Conjunctival fibroblasts enhance the survival and functional activity of peripheral blood eosinophils in vitro

PURPOSE. To examine the effect of human conjunctival fibroblasts on the sur vival and functional activity of human peripheral blood eosinophils. METHODS. Eosinophils were purified by negative immunoselection [magnetic ac tivated cell sorter (MACS), purity > 97%] from volunteers with mild atopia. Fibroblasts were cultured from conjunctival specimens of healthy donors. E osinophils were cultured on confluent monolayers of conjunctival fibroblast s or in culture medium alone. Eosinophil survival was evaluated by the tryp an blue exclusion test. Eosinophil adherence was assessed by counting the a ttached cells after washing the cultures. Eosinophil viability and adherenc e in coculture were also assessed in the presence of anti-granulocyte-macro phage colony-stimulating factor (GM-CSF), anti-interleukin (IL)-3, and anti -IL-5 neutralizing antibodies. Cocultured eosinophils were activated by lip opolysaccharide (LPS) after 4 days in culture, and eosinophil peroxidase (E PO) release was determined as a marker of their activation. RESULTS. Eosinophils cocultured with conjunctival fibroblasts had a signifi cantly increased viability) of 35.9% (P = 0.004) and 12.8% (P = 0.003) on d ays 4 and 8, respectively. Fibroblast-conditioned medium did not enhance th e 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 -i da ys with conjunctival fibroblasts induced higher EPO release than in freshly isolated eosinophils (P = 0.01). CONCLUSIONS. Human conjunctival fibroblasts induced prolonged survival and increased secretor) function of human peripheral blood eosinophils. Increas ed survival is partially mediated by IL-3, IL-5, and GM-CSF. The coculture of conjunctival fibroblasts with eosinophils can serve Is an in vitro syste m for the study of eosinophil behavior in the ocular surface and of cellula r interactions in allergic eye diseases.