-
`
`Analysis of Topical Cyclosporine Treatment
`of Patients With Dry Eye Syndrome
`
`Effect on Conjtmctival Lymphocytes
`
`Kathleen S. Kunert, MD; Aim 5. Tisdale, M5; Michael E. Stem, Pl1D;_l. A. Smith; Ilene K. Gipson, PhD
`
`Olaiulive: 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-Sjogren).
`
`Methods: Biopsy specimens were obtained at baseline
`and after 6 months of cyclosporine treatment from eyes
`of 32 patients with moderate to severe dry eye syn-
`drome; 19 were cyclosporine treated 00.05% cyclospor—
`ine, n= 13; 0.1% cyclosporine, n=6) and 13 were ve-
`hicle treated. Within this group there were 12 with Sjogren
`syndrome and 20 with non—Sjt")gren 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 CD11a and l-II_A—DR.
`
`tive for CD3, CD4, and CD8, while in vehicle-treated eyes,
`results showed increases in these markers, although these
`differences were not statistically significant. Following
`treatment with 0.05% cyclosporine, there was a signifi-
`cant decrease in the number ofcells expressing the lym-
`phocyte activation markers CD1 la (P<.05) and HLA—DR
`(P< .05}, indicating less activation of lymphocytes as com-
`pared with vehicle treatment. Within the Sjogren pa-
`tient subgroup, those treated with 0.05% cyclosporine
`also showed a significant decrease in the number ofcells
`positive for CD11a {P<.001) as well as CD3 (P403),
`indicating a reduction in number of activated lympho-
`cytes.
`
`Conclusion: Treatment of dry eye syndrome with topi-
`cal cyclosporine significantly reduced the numbers ofac—
`tivated lymphocytes within the conjunctiva.
`
`Ilosulls: In cyclosporine-treated eyes, biopsy results of
`conjunctivae showed decreases in the number ofcells posi-
`
`Arch Ophtltalmol. 2000;118:1489-I496
`
`
`
`ERATOCONJUNCTNITIS sicca
`(KCS), or dry eye syn-
`drome, is characterized by
`chronic dryness of the cor-
`nea and conjunctiva.' Pa-
`tients with KCS typically show symp-
`toms of ocular discomfort ranging from
`irritation to severe pain. Redness, burn-
`ing, itching, foreign body sensation, con-
`tact lens intolerance, photophobia, and
`blurred vision can occur?
`
`Although KCS can arise from vari-
`ous types of diseases, common to all is the
`involvement of immune—mediated or in-
`
`flammatory-mediated pathways.’ immu-
`nopathologic studies of the laerimal gland
`in patients with Sjogren syndrome show
`progressive lymphocytic infiltration, pri-
`marily consisting of CD4+ T and B cells.”
`This infiltration is believed to be respon-
`sible for the destruction of normal secre-
`
`tory function.“ Lymphocytic infiltration of
`the lacrimal gland has also been de-
`scribed in patients with non-Sjogren
`KCS.” Although the iminunopathologic
`
`analysis of the lacrinial gland has re-
`ceived considerable attention, less work
`has been done on pathological changes oc-
`curring in the ocular surface. The chronic
`dryness of the ocular surface in Sjogren
`syndrome has been attributed to deterio-
`ration of lacrimal gland function with de-
`creased tear production."-'0 However, in
`Sjogren syndrome, conjunctival epithe-
`lial and stromal T-cell infiltration (pre-
`dominantly CD3+ and CD4+ T lympho-
`cytes) has also been shown to occur along
`with drying of the ocular surface.""“
`Supporting a role for an immuno-
`pathogenesis of KCS are the reports ofac-
`tivated lymphocytes as demonstrated by
`expression of lymphocyte activation mark-
`ers such as l-ILA-DR (MHC class II) and
`lCAM—1 (intercellular adhesion mol-
`ecule-1) in the conjunctiva ofpatients with
`Sjfigren syndrorne.'3'” To date, there is
`little information on the effect of modu-
`
`lating these molecules in the conjunctiva
`of patients with Sjogren and non-—
`Sjogren syndrome.
`
`From the Schepens Eye
`Researcli institute and
`
`Departmciit cf Oplitlinlmology,
`Htiwrird Medical School,
`Boston, Mass (Drs Ktmert and
`Gipson, Ms Tisdale); Allergen,
`inc, Irvine, Calif (Dr Stern);
`and the National Eye institute,
`Betliesdri, Md (Ms Srnitli)_
`Dr Stem is an employee of
`Allergen inc.
