Cornea I2(6).'507—5Il, I993.
`
`© 1993 Raven Press. Ltd.. New York
`
`Effect of Topical Cyclosporin A on
`Conjunctival T Cells in Patients with
`Secondary SJogren’s Syndrome
`
`William J. Power, F.R.C.S., Paul Mullaney, F.R.C.S.I.,
`Michael Farrell, M.R.C.Path., and Louis M. Collum, F.R.C.S.
`
`The effect of topical cyclosporin A on conjunctival T cells
`was studied in nine patients with secondary Sj<'5gren‘s dis-
`ease. Patients had eonjunctival biopsies performed before
`and after a 6-week course of topical cyclosporin. Epithe-
`lium and substantia propria in the Sjogren’s patients be-
`fore treatment showed significantly more CD4+ cells
`than specimens taken from nine age- and sex-matched
`controls. Following treatment with topical cyclosporin,
`there was a significant reduction in the number of CD4 +
`cells in both the conjunctiva] epithelium and substantia
`propria. Despite the fact that the treatment resulted in
`immunopathological improvement,
`the clinical benefit
`was not as favorable. Our results suggest that topical cy-
`closporin may have a local immunosuppressive effect on
`the conjunctiva in patients with Sjogren’s disease.
`Key Words: Cyclosporin—-Sj6gren‘s syndrome—
`Monoclonal antibody—Conjunctival biopsy—T-cells.
`
`Cyclosporin A is a cyclic polypeptide of 11 amino
`acids. It is insoluble in water but can be dissolved in
`
`olive oil or sesame oil at 60°C. It inhibits T lympho-
`cyte response to mitogens or antigens and lympho-
`kine production by activated T lymphocytes (1,2).
`It has also been shown to block the production of
`interleukin 2 by activated T lymphocytes (3). In
`contrast, it has no effect on T suppressor lympho-
`cytes (2). The usefulness of cyclosporin A in tissue
`transplantation has been extensively investigated.
`It has been shown to be effective in increasing the
`survival time of renal (4,5), liver (6), and cardiac (7)
`transplants. It has also been used with success in
`preventing graft versus host disease in patients after
`bone marrow transplantation (8,9).
`
`Submitted July 22, 1992. Accepted January 5, 1993.
`From the Royal Victoria Eye and Ear Hospital, Dublin, Ire-
`land.
`Address correspondence and reprint requests to Dr. W.J.
`Power at The Royal Victoria Eye and Ear Hospital, Adelaide
`Road, Dublin 2, Ireland.
`
`In patients with chronic intraocular inflamma-
`tion, systemic cyclosporin has been used with some
`success, although side effects, particularly nephro—
`toxicity, were seen in a significant proportion of
`patients (10). Systemic cyclosporin has also been
`used with varying success in the prevention of cor-
`neal allograft rejection in high-risk patients (11,12),
`in the treatment of patients with Behcet’s disease
`(13-15), and in the treatment of Mooren’s ulcer (16).
`Recurrence of disease is not infrequent after discon-
`tinuing treatment, and long-term treatment may be
`necessary (10). In an effort to overcome the side
`effects associated with long-term systemic cyclo-
`sporin, workers have recently been studying the ef-
`fects of topical cyclosporin A on immunoreactive
`cells at a local level (17).
`Sjogren’s syndrome is an autoimmune disease
`characterized by lymphocytic infiltration of the
`exocrine glands and polyclonal B lymphocyte pro-
`liferation (18). It is generally accepted that the prin-
`cipal cell of this inflammatory infiltrate is an acti-
`vated T lymphocyte bearing the T helper-inducer
`phenotype (19). This finding, together with the fact
`that cyclosporin appears to have a inhibitory effect
`on these cells, prompted us to study the effect of
`topical cyclosporin on a group of patients with sec-
`ondary Sjogren’s syndrome. We report our results
`on the use of topical cyclosporin in these patients
`from a clinical and immunocellular perspective.
`
`PATIENTS AND METHODS
`
`Patients
`
`Patients were eligible for admission to the study if
`they had a severe tear film defect in association
`with a diagnosed connective tissue disorder. The
`
`507
`
`ALL 2016
`MYLAN PHARMACEUTICALS V. ALLERGAN
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`

`

`508
`
`W.J. POWER ET AL.
