|Figure 1. Eotaxin elicits vesicular transport-mediated release of IL-4 from eosinophils.|
Eosinophils are the predominant effector cells in allergic reactions. Release of eosinophil-specific granule contents contributes to inflammation associated with allergic immune responses.1 In addition to cationic granule proteins, eosinophil granules may contain preformed stores of cytokines and chemokines.2-4 For example, the prototypical Th2 cytokine IL-4, is synthesized and stored within eosinophil-specific granules.5-9 Eosinophil release of IL-4 may stimulate additional, or augment existing, immune responses in areas of eosinophil localization. The mechanism (s) involved in release of IL-4 from human eosinophil granules has previously not been identified. Bandeira-Melo et al.10 now demonstrate that chemokines acting via CCR3-mediated signaling pathways can rapidly mobilize these preformed stores of IL-4 from eosinophils via vesicular transport.
Eosinophil-specific granule contents can be selectively incorporated into small vesicles that traffic to the cell surface and are then released by "piecemeal" degranulation.11,12 The EliCell assay is a sensitive, immunofluorescent microscopic assay specifically designed to study this piecemeal degranulation process within eosinophils.13 It is a gel-phase dual antibody capture and detection assay based on microscopic observations of individual viable cells. Bandeira-Melo et al.10 use the EliCell assay to enumerate the proportion of cells releasing IL-4 and electronically quantitate the average relative amounts of IL-4 released extracellularly following exposure of human eosinophils to various eosinophil agonists.
Chemokines acting via CCR3-initiated signaling pathways can rapidly mobilize preformed IL-4 from within eosinophils.10 The CC chemokines eotaxin and RANTES do not induce IL-4 release from eosinophils at levels detectable by ELISA, but can elicit IL-4 release at levels detectable by the EliCell assay. The punctate pattern of IL-4 release at discrete cell surface sites is compatible with a vesicular transport-mediated process of release.
IL-5, a cytokine that controls the production, activation and localization of eosinophils, enhances eotaxin-stimulated mobilization of IL-4.14 Pretreatment of eosinophils with a neutralizing anti-CCR3 monoclonal antibody inhibits IL-4 release from IL-5/eotaxin-stimulated eosinophils. The IL-5/eotaxin-stimulated release of eosinophil IL-4 does not require new synthesis of IL-4 or other proteins that may contribute to the secretory process.10 Treatment of eosinophils with either actinomycin D (transcription inhibitor) or cycloheximide (protein synthesis inhibitor) does not affect the release of IL-4 induced by IL-5/eotaxin. By contrast, brefeldin A, an inhibitor of vesicle formation, substantially suppresses the amount of IL-4 released from IL-5/eotaxin-stimulated eosinophils.
Unlike CD4+ T cells, eosinophils contain preformed stores of IL-4 that are released via vesicular transport. Chemokines, by means of CCR3-mediated signaling, can rapidly induce this release. Although thelevels of IL-4 released may not be sufficient for immunoassay detection, the localized release of IL-4 may effectively influence immune responses in areas where eosinophils have concentrated.10