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Abraham Solomon, Russel Shmilowich, David Shasha, Joseph Frucht–Pery, Jacob Pe’er, Sergio Bonini, Francesca Levi–Schaffer; Conjunctival Fibroblasts Enhance the Survival and Functional Activity of Peripheral Blood Eosinophils In Vitro. Invest. Ophthalmol. Vis. Sci. 2000;41(5):1038-1044.
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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
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