`
`
`\«’\’\’\’\’\-'. .-\ RC1 {UPI IT} 1.-\ LM()l..CO M
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`
`SUBJECTS AND METHODS
`
`SUBJECTS
`
`Conjunctival biopsy specimens from 32 patients were ex-
`amined; l3 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, sponsor and investigator obligations, informed con-
`sent regulations, and the Declaration 0fHEl5It'1l(i. Potential
`patients signed a prescreening informed consent form and
`a second written informed consent form prior to actual en-
`rollment." The protocol for this study is described briefly
`here. Adult patients of either sex were eligible for partici-
`pation if they had a diagnosis of moderate to severe KCS
`at initial examination as defined by the following criteria:
`(1) Schirmer test results (without anesthesia) less than or
`equal to 5 rnmffi min in at least 1 eye (ifSchirmer 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 same eye); (2) sum ofcorneal and interpalpebral con-
`junctival staining greater than or equal to +5 in the same
`eye where corneal staining was greater than or equal to +2;
`(3) a baseline Ocular Surface Disease lndex” score of 0.]
`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 ifthey had par-
`ticipated in an earlier clinical trial with CsA ophthalmic
`emulsion or had used systemic or topical ophthalmic CsA
`within 90 days prior to the study. Other exclusion criteria
`were the presence or history of any systemic or ocular dis-
`order or condition (including ocular surgery, trauma, and
`disease); current or recent use of topical ophthalmic or sys-
`temie medications that could affect a dry eye condition;
`known hypersensitivity to any component of the drug or
`procedural medications such as stains or anesthetics;
`
`required contact lens wear during the study; recent (within
`1 month) or anticipated use of temporary punctal plugs dur-
`ing the study; permanent occlusion of lacrimal puricla within
`3 months of the study; or ifthe 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) or if their KCS was secondary to the destruction of
`conjunctiva] goblet cells or scarring.
`A retrospective diagnosis of Sjogren syndrome was used
`with modified criteria reported by Vitali et all’ to ensure
`that a consistent definition of Sjogren syndrome was as-
`signed to the patients enrolled. Diagnosis included pres-
`ence of at least one of the following autoantibodies in sera:
`antinuclear antibody (ANA), rheumatoid factor (RF), and
`Sjogren syndrome autoantibodies class SS—A (R0) 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 assigned artificial tears (REFRESH Lubricant Eye
`Drops; Allergan Inc) as needed up to month 4.
`Full-thickness conjunctiva] biopsy specimens of a 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 Schirmer tear test
`value (without anesthesia) and the worse sum of corneal
`and interpalpebral conjunctiva] 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 inferoternporal quadrant, also close to niidline.
`
`TISSUE PROCESSING FOR
`IMMUNOHISTUCHEMICAL ANALYSIS
`
`After removal, the baseline biopsy specimens were imme-
`diately frozen in OCT embedding compound ('i'issue—'l'ek;
`Miles Laboratories, Elkhart, Ind) in a cryomold (Miles
`Laboratories) and stored at —80°C until patient-matched
`
`Currently, administration of artificial tears is the most
`common therapy available for lubricating a dry ocular sur-
`face. This palliative treatment gives only temporary and in-
`complete symptomatic relief and does not address the cause
`of the symptoms, which may include immune-mediated
`inflammation of the ocular surface. Evidence of inflamma-
`
`tory processes in the pathogenesis of KCS led to the de-
`velopment of cyclosporine (CSA) as a first attempt to treat
`this condition therapeutically. Cyclospotine is an immu-
`nosuppressive agent commonly used systemically to treat
`inflammatory diseases such as psoriasis or rheumatoid ar-
`thritis or to prevent organ transplant rejection.” Topical
`CsA has been used as treatment of ocular conditions such
`
`as vernal keratoconjunctivitis, '5 corneal transplants, '° cor-
`neal ulcers,” and herpetic stromal keratitis.'3 The effect of
`this drug on inflammatory diseases is due to its ability to
`
`inhibit T—cell—mediated inflammation by preventing the ac-
`tivation of T cells (by antigen-presenting cells or
`cytokines) . '9-3° Activated T cells are responsible for the pro-
`duction of inflammatory substances such as cytokines,
`which lead to further tissue damage and, in turn, to the ac-
`tivation of more T cells and the production of even more
`inflammatory substances.
