The medium of the fibroblast monolayers was gently aspirated, and
eosinophils (1 × 105 cells in 200 μl
enriched medium (EM) consisting of RPMI-1640 [Biological Industries]
supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine, 1 mM HEPES, and 5% FCS) were added to each
well of confluent monolayers of conjunctival fibroblasts (96-well
plates) and incubated at 37°C in 5% CO2 for up
to 16 days. For extended periods of culturing, the culture medium was
changed every 4 days by gentle aspiration and addition of fresh medium.
Control samples consisted of eosinophils cultured in wells in the
absence of fibroblasts. For all the experiments, the cocultures were
repeated four times, each consisting of eosinophils and conjunctival
fibroblasts taken from four different individuals.
To assess whether the increased survival of eosinophils cultured with
fibroblasts was attributable to IL-3, IL-5, or GM-CSF, neutralizing
mouse anti-human monoclonal antibodies to these cytokines (R&D Systems,
Minneapolis, MN) were added to the fibroblast cultures when the
eosinophils were added (day 0 of the experiments). Antibodies were
added at a final concentration of 4 μg/ml each, found to be an
optimal concentration from pilot experiments in which two antibody
concentrations of 4 μg/ml and 40 μg/ml provided similar inhibition
values. In addition, to validate the neutralizing capacity of the
anti-cytokine antibodies, eosinophils were incubated with an optimal
concentration of recombinant human (rh)GM-CSF, rhIL-3, and rhIL-5 in
the presence of optimal neutralizing concentrations of, respectively,
anti-GM-CSF, anti-IL-3, and anti-IL-5. A nonrelevant monoclonal
antibody, human anti-CD3 antibody (Miltenyi) at a final concentration
of 4 μg/ml, was added as a control in these experiments.
In some experiments, freshly isolated eosinophils were cultured with
conjunctival fibroblast–conditioned medium. The conditioned medium was
obtained by culturing confluent monolayers of conjunctival fibroblasts
in DMEM-10% FCS for a period of 48 hours. To the eosinophils in
96-well plates (1 × 105/200 μl
EM) 200 μl conditioned medium was added, and the cells were
cultured for 96 hours.