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.
BACKGROUND: In general, the non-pyroglyphid mites Lepidoglyphus destructor and Blomia tropicalis show a different geographical distribution. Allergic sensitization to both species have been demonstrated in several investigations. However, whether this reflects cross-reactivity or dual sensitization is so far not known. OBJECTIVE: The aim of the study was to investigate the allergenicity and allergenic crossreactivity of L. destructor and B. tropicalis using sera from Sweden and Brazil. METHODS: Allergens in extracts of L. destructor and B. tropicalis were identified with SDS-PAGE and immunoblotting and the crossreactivity was studied by an immunoblot inhibition method. In addition to mite extracts, a recombinant major allergen of L. destructor, Lep d 2, was used. RESULTS: The extract prepared from L. destructor contained 21 IgE-binding components when using the Swedish or the Brazilian sera. A 15 kDa allergen was recognized by 85% of the Swedish sera and 78% of the Brazilian. The B. tropicalis extract exposed 23 IgE-binding components when the Brazilian sera were used and 19 when the Swedish sera were used. A total of 83% of the Brazilian sera and 80% of the Swedish sera identified a 14.5 kDa allergen. The IgE response of the Swedish serum pool to 10 B. tropicalis allergens was inhibited by L. destructor extract. Likewise, the response of the Brazilian serum pool to four different L. destructor allergens was inhibited by B. tropicalis extract. The recombinant Lep d 2 allergen inhibited 33% of the IgE binding of the Swedish serum pool to the 14.5 kDa allergen in the B. tropicalis extract. CONCLUSION: Crossreactivity with several proteins from L. destructor and B. tropicalis was demonstrated. The results suggest that a B. tropicalis 14.5 kDa allergen is antigenically crossreactive with recombinant L. destructor allergen Lep d 2.