`Clinical trials with this dmg have shown improve-
`ment in various objective measures of KCS such as cor-
`neal staining and Schirmer test values.“ To attempt to
`find tissue correlates in these patients, conjunctival bi-
`opsy specimens from patients with Sjogren and non-
`Sjogren KCS treated with CsA or vehicle were evaluated
`immunohistochemically for the presence of activated T
`cells (CD3+ [Pan-T cell], CD4+ [T helper cell], and CD8+
`[cytotoxic T cell]) and lymphocyte-activation markers
`
`
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`
`6-month biopsy specimens were obtained and similarly fro-
`zen. Six—micrortteter sections were taken from each block,
`mounted on gelatin-coated slides, and processed for im-
`munohistochemical analysis. Sectioning of tissue blocks and
`immunohistochemical experiments were performed as pairs
`of biopsies, pretreatment and posttreatment, to minimize
`differences due to experimental conditions.
`
`IMMUNOHISTOCHEMICAL ANALYSIS
`
`Irnrnunohistochemical staining 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), CD1 la (PharMingen, San Diego),
`and 1-ILA-DR (Phariviingen). Cryostat sections were fixed
`in cold acetone (-20°C) for 3 minutes and air dried at room
`temperature for 30 to 43 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 1 hour at room temperature in primary an-
`tibodies at concentrations derived empirically: CD3, 1,0
`pg/mL; CD4, 5.0 pg/mL; CD8, 25 pg/mL; CD1]a, 10.0
`pg/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 temperature in the sec-
`ondary antibody, fluorescein isothiocyanate—conjugated Af-
`finipure Donkey Anti-Mouse lgG Uackson lrnrnunoresearch,
`West Grove, Pa) at a dilution of 1/50. Sections were then
`rinsed itt PBS, mounted in Vectashield (Vector Labs, Bur-
`lingame, Calif), cover—slipped, and viewed under a 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 immunohistochemical
`analysis were run.
`Three separate images were acquired for each anti-
`body and biopsy specimen under a X 20 objective using a
`Spot acquisition program (Diagnostic lnstrutnents Inc). The
`first field selected for imaging was the field with the high-
`est number of positive cells, followed by images to the left
`
`and right of that area. In this manner the entire biopsy area
`was usually captured.
`
`COUNTING PROCEDURE
`
`Measurement of the entire area of epithelium and stroma
`(substantia propria) was achieved by tracing the area us-
`ing the lasso tool under the Adobe Photoshop computer
`program (Adobe Systems Inc, Sanjose, Calif). The total data
`area, measured in pixels, was acquired through the “Im-
`age: Histogram“ command in Photoshop. Two indepen-
`dent counts were recorded for cells positive for each anti-
`body within the traced area. Cells per unit area of pixels
`were adjusted to real unit area or cells per millimeter squared
`of real tissue area, based on 28.346 pixels per centimeter
`in Photoshop and the fact that 1 mm equals 67.8 cm equals
`1922 pixels at X20 magnification on the Nikon micro-
`scope. Data were recorded as cells per millimeter 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 continuous variables and the Fisher exact test
`for categorical variables.
`Percent changes in the number of cells expressing
`lymphocytic andfor lymphocyte activation markers were
`summarized using descriptive statistics (ie, satnple size,
`mean, SD, minimum, maximum, and median). A 1-way
`ANOVA with 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 Sjtigren and
`non-Sjogren subpopulations, excluding the 0.1% CsA
`treatment group in which there was only 1 patient in the
`Sjogren subset.
`
`(CD11a and I-ILA—DR) to further understand the under-
`lying mechanism of CsA treatment.
`
`%—
`PATIENT POPULATION
`
`The mean:SD age of our subjects was 59.0: 13.5 years
`(range, 28.8-84.2 years), including 2? 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 of cells positive for CD3, CD4, and CD8 following
`
`treatment with 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 baseline fol-
`lowing vehicle treatment.
`Figure I shows the percent change from baseline
`for cells expressing the lymphocytic markers (CD3, CD4,
`and CD8) after 6 months of treatment for the overall pa-
`tient population. Note that there was a reduction from
`baseline in the number of CD3-positive cells in the CsA-
`treated groups, while there was an increase from base-
`line in the vehicle-treated group. There was also an in-
`crease from baseline in the numbers of CD4--positive cells
`in the vehicle group, with a smaller increase in the 0.05%
`CsA group and a slight decrease in the 0.1% CsA group.
