Analysis of Topical Cyclosporine Treatment
`of Patients With Dry Eye Syndrome
`Effect on Conjunctival Lymphocytes
`
`Kathleen S. Kunert, MD; Ann S. Tisdale, MS; Michael E. Stern, PhD; J. A. Smith; Tene K. Gipson, PhD
`
`
`
`
`
`ERATOCONJUNCTIVITIS sicca
`analysis of the lacrimal gland has re-
`(KCS), or dry eye syn-
`ceived considerable attention, less work
`drome,is characterized by
`has been done on pathological changes oc-
`curring in the ocularsurface. The chronic
`chronic dryness of the cor-
`nea and conjunctiva.! Pa-
`dryness of the ocular surface in Sjogren
`tients with KCS typically show symp-
`syndromehas been attributedto deterio-
`toms of ocular discomfort ranging from
`rationof lacrimal gland function with de-
`irritation to severe pain. Redness, burn-
`creased tear production.?'© However, in
`ing, itching, foreign body sensation, con-
`Sjégren syndrome, conjunctival epithe-
`tact lens intolerance, photophobia, and
`lial and stromal T-cell infiltration (pre-
`blurred vision can occur.’
`dominantly CD3+ and GD4+ T lympho-
`cytes) has also been shownto occur along
`Although KCScan arise from vari-
`with drying of the ocular surface.2!
`ous types of diseases, commonto all is the
`involvement of immune-mediated or in-
`Supporting a role for an immuno-
`flammatory-mediated pathways.? Immu-
`pathogenesis of KCSarethe reports of ac-’
`Fromthe Schepens Eye
`nopathologic studies of the lacrimal gland
`tivated lymphocytes as demonstrated by
`ResearchInstitute and
`in patients with Sjogren syndrome show
`expression of lymphocyte activation mark-
`Department of Ophthalmology,
`progressive lymphocytic infiltration, pri-
`ers such as HLA-DR (MHC class II) and
`Harvard Medical School,
`marily consisting of CD4+ T andBcells,*5
`ICAM-1 (intercellular ‘adhesion mol-
`Boston, Mass (Drs Kunert and
`This infiltration is believed to be respon-
`ecule-1) in the conjunctiva of patients with
`Gipson, Ms Tisdale); Allergan,
`sible for the destruction of normal secre-
`Sjogren syndrome.’*3 To date, there is
`Inc, Irvine, Calif (Dr Stern);
`little information on the’effect of modu-
`tory function.® Lymphocytic infiltration of
`and the National Eye Institute,
`the lacrimal gland has also been de-
`Bethesda, Md (Ms Smith).
`latinig these molecules inthe conjunctiva
`scribed in patients with non-Sjégren
`of patients with Sjégren and non-
`Dr Sternis an employee of
`KCS."4 Although the immunopathologic
`Allergan Inc.
`Sjogren syndrome.
`
`Objective: To study the effect of topical cyclosporine
`on lymphocyte activation within the conjunctiva of
`patients with moderate to severe dry eye syndrome
`(Sjogren and non-Sjégren).
`
`Metheds: Biopsy specimens were obtained atbaseline
`and after 6 monthsof cyclosporine treatment from eyes
`of 32 patients with moderate to severe dry eye syn-
`drome; 19 were cyclosporinetreated (0.05% cyclospor-
`ine, n=13; 0.1% cyclosporine, n=6) and 13 were ve-
`hicle treated. Within this group there were 12 withSjogren
`syndrome and 20 with non-Sjégren syndrome. Biopsy
`tissue was analyzed using immunohistochemical local-
`ization of binding of monoclonal antibodies to lympho-
`cytic markers CD3, CD4, and CD8 as well as lympho-
`cyte activation markers CD11la and HLA-DR.
`
`Results: In cyclosporine-treated eyes, biopsyresults of
`conjunctivae showed decreases in the numberofcells posi-
`
`tive for CD3, CD4, and CD8,while in vehicle-treated eyes,
`results showedincreasesin these markers, although these
`differences were notstatistically significant. Following
`treatment with 0.05% cyclosporine, there was a signifi-
`cant decrease in the numberofcells expressing the lym-
`phocyteactivation markers CD11a (P<.05) and HLA-DR
`(P<.05), indicatingless activation oflymphocytes as com-
`pared with vehicle treatment. Withinthe Sjogren pa-
`tient subgroup, those treated with 0.05% cyclosporine
`also showed a significant decrease in the numberofcells
`positive for CD1la (P<.001) as well as CD3 (P<.03),
`indicating a reduction in numberof activated lympho
`cytes.
`
`Conclusion: Treatmentof dry eye syndromewithtopi-
`cal cyclosporinesignificantly reducedthe numbersofac-
`tivated lymphocytes within the conjunctiva.
