Flow cytometric analysis of inflammatory markers in KCS: 6-month treatmentwith topical cyclosporin A

PURPOSE. Immune-based inflammation has been observed as a common mechanism of keratoconjunctivitis sicca (KCS). In KCS-affected eves, upregulated expr ession of HLA DR and various immune- or apoptosis-related markers by conjun ctival epithelial cells has been demonstrated in an earlier study, by a tec hnique of flow cytometry in impression cytology (IC) specimens. The purpose of this study was to monitor the effects of topical cyclosporin A on the e xpression 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. F or 6 months, they randomly received 0.05% or 0.1% cyclosporin A of vehicle. Specimens were processed and analyzed in a masked manner by flow cytometry , using monoclonal antibodies directed to HLA DR, CD40, CD40 ligand, Fas, a nd the apoptotic marker APO2.7. Percentages of positive cells were calculat ed and levels of expression quantified after conversion into standardized u nits of fluorescence. RESULTS. One hundred fifty-eight patients had at least two IC specimens ava ilable for flow cytometry analysis. HLA DR expression, both in percentage o f 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 wit h baseline values, whereas vehicle did not induce any change in HLA DR expr ession over time. The 0.05% and 0.1% cyclosporin emulsions were significant ly more effective than the vehicle in reducing HLA DR at months 3 and 6 (0. 05%), and at month 6 (01%). CD40 expression was significantly reduced at mo nth 3 and partially at month 6, compared with baseline, with no reduction i n 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 i n the conjunctival epithelium of patients with KCS enrolled in a large mult icenter trial. Topical cyclosporin A strikingly reduced HLA DR and to a les ser extent, ether inflammatory and apoptotic markers, whereas the vehicle, used as a control tear substitute, had almost no effect. This study confirm s that cyclosporin A may be efficient in reducing conjunctival inflammation in moderate to severe RCS and is consistent with clinical results in this indication.