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

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.

Hypoxia causes IL‐8 secretion, Charcot Leyden crystal formation, and suppression of corticosteroid‐induced apoptosis in human eosinophils

“…Low oxygen tensions do not promote indiscriminate degranulation; indeed, EDN release was actually inhibited by hypoxia. Selective mobilization of pre-formed cytokines from eosinophil granule stores by 'piecemeal degranulation', a process that involves the trafficking of small vesicles directed by SNAP/SNARE interactions, has been observed previously, although not in response to hypoxia; for example, eotaxin can induce the rapid and selective secretion of IL-4 from eosinophils [53], while IFN-c promotes IL-3 and RANTES release [54,55].…”

Eosinophils and Th2 immunity: contemporary insights

“…Human eosinophils respond to multiple chemokine, cytokine and microbial stimuli with the very rapid and stimulus-specific secretion of a plethora of cytokines, including IL-4, IL-13 and IL-6. [31][32][33][34][35][36][37] Non-eosinophil innate cell sources of IL-4 include NK T cells, gd T cells, basophils and mast cells. Of these, NK T, gd T cells and mast cells reside primarily within tissues, whereas eosinophils and basophils are recruited from the blood into affected tissues.…”

Interleukin‐4 and RANTES expression in maturing eosinophils derived from human cord blood CD34+ progenitors

“…Slides were examined using a r 100 objective under a Leica confocal laser-scanning microscope (Heidelberg, Germany), as previously described. 6,17 Transmission electron microscopy This procedure was a modi®cation of a previously described technique. 18 Brie¯y, pelleted colonies of cells from day 21 of semisolid culture were ®xed in freshly prepared formaldehyde (2% in phosphate buffer, 0 .…”