular surface dis-
`ease, especially the chronic subclinical ocular surface inflam-
`mation of dry eye.
`Traditional therapies for keratoconjunctivitis have con-
`sisted of artificial 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 inflammatory 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 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 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-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, doxycy-
`cline, 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 hypoth-
`esized that one of the mechanisms of action of doxycycline in
`dry eye is the downregulation of the IL-1–mediated inflamma-
`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
`Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine
`serum (FBS), HEPES buffer, F12 (Ham’s), 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-b precursor, IL-1b– converting en-
`zyme (ICE), and IL-b mature enzyme-linked immunosorbent
`assay (ELISA) kits were from Cistron (Pine Brook, NJ); IL-1 a
`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 Ham’s F12 medium, supple-
`mented with 5% FBS, 0.5% dimethyl sulfoxide, 2 ng/ml EGF, 5
`mg/ml insulin, 5 mg/ml transferrin, 5 ng/ml selenium, 0.5 mg/ml
`hydrocortisone, 30 ng/ml cholera toxin A, 50 mg/ml gentami-
`cin, and 1.25 mg/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 mg/ml bacterial
`LPS alone or in combination with either 0.1 mg/ml MP or 10
`mg/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 280°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 280°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-1a, IL-1b, IL-1 RA, and ICE.
`
`RPA Template Construction
`Partial cDNAs for human IL-1a, IL-1b, IL-1 RA, and glyceralde-
`hyde-3-phosphate dehydrogenase (GAPDH) were prepared by
`reverse transcription–polymerase chain reaction (RT-PCR).
`PolyA1 RNA was isolated from cultured human corneal epi-
`thelial 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-1a(gene accession, X02531), IL-1b
`(gene accession, X02532), IL-1 RA (gene accession, M63099),
`and GAPDH (gene accession, NM 002046), according to the
`manufacturer’s instructions (Superscript II Reverse Transcrip-
`tion 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 59 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
`
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`2546
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`Solomon et al.
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`IOVS, August 2000, Vol. 41, No. 9
`
`TABLE 1. Primers Used in RNAse Protection Assay
`
`cDNA
`
`Size (bp)
`
`Upstream Primer (5*–3*)
`
`Downstream Primer-1 (5*–3*)
`
`Downstream Primer-2 (5*–3*)
`
`IL-1a
`
`IL-1b
`
`IL-1 RA
`
`GAPDH
`
`407
`
`348
`
`308
`
`188
`
`CAA GGA GAG CAT GGT GGT
`AGT AGC AAC CAA CG
`GCT ACG AAT CTC CGA CCA
`CCA CTA CAG C
`CCA TTC AGA GAC GAT CTG
`CCG ACC
`GAC ATC AAG AAG GTG GTG
`AAG CAG GC
`
`GCA CTG GTT GGT CTT CAT
`CTT GGG C
`CCT TGT ACA AAG GAC ATG
`GAG AAC ACC
`GCT TGT CCT GCT TTC TGT
`TCT CGC
`
`TAG TGC CGT GAG TTT CCC
`AGA AGA AGA GGA GG
`CTT ATC ATC TTT CAA CAC
`GCA GGA CAG G
`CTG TCT GAG CGG ATG AAG
`GCG AAG C
`CCA AAT TCG TTG TCA TAC
`CAG GAA ATG AGC
`
`mRNA-radiolabeled antisense RNA hybrids are treated with
`RNase specific for single-stranded RNA. Protected hybrids can
`then be resolved and quantified using gel electrophoresis and
`autoradiography. Radiolabeled antisense RNA was transcribed
`using the a kit (Maxiscript T7; Ambion) and labeling with
`[a-32P]CTP (800 Ci/mmol). Plasmids were digested at a unique
`BamHI site upstream of the cloned cDNAs. RNA probes were
`generated for IL-1a, IL-1b, 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 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 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-1a, IL-1b, IL-1 RA,
`and ICE
`
`RNase protection assays were performed (Direct Protect Sys-
`tem; Ambion) as described by the manufacturer. Briefly, cul-
`tured human corneal epithelial cells were resuspended in lysis
`buffer at approximately 107 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 ml of cell lysate, 105 cpm of each
`cytokine probe (specific activity, 5 3 10 5 cpm/mg) and 4 3
`104 cpm of the GAPDH probe (specific activity, 5 3 10 4
`cpm/mg) 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 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-1a, IL-1b, 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 6 SEM of relative mRNA amounts from three
`or four experiments.
