Pro- and Anti-inflammatory Forms of Interleukin-1 in
`the Tear Fluid and Conjunctiva of Patients with
`Dry-Eye Disease
`
`Abraham Solomon,1 Dilek Dursun,1 Zuguo Liu,1,2 Yuhuan Xie,1,2 Angelo Macri,1 and
`Stephen C. Pflugfelder1
`
`PURPOSE. To compare the expression of the pro- and anti-
`inflammatory forms of interleukin (IL)-1 in the tear fluid and
`conjunctival epithelium of normal eyes and those with dry-eye
`disease.
`METHODS. The concentrations of IL-1␣, IL-1␤ (precursor and
`mature forms), and IL-1 receptor antagonist (IL-1Ra) were mea-
`sured by ELISA in tear fluid samples obtained from normal
`individuals and patients with dry eye who had rosacea-associ-
`ated meibomian gland disease (MGD) or Sjo¨gren’s syndrome
`(SS) aqueous tear deficiency (ATD). These cytokines were also
`measured in normal tear fluid before and after nasal stimulation
`to induce reflex tearing. The relative expression of these cyto-
`kines was evaluated in conjunctival impression cytology spec-
`imens and conjunctival biopsy tissue obtained from normal
`subjects and SS ATD–affected patients using immunofluores-
`cent staining. Matrix metalloproteinase (MMP)-9 concentration
`and activity in the tear fluid were evaluated with gelatin zy-
`mography and with an MMP-9 activity assay kit, respectively.
`RESULTS. Compared with normal subjects, the concentration of
`IL-1␣ and mature IL-1␤ in the tear fluid was increased, and the
`concentration of precursor IL-1␤ was decreased in patients
`with MGD (P ⬍ 0.05, P ⫽ 0.02, and P ⬍ 0.01, respectively) and
`SS ATD (P ⬍ 0.001, P ⫽ 0.02, and P ⬍ 0.001, respectively).
`There was no significant change in the concentration of IL-1␣,
`precursor IL-1␤, and IL-1Ra in reflex tear fluid, indicating that
`the lacrimal glands may secrete these cytokines. The activity of
`MMP-9, a physiological activator of IL-1␤, was significantly
`elevated in the tear fluid of both dry-eye groups compared with
`normal subjects. A strong positive correlation was observed
`between the intensity of corneal fluorescein staining and the
`tear fluid IL-1␣ concentration (r2 ⫽ 0.17, P ⬍ 0.02) and the
`mature-to-precursor IL-1␤ratio (r2 ⫽ 0.46, P ⬍ 0.001). Positive
`immunofluorescent staining for IL-1␣, mature IL-1␤, and IL-1Ra
`was observed in a significantly greater percentage of conjunc-
`tival cytology specimens from eyes with SS ATD than in those
`from normal eyes (P ⬍ 0.01 for IL-1␣, P ⬍ 0.009 for mature
`IL-1␤, and P ⬍ 0.05 for IL-1Ra).
`
`From the 1Ocular Surface and Tear Center, Bascom Palmer Eye
`Institute, Department of Ophthalmology, University of Miami School of
`Medicine, Florida; and the 2Zhongshan Ophthalmic Center, Sun Yat-
`sen University of Medical Sciences, Guangzhou, China.
`Supported in part by Fight-for-Sight Postdoctoral Fellowship
`PD98025; Grant EY11915, National Eye Institute, Bethesda, Maryland;
`an unrestricted grant from Research to Prevent Blindness; and the Drs.
`David and Maureen Smith Ocular Surface and Tear Research Fund.
`Submitted for publication February 2, 2001; revised April 20,
`2001; accepted May 15, 2001.
`Commercial relationships policy: N.
