Cornea 19(4): 492–496, 2000.
`
`© 2000 Lippincott Williams & Wilkins, Inc., Philadelphia
`
`Interleukin-6 Levels in the Conjunctival Epithelium
`of Patients with Dry Eye Disease Treated with
`Cyclosporine Ophthalmic Emulsion
`
`Kathleen Turner, D.V.M., Stephen C. Pflugfelder, M.D., Zhonghua Ji, M.D.,
`William J. Feuer, M.S., Michael Stern, PhD, and Brenda L. Reis, PhD
`
`Purpose. To evaluate interleukin-6 (IL-6) levels in the conjuncti-
`val epithelium of patients with moderate to severe dry eye disease
`before and after treatment with cyclosporin A ophthalmic emul-
`sion (CsA) or its vehicle. Methods. Conjunctival cytology speci-
`mens were obtained from a subset of patients enrolled in a 6-month
`randomized, double-masked clinical trial of the efficacy and safety
`of topical CsA at baseline and after 3 and 6 months of B.I.D.
`treatment with 0.05% cyclosporine emulsion (n ⳱ 13), 0.1% cy-
`closporine emulsion (n ⳱ 8), or vehicle (n ⳱ 10). RNA was
`extracted and a competitive reverse transcriptase polymerase chain
`reaction (RT-PCR) was used to evaluate the levels of mRNA en-
`coding the inflammatory cytokine IL-6 and a housekeeping gene,
`G3PDH. Levels of IL-6 and G3PDH were measured and com-
`pared. Results. There was no change from baseline in the level of
`G3PDH after 3 or 6 months in any group. IL-6 normalized for
`G3PDH (IL-6/G3PDH ratio) was not different from baseline at 3
`months but showed a significant decrease from baseline in the
`group treated with 0.05% CsA (p ⳱ 0.048) at 6 months. No
`significant between-group differences were noted and no correla-
`tion was observed between the change in IL-6/G3PDH and corneal
`fluorescein staining. Conclusions. This preliminary, small-cohort
`study showed a decrease in IL-6 in the conjunctival epithelium of
`moderate to severe dry eye patients treated with 0.05% CsA for 6
`months. The observed decrease suggests that dry eye disease in-
`volves immune-mediated inflammatory processes that may be de-
`creased by treatment with topical ophthalmic cyclosporine.
`Key Words: Conjunctiva—Cyclosporin A—Dry eye disease—
`Interleukin-6—Keratoconjunctivitis sicca.
`
`Specific evidence of chronic immune activation of the conjunc-
`tival epithelium in patients with dry eye suggests that cell-
`mediated inflammatory processes may play an important role in
`the pathogenesis of dry eye disease.1–4 These immunopathologic
`findings include increased expression of immunologic adhesion
`molecules (i.e., HLA class II and ICAM-1 antigens) and inflam-
`
`Submitted September 13, 1999. Revision received January 8, 2000. Ac-
`cepted January 15, 2000.
`From the Bascom Palmer Eye Institute (K.T., S.C.P., Z.J., W.J.F.), De-
`partment of Ophthalmology, University of Miami School of Medicine,
`Miami, Florida; and Allergan, Inc. (M.S., B.L.R.), Irvine, California,
`U.S.A.
`Supported by Allergan Inc., 2525 Dupont Drive, Irvine, CA 92713-9534,
`U.S.A.
`Address correspondence and reprint requests to S.C. Pflugfelder, M.D.
`Bascom Palmer Eye Institute, 900 N.W. 17th Street, Miami, FL 33136,
`U.S.A.
`
`matory cytokines (IL-1, IL-6, IL-8, and TNF-␣) in these pa-
`tients.5–7 Among the inflammatory cytokines evaluated, levels of
`interleukin-6 (IL-6) showed the greatest elevation in eyes with dry
`eye disease when compared to normal eyes.6
`Further evidence that immune-mediated inflammatory processes
`are involved in the pathogenesis of dry eye disease comes from
`reports that topical use of the immunomodulatory agent cyclospor-
`ine can improve the signs and symptoms of dry eye.8–10 However,
`it has not been determined whether the use of topical cyclosporine
`can cause measurable decreases in any of the inflammatory mark-
`ers that are associated with dry eye disease.
