Eosinophils altered phenotypically and primed by culture with granulocyte/macrophage colony-stimulating factor and 3T3 fibroblasts generate leukotriene C4 in response to FMLP

Abstract

Normodense eosinophils failed to generate leukotriene C4 (LTC4) in response to incremental concentrations of FMLP but did produce LTC4 when stimulated with calcium ionophore A23187. Normodense eosinophils, maintained in culture with 10(-11) M granulocyte/macrophage colony-stimulating factor (GM-CSF) in the presence of 3T3 fibroblasts, became responsive to transmembrane stimulation with FMLP by day 4 with a maximal effect by day 7. After 7 d of culture, hypodense eosinophils stimulated with 2 x 10(-7) M FMLP generated 26 ng LTC4/10(6) cells, and LTC4 biosynthesis was blocked by N-tertbutoxy-carbonyl-L-methionyl-L-leucyl-L-phenylalanine (N-t-BOC-MLP). Neither calcium ionophore stimulation of LTC4 from endogenous arachidonic acid nor substrate-initiated production of LTC4 from incorporated LTA4 changed when eosinophils were cocultured with GM-CSF and 3T3 fibroblasts. Furthermore, when incubated with 10(-6) M FMLP, normodense eosinophils generated no net superoxide measured by the reduction of cytochrome c, whereas replicate eosinophils cultured for 7 d with 10(-11) M GM-CSF and 3T3 fibroblasts reduced a net of 17 nmol of cytochrome c/10(6) cells. These studies suggest that primed and phenotypically altered eosinophils present at an extravascular site may exert pathobiologic effects by responding to soluble ligands in the tissues.