`
`Arch Ophthalmol. 2000;118:1489-1496
`
`(REPRINTED) ARCH OPHTHALMOL/VOL 118, NOV 2000
`1489
`
`WWW. ARCHOPHTHALMOL,COM
`
`©2000 American Medical Association. All rights reserved,
`Downloaded From: http://archopht.jamanetwork.com/ by a University of Michigan User on 01/25/2016
`
`EXHIBIT 1012
`
`

`

`
`
`
`
`
`
`SUBJECTS AND METHODS
`
`SUBJECTS
`
`required contact lens wear during the study, recent (within
`1 month) oranticipateduse of temporary punctal plugs dur-
`ing the study; permanentocclusion of lacrimal puncta within
`3 monthsof the study;orif the patients were pregnant,lac-
`tating, or planning a pregnancy. Patients were also ex-
`cluded if they appeared to have end-stage lacrimal gland
`disease (Schirmer reading with nasal stimulation <3 mm/5
`min) orif their KCS was secondary to the destruction of
`conjunctival goblet cells or scarring.
`A retrospective diagnosis of Sjogren syndrome was used
`with modified criteria reported by Vitali et al” to ensure
`that a consistent definition of Sjogren syndrome was as-
`signed to the patients enrolled. Diagnosis includedpres-
`ence of atleast oneof the following autoantibodiesin sera:
`antinuclear antibody (ANA), rheumatoid factor (RF), and
`Sjogren syndromeautoantibodiesclass SS-A (Ro)and class
`SS-B (La). In addition, oral and ocular symptoms were used
`to classify patients with Sjogren syndrome.
`Patients instilled 1 drop of 0.05% or 0.1% CsA oph-
`thalmic emulsions or vehicle of CsA ophthalmic emulsion
`twice daily in each eye for 6 months; once on waking in
`the morning and once at bedtime. Patients were allowed
`to use assignedartificial tears (REFRESH Lubricant Eye
`Drops; Allergan Inc) as needed up to month4.
`Full-thickness conjunctival biopsy specimens ofa stan-
`dard size (2-3 mm) were removed from the “worse” eye
`by surgeons following standard procedure. The worse eye
`was defined as the eye with the worse Schirmertear test
`value (without anesthesia) and the worse sum of corneal
`and interpalpebral conjunctival staining. If both eyes were
`comparable, then the right eye was used. At the baseline
`visit, the conjunctival biopsy specimen was obtained from
`the inferonasal quadrant close to midline. At the 6-month
`visit, the sample was removed from the same eye but from
`the inferetemporal quadrant, also close to midline.
`
`Conjunctival biopsy specimens from32 patients were ex-
`amined; 13 patients were treated with 0.05% CsA, 6 with
`0.1% CsA, and 13 with vehicle alone. This subject group
`was randomly chosen from a double-masked, vehicle-
`controlled clinical study designed by Allergan,Inc, Irvine,
`Calif, to investigate the efficacy and safety of topical CsA
`in the treatment of moderate to severe KCS.The study
`was conducted in compliance with Good Clinical Prac-
`tices, investigational site institutional review board regu-
`lations, sponsorandinvestigator obligations, informed con-
`sent regulations, and the Declaration of Helsinki. Potential
`patients signed a prescreening informed consent form and
`a second written informed consentform priorto actualen-
`rollment.”! Theprotocolfor this study is described briefly
`here. Adult patients of either sex wereeligible for partici-
`pation if they had a diagnosis of moderate to severe KCS
`atinitial examination as defined bythe following criteria:
`(1) Schirmertest results (without anesthesia) less than or
`equal to 5 mm/5 minin at least 1 eye (if Schirmer test re-
`sults without anesthesia equaled 0 mm/5 min, then Schirmer
`test results with nasal stimulation had to be >3 mm/5 min
`in the sameeye); (2) sum of corneal andinterpalpebral con-
`junctival staining greater than or equal to +5 in the same
`eye where cornealstaining was greater than or equalto +2;
`(3) a baseline Ocular Surface Disease Index”score of0.1
`with no more than 3 responses of “not applicable”; and (4)
`a score greater than or equal to 3 on the Subjective Facial
`Expression Scale." Signs and symptoms must have been
`present despite conventional management.