`The purpose of this study was to evaluate the level of the in-
`flammatory cytokine IL-6 in the conjunctiva of patients with kera-
`toconjunctivitis sicca enrolled in a randomized trial of two differ-
`ent concentrations of cyclosporin A ophthalmic emulsion (CsA)
`and a castor oil emulsion vehicle. IL-6 was chosen because pre-
`vious studies have shown that the level of IL-6 mRNA is elevated
`in the conjunctival epithelium of patients with Sjo¨gren’s syndrome
`keratoconjunctivitis sicca.5,6 Furthermore, the level of IL-6 mRNA
`was found to decrease in bronchoalveolar lavage cells obtained
`from patients with acute lung transplant rejection who were treated
`with aerosolized cyclosporine.11
`
`PATIENTS AND METHODS
`
`Patients
`Evaluation of the level of IL-6 mRNA in the conjunctiva was
`performed on conjunctival cytology specimens from a subset of
`patients enrolled in a prospective, double-masked, vehicle-
`controlled, multi-center clinical trial of topical cyclosporine emul-
`sion (CsA) for the treatment of dry eye disease.9 Adult patients of
`either sex were eligible for participation if they presented with a
`diagnosis of moderate to severe dry eye disease as defined by the
`following criteria:
`1) Schirmer test without anesthesia of ⱕ5 mm/5 minutes in at
`least one eye (If Schirmer test without anesthesia ⳱ 0 mm/5
`minutes, then Schirmer with nasal stimulation had to be > 3
`mm/5 minutes in the same eye.);
`2) sum of corneal and interpalpebral conjunctival staining of ⱖ +5
`in the same eye where corneal staining was ⱖ +2;
`3) a baseline Ocular Surface Disease Index娀 (OSDI娀) score of 0.1
`with no more than three responses of “not applicable;” and
`4) a score of ⱖ 3 on the Subjective Facial Expression Scale.
`The OSDI娀 is a questionnaire consisting of 12 questions (each
`
`492
`
`ARGENTUM - EX. 1014, p. 001
`
`

`
`CONJUNCTIVAL IL-6 IN DRY EYE DISEASE
`
`493
`
`FIG. 1. Ethidium bromide-stained agarose gel. Initial PCR was performed using high and low concen-
`trations of mimics for G3PDH (10−3 and 10−4 attomole/µL), and IL-6 (10−5 and 10−6 attomole/µL). The
`percentage of product (G3PDH or IL-6) per total amplified product (gene + mimic) in the lane is given
`at the bottom. amol = attomole.
`
`rated from 0 ⳱ none of the time to 4 ⳱ all of the time) for the
`evaluation of the impact of a patient’s dry eye disease on his or her
`vision-related functioning. The overall score was calculated by
`dividing the sum of the responses for all questions answered by the
`total possible score and then multiplying by 100. Thus, overall
`scores ranged from 0 ⳱ no disability to 100 ⳱ complete disabil-
`ity.
`To be eligible for enrollment, signs and symptoms of dry eye
`disease must have presented despite conventional management,
`which may have included artificial tear drops, gels and ointments,
`sympathomimetic agents, parasympathomimetic agents, and punc-
`tal occlusion. Eligible patients were enrolled if they were deemed
`capable of following the study protocol and were considered likely
`to complete the treatment period and to return for all scheduled
`visits, if they had normal lid position and closure, and if they had
`a best-corrected early treatment of diabetic retinopathy study vi-
`sual acuity score of +0.7 logmar or better in each eye.
