Rapid Mobilization of Intracellularly Stored RANTES in Response to Interferon-γ in Human Eosinophils

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Abstract
The CC chemokine RANTES is synthesized, stored, and upregulated in response to interferon-γ (IFN-γ) in human peripheral blood eosinophils. In this report, we propose that RANTES is rapidly mobilized from eosinophil crystalloid granules during agonist-induced degranulation. We stimulated purified eosinophils (>99%) from atopic asthmatics with 500 U/mL IFN-γ to analyze the kinetics of mobilization and release of RANTES (0 to 240 minutes). We used subcellular fractionation, immunogold analysis, two-color confocal laser scanning microscopy (CLSM), and enzyme-linked immunosorbent assay (ELISA) to trace the movement of eosinophil-derived RANTES from intracellular stores to release. RANTES was rapidly mobilized (10 minutes) and released after 120 minutes of stimulation (80 ± 15 pg/mL per 2 × 106 cells). RANTES appeared to be stored in at least two intracellular compartments: the matrix of crystalloid granules, detected by major basic protein and eosinophil peroxidase activities, and a specialized small secretory vesicle present in light membrane fractions. The extragranular RANTES was mobilized more rapidly than that of crystalloid granules during IFN-γ stimulation. This effect was not observed in eosinophils treated with IFN-, interleukin-3 (IL-3), IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), or genistein followed by IFN-γ. Our findings suggest that RANTES may be mobilized and released by piecemeal degranulation upon stimulation, involving transport through a putative pool of small secretory vesicles.