`
`Immunodetection of IL-1b Precursor, IL-1b
`Mature, IL-1a, 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 280°C until assayed. The concentrations of IL-1b
`precursor and IL-1b 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 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 (7.5–203
`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 peroxidase– conjugated 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
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`Doxycycline Inhibition of IL-1 in Corneal Epithelium 2547
`
`(A) RNase protection as-
`FIGURE 1.
`say of RNA extracted from primary
`cultured human corneal epithelial
`cells. Cells were treated with either
`10 mg/ml bacterial LPS alone or in
`combination with either 0.1 mg/ml
`MP (LPS 1 MP) or 10 mg/ml doxycy-
`cline (LPS 1 Doxy).
`(B) mRNA
`amounts for IL-1a, IL-1b, and IL-1 RA
`corrected for the different amounts
`of GAPDH. Data are the mean 6 SEM
`of four different experiments on pri-
`mary cultures from four different do-
`nor corneas. A significant increase in
`the mRNA amounts of IL-1b was ob-
`served after treatment with LPS, with
`a subsequent significant decrease to
`the control
`level when either MP
`or doxycycline was added to LPS
`(*P 5 0.037, ANOVA). Similar nonsig-
`nificant changes occurred in the
`IL-1a mRNA expression. The mRNA
`amounts of IL-1 RA were significantly
`increased by LPS (**P 5 0.017,
`ANOVA) but were not markedly
`changed with the addition of MP or
`doxycycline.
`
`then exposed to x-ray film (Eastman Kodak, Rochester, NY)
`from 30 seconds to 3 minutes.
`
`IL-1b Activity Assay
`To evaluate the functional activity of IL-1bsecreted 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-1b.6
`IL-1b 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 cul-
`tured 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 de-
`scribed.17 Briefly, the central corneas of donor eye bank eyes
`were isolated with a 6-mm trephine after removal of the epi-
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`IOVS, August 2000, Vol. 41, No. 9
`
`FIGURE 2. Cellular protein amounts
`of the precursor (A) and the ma-
`ture (B) forms of IL-1b 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 1 MP, or
`LPS 1 doxycycline (LPS 1 Doxy).
`Protein amounts are expressed in
`picograms per milligram of total
`cellular protein assayed in corre-
`sponding cellular lysates. Data are
`the mean 6 SEM from four inde-
`pendent experiments performed
`in HLE cultures from four different
`donor corneas. LPS induced a sig-
`nificant increase in the intracellu-
`lar concentration of the mature
`IL-1b protein, with subsequent de-
`crease when MP or doxycycline
`was added (*P 5 0.035, ANOVA).
`
`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
`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 3 105 cells per well. After 5 days
`in culture, on confluence, cultures were switched to a serum-
`free medium containing DMEM supplemented with 5 mg/ml
`insulin, 5 mg/ml transferrin, 5 ng/ml selenium, 50 mg/ml gen-
`tamicin, and 1.25 mg/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-1b with one
`
`of the following: D-ITS alone, recombinant human (rh)-mature
`IL-1b (10 ng/ml), rh-pre-IL-1b (10 ng/ml), and rh-pre-IL-1b (10
`ng/ml) with matrix MMP-9 (1 mg/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 1 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 fibroblasts, two additional treatments were
`added: SHEM with LPS (10 mg/ml; SHEM-LPS) and SHEM with
`LPS and doxycycline (10 mg/ml each; SHEM-LPS 1 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.