`Patients were excluded from the study if they had used systemic
`or topical ophthalmic cyclosporine within 90 days before the
`study. Other exclusion criteria included the presence or history of
`any systemic or ocular disorder or condition (including ocular
`surgery, trauma, and disease) that could possibly interfere with the
`interpretation of the study results; current or recent use of topical
`ophthalmic or systemic medications that could affect a dry eye
`condition; known hypersensitivity to any component of the study
`or procedural medications; required contact lens wear during the
`study; recent (within 1 month) or anticipated use of temporary
`
`punctal plugs during the study; permanent occlusion of lacrimal
`puncta within 3 months of the study; or if they were pregnant,
`lactating, or planning a pregnancy. Patients were also excluded if
`they appeared to have end stage lacrimal gland disease (Schirmer
`reading with nasal stimulation of < 3 mm/5 minutes) or if their dry
`eye disease was secondary to the destruction of conjunctival goblet
`cells or scarring. Any patient who no longer met the criteria for
`moderate to severe dry eye (as defined above) after completing the
`2-week run-in phase was excluded from enrollment in the treat-
`ment phase of the study.
`Conjunctival epithelial samples were obtained at baseline, at 3
`months, and at 6 months from four participating sites in the United
`States. Informed consent was obtained from all patients before the
`study. The study complied with the Tenets of the Declaration of
`Helsinki. Conjunctival epithelial samples were obtained by im-
`pression debridement with a nitrocellulose membrane from the
`temporal bulbar conjunctiva of the “worse” eye. The “worse” eye
`was defined as the eye with the worse Schirmer tear test value
`(without anesthesia) and the worse sum of corneal and interpalpe-
`bral conjunctival staining. If both eyes were comparable, then the
`right eye was used. The membranes were placed in Trizol RNA
`lysis buffer (Life Technologies, Rockville, MD, U.S.A.) and im-
`mediately frozen at −80°C. Samples were shipped in dry ice by a
`commercial overnight delivery service to the central laboratory at
`the University of Miami School of Medicine. Upon arrival,
`samples were placed in a −80°C freezer until the RNA was ex-
`tracted.
`
`Cornea, Vol. 19, No. 4, 2000
`
`ARGENTUM - EX. 1014, p. 002
`
`

`
`494
`
`K. TURNER ET AL.
`
`FIG. 2. Example of final gels for each patient RNA
`sample. G3PDH primers were reacted with 10−4 at-
`tomole/µL mimic, and IL-6 with 10−6 attomole/µL
`mimic. The upper band in each lane is the amplified
`product and the lower band is the amplified mimic.
`Lanes 1, 3, and 5 are G3PDH and the G3PDH mimic;
`lanes 2, 4, and 6 are IL-6 and the IL-6 mimic. Lanes
`1 and 2 are baseline, lanes 3 and 4 are month 3,
`lanes 5 and 6 are month 6, lane 7 is cDNA-(PCR
`reaction mixture without cDNA), and lane 8 is the
`DNA marker IX (Boehringer,
`Indianapolis,
`IN,
`U.S.A.).
`
`RNA Extraction and cDNA Synthesis
`RNA was extracted from the conjunctival epithelial samples
`using a previously reported protocol.5 Complementary DNA
`(cDNA) was synthesized with a first strand cDNA synthesis kit
`(Boehringer, Indianapolis, IN, U.S.A.) using 1 ␮g of total RNA.
`Reverse transcriptase negative controls were performed for each
`RNA sample. The efficiency of cDNA synthesis was assessed by
`evaluating the level of cyclophilin mRNA in each sample by poly-
`merase chain reaction (PCR) amplification using 35 cycles. We
`added 2 ␮L of cDNA to the PCR reaction mixture containing 5 ␮L
`of 10× reaction buffer, 1 ␮L deoxy-nucleotide triphosphates (10
`mmol/L deoxyadenosine triphosphate, 10 mmol/L deoxycytidine
`triphosphate, 10 mmol/L deoxyguanosine triphosphate, and 10
`mmol/L deoxythymidine triphosphate), 39.7 ␮L sterile water, 0.3
`␮L Taq DNA polymerase (5 units/␮L), and 2 ␮L of primers spe-
`cific for cyclophilin.12 The upstream cyclophilin primer sequence
`is 5⬘ ATG GTT AAC CCC ACC GTG TTC GAC 3⬘, and the
`
`downstream primer sequence is 5⬘ CTG GAT TGC AGA GTT
`AAG TTT 3⬘. Cyclophilin is a constitutively expressed gene in all
`human cells that serves as the cyclosporine-binding protein.13 The
`PCR products were separated by 1.6% agarose gel electrophoresis
`and the intensity of the band was visually graded from 0 (no band)
`to 4+ (strong). cDNA samples that yielded 2+, 3+, or 4+ bands were
`used for competitive reverse transcriptase polymerase chain reaction
`(RT-PCR) analysis of IL-6 and G3PDH, a housekeeping gene, levels.