`Patients were excluded from the study if they had par-
`ticipated in an earlier clinical trial with CsA ophthalmic
`emulsion or had used systemic or topical ophthalmic CsA
`within 90 daysprior to the study. Other exclusioncriteria
`were the presence or history of any systemic or ocular dis-
`order or condition (including ocular surgery, trauma, and
`disease); currentor recent use of topical ophthalmic orsys-
`After removal, the baseline biopsy specimens were imme-
`diately frozen in OCT embedding compound(Tissue-Tek;
`temic medications that could affect a dry eye condition;
`Miles Laboratories, Elkhart, Ind) in a cryomold (Miles
`known hypersensitivity to any component of the drug or
`procedural medications such as stains or anesthetics;
`Laboratories) and stored at -80°C unul patient-matched
`
`
`
`TISSUE PROCESSING FOR
`IMMUNOHISTOCHEMICAL ANALYSIS
`
`inhibit T-cell-mediatedinflammation by preventingthe ac-
`Currently, administration ofartificial tears is the most
`common therapy available for lubricating a dry ocular sur-
`tivation of T cells (by antigen-presenting cells or
`face. This palliative treatment gives only temporary and in-
`cytokines).!°”° Activated T cells are responsible for the pro-
`complete symptomatic reliefand does not address the cause
`duction of inflammatory substances such as cytokines,
`whichlead to furthertissue damageand,in tum,to the ac-
`of the symptoms, which may include immune-mediated
`inflammation ofthe ocular surface. Evidence of inflamma-
`tivation of more T cells and the production of even more
`inflammatory substances.
`tory processes in the pathogenesis of KCSled to the de-
`Clinical trials with this drug have shown improve-
`velopmentof cyclosporine (CsA) asa first attemptto treat
`ment in various objective measures of KCS such ascor-
`this condition therapeutically. Cyclosporine is an immu-
`neal staining and Schirmertest values.7! To attempt to
`nosuppressive agent commonlyused systemically to treat
`inflammatory diseases such as psoriasis or rheumatoid ar-
`find tissue correlates in these patients, conjunctival bi-
`opsy specimens frompatients with Sjogren and non-
`thritis or to prevent organ transplant rejection." Topical
`CsA has been usedas treatment of ocular conditions such
`Sjogren KCStreated with CsA or vehicle were evaluated
`immunohistochemically for the presence of activated T
`as vernal keratoconjunctivitis,’° commeal transplants,'* cor-
`neal ulcers,”’ and herpetic stromal keratitis.'® The effect of
`cells (CD3+ [Pan-T cell], CD4+ [T helpercell], and CD8+
`[cytotoxic T cell]) and lymphocyte-activation markers
`this drug on inflammatory diseases is due to its ability to
` ae
`WWW.ARCHOPHTHALMOL.COM
`(REPRINTED) ARCH OPHTHALMOL/VOL 118, NOV 2000
`1490
`
`©2000 American Medical Association, All rights reserved.
`Downloaded From: http;//archopht.jamanetwork.com/ by a University of Michigan User on 01/25/2016
`
`

`

`
`
`
`
`6-monthbiopsy specimens were obtained andsimilarly fro-
`zen. Six-micrometersections were taken from each block,
`mounted on gelatin-coatedslides, and processed for im-
`munohistochemicalanalysis. Sectioningoftissue blocks and
`immunchistochemical experiments were performed as pairs
`of biopsies, pretreatment and posttreatment, to minimize
`differences due to experimental conditions.
`
`IMMUNOHISTOCHEMICAL ANALYSIS
`
`and rightof thatarea.In this mannertheentire biopsyarea
`was usually captured.
`
`COUNTING PROCEDURE
`
`Measurementofthe entire area of epitheliumand stroma
`(substantia propria) was achieved by tracing the area us-
`ing the lasso tool under the Adobe Photoshop computer
`program (AdobeSystemsInc, SanJose, Calif). The total data
`area, measuredin pixels, was acquired through the “Im-
`age: Histogram” command in Photoshop. Two indepen-
`dent counts were recordedfor cells positive for each anti-
`body within the traced area. Cells per unit area of pixels
`were adjustedto real unit area or cells per millimeter squared
`ofreal tissue area, based on 28.346 pixels per centimeter
`in Photoshop and thefact that 1 mmequals 67.8 cm equals
`1922 pixels at X20 magnification on the Nikon micro-
`scope. Data were recordedas cells permillimeter squared
`for all markers, and statistical analysis was based on these
`measurements,
`
`STATISTICAL METHODS
`
`Baseline characteristics were tabulated and summarized by
`treatment groups. Overall differences among treatment
`groups were tested using a 2-way analysis of variance
`(ANOVA)for continuousvariables and the Fisher exact test
`for categorical variables,
`Percent changes in the numberofcells expressing
`lymphocytic and/or lymphocyteactivation markers were
`summarized using descriptive statistics (ie, sample size,
`mean, SD, minimum, maximum, and median). A l-way
`ANOVAwith main effect for treatment was used to test
`for differences in percent change from baseline and
`ratios among treatment groups by visit. If the test for
`among-group differences in main effect was significant,
`then all 3 pairwise comparisons were made. Within-
`group changes from baseline were analyzed by the
`paired t test method.
