Nakajima, Hirokazu

Abstract To study the capacity and regulation of cytokine production by normal peripheral blood eosinophils, we isolated eosinophils from healthy individuals and stimulated them with immobilized Ig or TNF-α, with or without exogenous IL-5. By reverse transcription-PCR, uncultured, freshly isolated eosinophils constitutively expressed mRNA for IL-4, IL-10, and TGF-β1. Eosinophils stimulated by immobilized secretory IgA, immobilized IgA, immobilized IgG, or TNF-α for 3 h expressed mRNA encoding IL-3, IL-4, IL-8, IL-10, granulocyte-macrophage CSF, TNF-α, TGF-β, and RANTES. The mRNA for IL-2, IL-5, or IFN-γ was not detected. IL-4 and IL-10 protein, but not IL-8, were measurable in lysates of fresh eosinophils or eosinophils cultured with medium alone for 24 h. Eosinophils incubated with immobilized Ig or TNF-α released IL-8 protein into the supernatants. In contrast, IL-4 and IL-10 proteins were not detectable. Soluble secretory IgA immune complexes also induced degranulation, as measured by eosinophil-derived neurotoxin, and IL-8 release, but not IL-4 or IL-10 release, from eosinophils. Release of IL-8 protein and storage of IL-4 and IL-10 proteins were enhanced by exogenous IL-5 and inhibited by a transcription inhibitor, actinomycin D. Degranulation of stored granule proteins was not affected by actinomycin D. Therefore, normal peripheral blood eosinophils can transcribe and synthesize several cytokines, including IL-4, IL-8, and IL-10; some are stored, and some are released. These cytokines may play important roles in modulating immune responses in diseases associated with eosinophils.

Abstract Use of the murine monoclonal antibodies EG1 and EG2 has been based on the assumption that EG2 recognizes activated eosinophils. We examined the reactivity of EG1 and EG2 with eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), and stimulated and nonstimulated eosinophils from normal donors. By radioimmunoassay, EG1 recognized only ECP, whereas EG2 recognized both ECP and EDN. By Western blot, EG1 reacted with ECP, EG2 reacted with both ECP and EDN, but EG2 could not distinguish between lysates of stimulated and nonstimulated eosinophils. By immunofluorescence, EG1 and EG2 at 20 μg/mL stained 95–100% of nonstimulated eosinophils, regardless of fixative; EG1 and EG2 at 0.1 μg/mL stained 61–90% of acetone- and paraformaldehyde-fixed and only 5–21% of methanol-fixed nonstimulated eosinophils. Thus, the reactivity of EG1 and EG2 with eosinophils depends on the method of fixation and antibody concentration; and EG2, in contrast to previous reports, cannot reliably discriminate between resting and activated eosinophils. J. Leukoc. Biol. 66: 447–454; 1999.