`cDNA samples that yielded 0 or 1+ bands were resynthesized.
`
`Semiquantitative Analysis of mRNA Levels by
`Competitive RT-PCR
`The relative levels of RNA transcripts encoding the inflamma-
`tory cytokine IL-6 or the housekeeping protein G3PDH were
`evaluated by a competitive PCR technique. This technique was
`performed by adding 2 ␮L of sample cDNA to the PCR mixture
`described above, containing 2 ␮L of primers specific for IL-6 or
`
`TABLE 1. Average percent of product
`
`Cyclosporine
`dose
`
`Vehicle
`
`0.05%
`
`0.10%
`
`% G3PDH in lane
`
`Mimic 10−3
`attomole
`
`Mimic 10−4
`attomole
`
`Mo
`
`Mean
`
`0
`3
`6
`0
`3
`6
`0
`3
`6
`
`47
`31
`32
`46
`27
`53
`37
`29
`24
`
`Std.
`dev.
`
`20
`33
`22
`22
`22
`18
`20
`25
`17
`
`Mean
`
`74
`48
`70
`70
`46
`77
`57
`35
`40
`
`Std.
`dev.
`
`19
`43
`28
`32
`43
`16
`31
`38
`22
`
`% IL-6 in lane
`
`Mimic 10−5
`attomole
`
`Mimic 10−6
`attomole
`
`p value
`
`Mean
`
`0.002
`0.051
`0.001
`0.005
`0.026
`0.003
`0.012
`0.388
`0.025
`
`21
`17
`4
`34
`26
`34
`12
`16
`12
`
`Std.
`dev.
`
`13
`18
`6
`26
`22
`27
`15
`19
`16
`
`Mean
`
`41
`29
`9
`61
`41
`53
`37
`31
`32
`
`Std.
`dev.
`
`27
`24
`10
`30
`34
`28
`17
`25
`26
`
`p value
`
`0.008
`0.005
`0.091
`0.005
`0.081
`0.004
`0.002
`0.003
`0.009
`
`Cornea, Vol. 19, No. 4, 2000
`
`ARGENTUM - EX. 1014, p. 003
`
`

`
`CONJUNCTIVAL IL-6 IN DRY EYE DISEASE
`
`TABLE 2. Change in percent G3PDH from baseline
`
`Group
`
`Vehicle
`0.05% CsA
`0.10% CsA
`
`3-mo
`mean
`
`−12
`−15
`−4.4
`
`SD
`
`31
`33
`27
`
`p value
`
`0.4
`0.2
`0.6
`
`Range
`
`−61 to 17
`−72 to 28
`−45 to 47
`
`6-mo
`mean
`
`13
`3.7
`12
`
`SD
`
`25
`27
`33
`
`p value
`
`0.14
`0.6
`0.3
`
`495
`
`Range
`
`−15–21
`−38–64
`−55–35
`
`G3PDH.6 Both the IL-6 and G3PDH PCR reactions were run for
`35 cycles. The primer sequences for IL-6 and G3PDH were chosen
`to span an intron to confirm that the PCR product was due to
`amplification of cDNA, not genomic DNA. The size of the am-
`plified IL-6 cDNA product was 627 base pairs, and the size of the
`amplified G3PDH cDNA product was 983 base pairs. Each reac-
`tion tube used to amplify the IL-6 cDNA was spiked with a spe-
`cifically designed “mimic” template (Clontech, Palo Alto, CA,
`U.S.A.) that contained DNA sequences that were complimentary
`to the IL-6 PCR primers. These primer-specific sequences were
`ligated to the 5⬘ and 3⬘ ends of a nonspecific DNA fragment (in
`this case, a portion of the v-erbB gene). The mimic templates
`compete with the IL-6-specific cDNA sequences in the conjunc-
`tival epithelial specimens for the reaction primers and they have a
`different length (in base pairs) after PCR amplification than the
`cytokine amplification product. The added mimics also served as
`internal controls in each reaction tube. Two concentrations of
`mimic (10−5 and 10−6 attomole/␮L) were used for each sample.