`The same analysis was performed on Sjégren and
`non-Sjégren subpopulations, excluding the 0.1% CsA
`- treatment group in which there was only 1 patient in the
`Sjogren subset.
`
`Immunohistochemicalstaining for lymphocytic markers as
`well as lymphocyte activation markers was conducted us-
`ing monoclonal. antibodies to CD3 (PharMingen,San Diego,
`Calif), CD4 (Becton-Dickinson,SanJose, Calif}, CD8 (Bec-
`ton-Dickinson, SanJose), CD1lla (PharMingen, San Diego),
`and HLA-DR (PharMingen). Cryostat sections were fixed
`in cold acetone (~20°C) for 3 minutes andair dried at room
`temperature for 30 to 45 minutes, They were then rinsed
`in 3 changes of phosphate-buffered saline (PBS) and incu-
`bated in PBS with 1% bovine serum albumin (BSA) (Sigma
`Chemical Co, St Louis, Mo) for 10 minutes. Sections were
`incubated for | hour at room temperature in primary an-
`tibodies at concentrations derived empirically: CD3, 1.0
`pg/mL; CD4, 5.0 pg/mL; CD8, 2.5 ng/mL; CD11a, 10.0
`ng/mL, and HLA-DR, 1.0 pg/mL.Sections were rinsed in
`PBS alone, followed by 10 minutes in PBS with 1% BSA be-
`fore incubation for 1 hour at room temperaturein the sec-
`ondaryantibody,fluorescein isothiocyanate~conjugated Af-
`finipure Donkey Anti-Mouse IgG (Jackson Immunoresearch,
`West Grove, Pa) at a dilution of 1/50. Sections were then
`rinsed in PBS, mounted in Vectashield (Vector Labs, Bur-
`lingame, Calif), cover-slipped, and viewed undera micro-
`scope (Eclipse E800; Nikon, Melville, NY) interfaced with
`a digital camera (Spot Digital Camera; Diagnostic Instru-
`ments Inc, Micro Video Instruments, Avon, Mass). Sec-
`ondary antibody controls omitting the primary antibody
`for all biopsy specimens for each immunochistochemical
`analysis were run,
`Three separate images were acquired for each anti-
`body and biopsy specimen under a X20 objective using a
`Spot acquisition program (Diagnostic Instruments Inc). The
`first field selected for imaging wasthefield with the high-
`est numberofpositive cells, followed by imagesto theleft
`
`
`
`(CD11a and HLA-DR)to further understand the under-
`lying mechanism of CsA treatment.
`
`az
`PATIENT POPULATION
`
`The mean+SDage of our subjects was 59.0+13.5 years
`(range, 28.8-84.2 years), including 27 women and 5 men.
`Within this group, there were 12 Sjogren and 20 non-
`Sjogren patients,
`
`LYMPHOCYTIC MARKERS
`
`In general, there was a decrease from baseline in the num-
`ber ofcells positive for CD3, CD4, and CD8 following
`
`treatmentwith either concentration of CsA. The only ex-
`ception was that there was a mean increase from base-
`line in the CD4-positive T helper cell population follow-
`ing 0.05% CsA treatment. In comparison,all cells positive
`for the lymphocytic markers increased from baselinefol-
`lowing vehicle treatment.
`Figure ¥ showsthe percent change from baseline
`for cells expressing the lymphocytic markers (CD3, CD4,
`and CD8)after 6 monthsoftreatmentfor the overall pa-
`tient population. Note that there was a reduction from
`baseline in the number of CD3-positivecells in the CsA-
`treated groups, while there was an increase from base-
`line in the vehicle-treated group. There wasalso anin-
`crease from baseline in the numbers of CD4-positive cells
`in the vehicle group, with a smaller increase in the 0.05%
`CsA group anda slight decrease in the 0.1% CsA group.
`
`(REPRINTED) ARCH OPHTHALMOL/VOL 118, NOV 2000
`1491
`
`WWW. ARCHOPHTHALMOL.COM
`
`©2000 American Medical Association. All rights reserved.
`Downloaded From:http://archopht.jamanetwork.com/ by a University of Michigan User on 01/25/2016
`
`

`

`
` EF] 0.05% CsA
`
`
`EB 0.1% CsA
`
`
`Ef] Vehicle
`
`
`
`
`
`
`
`
`
`0 tomes
`
`
`
`-60
`~100
`
`
`
`
`
`
`
`
`
`
`1404
`
` =] 0.05%CsA
`1204
`EZ 0.1% CsA
`ad Vehicle
`
`
`
`100+
`
`D>oooSs n
`
`
`
`
`
`
`
`
`
`
`
`
`Cb3
`
`cb4
`
`cD8
`
`Figure 1, Percent change forcells positive for the lymphocytic markers CD3,
`€D4, and CD8in the overall patient population. Values presented are mean
`percent change+ SE from baseline at month 6. CsA indicates cyclosporine.