`The publication costs of this article were defrayed in part by page
`charge payment. This article must therefore be marked “advertise-
`ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`Corresponding author: Stephen C. Pflugfelder, Cullen Eye Insti-
`tute, 6565 Fannin, NC 205, Houston, TX 77030. stevenp@bcm.tmc.edu
`
`Investigative Ophthalmology & Visual Science, September 2001, Vol. 42, No. 10
`Copyright © Association for Research in Vision and Ophthalmology
`
`CONCLUSIONS. Dry-eye disease is accompanied by an increase in
`the proinflammatory forms of IL-1 (IL-1␣and mature IL-1␤) and
`a decrease in the biologically inactive precursor IL-1␤ in tear
`fluid. Increased protease activity on the ocular surface may be
`one mechanism by which precursor IL-1␤ is cleaved to the
`mature, biologically active form. The conjunctival epithelium
`appears to be one source of the increased concentration of IL-1
`in the tear fluid of patients with dry-eye disease. These results
`suggest that IL-1 may play a key role in the pathogenesis of
`keratoconjunctivitis sicca. (Invest Ophthalmol Vis Sci. 2001;
`42:2283–2292)
`
`T here is increasing evidence that dry eye is accompanied by
`
`subclinical ocular surface inflammation. The evidence for
`this inflammation includes increased expression of immune
`activation markers, such as HLA-DR, intercellular adhesion mol-
`ecule (ICAM)-1, and CD-40, by the conjunctival epithelium and
`infiltration of the conjunctiva by inflammatory cells.1–7 The
`importance of inflammation in the pathogenesis of dry eye is
`underscored by reports that the signs and symptoms of dry eye
`markedly improve with anti-inflammatory therapies such as
`glucocorticosteroids and cyclosporin.8,9
`Our group has previously evaluated the levels of the inflam-
`matory cytokines that are capable of modulating the expres-
`sion of these inflammatory markers and of stimulating leuko-
`cyte chemotaxis onto the ocular surface of dry eyes. Our
`studies showed that the levels of RNAs encoding a number of
`different inflammatory cytokines, including interleukin (IL)-1,
`-6, and -8 and TNF-␣, were elevated in the conjunctival epithe-
`lium of patients with Sjo¨gren’s syndrome (SS) keratoconjunc-
`tivitis sicca (KCS) compared with normal subjects.10 The levels
`of IL-1 and -8 RNA are directly correlated with the intensity of
`corneal fluorescein staining and are inversely correlated with
`conjunctival goblet cell density. In a subsequent study, we
`found that
`the concentration of matrix metalloproteinase
`(MMP)-9, the principal MMP enzyme produced by the corneal
`epithelium and a key factor in the pathogenesis of sterile
`corneal ulceration, increases as tear clearance decreases.11 The
`mechanism by which these inflammatory and matrix-degrading
`factors are upregulated in dry-eye disease has not been estab-
`lished.
`The proinflammatory cytokine IL-1 is an important mediator
`of inflammation and immunity.12 IL-1 has been implicated in
`the pathogenesis of human inflammatory diseases, such as
`septic shock, rheumatoid arthritis, and periodontitis,13–15 as
`well as the corneal and ocular surface diseases rosacea, bullous
`keratopathy,
`keratoconus,
`and
`sterile
`corneal
`ulcer-
`ation.11,16 –17 Both proinflammatory forms of IL-1 (IL-1␣ and
`-1␤) are multifunctional cytokines that in general produce
`similar biological effects, although these may vary among dif-
`ferent cell types and organ systems.19,20 IL-1 is a potent inducer
`of other inflammatory cytokines such as IL-6 and -8, TNF-␣, and
`granulocyte-macrophage
`colony-stimulating
`factor
`(GM-
`CSF).21,22 It also stimulates production of MMP enzymes by
`epithelial and inflammatory cells.23 Both IL-1␣ and -1␤ are
`
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`IOVS, September 2001, Vol. 42, No. 10
`
`TABLE 1. Demographic Characteristics of Study Patients
`
`Group
`
`Normal 1
`Normal 2
`SS ATD
`MGD
`
`n
`
`10
`7
`9
`13
`
`Gender
`
`Age
`
`6 women, 4 men
`4 women, 3 men
`8 women, 3 men
`7 women, 6 men
`
`32 ⫾ 6.6*
`46 ⫾ 8.6†
`68 ⫾ 9.1
`55 ⫾ 16.3
`
`Age
`Range
`
`24–44
`35–57
`53–80
`25–73
`
`Age is expressed as mean years ⫾ SD.
`* Normal 1 vs SS ATD P ⬍ 0.001; Normal 1 vs MGD P ⫽ 0.0004.
`† Normal 2 vs SS ATD P ⫽ 0.001; Normal 2 vs MGD P ⫽ 0.36.