`
`diagnosis of connective tissue disease was made by
`either a clinical immunologist or rheumatologist on
`the basis of clinical, laboratory, and radiological
`findings (Table 1). For the purpose of the study,
`patients were deemed to have a severe tear film
`defect when they had a Schirmer test done with
`topical anaesthesia of 0 on two separate occasions,
`abnormal tear film break up time (<5 s), had posi-
`tive rose bengal staining of conjunctiva and cornea,
`and had symptoms not controlled on maximum oc-
`ular lubricants. Six of the patients had punctal oc-
`clusion performed on both upper and lower lids in
`the past. All patients were using preservative-free
`ocular lubricants. Six of the patients also had xero-
`stomia, defined as symptoms of dry mouth with as-
`sociated dysphagia.
`The following were excluded from the study:
`uniocular patients, those who had received either
`topical or systemic cyclosporin in the past, and any-
`one who was currently on immunosuppressive ther-
`apy. Informed consent was obtained from each pa-
`tient.
`
`On entry into the study, patients had a full-
`thickness inferonasal bulbar conjunctival biopsy
`performed 4 mm from the limbus under local anaes-
`thesia. Two weeks later, when the eye had settled,
`patients were started on 2% cyclosporin A drops in
`olive oil to be used four times daily in both eyes.
`During the next 6 weeks, patients were seen on
`three occasions. At each visit, patients were ques-
`tioned as to whether their symptoms had improved,
`deteriorated or had remained static. Symptoms of
`dryness, grittiness, and burning were scored on a
`four-point scale (0 = absent, 1 = mild, 2 = mod-
`erate, 3 = severe). Pretreatment scores were com-
`pared to the final examination scores. An attempt
`was made to assess the effect of treatment on the
`
`degree of epithelial damage as indicated by staining.
`The amount of rose bengal staining was assessed as
`absent, mild, moderate, or severe and scored 0, 1,
`2, or 3, respectively, as described by van Bij-
`sterveld (20). Any evidence of toxicity that could
`have been attributed to the treatment was noted. In
`
`Patient
`
`cwNmm¢wm4
`
`Age
`59
`30
`81
`57
`53
`64
`73
`72
`39
`
`TABLE 1. Clinical findings
`
`Sex
`
`Xerophthaimla
`
`Xerostomia
`
`mggmmmmnm
`
`+++++++++
`
`|+|+l++++
`
`Diagnosis
`SLE
`MCTD
`HA
`HA
`HA
`MCTD
`HA
`HA
`RA
`
`SLE, systemic lupus erythematosus; MCTD. mixed connective tis-
`sue disease; HA, rheumatoid arthritis.
`
`Cornea, Vol. 12, No.6, 1993
`
`particular, evidence of corneal epithelial toxicity
`and allergic reactions was looked for. No attempt
`was made to score these adverse events. At the end
`
`of the 6 weeks of treatment, a conjunctival biopsy
`adjacent to the previous site was repeated and the
`treatment discontinued. Nine age- and sex-matched
`patients undergoing cataract or strabismus surgery
`also had conjunctival biopsies taken in an identical
`manner for comparison. These patients all had nor-
`mal Schirmer testing, had no evidence of rose ben-
`gal staining, and, apart from either cataract or stra-
`bismus, had a normal ocular examination. None of
`the control patients were on any ocular medica-
`tions.
`
`Methods
`
`Biopsy material was snap frozen using L.I.P.
`Freeze (Equipment and Services, Galway, Ireland)
`instant freezing aerosol. Seven-micron frozen sec-
`tions were air dried for 4 h. Subsequently, sections
`were fixed in acetone for 10 min at room tempera-
`ture and allowed to air dry for 5 min, following
`which they were immersed in tris buffered saline
`(TBS). The primary antisera (Ortho Pharmaceuti-
`cals, Dublin, Ireland) were applied for 1 h (anti-CD4
`at a dilution of 1 in 50 and anti-CD8 at a dilution of
`1 in 200). Sections were then washed three times in
`TBS for 15 min each wash and then incubated with
`
`rabbit anti—mouse antibody (Dako, Copenhagen,
`Denmark) 1 in 200 for 30 min. Following subsequent
`washing for 15 min, sections were incubated with
`avidin biotin complex for 30 min and following fur-
`ther washing were developed in diamino benzidine.
`The primary antibodies used were the CD4 anti-
`body to identify T-helper/inducer cells (CD4+
`cells) and the CD8 antibody to identify T-suppres-
`sor/cytotoxic cells (CD8+ cells). Positive controls
`were performed with fresh tissue obtained from ton-
`sillectomy specimens and were processed in the
`same way as described for the conjunctival speci-
`mens. Negative controls consisted of replacing the
`primary antibody with TBS. Sections were counter
`stained with haematoxylin, and were then dehy-
`drated, cleared, and mounted in DPX (a xylene-
`based neutral mounting medium). The processed
`sections were read in a masked fashion using a Ni-
`kon microscope with an attached eye piece grati-
`cule. Using a X40 objective, the number of cells
`labelled with each monoclonal antibody was
`counted for at least 10 different fields in both the
`
`epithelium and substantia propria. Cells were con-
`sidered positive if they showed a dense dark black-
`bluish ring on the cellular membrane. The total
`number of stained cells was expressed as the num-
`ber of cells per mmz.