`
`
`
`
`Mean%Change
`
`
`
`
`
`
`
`
`~ Non-Sjogren Syndrome
`Sjdgren Syndrome
`CD3
`
`Figure 2. Percent change for CD3-positive cells from the Sjégren syndrome
`and non-Sjégren syndrome subpopulations. Values presented are mean
`percent changeSEfrom baseline at month 6. The P valueis relative to
`pairwise-comparisons from 1-way analysis of variance. CsA indicates
`cyclosporine.
`
`The CD8-positivecells exhibited the same pattern as CD3-
`positive cells but with less of a decrease from baseline
`following CsA andless of anincrease from baselinefol-
`lowingvehicle treatment. However, the changefrom base-
`line in the number of T lymphocytes (CD3+, CD4+, and
`CD8+) didnot reachstatistical significance, either among
`or within treatment groups (Figure 1).
`Within the Sjogren subgroup, 0.5% CsA treatment
`resulted in significantly greater (P<.03) decreases in CD3-
`positive cells than did vehicle. The CD3-positivecells de-
`creased from baselinein all treatment groups amongthe
`non-Sjégren subgroup. However, this decrease was not
`Statistically significant in either group (Figure 2).
`
`LYMPHOCYTE-ACTIVATION MARKERS
`
`In general, there was a decrease from baseline in the num-
`berof cells positive for lymphocyte activation markers
`CD1la and HLA-DR following CsA treatment com-
`pared with an increase from baseline in thesecells fol-
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`cDita
`
`HLA-DR
`
`Figure 3. Percent changefor cells positive for the lymphocyte activation
`markers CD11a and HLA-DR in the overall patient population. Values
`presented are mean percent changex SE from baseline at month 6. The P
`values are relative to pairwise comparisons (P<.05) and within-group
`differences (P<,03). CsA indicates cyclosporine.
`
`
`
`
`
`
`100
`~120|
`
`
`
`
`
`
`
`0.05% CsA
`
`a
`Vehicle
`
`—
`~~
`Non-Sjégren Syndrame
`Sjégren Syndrome
`CDila
`
`Figure 4. Percent change for CD11a-positive cells from the Sjdgren
`syndrome and non—Sjégren syndrome subsets. Values presented are mean
`percent changeSEfrom baseline at month 6. The P value is relative to
`pairwise comparisons from 1-way analysis of variance. CsA indicates
`cyclosporine.
`
`lowing vehicle treatment for the overall patient popula-
`tion.
`
`Statistical analysis revealed a significant among-’
`group difference in change from baselinefor cells ex-
`pressing CD1la (P=.04) and HLA-DR (P=.02) for the
`overall patient population. Pairwise comparisons showed
`significant reductions with 0.05% CsA treatment com-
`pared with treatmentwithvehicle incells positive for both
`markers CDlla (P=.05) and HLA-DR (P=.016)
`(Figure 3). Furthermore, a comparison within indi-
`vidual treatment groups, comparing pretreatmentto post-
`treatmentresults, revealed a statistically significant de-
`crease from baseline forHLA-DRin the 0.05% CsA group
`(P=.03) (Figure 3).
`Within the Sjégren subgrouptreated with 0.5% CsA,
`there weresignificantly greater (P<.001)decreasesin cells
`positive for CD1lla than in vehicle. There was a de-
`crease frombaselinein both treatment groups (CsA and
`vehicle) among the non-Sjégren subgroup (Figure 4),
`This decrease did not reach statistical significance.
`
`
`
`(REPRINTED) ARCH OPHTHALMOL /VOL 118, NOV 2000
`1492
`
`WWW. ARCHOPHTHALMOLCOM
`
`©2000 American MedicalAssociation. All rights reserved.
`Downloaded From: http://archopht.jamanetwork.com/ by a University of Michigan User on 01/25/2016
`
`

`

`Pretreatment
`
`Cc Posttreatment
`
`Figure 5. /mmunofluorescence micrographs demonstrating cells positive for the lymphocyte activation marker CD11a in conjunctival biopsy specimens of
`patients with non—Sjégren keratoconjunctivitis sicea pretreatment and posttreatment with (A and B) 0.05% cyclosporine and (C and D) vehicle. The number of
`positive cells within epithelium and substantia propria in the
`cyclosporine-treated group decreased, while the numberin the vehicle-treated biopsy sample
`increased (bar=25 pm).