`
`synthesized as precursor proteins with a molecular mass of
`approximately 33 kDa.19 The precursor and the mature 17-kDa
`forms of IL-1␣ are both biologically active.19 In contrast, the
`precursor form of IL-1␤ possesses minimal biological activity
`and requires cleavage to the 17-kDa mature form to become
`active.19 This conversion occurs within cells by IL-1␤– convert-
`ing enzyme (also known as ICE or caspase 1) and in the
`extracellular environment by a number of proteases, including
`leukocyte elastase, granzyme A, and MMP-2 and -9.12,24,25
`Among a number of different MMPs evaluated, MMP-9 was
`found to be the most efficient activator of precursor IL-1␤.25
`IL-1Ra is a cytokine that inhibits the activities of the proin-
`flammatory forms of IL-1 by competitively binding to the type
`1 IL-1 receptor.26 Administration of IL-1Ra has been found to
`be clinically beneficial in the treatment of arthritis and preven-
`tion of corneal transplant rejection in experimental mod-
`els.14,27 Both proinflammatory forms (IL-1␣ and -1␤) and the
`anti-inflammatory form (IL-1Ra) of IL-1 have been detected in
`the human corneal epithelium.28 –31 IL-1Ra has also been de-
`tected in human conjunctival epithelial cells.32 IL-1␣ and -1␤
`have also been detected in human tear fluid.11,33
`We hypothesized that increased concentration and/or activ-
`ity of IL-1 could be an initiating factor for the observed ocular
`surface immunopathology of dry eye. This study was designed
`to test this hypothesis by comparing the concentrations of
`IL-1␣, inactive precursor and active mature IL-1␤ and IL-1Ra in
`tear fluid samples obtained from patients with dry-eye disease
`who had rosacea-associated meibomian gland disease (MGD)
`or Sjo¨gren’s syndrome (SS) aqueous tear deficiency (ATD) and
`normal asymptomatic subjects. The relative levels of expres-
`sion of the IL-1 family of cytokines in the conjunctival epithe-
`lium of normal subjects and patients with SS ATD, the dry-eye
`condition that has been reported to involve the most severe
`
`KCS,34 were compared using immunofluorescent staining. The
`activity of MMP-9, a protease that activates precursor IL-1␤ in
`the extracellular environment was also evaluated. Finally, the
`tear fluid concentrations of lactoferrin, a protein secreted by
`the lacrimal glands into human tears that inhibits the formation
`of reactive oxygen species that can damage cells and promote
`production and release of IL-1 was measured.35,36
`
`MATERIALS AND METHODS
`
`Materials
`Rabbit anti-human polyclonal antibodies specific for IL-1␣, the precur-
`sor and mature forms of IL-1␤, and recombinant precursor and mature
`human IL-1␤and ELISA kits for the precursor and mature forms of IL-1␤
`were purchased from Cistron (Pine Brook, NJ); ELISA kits for IL-1␣and
`IL-1Ra and recombinant human IL-1␣ and IL1-Ra and polyclonal anti-
`sera for IL-1Ra from R&D Systems (Minneapolis, MN); FITC-conjugated
`secondary antibodies from Caltag Laboratories (Burlingame, CA); and
`Texas red-conjugated secondary antibodies from Molecular Probes
`(Eugene, OR).
`
`Patient Selection
`This study was conducted according to a protocol approved by the
`University of Miami School of Medicine Institutional Review Board and
`in accordance with the tenets of the Declaration of Helsinki. Informed
`consent was obtained from participants after the nature and possible
`consequences of the study were explained.
`Tear fluid samples were obtained from 9 patients with primary SS
`ATD, 1 patient with non-SS ATD, 13 patients with rosacea-associated
`MGD, and 17 normal subjects. The demographic characteristics of
`these patients are presented in Table 1. One group of normal subjects
`consisted of six women and four men who had no history of eye
`disease or ocular surgery, did not use eye drops, and had no ocular
`irritation symptoms. All the subjects had a Schirmer 1 test score greater
`than 15 mm, normal meibomian glands, and no corneal fluorescein
`staining. This group was used for evaluating tear proteins in unstimu-
`lated tear fluid. A second group of asymptomatic normal subjects (four
`women and three men) who met the same criteria was used for
`evaluating the effects of reflex tearing on the concentration of the IL-1
`family of cytokines in tear fluid. All normal subjects were recruited
`from the employees of the Bascom Palmer Eye Institute.
`The SS group consisted of eight women and one man. Diagnosis of
`primary SS was based on criteria proposed by Fox et al.37 and included
`the following: (1) a Schirmer 1 test score of 5 mm or less in at least one
`eye, (2) interpalpebral zone fluorescein dye staining of the conjunctiva
`
`TABLE 2. Concentrations of IL-1 Family Cytokines in Unstimulated Tear Fluid Obtained from Normal
`Subjects and Patients with MGD and SS ATD
`
`Cytokine
`
`Normal
`(n ⴝ 10)
`
`MGD
`(n ⴝ 13)
`
`SS
`(n ⴝ 9)
`
`IL-1␣
`
`Precursor IL-1␤
`
`Mature IL-1␤
`
`43.1 ⫾ 24
`
`253.7 ⫾ 90
`
`443.3 ⫾ 128.5
`
`379.2 ⫾ 73
`
`54.6 ⫾ 16
`
`29.8 ⫾ 10
`
`187.7 ⫾ 72
`
`21.2 ⫾ 10
`
`80.9 ⫾ 22
`
`Precursor-mature IL-1␤
`IL-1 Ra (⫻105)
`IL-1 Ra/IL-1␣ (⫻103)
`
`19.1 ⫾ 5
`2.95 ⫾ 1.27
`27.86 ⫾ 20.57
`
`1.17 ⫾ 0.7
`9.40 ⫾ 2.54
`6.95 ⫾ 6.6
`
`0.35 ⫾ 0.2
`23.96 ⫾ 12.35
`5.01 ⫾ 6.28
`
`IL-1 Ra/mature IL-1␤ (⫻103)
`
`40.5 ⫾ 18.39
`
`14.28 ⫾ 42.59
`
`70.29 ⫾ 44.15
`
`P
`
`*⬍0.05
`†⬍0.001
`*⬍0.01
`†⬍0.0001
`*0.02
`†0.02
`*†0.0001
`*†NS
`*0.012
`†0.007
`*0.0016
`†NS
`
`Data are expressed as mean picograms per milliliter.