`PCR reactions for G3PDH were also spiked with two concentra-
`tions (10−3 and 10−4 attomole/␮L) of a mimic with G3PDH
`primer-specific sequences. PCR reactions for IL-6 and G3PDH
`were performed on baseline (month 0), month 3, and month 6
`samples, simultaneously for each subject. The levels of PCR prod-
`ucts were evaluated by 1.6% agarose gel electrophoresis. Gels
`were stained with ethidium bromide (10 mg/mL) and were photo-
`graphed with a Polaroid camera (Cambridge, MA, U.S.A.). Base-
`line, month 3, and month 6 PCR products for IL-6 and G3PDH
`(and their mimics) from each subject were run together on one
`agarose gel (Fig. 1). Two final gels (one for the lower and the other
`for the higher mimic concentration) were run for each subject (Fig.
`2). The mimic concentration yielding measurable bands for IL-6
`and G3PDH that allowed comparisons between at least two time
`points (baseline to month 3, or baseline to month 6, or month 3 to
`month 6) was used for the final analysis.
`Photographs of each gel were scanned with a Hewlett Packard
`
`TABLE 3. Change in %IL-6/%G3PDH ratio from baseline
`
`CsA 0.05%
`
`CsA 0.1%
`
`Vehicle
`
`P valuea
`
`ScanJet (Palo Alto, CA, U.S.A.). The integrated optical density of
`the IL-6, G3PDH, and their mimic bands in the scanned images
`was measured. Levels of IL-6 and G3PDH in each lane were
`expressed as a fraction of total amplified product in each lane (e.g.,
`the percentage of IL-6 ⳱ IL-6/[IL-6 + IL-6 mimic]). Levels of
`G3PDH in each treatment group were compared over time to de-
`termine whether the amount of G3PDH changed significantly with
`CsA treatment. Levels of IL-6 were evaluated at each time point
`(baseline, month 3, and month 6) in relation to the level of G3PDH
`in each sample by dividing the percentage of IL-6 by the G3PHD
`(normalized IL-6).
`
`Statistical Analysis
`A two-tailed paired Student t test was used to confirm that IL-6
`and G3PDH, as a percentage of the product plus mimic total,
`decreased with sample dilution for each group at each follow-up
`period. Within group comparisons of G3PDH and IL-6, changes
`were performed with the two-tailed paired Student t test. Changes
`in the normalized IL-6 percentage from baseline to 3- and 6-month
`follow-up were calculated for each subject and between-group
`differences in the extent of change were examined with one-way
`analysis of variance.
`
`RESULTS
`
`Mimic as a Competitive Internal Control
`Mimics were added to the PCR reactions to act both as a posi-
`tive internal control for the PCR reaction and as a competitive
`template used for calculating the percentage of amplified cytokine
`or housekeeping protein. To confirm the competitive nature of the
`mimic, the percentage of IL-6 and G3PDH in each lane was com-
`pared using high and low mimic concentrations. For each treat-
`ment group and time point, the percentage of IL-6 and G3PDH
`increased as the concentration of added mimic decreased (Table 1).
`
`Changes from Baseline
`There were no significant differences between G3PDH levels at
`months 0, 3, or 6 in any treatment group (Table 2). This demon-
`strates that the levels of the housekeeping protein (G3PDH) are not
`changed by CsA treatment.
`The levels of IL-6 were then normalized to the G3PDH levels to
`identify treatment-related changes in IL-6 mRNA levels. At 6
`months posttreatment, a significant decrease in the ratio of the
`percentage of IL-6 to G3PDH was observed in the group treated
`with 0.05% CsA, but not in the vehicle or 0.10% CsA treatment
`groups (Table 3). The ratio of the percentage of IL-6 to G3PDH was
`not significantly different from baseline at month 3 in any treatment
`group. There were no significant differences in the between-group
`ratios of the percentage of IL-6 to the percentage of G3PDH.
