Rapid mobilization of intracellularly stored RANTES in response to interferon-gamma in human eosinophils.
The CC chemokine RANTES is synthesized, stored, and upregulated in response to interferon-gamma (IFN-gamma) 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-gamma 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 x 10(6) 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-gamma stimulation. This effect was not observed in eosinophils treated with IFN-alpha, interleukin-3 (IL-3), IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), or genistein followed by IFN-gamma. 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.
Lacy
P
P
Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada.
Mahmudi-Azer
S
S
Bablitz
B
B
Hagen
S C
SC
Velazquez
J R
JR
Man
S F
SF
Moqbel
R
R
D000970:Antineoplastic Agents / Q000494:pharmacology*; D001249:Asthma / Q000276:immunology; D002478:Cells, Cultured; D018946:Chemokine CCL5 / Q000378:metabolism*; D016207:Cytokines / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D004804:Eosinophils / Q000276:immunology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007150:Immunohistochemistry; D007371:Interferon-gamma / Q000494:pharmacology*
D000970:Antineoplastic Agents; D018946:Chemokine CCL5; D016207:Cytokines; D007371:Interferon-gamma