`* Normal vs MGD.
`† Normal vs SS.
`
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`IL-1 Cytokines in Keratoconjunctivitis Sicca
`
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`
`(A) Correlation between
`FIGURE 1.
`IL-1␣ concentration in tears and cor-
`neal fluorescein staining scores. (B)
`Correlation between mature-precur-
`sor IL-1␤ ratio in tears and corneal
`fluorescein staining scores.
`
`and cornea, (3) xerostomia, (4) elevated serum autoantibody titers
`(antinuclear antibody and/or rheumatoid factor titer ⱖ1:160), and (5)
`symptoms of moderate to severe ocular irritation.37 The patient with
`non-SS ATD was a 67-year-old woman who had a Schirmer 1 test score
`of 4 mm in both eyes and interpalpebral corneal and conjunctival
`staining, but did not have xerostomia or serum autoantibodies.
`The MGD group consisted of seven women and six men with the
`chief symptoms of ocular irritation and/or redness that was diagnosed
`as ocular rosacea with MGD, according to previously reported crite-
`ria.38 Corneal fluorescein staining was graded using a previously re-
`ported method.39 The cornea was examined under blue-light illumina-
`tion 2 minutes after instillation of 5 ␮l 2% fluorescein into the tear film.
`The intensity of the corneal fluorescein staining was graded in each of
`four quadrants on the cornea (temporal, nasal, superior, inferior) using
`
`a standardized four-point scale (0, no staining; 1, mild; 2, moderate; and
`3, intense). The range of staining scores was 0 to 12. Patients with dry
`eye were excluded if they had any clinical signs of external ocular
`infection, including staphylococcal blepharitis or bacterial conjuncti-
`vitis.
`
`Tear Fluid Collection and Sample Extraction
`
`For experiments comparing the concentrations of proteins in unstimu-
`lated tear fluid, a tear fluid sample was collected from the inferior tear
`meniscus of both eyes, causing the least irritation possible, using a
`preweighed polyester wick (Transorb rods; American Filtrona, Rich-
`mond, VA) to obtain the sample, as previously described.11,40 The
`volume of collected tears was determined by reweighing the rods
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`
`IOVS, September 2001, Vol. 42, No. 10
`
`TABLE 3. Poststimulation Tear Fluid Concentrations of IL-1␣, Precursor IL-1␤, and IL-1Ra in Seven
`Normal Subjects
`
`Cytokine
`
`Prestimulation
`
`Poststimulation 1
`(1 minute)
`
`Poststimulation 2
`(5 minutes)
`
`IL-1␣
`Precursor IL-1␤
`IL-1 Ra (⫻104)
`
`53.6 ⫾ 26.8†
`712 ⫾ 395‡
`23.81 ⫾ 11.25§
`
`35.2 ⫾ 10.0
`92.1 ⫾ 68
`22.13 ⫾ 81.37
`
`108.7 ⫾ 48.6
`384.7 ⫾ 188
`113.67 ⫾ 73.65
`
`P *
`
`NS
`NS
`NS
`
`Data are expressed as mean picograms per milliliter.
`* NS ⫽ no significant within group differences by ANOVA and no significant differences between the
`prestimulation concentration and either poststimulation concentration or between the 1- and 5-minute
`poststimulation concentrations, by the Wilcoxon test.
`† P ⫽ 0.01 compared with MGD; P ⫽ 0.006 compared with SS ATD (Table 2).
`‡ P ⫽ 0.03 compared with MGD; P ⫽ 0.05 compared with SS ATD (Table 2).
`§ P ⫽ 0.01 compared with MGD; P ⫽ 0.04 compared with SS ATD (Table 2).