`
`D
`
`
`
`Figure 5 and Figure 6 showa representative set
`of immunofluorescence micrographsforcells positive for
`the markers CD11a and HLA-DRfrom the non-Sjogren
`subgroup treated with 0.05% CsA or vehicle. Figure 7
`shows immunofluorescence micrographsforcells posi-
`tive for the markers CD3 and CD1 1a from patients with
`Sjogren KCS treated with 0.05% CsA.
`
`These findings provide additional evidence that in-
`flammation plays a role in the pathogenesis of KCS and
`suggests that modulating the underlying immunere-
`sponse may prove moreefficacious in the treatment of
`KCSthan thefrequentuseofartificial tears. Topical CsA
`has been successfully used for the treatment of canine
`dry eye for many years. Studies in the canine KCS model
`have demonstrated that CsA decreases the conjunctival
`——iiim—¥§
`and lacrimal gland lymphocytic infiltrates.***°
`However, there have beenonly a limited numberof
`In the presentstudy, immunohistochemicalanalysis was
`reports on the use of topical CsA in the treatmentof dry
`used to evaluate changes in the presence of cells posi-
`eye syndrome in humans*”” with only 1 attempt to look
`tive for lymphocytic and lymphocyte activation mark-
`at the effect of the treatmentat a cellular level.*° Power
`ers in conjunctival biopsy specimensofpatients with mod-
`et al? reported a significant reduction in CD4-positive
`erate to severe KCS, following treatrnent with 0.05% CsA,
`T lymphocytesin both the conjunctival epithelium and
`0.1% CsA, or vehicle. We found that CsA treatmentre- ©
`the substantia propria of patients with secondary Sjé-
`duced the numberof activated T lymphocytes within the
`gren syndrome compared with non—dryeye controlsfol-
`ocular surface of patients with and without Sjogren syn-
`lowing treatment with CsA. The present study also dem-
`drome. After 6 monthsof treatment with 0.05% CsA,sta-
`onstrated a significant decrease in CD3-positivecells after
`tistically significant decreases were seen in cells positive
`6 months of 0.05% CsA treatmentin patients with Sjo-
`for CD11a and HLA-DR compared with those in vehicle
`gren syndrome.
`for the overall patient population. Within the Sjogren pa-
`Furthermore, the numberof cells positive for CD1la
`tient subgroup treated with 0.05% CsA,there were also
`and HLA-DR, which are lymphocyte activation mark-
`significantly greater decreases than with vehicle in the
`ers, decreased significantly in patient populationstreated
`numberof cells positive for CD3 and CD1 1a.
`with CsA. HLA-DRisa class ILmajor histocompatibility
`
`(REPRINTED) ARCH OPHTHALMOL /VOL 118, NOV 2000
`1493
`
`WWW. ARCHOPHTHALMOL.COM
`
`©2000 American Medical Association, All rights reserved.
`Downloaded From: http://archopht.jamanetwork.com/ by a University of Michigan User on 01/25/2016
`
`

`

`
`
`Pretreatment
`
`Posttreatment
`
`E
`
`F
`
`Figure 6. /mmunotluorescence micrographs demonstrating cells positive for HLA
`-DR in conjunctival biopsy specimens ofpatients with non-Sjégren
`keratoconjunctivitis sicca pretreatment and posttreatment with (A and 8) 0.05%
`cyclosporine and (C and D) vehicle. A decrease in the number ofpositive cells
`within epithelium and substantia propria in the 0.05% cyclosporine-treated group is apparent compared with an increase in numberin the vehicle-treated biopsy
`sample. E and F, Example ofa negative controlfor a vehicle biopsy in
`which the primary antibody was omitted. Bar=25 um (A-C).
`
`complex antigen thatis expressed in inflamed regions and
`serves as a ligandfor the T-cell receptor. CD4+ T lym-
`phocytes are activated through a signal from HLA-DR mol-
`ecules of antigen-presentingcells.>! Immunopathologic
`studies show evidence of immuneactivation of the con-
`junctival epithelium in Sjégren syndrome. Compared with
`control eyes, a significantly greater percentage of con-
`junctival epithelial cells from patients with Sjégren syn-
`drome express the HLA-DR molecule.Hingorani et
`al” report a decrease in HLA-DRexpression oncells in
`the substantia propria ofpatients with atopic keratocon-
`junctivitis following 3 monthsof treatment with CsA. In
`
`confirmation of these findings, the data presented here
`demonstrate a reduction in the numberofcells positive
`for the lymphocyte activation marker HLA-DR after 6
`monthsof 0.05% CsA treatment.
