Flow cytometric analysis of inflammatory markers in KCS: 6-month treatment with topical cyclosporin A.
OBJECTIVE
Immune-based inflammation has been observed as a common mechanism of keratoconjunctivitis sicca (KCS). In KCS-affected eyes, upregulated expression of HLA DR and various immune- or apoptosis-related markers by conjunctival epithelial cells has been demonstrated in an earlier study, by a technique of flow cytometry in impression cytology (IC) specimens. The purpose of this study was to monitor the effects of topical cyclosporin A on the expression of these markers throughout a 6-month period of treatment.


METHODS
Patients with moderate to severe KCS included in a large European multicenter clinical trial (Cyclosporin Dry Eye Study, Allergan, Irvine, CA) underwent collection of IC specimens at baseline, month 3, and month 6. For 6 months, they randomly received 0.05% or 0.1% cyclosporin A or vehicle. Specimens were processed and analyzed in a masked manner by flow cytometry, using monoclonal antibodies directed to HLA DR, CD40, CD40 ligand, Fas, and the apoptotic marker APO2.7. Percentages of positive cells were calculated and levels of expression quantified after conversion into standardized units of fluorescence.


RESULTS
One hundred fifty-eight patients had at least two IC specimens available for flow cytometry analysis. HLA DR expression, both in percentage of positive cells and level of expression, was highly significantly reduced after 0.05% and 0.1% cyclosporin A treatment at months 3 and 6 compared with baseline values, whereas vehicle did not induce any change in HLA DR expression over time. The 0.05% and 0.1% cyclosporin emulsions were significantly more effective than the vehicle in reducing HLA DR at months 3 and 6 (0.05%), and at month 6 (0.1%). CD40 expression was significantly reduced at month 3 and partially at month 6, compared with baseline, with no reduction in patients who received the vehicle. CD40 ligand expression also decreased at months 3 and 6 in patients taking both concentrations of cyclosporin A. APO2.7 expression was significantly increased in all three groups, whereas percentage of Fas-positive cells decreased only in patients treated with 0.05% cyclosporin A at months 3 and 6.


CONCLUSIONS
Flow cytometry provided an objective technique to monitor the effects of topical cyclosporin A on immune- and apoptosis-related markers in the conjunctival epithelium of patients with KCS enrolled in a large multicenter trial. Topical cyclosporin A strikingly reduced HLA DR and to a lesser extent, other inflammatory and apoptotic markers, whereas the vehicle, used as a control tear substitute, had almost no effect. This study confirms that cyclosporin A may be efficient in reducing conjunctival inflammation in moderate to severe KCS and is consistent with clinical results in this indication.
Brignole
F
F
Laboratory of Immunohematology and the. Department of Ophthalmology, Ambroise Paré Hospital, APHP, University of Paris-V René Descartes, Boulogne, France.
Pisella
P J
PJ
De Saint Jean
M
M
Goldschild
M
M
Goguel
A
A
Baudouin
C
C
D000287:Administration, Topical; D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015415:Biomarkers / Q000032:analysis; D019013:CD40 Antigens / Q000378:metabolism*; D023201:CD40 Ligand / Q000378:metabolism*; D003315:Cornea / Q000187:drug effects / Q000378:metabolism; D016572:Cyclosporine / Q000008:administration & dosage / Q000627:therapeutic use*; D004311:Double-Blind Method; D005260:Female; D005434:Flow Cytometry; D006684:HLA-DR Antigens / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D007166:Immunosuppressive Agents / Q000008:administration & dosage / Q000627:therapeutic use*; D007638:Keratoconjunctivitis Sicca / Q000188:drug therapy* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009883:Ophthalmic Solutions / Q000008:administration & dosage / Q000627:therapeutic use; D029867:Xenopus Proteins; D019014:fas Receptor / Q000378:metabolism*
D015415:Biomarkers; D019013:CD40 Antigens; D006684:HLA-DR Antigens; D018398:Homeodomain Proteins; C078105:Hox 2.7 protein, Xenopus; D007166:Immunosuppressive Agents; D009883:Ophthalmic Solutions; D029867:Xenopus Proteins; D019014:fas Receptor; D023201:CD40 Ligand; D016572:Cyclosporine