`
`immediately after tear collection (model GA110 scale; Ohaus, Florham
`Park, NJ). Wicks were then placed into the end of a micropipette tip
`located within a 0.5-ml tube (Eppendorf, Fremont, CA) as described by
`Jones et al.40 For experiments evaluating the concentrations of IL-1
`cytokines in reflex tear fluid, an unstimulated tear fluid sample was
`collected, and reflex tearing was induced by placing a dry cotton-
`tipped applicator under the middle nasal turbinate, advancing it until
`the nasal membrane of the ethmoid sinus was reached, then rotating it
`for 1 minute as previously described.41 Tear fluid was then collected
`from the ipsilateral eye 1 and 5 minutes after stimulation.
`Tears were extracted from the saturated wicks by centrifuging
`them at 12,000 rpm for 5 minutes within the pipette tip after adding a
`volume of buffer (50 mM Tris/HCl, 0.15 M NaCl, 10 mM CaCl2, 0.005%
`Brij35, 0.02% sodium azide [pH 7.5]) 10 times greater than the original
`volume of the tear sample. The rods and pipette tips were carefully
`removed and the tear fluid aspirated. Tear fluid from both eyes was
`combined. This method resulted in a final tear dilution factor of 1:11
`for the ELISA, gelatin zymography, and the MMP-9 activity assay. Tear
`samples were placed in numbered 500-␮l tubes (Eppendorf) and
`stored at ⫺80°C for 3 to 7 days until they were used.
`Subjects with dry eye were not receiving any eye treatment other
`than nonpreserved artificial tears, and they were instructed not to
`instill any eye drops on the day the tear collection was performed. Tear
`collection was repeated on a separate day in all subjects, and both tear
`collections were performed in the morning. Tear fluid from one tear
`collection was used for cytokine ELISAs, and tear fluid from the second
`collection was used for the lactoferrin ELISA, gelatin zymography, and
`the MMP-9 activity assay.
`
`IL-1␣, -1␤, and -1Ra and Lactoferrin ELISAs
`The concentrations of IL-1 family cytokines were determined with
`commercial ELISA kits. For these assays, tear samples were diluted in
`
`ELISA buffer (supplied by the manufacturer) to a final volume of 100 to
`200 ␮l. These assays were performed as recommended by the manu-
`facturer. Tear lactoferrin concentration was measured with an immu-
`noassay (Touch Tear MicroAssay System; Touch Scientific, Raleigh,
`NC).
`
`Gelatin Zymography and MMP-9 Activity Assay
`
`Gelatinase level in the tear fluid was measured by gelatin zymography,
`as previously described.11 Diluted tear samples (all at a dilution of 1:11)
`were incubated with SDS-gel sample buffer for 30 minutes at room
`temperature and analyzed by electrophoresis on a 10% SDS-polyacryl-
`amide gel containing 1 mg/ml gelatin. After electrophoresis, the pro-
`teins were renatured by removing SDS from the gel using two washes
`of 0.25% Triton X-100 (30 minutes per wash). This was followed by an
`18-hour incubation at 37°C in the digestion buffer consisting of 50 mM
`Tris-HCl (pH 7.4) containing 0.15 M NaCl, 10 mM CaCl2, 2 ␮M ZnSO4,
`1 mM phenylmethylsulfonyl fluoride (PMSF), 0.005% Brij35, and 0.02%
`sodium azide. After this incubation, the gel was briefly rinsed in
`distilled water and stained with 0.25% Coomassie brilliant blue R250
`prepared in 40% isopropanol solution for 1 hour. The gel was
`destained with 7% acetic acid. Gelatinase activity in the gel was visible
`as a clear area in the blue background, indicating an area where the
`gelatin had been digested. The minimum sensitivity of this technique
`for detecting gelatinase B is 0.05 ng/lane. The molecular weight of
`gelatinases in the tear fluid was determined from molecular weight
`standards (prestained broad range standards; Bio-Rad, Hercules, CA)
`and 0.1 ng purified rabbit 92-kDa progelatinase B (Oncogene Research,
`Cambridge, MA) that were run in separate lanes on the gel. These gels
`were photographed with a camera (Polaroid, Cambridge, MA), and the
`photographs were scanned (Scan Jet 4C scanner; Hewlett-Packard,
`Palo Alto, CA) into a computer.
`
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`FIGURE 2.
`concentra-
`Lactoferrin
`tion in tears from normal subjects,
`patients with MGD, and patients
`with SS ATD. *P ⬍ 0.01 compared
`with patients with SS.
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`IOVS, September 2001, Vol. 42, No. 10
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`IL-1 Cytokines in Keratoconjunctivitis Sicca
`
`2287
`
`FIGURE 3. Gelatin zymogram of tear
`samples from a normal subject, two
`patients with MGD, a patient with
`non-SS ATD, and two patients with
`SS ATD. The 92-kDa pro-MMP-9 and
`72-kDa pro-MMP-2 bands are marked.