`CD11a/LFA-1 dymphocyte function-associated an-
`tigen) is associated with adhesion of lymphocytes, mac-
`rophages, and granulocytes andis a ligand ofintercel-
`lular adhesion molecule-1 (ICAM-1), which supports the
`binding of lymphocytes to antigen-presenting cells.#
`CD1 1a is up-regulated during activation of human lym-
`phocytes and, with its ligand ICAM-1, plays an impor-
`tant role in cell-to-cell interactions andcell migration of
`
`(REPRINTED) ARCH OPHTHALMOL/VOL 118, NOV 20001494
`
`WWW. ARCHOPHTHALMOL.COM
`
`©2000 American Medical Association. All rights reserved.
`Downloaded From:http://archopht.jamanctwork.com/ by a University of Michigan User on 01/25/2016
`
`

`

`
`
`Posttreatment D
`
`Pretreatment
`
`c
`
`A
`
`Figure 7. /mmunofluorescence micrographs demonstrating cells positive for (A and B) CD3and (€ and D) CD11ain conjunctival blopsy specimens ofpatients
`with Sjégren keratoconjunctivitis sicca pretreatment and posttreatment with 0.05%
`cyclosporine. Note the decrease in numberofpositive cells within the
`epithelium and substantia propria in the posttreatment biopsy specimens (bar=
`25 yim).
`
`
`
`lymphocytesinto the surroundingtissue such as the con-
`junctival epithelium and substantia propria*3’ Cyclo-
`sporine has been shownto regulate immune-based in-
`flammationwithin epithelialtissues by inhibiting ICAM-1
`production.Our data supporttheseresults, showingre-
`duced immuneactivation by meansof a decrease in the
`numberofcells positive for CD1 la after a 6-month course
`of 0.05% topical CsA treatment.
`Part of the beneficial effect of CsA mightbe due to
`the reduction in T-cell activation asillustrated by a de-
`crease ofcells positive for HLA-DR. By preventing the
`migration of new lymphocytes into the conjunctiva, as
`suggested by the reduction in CD11a-positive cells, CsA
`may help to reduce the inflammatory process. The fact
`that the data show a reduction in positive cells mainly
`for the lymphocyte activation markers CDlla and
`HLA-DRsuggests that CsA is promoting lymphocytes to
`a more quiescentstatus rather than eliminating present
`lymphocytes. This might explain why the change from
`baseline in the numberof T lymphocytes (CD3+, CD4+,
`and CD8+) did not reachstatisticalsignificance for the
`overall patient population. However, anothercontribut-
`ing factor may be the small patient number and high vari-
`ability within each treatmentgroup.
`
`These results provide further evidence that topical
`use of CsA may havea local immunoregulatory effect on
`inflammation in the conjunctiva ofpatients with dry eye
`syndrome. This effect is evident in the reduction of the
`numberofcells positive for lymphocyte activation mark-
`ers. In preventing the activation of T cells in the con-
`junctiva, topical administration of CsA may interrupt an
`ongoing immunereaction, Even though the reduction
`in Pan-T cells (CD3) and CD11a seemsto be larger in
`patients with Sjogren syndrome, our data on the lym-
`phocyte activation markers, especially HLA-DR, pto-
`vide evidence that CsA treatmentis providing benefits
`for both types of dry eye syndrome. The larger effect in
`patients with Sjogren syndrome maybe dueto the fact
`that there are greater numbers of lymphocytic infil-
`trates in patients with this disease.
`In conclusion,this study demonstrates a reduction in
`activated lymphocytes with topical CsA usein patients with
`moderate to severe KCS. Consistent with these findings,
`clinical symptoms of KCSalso appearto improve with the
`use of CsA in the overall patient population of the multi-
`center study conductedby Allergan Inc.”" This suggests that
`CsA treatmentmayhelp to reduce the pathophysiological
`factors contributing to the development of KCS.
`
`(REPRINTED) ARCH OPHTHALMOL/VOL118, NOV 2000
`1495
`
`WWW. ARCHOPHTHALMOL.COM
`
`©2000 American Medical Association. All rights reserved.
`Downloaded From: http://archopht.jamanctwork.com/ by a University of Michigan User on 01/25/2016
`
`

`

`
`
`Accepted for publication March 27, 2000.
`The authors wish to thank specifically Brenda L.Reis,
`PhD (Allergan, Inc), for her help and input in organizing
`this project and Bruce R. Ksander, PhD (Schepens Eye Re-
`search Institute), for his critical review of the manuscript.
`This project was supported by AllerganInc, Irvine, Calif.
`Reprints: Ilene K. Gipson, PhD, Schepens Eye Re-
`search Institute, Boston, MA 02114 (e-mail: gipson
`@vision.eri.harvard.edu).
