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1 From the Department of Ophthalmology, Hadassah University Hospital; and the 2 Department of Pharmacology, School of Pharmacy, Hadassah Medical School, Hebrew University, Jerusalem, Israel; the 3 Institute of Experimental Medicine, Consiglio Nationale delle Ricerche, Rome, Italy; and 4 Clinical Immunology and Allergology, Second University of Naples, Italy.
PURPOSE. To examine the effect of human conjunctival fibroblasts on the survival and functional activity of human peripheral blood eosinophils.
METHODS. Eosinophils were purified by negative immunoselection [magnetic activated cell sorter (MACS), purity > 97%] from volunteers with mild atopia. Fibroblasts were cultured from conjunctival specimens of healthy donors. Eosinophils were cultured on confluent monolayers of conjunctival fibroblasts or in culture medium alone. Eosinophil survival was evaluated by the trypan blue exclusion test. Eosinophil adherence was assessed by counting the attached cells after washing the cultures. Eosinophil viability and adherence in coculture were also assessed in the presence of anti-granulocyte-macrophage colony-stimulating factor (GM-CSF), anti-interleukin (IL)-3, and anti-IL-5 neutralizing antibodies. Cocultured eosinophils were activated by lipopolysaccharide (LPS) after 4 days in culture, and eosinophil peroxidase (EPO) release was determined as a marker of their activation.
RESULTS. Eosinophils cocultured with conjunctival fibroblasts had a significantly increased viability of 35.9% (P = 0.004) and 12.8% (P = 0.003) on days 4 and 8, respectively. Fibroblast-conditioned medium did not enhance the survival of eosinophils. The increase in eosinophil survival in coculture was partially inhibited by anti-GM-CSF (P = 0.019), anti-IL-3 (P = 0.033), or anti-IL-5 (P = 0.011), whereas eosinophil adherence was reduced by anti-GM-CSF alone (P = 0.034). LPS activation of eosinophils cultured for 4 days with conjunctival fibroblasts induced higher EPO release than in freshly isolated eosinophils (P = 0.01).
CONCLUSIONS. Human conjunctival fibroblasts induced prolonged survival and increased secretory function of human peripheral blood eosinophils. Increased survival is partially mediated by IL-3, IL-5, and GM-CSF. The coculture of conjunctival fibroblasts with eosinophils can serve as an in vitro system for the study of eosinophil behavior in the ocular surface and of cellular interactions in allergic eye diseases.
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