Ag-specific recognitiong activationg and effector function of T cells in the conjunctiva with experimental immune-mediated blepharoconjunctivitis

PURPOSE. To investigate antigen (Ag) specificity, activation, and effector function of the Ag-specific T cells involved in the development of experimental immune-mediated blepharoconjunctivitis (EC), an experimental conjunctivitis. METHODS. EC was induced in Brown Norway rats by injection of ovalbumin (OVA)-specific T cells followed by OVA challenge with eye drops. Eyes, including the conjunctivas, were harvested at different time points after challenge. The dependence of EC onset. on the challenging Ag was assessed by challenge with an irrelevant Ag or stimulatory OVA peptides. To show the infiltration of transferred T cells into the conjunctiva, T cells were labeled with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) before transfer. The activation of T cells in the conjunctiva was assessed by measuring phosphorylation of Lck-associated molecules by Western blot analysis. Conjunctivas were also examined by immunohistochemistry and used for reverse transcription-polymerase chain reaction to determine the phenotype of the infiltrating cells and cytokine, chemokine, and chemokine receptor expression. To investigate infiltration of non Ag-specific T cells into the conjunctiva, ragweed (RW)-primed lymphocytes were transferred into OVA-specific T-cell receptor transgenic (DO11.10) mice; The mice were then challenged with RW and the conjunctivas were harvested for immunohistochemistry to detect T cells derived from DO11.10 mice. RESULTS. EC was induced only when challenged with OVA protein or stimulatory OVA peptides, and CFSE-labeled transferred cells were found in the conjunctiva. Phosphorylation of Lck and an 85-kDa Lck-associated molecule were observed in the conjunctiva 6 hours after challenge. Many cytokines and chemokines began to be expressed at 6 hours, and individual expression patterns over time correlated well with the infiltration patterns of different inflammatory cells. In DO11.10 mice that received RW-primed lymphocytes, T cells derived from the recipient mice infiltrated the conjunctiva after RW challenge. CONCLUSIONS. Ag-specific T cells initiate EC by first infiltrating the conjunctiva, where they become activated by the specific Ag in the conjunctiva.