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`Doxycycline Inhibition of IL-1 in Corneal Epithelium 2549
`
`FIGURE 3.
`Supernatant protein
`amounts of the precursor (A) and
`the mature (B) forms of IL-1b 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 significant increase
`in the extracellular concentration of
`the mature IL-1b protein, with sub-
`sequent decrease when MP or doxy-
`cycline was added. (*P 5 0.026,
`ANOVA). The IL-1b mature-to-pre-
`cursor ratio of protein concentra-
`tions (C) was markedly reduced after
`the addition of either MP or doxycy-
`cline.
`
`Measurement of ICE Activity
`
`The specific activity of ICE in cell lysates was measured, as
`previously described,18 by incubating lysates with the flu-
`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 37°C. Fluorescence was
`determined with a fluorometer at 360 nm (excitation) and
`530 nm (emission).
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`FIGURE 4. Functional activity of IL-1bmeasured by MMP-1 and -3 secretion from corneal fibroblasts treated with conditioned media from HLE. (A)
`MMP-1 secretion from corneal fibroblasts treated with serum-free medium (D-ITS), rh-mature IL-1b (mIL-1b), rh-precursor IL-1b (pIL-1b), and
`precursor IL-1b treated with MMP-9 (pIL-1b 1 MMP-9). Both mIL-1b and pIL-1b 1 MMP-9 significantly increased MMP-1 protein expression, when
`compared with D-ITS alone (*P 5 0.03, t-test). (B) MMP-1 secretion from corneal fibroblasts treated with conditioned media (CM) from HLE cultures
`that had been treated with SHEM alone (CM-SHEM), LPS (CM-LPS), LPS 1 doxycycline (CM-LPS 1 doxy), or doxycycline alone (CM-doxy). Corneal
`fibroblasts were also directly treated with medium containing LPS (SHEM-LPS) or LPS 1 doxycycline (SHEM-LPS 1 doxy). A significant increase in
`MMP-1 secretion was noted with CM-LPS compared with CM-SHEM, indicating that the IL-1b protein from HLE cultures was functionally active
`(*P 5 0.01, t-test). (C) MMP-3 secretion from corneal fibroblasts treated as in (A). Both mIL-1b and pIL-1b 1 MMP-9 significantly increased MMP-3
`protein expression, when compared with D-ITS alone (*P 5 0.005, **P 5 0.003, respectively). (D) MMP-3 secretion from corneal fibroblasts treated
`as in (B). Whereas CM-LPS significantly increased MMP-3 secretion compared with CM-SHEM (*P 5 0.04), CM-doxy decreased MMP-3 secretion
`(**P 5 0.01), both indicative of the activity of IL-1b protein derived from the HLE cultures. A significant decrease of MMP-3 after direct treatment
`with SHEM-LPS 1 doxy (***P 5 0.03) is a result of the direct effect of doxycycline on MMP-3. Data are expressed as treatment means 6 SEM from
`three independent experiments derived from conditioned media from three HLE cultures.
`
`Statistical Analysis
`Results are expressed as mean 6 SEM of at least three experi-
`ments 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.
`
`RESULTS
`
`Expression of the IL-1 Genes in the Human
`Corneal Epithelium
`All three genes of the IL-1 family: IL-1a, IL-1b, 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-1bwere
`found in these cultured epithelial cells and their supernatants.
`Considerably higher concentrations of the mature IL-1b were
`found within the cells (Figs. 2A, 2B), whereas higher precursor
`IL-1b concentrations were found in the extracellular environ-
`ment (Figs. 3A, 3B).
`
`Regulation of IL-1b by Endotoxin, Doxycycline,
`and MP
`The IL-1b mRNA was expressed at very low levels in the
`nonactivated corneal epithelium. However, when stimulated
`it was significantly upregulated (sevenfold; P 5
`with LPS,
`0.037) when compared with its baseline level (Figs. 1A, 1B).