`Sample SS2 (right lane) was obtained
`from a 75-year-old patient with SS
`who had a 30-year history of bilateral
`recurrent sterile corneal ulceration
`with perforation.
`
`MMP-9 activity in tear fluid was measured with an MMP-9 activity
`assay system (Biotrak; Amersham Pharmacia Biotech, Piscataway, NJ),
`according to a previously published protocol.42 This colorimetric assay
`captures MMP-9 in the tear fluid and measures its activity in cleaving a
`modified prodetection enzyme and the subsequent cleavage of its
`chromogenic peptide substrate.43
`
`Immunofluorescent Staining of Conjunctival
`Impression Cytology and Conjunctival Biopsy
`Specimens
`Impression Cytology. The expression of four forms of IL-1
`(IL-1␣, precursor and mature IL-1␤, and IL-1Ra) was evaluated in
`conjunctival impression cytology specimens obtained from 6 ideal
`normal subjects and 16 patients with SS ATD.
`Impression cytology was performed by lightly pressing a mem-
`brane (Biopore; Millipore, Bedford, MA) against the nasal, inferior, and
`temporal bulbar conjunctiva, 1 mm behind the limbus after instillation
`of topical anesthesia (0.5% proparacaine hydrochloride). The mem-
`branes were placed in a container and stored at ⫺80°C until they were
`processed. Before staining, these membranes were fixed with cold
`methanol for 10 minutes and were then blocked for 20 minutes with
`PBS containing 2% fetal bovine serum (FBS) to prevent nonspecific
`staining. The cytology specimens obtained from the nasal and temporal
`bulbar conjunctiva were bisected, and one of the four primary anti-
`bodies (IL-1␣, precursor and mature IL-1␤ and -1Ra) was applied to
`
`each membrane for 1 hour at room temperature in a moist chamber.
`Membranes were rinsed three times with PBS and incubated for 1 hour
`with FITC-conjugated secondary antibody. The cytology specimens
`obtained from the inferior bulbar conjunctiva was treated only with
`secondary antibody and served as a negative control. Membranes were
`washed three times with PBS, placed on a glass microscope slide, and
`covered with nonfade mounting medium (Fluoromount-G; Southern
`Biotechnology Associates, Birmingham, AL) and a glass coverslip. Spec-
`imens were examined and photographed with a microscope (Axiophot
`II; Nikon, Tokyo, Japan) using TMAX 400 film (Eastman Kodak, Roch-
`ester, NY). The fluorescein staining was visually graded negative if the
`staining was less than or equal to the secondary antibody control and
`positive if it was greater than the secondary antibody control by two
`independent observers.
`Conjunctival Biopsy. Samples were taken from the superior or
`superotemporal bulbar conjunctiva of three normal subjects with
`Schirmer 1 test scores of 15 mm or more, no corneal fluorescein
`staining, and no lid or conjunctival inflammation, and three patients
`with SS KCS during cataract surgery. Tissue specimens were placed in
`DMEM (Life Technologies, Gaithersburg, MD) for transport and then
`were embedded in optimal cutting temperature (OCT) compound
`(Tissue Tek, Elkhart, IN), rapidly frozen in liquid nitrogen, and stored
`at ⫺70°C. Within 72 hours, serial 4- to 5-␮m-thick sections were cut.
`Indirect immunofluorescence staining on tissue sections was per-
`formed by a previously reported technique,44 using polyclonal anti-
`
`FIGURE 4. MMP-9 activity in tear
`samples of normal subjects, patients
`with MGD, and patients with SS ATD,
`measured by an MMP-9 activity assay.
`*P ⬍ 0.001 compared with normal
`subjects.
`
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`2288
`
`Solomon et al.
`
`IOVS, September 2001, Vol. 42, No. 10
`
`TABLE 4. Expression of IL-1 Cytokines in Conjunctival Impression
`Cytology Specimens from Normal Subjects and Patients with SS KCS
`
`Normal Eyes
`
`SS KCS
`
`Antibodies
`
`Eyes
`(n)
`
`IL-1␣
`Negative
`Positive
`Total
`Precursor
`IL-1␤
`Negative
`Positive
`Total
`Mature IL-
`1␤
`Negative
`Positive
`Total
`IL-1 Ra
`Negative
`Positive
`Total
`
`6
`0
`6
`
`2
`4
`6
`
`4
`2
`6
`
`6
`0
`6
`
`* Fisher exact test.