`
`
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`
`140
`
`120
`
`100
`
`no5
`
`Meanvachange 838
`
`
`
`:332ru.1:
`73=ru:1:
`
`E
`
`$2
`
`Figure 3. Percent change for ceits positive for the iympnocyte activation
`markers CD1 Ia and HtA—DH in the overaii patient popoiation. liaioes
`presented are mean percent cnange:SE from baseiine at month 6. The P
`vetoes are reiative to pairwise comparisons (P<.tJ5) and within-group
`differences (P< .03). Csil indicates cvciosporine.
`
`Non—Siogren Syndrome CD113
`D3
`
`El o_o5°r. can
`U Vehicle
`
`Sjogren Syndrome
`
`Figure 4. Percent change for CD3‘ ta—posi'tive ceiis from the Sidnren
`syndrome and non—Sidgren syndrome sunsets. Vaioes presented are mean
`percent change:SE from oaseiine at month 6. The P veins is relative to
`pairwise comparisons from 1-way anaivsis of variance. [ISA indicates
`cyciosporine.
`
`lowing vehicle treatment for the overall patient popula-
`tion.
`
`Statistical analysis revealed a significant among-
`group difference in change from baseline for cells ex-
`pressing CD113 (P=.0‘l-) and I-lI_A—DR (P=.O2) for the
`overall patient population. Pairwise comparisons showed
`significant reductions with 0.05% CsA treatment com-
`pared with treatment with vehicle in cells positive for both
`markers CD112 (P=.05) and HLA-DR (P=.016)
`(Figure 3). Furthermore, a comparison within indi-
`vidual treatment groups, comparing pretreatment to post-
`treatment results, revealed a statistically significant de-
`crease from baseline for E-lLA—DR in the 0.05% CSA group
`(Pg _@3} (Figure 3).
`Within the Sjogren subgroup treated with 0.5% CsA,
`there were significantly greater (P<.001) decreases in cells
`positive for CD113 than in vehicle. There was a de-
`crease from baseline in both treatment groups (CsA and
`vehicle) among the non-Sjégren subgroup (Figure 4).
`This decrease did not reach statistical significance.
`
`C-D8
`
`Figure 1 . Percent change for ceiis positive for the tympnocytic markers CD3,
`CD4, and CBS in tire overait patient popotat.-"on. vaioes presented are mean
`percent cirangersf from baseiine at month 6. 6521 indicates cvciosporine.
`
`P<.03
`
`MG
`to —
`D -.
`-10 _
`.20 _
`-30 -
`
`%E
`
`E:
`33 -40 -
`E -50 -
`E
`_w _
`_}0 _
`
`CI 0.05% CSA
`El vehicle
`
`.31]
`_9,,] _
`«we
`ItIon—Sjogren Syndrome
`Siogren Syndrome
`003
`Figure 2. Percent cnange for CD3-positive cetis from the Sjdgren syndrome
`and non—Sidgren syndrome sebnopoiat.-‘ons. Vetoes presented are mean
`percent cnanoessf from oaseiine at month 5. The P vaioe is reiative to
`pairwise comparisons from 1-way anaiysis of variance. CsA indicates
`cyciosporine_
`
`The CD8-positive cells exhibited the same pattern as CD3-
`positive cells but with less of a decrease from baseline
`following CSA and less of an increase from baseline fol-
`lowing vehicle treatment. However, the change from base-
`line in the number DfT lymphocytes (CD3+, CD4+, and
`CD8+) did not reach statistical 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—positive cells de-
`creased from baseline in all treatment groups among the
`non—Sjogren 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-
`ber of cells positive for lymphocyte activation markers
`CD1la and l-ILA-DR following CsA treatment com-
`pared with an increase from baseline in these cells fol-
`
`
`\«’\’\«’\’\’\-'. ARCIiUP|'|T1i.-\l_M()L.CUi\-I
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`

`
`Pretreatment
`
`Pu sttrealmenl
`
`Figure 5. tmmonoiiuoresoenoe miorograpits demonstrating oeiis positive for the iyrnpiioeyte activation marker CD? fat in eorritinetitrai biopsy specimens of
`patients with non—S,itilgren i<eratooonionciivi'tis sioca pretreatment and posrireatment with {A and B) 0.05% cyciosporine and ((2 and 5') vehicle. Tire number of
`posititre cetis witnin epitrteiium and sobstantia propria in the eyciosporine-treated group decreased, wniie tne ntrmoerirt tire venicie-treated biopsy sampie
`increased (bar=25 pm).