`
`2
`
`

`

`TOPICAL CYCLOSPORIN IN SECONDARY SJOGREN’S SYNDROME
`
`509
`
`Means and standard errors of the mean were cal-
`
`DISCUSSION
`
`culated for both cell types in the normals and in the
`Sj6gren’s' group before and after treatment. Com-
`parisons between groups were analyzed using the
`Wilcoxon two-sample rank sum test.
`
`RESULTS
`
`Nine patients with Sj6gren’s disease were en-
`tered into the study. The mean cell numbers in the
`epithelium and substantia propria are shown in Ta-
`bles 2 and 3. There was a significantly greater num-
`ber of CD4+ cells in the epithelium and substantia
`propria in the Sj6gren’s group before treatment
`when compared with the normals. There was no
`significant difference in the number of CD8+ cells
`in the epithelium of the normals versus the pre
`treatment Sj6gren’s and although there was a
`greater number of CD8 + cells in the substantia pro-
`pria, this was not statistically significant.
`Following treatment, there was a significant re-
`duction in CD4+ cells, in both the epithelium and
`substantia propria in the Sj6gren’s patients. There
`was no significant change in the number of CD8+
`cells following treatment.
`Table 4 shows the effect of treatment on the cho-
`
`sen clinical parameters. There was a significant im-
`provement in the symptom of dryness, but there
`was also a definite deterioration in the symptom of
`burning. This was experienced by all patients.
`When questioned about this, all patients felt that
`this was directly related to the drops. All patients
`experienced burning following instillation of the
`drops lasting from a few minutes to a maximum of
`30 min. Patients were asked if this burning would
`discourage them from continuing treatment after
`the study period. Seven of the nine said they would
`prefer not to continue with the treatment on com-
`pletion of the study because of the discomfort they
`experienced after instilling the drops. Two patients
`developed a “hurricane” epitheliopathy that per-
`sisted throughout the course of treatment. In both
`patients, the epitheliopathy resolved within 2 weeks
`of discontinuing the treatment. There were no other
`side effects noted.
`
`The aim of this study was to look at conjunctiva]
`lymphocyte subpopulations in patients with
`Sj<'5gren‘s disease before and after treatment with
`topical cyclosporin. We have shown that a 6-week
`course of topical cyclosporin significantly de-
`creased the numbers of CD4 + cells in both the ep-
`ithelium and substantia propria. Dalavanga et al.
`(21) demonstrated a reduction in the number of
`CD4 + cells in labial minor salivary gland tissue fol-
`lowing systemic cyclosporin A treatment in a group
`of patients with Sj6gren’s syndrome. However, like
`the authors of the present study, they were disap-
`pointed in the clinical improvement produced with
`the treatment. In a study on the effects of topical
`cyclosporin in nickel hypersensitivity, Aldridge et
`al. (22) demonstrated a marked diminution in lym-
`phocyte infiltration in skin biopsies adjacent to con-
`tact reactions following treatment with topical cy-
`closporin 5%. They were unable to detect systemic
`absorption of the drug and therefore suggested that
`the effect was produced by local alteration of the
`immune response.
`There have been few reports on the use of topical
`cyclosporin in the treatment of Sj6gren’s syndrome
`(23). In a series of 36 sequential cases of canine
`keratoconjunctivitis, Kaswan et al.
`(23) demon-
`strated a significant increase in tear production and
`a progressive reduction of superficial corneal
`neovascularisation with topical cyclosporin usage.
`Additional benefits that were seen with the treat-
`
`ment included reduced mucopurulent conjunctivi-
`tis, rapid healing of persistent corneal ulcers, and
`reduced dependence on frequent topical lubricants.
`There was no attempt made to look at the effect of
`the treatment at a cellular level.
`
`Our results provide further evidence that topical
`cyclosporin may have a local immunoregulatory ef-
`fect on human conjunctiva. Antigen stimulus to
`T-cells is required continually to maintain an active
`immune response (24). In interfering with the early
`helper T-cell response, topical administration of cy-
`closporin can interrupt an ongoing immune reac-
`tion. We believe that topical cyclosporin may work
`
`TABLE 2. Lymphocyte subpopulations in conjunctiva! epithelium“
`
`Sjogren's (n = 9)
`
`
`
`p (before vs
`after treatment)
`
`Normals (n = 9)
`
`Before treatment
`
`After treatment
`
`Cell type
`T helper/inducer cells
`(CD4+)
`T cytotoxic/suppressor
`
`cells (CD8+) 0.17 3.131078 (1.8-4.8) 2.87 1 0.96 (1.3—5.1) 3.27 : 081 (2.1—5.2)
`
`2.42: 0.31 (1.6—3.1)
`
`5.61 : 1.63 (3.4—7.1)
`
`2.78 1 0.29 (1.5-3.5)
`
`0.018
`
`
`
`
`
`
`
`" Number of cells/mm“ : SEM. The range Is given in brackets.