`
`%
`
`100
`0
`100
`
`33.33
`66.67
`100
`
`66.67
`33.33
`100
`
`100
`0
`100
`
`Eyes
`(n)
`
`%
`
`P *
`
`5
`11
`16
`
`0
`15
`15
`
`1
`15
`16
`
`8
`8
`16
`
`31.25
`68.75
`100
`
`0
`100
`100
`
`6.25
`93.75
`100
`
`50
`50
`100
`
`0.012
`
`0.071
`
`0.009
`
`0.051
`
`bodies for IL-1␣, -1␤, and -1Ra at a concentration of 50 ␮g/ml and
`FITC-labeled secondary antibodies. The specificity of these antibodies
`was evaluated by neutralization with their corresponding recombinant
`human cytokine proteins according to the manufacturer’s instructions.
`Primary antibodies were incubated for 1 hour at 37°C (0.002 ␮g/ml
`anti-IL-1␣ or anti-IL-1␤ antibodies with 50 pg/ml of their respective
`recombinant proteins and 1 ␮g/ml anti-IL-1Ra antibody with 50 ng/ml
`of its recombinant protein), before applying the mixture to tissue
`sections. Some sections were treated with the secondary antibody
`alone as a negative control. Slides were photographed as for the assays
`described earlier.
`
`Statistics
`Student’s t-test or Mann-Whitney test were used when appropriate for
`comparing the ELISA results between groups. One-way ANOVA and
`Wilcoxon paired tests were used to compare pre- and poststimulation
`tear cytokine concentrations. The ELISA data are expressed as means ⫾
`SD, and the differences were considered statistically significant at P ⬍
`0.05. The t-distribution was used to determine whether correlation
`coefficients were different from zero.45
`
`RESULTS
`
`Profile of IL-1 Cytokines in Tear Fluid
`The concentrations of the IL-1 family of cytokines in basal tear
`fluid collected from normal subjects and patients with MGD
`and SS ATD are presented in Table 2. The concentration of
`IL-1␣ was significantly higher in the tear fluid of both dry-eye
`groups than in the normal group. The precursor form of IL-1␤
`was the predominant form of this cytokine in normal tear fluid
`with a mean precursor-to-mature ratio of 19.1 ⫾ 14.2. IL-1Ra
`was also detected in normal tear fluid with a ratio to IL-1␣ and
`precursor IL-1␤ of more than 27,000 and more than 40,000,
`respectively. Compared with normal eyes, there was a statisti-
`cally significant increase in the concentration of mature IL-1␤
`and a decrease in the concentration of precursor IL-1␤ in the
`tear fluid of patients with MGD and SS ATD. These changes
`resulted in significantly lower ratios of the precursor-to-mature
`forms of IL-1␤in both dry-eye groups. There was no significant
`difference in the concentration of IL-1Ra among the three
`
`groups; however, the ratio of IL-1Ra to IL-1␣ was significantly
`lower in the two dry-eye groups and the IL-1Ra-to-IL-1␤ ratio
`was significantly decreased in the MGD group compared with
`the control group. A strong correlation was observed between
`the clinical intensity of corneal fluorescein staining and the log
`of the tear IL-1␣ concentration (Fig. 1A) and between the
`corneal fluorescein staining score and the precursor-mature
`IL-1␤ ratio (Fig. 1B).
`
`Concentrations of IL-1 Cytokines in Reflex
`Tear Fluid
`One minute after stimulation of the nasal mucosa, a 1.5-fold
`decrease of the tear fluid IL-1␣ concentration, a 7.7-fold de-
`crease in precursor IL-1␤ concentration, and a minimal de-
`crease in IL-1Ra concentration was observed. However, 5 min-
`utes after stimulation, the concentrations of all three cytokines
`increased compared with the 1-minute levels: IL-1␣(threefold),
`IL-1␤ (fourfold), and IL-1Ra (fivefold). Within-group (ANOVA)
`and paired (Wilcoxon test) comparisons between the pre-
`stimulation concentration and either the poststimulation con-
`centration or between the 1 and 5 minute poststimulation
`concentrations were not statistically significant (Table 3). Be-
`cause the concentration of mature IL-1␤was near or below the
`level of detection of our immunoassay in unstimulated and
`stimulated tear fluid obtained from these normal subjects, no
`meaningful statistical comparison could be made.
`
`Lactoferrin Concentration in Tear Fluid
`The tear fluid concentration of lactoferrin was 1.35 ⫾ 0.07
`␮g/ml in normal subjects, 1.16 ⫾ 0.16 ␮g/ml in patients with
`MGD (no significant difference), and 0.08 ⫾ 0.04 ␮g/ml in
`patients with SS (P ⫽ 0.001 when compared with either nor-
`mal subjects or MGD; Fig. 2).