`
`Cornea, Vol. 19, No. 4, 2000
`
`Day 0b
`
`1.144 ± 1.005
`(n = 14)
`Change from baseline
`Month 3
`0.036 ± 0.643
`n = 10
`p = 0.862
`−0.626 ± 1.025
`n = 13
`p = 0.048c
`
`Withinc
`Month 6
`
`Within
`
`1.401 ± 1.198
`(n = 12)
`
`0.846 ± 0.663
`(n = 10)
`
`2.269 ± 5.524
`−0.095 ± 1.430
`n = 7
`n = 10
`p = 0.319
`p = 0.839
`−0.384 ± 1.248 −0.460 ± 0.749
`n = 8
`n = 10
`p = 0.084d
`p = 0.413
`
`0.438
`
`0.221
`
`0.853
`
`a Among-group p values from one-way analysis of variance.
`b Values are mimic corrected ratios of IL-6 to G3PDH.
`c Within-group p value from paired-t-test.
`Note. CsA = cyclosporine ophthalmic emulsion. Values shown are
`mean ± standard deviation. A negative value indicates a decrease
`from baseline.
`
`ARGENTUM - EX. 1014, p. 004
`
`

`
`496
`
`K. TURNER ET AL.
`
`DISCUSSION
`
`REFERENCES
`
`The most important finding of the present study was that nor-
`malized IL-6 levels significantly decreased from baseline after 6
`months of treatment with 0.05% CsA, whereas the decreases seen
`after treatment with either 0.1% CsA or vehicle were not statisti-
`cally significant. The decrease in this inflammatory factor by cy-
`closporine is consistent with the results of another study conducted
`on conjunctival biopsies taken from a separate cohort of patients
`that participated in this multi-centered clinical trial. Kunert et al.14
`demonstrated that there was a significant decrease in the immune
`activation markers HLA-DR and CD11a after 6 months of treat-
`ment with 0.05% CsA, whereas these same markers increased in
`the vehicle group. Other studies have demonstrated that increased
`levels of IL-6, and other inflammatory markers, are associated
`with dry eye disease.5–7,15 For example, a study by Pflugfelder et
`al.6 used the same assay as the present study to demonstrate that
`the levels of IL-1␣, IL-6, IL-8, TNF-␣, and TNF-␤1 RNA found
`in the conjunctival epithelium of Sjo¨gren’s syndrome patients were
`significantly greater than those found in control patients. However,
`the present study and the other study performed on this patient
`population14 are the first to demonstrate that a topical ophthalmic
`treatment for dry eye disease (cyclosporin A) can significantly
`decrease several markers of inflammation.
`In the present study, no statistically significant between-group
`differences were seen in IL-6 levels. Thus, it is not possible to rule
`out regression to the mean as the source of the IL-6 decrease.16
`However, a more likely explanation may be a large therapeutic
`effect observed in the vehicle group. Disruption of epidermal bar-
`rier function results in increased levels of several inflammatory
`cytokines, including TNF-␣, IL-1, and IL-6, in the epidermis.17 A
`therapeutic effect of the oil-in-water vehicle of this cyclosporine
`emulsion might be expected, as topical application of certain lipid
`mixtures can accelerate epidermal barrier recovery after defined
`barrier insults in mice.18
`A very small amount of RNA was obtained from the samples
`provided for this study. Consequently, a highly sensitive competi-
`tive RT-PCR assay was used to evaluate the levels of IL-6 and a
`constituently expressed gene, G3PDH. The assay was designed to
`determine the relative amount of each of these mRNAs in the
`sample when compared to a competitive template (mimic) that was
`added to each of the PCR reaction tubes. The validity of this
`approach is supported by the fact that the levels of G3PDH were
`not affected by either the study medication or the vehicle.
`In conclusion, the findings of the present study demonstrate that
`topical CsA decreases the levels of IL-6, an inflammatory cytokine
`that is upregulated in the conjunctiva of patients suffering from dry
`eye disease.
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`Cornea, Vol. 19, No. 4, 2000
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