`
`——_—___§
`
`BD>,
`
`oo
`
`>
`
`. Gilbard JP. Dry eye disorders. In: Albert DM, Jacobiec FA, eds. Principles and
`Practice ofOphthaimology—Clinical Practice. Philadelphia, Pa: WB Saunders Co;
`1994:257-276.
`. Lubniewski AJ, Nelson JD. Diagnosis and managementof dry eye and ocular
`surface disorders. Ophthalmol Clin North Am. 1990;3:575-594,
`. Stern ME, Beuerman RW, Fox R, Gao J, Mirchetf AK, Pflugfelder SC. The pa-
`thology ofdry eye:the interaction betweenthe ocular surface andlacrimal glands.
`Cornea. 1998;17:584-589,
`. Matsumoto I, Tsubota K, Satake Y, et al. Common T cell receptor clonotype in
`lacrimal glands and labial salivary glands from patients with Sjégren’s syn-
`drome, J Clin Invest, 1996;97:1969-1977.
`. Pepose JS, Akata RF, Pflugfelder SC, Voigt W. Mononuclearcell phenotypes and
`immunoglobulin gene rearrangementsin lacrimal gland biopsies from patients
`with Sjégren’s syndrome. Ophthalmotogy. 1990;97:1599-1605.
`. Sanders MD, Graham EM. Ocular disorders associated with systemic diseases.
`In: Vaughan DAT, ed. General Ophthalmotogy. Los Altos, Calif: Lange Medical
`Publications; 1986:262-301.
`. Damato BE, Allan D, Murray SB, Lee WR. Senile atrophy of the human lacrimal
`gland; the contribution of chronic inflammatory disease. Br / Ophthalmol. 1984;
`68:674-680,
`. Williamson J, Gibson AA, Wilson T,Forrester JV, Whaley K, Dick WC. Histology
`of the lacrimal gland in keratoconjunctivitis sicca. Br J Ophthalmol. 1973:57:
`852-858.
`. Pflugfelder SC, Huang AJ, Feuer W, Chuchovski PT, Pereira IC, Tseng SC, Con-
`junctival cytologic features of primary Sjégren’s syndrome. Ophthatmatoagy, 1990;
`97:985-991.
`Hikichi T, Yoshida A, Tsubota K. Lymphocyticinfiltration of the conjunctiva and the
`salivary gland in Sjégren’s syndrome[letter]. Arch Ophthalmol. 1993;111:21-22.
`Raphael M, Bellefgih S, Piette JCH, Le Hoang PH, Debre P, Chomette G. Con-
`junctival biopsy in Sjégren's syndrome: correlations between histological and
`immunchistochemical features. Histopathology. 1988;13:191-202.
`. Tsubota K, Fujihara T, Saito K, Takeuchi T. Conjunctival epithelium expression
`of HLA-DRin dry eye patients. Ophthalmologica. 1999;213;16-19,
`. Jones DT, MonroyD, JiZ, Atherton SS,Pflugfelder SC. Sjgren’s syndrome:cy-
`tokine and Epstein-Barr viral gene expression within the conjunctival epithe-
`lium. /nvest Ophthalmol Vis Sci. 1994;35:3493-3504,
`» Koo J, Lee J. Cyclosporine: whatclinicians need to know. Dermatol Clin. 1995;
`13:897-907.
`. Secchi AG, Leonardi A, TagnanS. Intraoperative use of mitomycin C and post-
`operative use of topical cyclosporine A in the treatment of tarsal vernal kerato-
`conjunctivitis. /nvest Ophthalmol Vis Sei. 1997;38(suppl):S1077.
`Zhao JC, Jin XY. Local therapy of cornealallograft rejection with cyclosporine.
`Am J Ophthalmol. 1995;119:189-194.
`Zhao JC, Jin XY. Immunological analysis and treatment of Mooren’s ulcer with
`cyclosporin A applied topically. Cornea. 1993;12:481-488,
`Giindiiz K, Ozdemir 6.Topical cyclosporin as an adjunct to topical acyclovir treat-
`ment in herpetic stromal keratitis. Ophthalmic Res, 1997;:29:405-408,
`
`co
`
`10.
`
`11.
`
`16,
`
`17.
`
`18.
`
`19.
`
`20.
`
`21,
`
`22,
`
`23.
`
`24,
`
`25.
`
`26.
`
`27.
`
`28.
`
`29,
`
`30.
`
`3t.
`
`32.
`
`33.
`
`34.
`
`35.
`
`36,
`
`37,
`
`38.
`
`Stern ME, Beuerman RW, Fox RI, Gao J, Mircheff AK, Pflugfelder SC. A unified
`theory of the role of the ocular surface in dry eye. Adv Exp Med Biol. 1998;438:
`643-651.
`Borel JF, Baumann G, Chapman |, et al. In