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`Doxycycline Inhibition of IL-1 in Corneal Epithelium 2551
`
`FIGURE 5. Cellular protein amounts of
`IL-1a (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 1 MP, and
`LPS 1 doxy induced a significant in-
`crease in the intracellular concentra-
`tion of the mature IL-1b protein, when
`compared with control. (*P , 0.001,
`ANOVA).
`
`The addition of either MP or doxycycline to the culture signif-
`icantly inhibited the LPS-induced upregulation (P 5 0.037) and
`reversed IL-1b expression almost to its basal level.
`The cellular protein concentration of pre-IL-1b was low,
`varying between 6.4 6 6.9 pg/mg protein in cells cultured in
`medium alone and 23.3 6 13.6 pg/mg protein in cells stimu-
`lated with LPS (Fig. 2A). The concentration of pre-IL-1b was
`not affected by MP or doxycycline.
`Considerably higher levels of IL-1b mature protein were
`found within the cells (Fig. 2B). A significant increase of IL-1b
`mature was observed after treatment with LPS (from 284.8 6
`50.8 to 550.6 6 89.1 pg/mg protein; P 5 0.035, ANOVA). This
`LPS-induced elevation was significantly inhibited by the addi-
`tion of either MP (397.7 6 38.1 pg/mg protein), or doxycycline
`(427 6 47.05 pg/mg protein; P 5 0.035, ANOVA).
`
`The supernatant concentrations of IL-1bprecursor protein
`showed an increase from 13.24 6 6.31 pg/mg protein in
`untreated cells to 42.17 6 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 de-
`crease in the supernatant concentration of pre-IL-b. In contrast,
`the mature form of IL-b in culture supernatant significantly
`increased after LPS treatment (from 3.47 6 1.42 to 11.29 6
`3.96 pg/mg protein), and decreased to baseline when either
`MP (4.71 6 1.41pg/mg protein) or doxycycline (2.9 6 0.61
`pg/mg protein) was added to LPS-treated cultures (P 5 0.026,
`ANOVA; Fig. 3B). The IL-1b 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-1b (Fig. 3C).
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`
`FIGURE 6.
`Supernatant protein amounts
`of IL-1a (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 1
`MP, and LPS 1 doxy induced a border-
`line significant increase in the extracel-
`lular concentration of the mature IL-1a
`protein, when compared with control
`(*P 5 0.055, ANOVA). The IL-1 RA
`protein secretion was significantly in-
`creased by both LPS 1 Mp and LPS 1
`doxy (**P , 0.001, ANOVA). The IL-1
`RA to IL-1a 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.
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`
`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 1 MP,
`LPS 1 doxycycline (LPS 1 doxy), or
`the combination of LPS with MP and
`doxycycline (LPS 1 MP 1 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.
`
`Functional Activity of IL-1b Secreted from
`Cultured Corneal Epithelium
`Mature IL-1b, but not precursor IL-1b, 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-1b bioactivity (Figs. 4A, 4C). Prein-
`cubation of precursor IL-1b with MMP-9, an MMP that is
`known to process precursor IL-1b to the mature biologically
`active form,19 resulted in MMP-1 and MMP-3 levels comparable
`to those observed with mature IL-b. MMP-1 and -3 were de-
`tected in corneal fibroblast cultures treated with HLE-condi-
`tioned media (Figs. 4B, 4D). Significantly increased production
`of these MMPs was observed when the LPS-treated HLE-condi-
`tioned media were added. Treatment of the fibroblast cultures
`with conditioned media from LPS 1 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.
`
`Regulation of IL-1a by Endotoxin and MP
`The mRNA of IL-1ashowed 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-1a were not statistically significant, al-
`though they followed a trend similar to that of IL-1b.
`The intracellular IL-1a protein concentration significantly
`increased from 525 6 151 to 1134 6 155 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-1a concentration from 3.4 6 1.2 to
`10.7 6 2.7 pg/mg protein (P 5 0.055), but neither MP nor
`doxycycline affected the concentration of this cytokine. The
`cellular concentration of IL-1 a 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 5 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 significan