`
`Cornea, Val. 12. No. 6. I993
`
`3
`
`

`

`510
`
`W.J. POWER ET AL.
`
`TABLE 3. Lymphocyte subpopulations in con/unctival substantia propria‘
`
`Sj6gren's (n = 9)
`
`p (before vs
`
`Cell type
`Normals (n = 9)
`Before treatment
`After treatment
`after treatment)
`T helper/inducer cells
`(CD4+)
`T cytotoxic/suppressor cells
`
`(CD8+) 0.29 8.34 :2 2.12 (6.1—10.9) 13.15 1 2.34 (9.1—16.4) 11.87 2* 1.57 (8.8—13.3)
`
`6.92 : 1.71 (3.9—8.7)
`
`21.66 : 3.12 (1 5.2-2-1.7)
`
`10.49 1 1.49 (8.7—13.1)
`
`0.02
`
`
`
`
`
`
`
`" Number of cells/mm? 1 SEM. The range is given in brackets.
`NS. not significant.
`
`in patients with Sj6gren’s syndrome because of its
`local immunosuppressive effect and not because of
`systemic absorption. Studies have shown that the
`topical application of drops does not produce de-
`tectable levels in the plasma (25,26). Furthermore,
`Foets et al. (27) demonstrated that topical cyclo-
`sporin applied to keratoplasties in rabbits protected
`the graft when applied to the recipient eye but not
`when applied to the fellow eye.
`The commonest side effect noted in the study was
`burning. All patients complained of this, although
`no patient discontinued treatment because of it.
`However, our study population may not be a rep-
`resentative group, as all our patients were highly
`motivated in their efforts to find a treatment which
`
`would help alleviate their symptoms. Two patients
`developed an epitheliopathy that resolved on dis-
`continuation of the treatment when they had com-
`pleted the study. It is not certain whether this epi-
`theliopathy is due to a direct toxic effect of the cy-
`closporin or the olive oil on the corneal epithelium
`or whether it is a feature of deficient corneal wet-
`
`ting. Versura (28) used scanning electron micros-
`copy to look at the effect of cyclosporin on the cor-
`neal epithelium of rabbits. She detected small focal
`
` Score change No. of patients
`
`TABLE 4. Change in clinical parameters
`following treatment‘
`
`Dryness
`0
`2
`+1
`7
`p = 0.016
`-1
`0
`+1
`p = 0.125
`-1
`-2
`-3
`p = 0.004
`+1
`0
`-1
`p = 1.00
`
`Grittiness
`
`Burning
`
`Fl.B. Staining
`
`1
`2
`6
`
`3
`4
`2
`
`3
`4
`2
`
`‘’ For each parameter, the difference in scores before and
`after treatment is given (+, improvement; —. deterioration).
`A two-tailed sign test was used to determine significance.
`
`Cornea, Vol. 12, Na. 6, I993
`
`areas where the normal arrangement of epithelial
`cells was lost. These changes were present in both
`the cyclosporin treated rabbits and in those receiv-
`ing olive oil only, suggesting that the oil as a vehicle
`may be responsible for the epithelial changes seen.
`It is also noteworthy that Kaswan (23) noted a de-
`crease in the frequency of irritative reactions when
`corn oil was used instead of olive oil. The effects of
`
`long-term usage of topical cyclosporin has not been
`studied, but it has been shown that topically applied
`cyclosporin does not inhibit corneal healing in the
`rabbit and guinea pig eye with short-term usage
`(29).
`We have demonstrated that the topical adminis-
`tration of 2% cyclosporin four times daily produces
`an immunopathological improvement in patients
`with Sj6gren’s disease. However, the clinical ben-
`efit was disappointing, mainly because of local irri-
`tation following use of the drug. This may have
`been related to the vehicle used to administer the
`
`drug. The use of cyclosporin in this condition
`clearly warrants further evaluation, and the results
`of a multicenter clinical trial evaluating the use of
`2% cyclosporin ointment in patients with kerato—
`conjunctivitis sicca, currently underway in the
`United States, are keenly awaited.
`
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`
`5
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