`
`Figure 5 and Figure 6 show a representative set
`of immunofluorescence micrographs for cells positive for
`the markers CD1la and HLA-DR from the non-Sjogren
`subgroup treated with 0.05% CsA or vehicle‘ Figure 7
`shows immurtofluorescence micrographs for cells posi-
`tive for the markers CD3 and CD1 la from patients with
`Sjtigren KCS treated with 0.05% CSA.
`
`%—
`
`In the present study, immunohistochemical analysis was
`ttsed to evaluate changes in the presence of cells posi-
`tive for lymphocytic and lymphocyte activation mark-
`ers in conjunctiva] biopsy specimens ofpatients with mod-
`erate to severe KCS, following treatment with 0.05% CSA.
`0.1% CsA, or vehicle. We fottnd that CsA treatment re-
`dttced the number ofactivated T lymphocytes within the
`ocular surface of patients with and without Sjogren syn-
`drome. After 6 months of treatment with 0.05% CsA, sta-
`tistically significant decreases were seen in cells positive
`for CD11a and l-lLA—DR compared with those in vehicle
`for the overall patient population. Within the Sjogren pa-
`tient subgroup treated with 0.05% CSA, there were also
`significantly greater decreases than with vehicle in the
`number of cells positive for CD3 and CD1 la.
`
`These findings provide additional evidence that in-
`flammation plays a role in the pathogenesis of KCS and
`suggests that modulating the underlying immttne re-
`sponse may prove more efficacious in the treattnent of
`KCS than the frequent use of artificial tears. Topical CSA
`has been successfully used for the treatment of canine
`dry eye for many years. Studies in the canine KCSn1odel
`have demonstrated that CsA decreases the conjunctival
`and lacrimal gland lymphocytic infiltrates.“'3"
`However, there have been only a limited numher of
`reports on the use of topical CsA in the treatment ofdry
`eye syndrome in humans37'3° with only I attempt to look
`at the effect of the treatment at a cellular level.” Power
`
`et all“ reported a significant reduction in CD4-positive
`T lympltocytes in both the conjunctival epitheliutn and
`the suhstantia propria of patients with secondary Sjo-
`gren syndrome compared with t1on—dry eye controls fol-
`lowing treatment with CSA, The present study also dem-
`onstrated a significant decrease in CD3—positive cells after
`6 months of 0.05% CsA treatment in patients with Sjo-
`gren syndrome.
`Furthermore. the number ofcells positive for CD] la
`and HLA—DR, which are lytnphocyte activation mark-
`ers, decreased significantly in patient populations treated
`with CsA. HLA-DR is a class ll major histocompatibility
`
`
`\\-'\\-'\\"..-\R('.l [UPI IT] l.-\l..\-I('t[..(ItJ.\=|
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`ARGENTUM - EX. 1012, p. 005
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`

`
`Pretreatment
`
`Postlreatment
`
`Figure ti. tmmunottuoresoence miorograpns denionstrating eetts positive for HLA—DR in conjunctiva! biopsy specimens of patients with non—Siogren
`keratooonionotivitis sieoa pretreatment and posttreatment with (A and B) 0.05% oyciosporine and (C and DJ veniote. A decrease in tne number of positive ceits
`vvittiin eoittreiiurn and sutistantia uropria in the 0.05% cyciosporine—treated group is apparent compared with an increase in number in the veniote-treated biopsy
`sampie. E and F, Exampte of a negative controt for a vettiste biopsy in tvttictt ttte primary antibody was omitted. Bar=25 urn {A-C).
`
`complex antigen that is expressed in inflamed regions and
`serves as a ligand for the T—cell receptor. CD4+ T lym-
`phocytes are activated through a signal from HLA-DR mol-
`ecules ol antigen—presenting cells. ” ltnmunopathologic
`stutlies show evidence of immune activation of the con-
`
`junctival epithelium in Sj ogren syndrome, Compared with
`control eyes. a significantly greater percentage of con-
`junetival epithelial cells from patients with Sjtigren syn-
`drome express the Hi_A—DR molecule.”-‘-’ Hingorani et
`al” report a decrease in HLA—DR expression on cells in
`the suhstantia propria of patients with atopic keratoeon—
`junetivitis following 3 months oftreatment with CSA. In
`
`confirmation of these findings, the data presented here
`demonstrate a reduction in the number of cells positive
`for the lymphocyte activation marker HLA-DR after 6
`months of 0.05% CsA treatment.