`
`MMP-9 Level and Activity in Tear Fluid
`Minimal or no 92-kDa pro-MMP-9 was observed by gelatin
`zymography of tear fluid samples taken from normal control
`subjects (representative sample shown in Fig. 3, left lane). In
`contrast, greater levels of pro-MMP-9 were found in tear fluid
`samples taken from patients with dry eye who had MGD,
`patients with non-SS ATD, and those with SS (Fig. 3). The
`strongest bands were observed in a 75-year-old patient with SS
`who had a 30-year history of bilateral recurrent sterile corneal
`ulceration with perforation (Fig. 3, right lane, SS2).
`To confirm the zymography results, a quantitative MMP-9
`activity assay detected an activity of 7.2 ⫾ 2.1 U/mg in normal
`control subjects, which was significantly increased to 473.1 ⫾
`173.5 U/mg in patients with MGD (66-fold, P ⬍ 0.0001) and to
`651.7 ⫾ 208.3 U/mg in patients with SS (90-fold, P ⬍ 0.0001;
`Fig. 4).
`
`Immunostaining of the IL-1 Cytokines in
`Conjunctival Impression Cytology Specimens
`Impression cytology was used to evaluate the relative levels of
`expression of IL-1␣, precursor IL-1␤, mature IL-1␤, and IL-1Ra
`in apical conjunctival epithelium specimens obtained from six
`ideal normal subjects and 16 patients with SS KCS by impres-
`sion de´bridement with a membrane (Biopore; Millipore). IL-1␣
`and -1Ra could not be immunodetected in the normal conjunc-
`tival epithelium, and the mature form of IL-1␤ was detected in
`only 33% of normal samples. Positive immunofluorescent stain-
`ing for IL-1␣, mature IL-1␤, and IL-1Ra was observed in a
`significantly greater percentage of conjunctival cytology spec-
`imens from eyes with SS ATD than in those from normal eyes
`(P ⬍ 0.01 for IL-1␣, P ⬍ 0.009 for mature IL-1␤, and P ⬍ 0.05
`for IL-1Ra; Table 4, Fig. 5).
`
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`IOVS, September 2001, Vol. 42, No. 10
`
`IL-1 Cytokines in Keratoconjunctivitis Sicca
`
`2289
`
`FIGURE 5. Expression of
`the IL-1
`family of cytokines in apical conjunc-
`tival epithelial
`impression cytology
`samples from normal subjects and
`patients with SS ATD. (A, B) Staining
`with IL-1␣–specific antibody in cytol-
`ogy membranes from normal (A) and
`SS ATD (B) eyes; (C, D) staining with
`precursor IL-1␤–specific antibody in
`cytology membranes from normal
`(C) and SS ATD (D) eyes; (E, F) stain-
`ing with mature IL-1␤–specific anti-
`body in cytology membranes from
`normal (E) and SS ATD (F) eyes; (G,
`H) staining with IL-1Ra–specific anti-
`body in cytology membranes from
`normal (G) and SS ATD (H) eyes.
`Original magnification, ⫻200.
`
`Immunostaining of the IL-1 Cytokines in
`Conjunctival Biopsy Specimens
`The antibodies for IL-1␣ and -1Ra stained the epithelial cells in
`conjunctival specimens obtained from patients with SS, with
`the strongest staining observed in the superficial cell layers
`(Figs. 6A, 6G). Isolated IL-1Ra–positive cells were scattered
`throughout the conjunctival epithelium (Fig. 6G). The identity
`of these cells could not be determined by phase microscopy;
`however, their staining pattern was similar to that of inflam-
`matory cells located in the conjunctival stroma just below the
`epithelial basement membrane. The antibody for IL-1␤ stained
`the superficial conjunctival epithelium, although to a lesser
`degree than IL-1␣and -1Ra (Fig. 6D). Immunofluorescent stain-
`ing for all three forms of IL-1 was markedly reduced after
`preincubation of these antibodies with their corresponding
`recombinant cytokine proteins (Figs. 6B, 6E, 6H), which indi-
`cates the specificity of the antibodies. Lower intensity staining
`for IL-1␣ and -1Ra was still observed in the superficial layers of
`the conjunctival epithelium after preincubation. This may be
`
`attributed to increased concentrations of these cytokines in the
`tightly compacted superficial metaplastic epithelial cells or to
`absorption of these cytokines from the tear film. Minimal or no
`staining was observed in conjunctival specimens stained with
`the secondary antibody alone (Figs. 6C, 6F, 6I).
`No staining above the background level was observed for
`these three antibodies in conjunctival sections obtained from
`normal subjects (Fig. 7).
`
`DISCUSSION
`In this study we found that IL-1␣, the precursor and mature
`forms of IL-1␤, and IL-1