Identification of local Th2 and Th0 lymphocytes in vernal conjunctivitis by cytokine flow cytometry

PURPOSE. Th2 lymphocytes may play a key role in the development of allergic diseases such as vernal keratoconjunctivitis (VKC). Cytokine flow cytometr y of tear samples was used to identify the phenotypical and functional prop erties of lymphocytes at the actual site of the allergic reaction. METHODS. Tear and blood samples were obtained from patients affected by act ive VKC (n = 12) and from normal control subjects (n = 10). Tears were obta ined after gentle scraping of the tarsal and bulbar conjunctiva. Tear and b lood samples were placed in a solution of brefeldin-A, phorbol myristate ac etate (PMA), ionomycin, and RPMI for 4 hours and then processed for flow cy tometry. Lymphocytes were marked with the monoclonal antibodies, anti-IFN-g amma and anti-interleukin (IL)-4. Levels of IL-4, IL-2, IFN-gamma, IL-2R, t otal IgE, eosinophil cationic protein (ECP), eosinophil protein X/neurotoxi n (EPX), and myeloperoxidase (MPO) were also evaluated in serum. RESULTS. Expression of IL-4 was observed in 9.2% +/- 9.5% of lymphocytes in tears of patients with VKC. Of the 12 patients with VKC, 8 (67%) had tear lymphocytes positive for IL-4 (Th2). Two patients (17%) had a double popula tion of lymphocytes: One was positive for Th2, and the other was positive f or both IL-4 and IFN-gamma (Th0). One patient (8%) was positive for IFN-gam ma (Thl) only, and one patient was negative for both ILs. No differences in the percentage of Th2 lymphocytes were found between tarsal and limbal pat ients. The percentage of Th2 lymphocytes was significantly correlated with the severity of the disease. No positive lymphocytes were found in tears of control subjects. Eosinophils, serum IgE, ECP, and EPX were all significan tly higher in VKC than in control subjects. CONCLUSIONS. In ocular allergic diseases, local lymphocytes expressed the T h2 phenotype and, to a lesser degree, the Th0 phenotype. Although results o f systemic allergic markers can be inconclusive in patients with VKC, flow cytometry demonstrated a local lymphocyte phenotype that call account for t he clinical and histologic abnormalities of VKC.