`
`CD1 ta./LFA—1 (iyinphoeyle function—assoeiated an-
`tigen) is associated with adhesion of lymphocytes, mac—
`rophages, and granulocytes and is a ligand of intercel-
`lular adhesion molecule—1 (lCAM— l ), which supports the
`binding of lymphocytes to antigen-presenting cells.“
`CD1la is up—regulated during activation of human lyin-
`phoeytes and, with its ligand lCAM—1, plays an i1npor—
`tant role in cell-to-cell interactions and cell migration of
`
`
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`

`
`Pretreatm en!
`
`Posttreatm en:
`
`Figure 3'. lmmunotiuorescence micrograpits demonstrating cells pt;-sitilre for (A and 5‘) CD3 and {G and 0) CD1 la in t:ort;'uncti'vai biopsy speoimens of patients
`with Sitigren iteratoconiunctivitis sicca prerreatmentanri posttreatment with 0.05% cyctasporine. were the decrease in number otpasitive cells within the
`epithelium and stibsiantia propria in the posttreatment biopsy specimens (bar=25 pm}.
`
`lymphocytes into the surrounding tissue such as the con-
`junctival epithelium and substantia propria.“'3'_' Cyclo-
`sporine has been shown to regulate immune—based in—
`flamrnation within epithelial tissues by inhibiting [CAM-1
`production.“ Our data support these results, showing re-
`duced immune activation by means of a decrease in the
`number ofcells positive for CD1la after a 6-month course
`of 0.05% topical CsA treatment.
`Part of the beneficial effect of CsA might be due to
`the reduction in T—cell activation as illustrated by a de—
`crease of cells positive for HLA—DR. By preventing the
`migration of new lymphocytes into the conjunctiva, as
`suggested by the reduction in CD1 ] a-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 CD11a and
`1-{LA-DR suggests that C513. is promoting lymphocytes to
`a more quiescent status rather than eliminating present
`lymphocytes. This might explain why the change from
`baseline in the number ofT lymphocytes (CD3+, CD4+,
`and CD8+) did not reach statistical significance for the
`overall patient population. However, another contribut-
`ing factor may be the small patient nttmber and high vari-
`ability vvithin each treatment group.
`
`These results provide further evidence that topical
`use of CsA may have a local intmunoregulatory effect on
`inflammation in the conjunctiva of patients with dry eye
`syndrome. This effect is evident in the reduction of the
`number ofcells positive for lymphocyte activation mark-
`ers. In preventing the activation of T cells in the con-
`junctiva, topical administration ofCsA may interrupt an
`ongoing immune reaction. Even though the reduction
`in Pan—T cells (CD3) and CD11a seems to be larger‘ in
`patients with Sjogren syndrome. our data on the lym-
`phocyte activation ma1'l<ers. especially HLA—DR. pro-
`vide evidence that CsA treatment is providing benefits
`for both types of dry eye syndrome. The larger effect in
`patients with Sjogren syndrome may be due to 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 Csit use in patients with
`moderate to severe KCS. Consistent with these findings.
`clinical symptoms of KCS also appear to improve with the
`use of CsA in the overall patient population of the multi-
`center study conducted by Allergan inc.“ This suggests that
`CsA treatment may help to redttce the pathophysiological
`factors contributing to the development of I-(CS.
`
`
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`ARGENTUM - EX. 1012, p. 007
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`ARGENTUM - EX. 1012, p. 007
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`

`
`Accepteclfor publication March 27, 2000.
`The authors wish to thanh specifically Brenda L. Reis,
`PhD (Allergan, Irlc),for her help and input in organizing
`this project and Bruce R. Ksancier, PhD (Schepens Eye Re-
`search Institute), for his critical review of the manuscript.
`This project was supported by Allergen Inc, Irvine, Calif
`Reprints: Ilene K. Gipson, PhD, Schepens Eye Re-
`search Institute. Boston. MA 02114 ('e-mail: gipson
`@vision.eri.harvard.edu).
`
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