Abstract
Human eosinophils release numerous cytokines that are pre-synthesized and stored within their cytoplasmic-specific (secretory) granules. For example, high levels of interferon-gamma (IFN-γ) are constitutively expressed in these cells, but the intracellular compartments involved in the transport and release of this cytokine remain to be established. In this work, we used a single-cell approach to investigate the subcellular localization of IFN-γ in human eosinophils stimulated or not with tumor necrosis factor alpha (TNF-α) or CC-chemokine ligand 11 CCL11 (eotaxin-1), inflammatory mediators that induce eosinophil activation and secretion. A pre-embedding immunonanogold transmission electron microscopy (TEM) technique that combines optimal epitope preservation and access to membrane microdomains was applied to detect precise localization of IFN-γ in combination with computational quantitative analyses. In parallel, degranulation processes and formation of eosinophil sombrero vesicles (EoSVs), large transport carriers involved in the transport of granule-derived cytokines, were investigated. Quantitative TEM revealed that both CCL11 and TNF-α-activated eosinophils significantly increased the total number of EoSVs compared to the unstimulated group, indicating that this vesicular system is actively formed in response to cell activation. Ultrastructural immunolabeling identified a robust pool of IFN-γ on secretory granules in both unstimulated and stimulated cells. Moreover, EoSVs carrying IFN-γ were seen around or/and in contact with secretory granules and also distributed in the cytoplasm. Labeling was clearly associated with EoSV membranes. The total number of IFN-γ-positive EoSVs was significantly higher in stimulated compared to unstimulated cells, and these labeled vesicles had a differential distribution in the cytoplasm of activated cells, being significantly higher in the cell periphery compared with the inner cell, thus revealing intracellular IFN-γ mobilization for release. IFN-γ extracellular labeling was found at the cell surface, including on extracellular vesicles. Our results provide direct evidence that human eosinophils compartmentalize IFN-γ within secretory granules and identify, for the first time, a vesicular trafficking of IFN-γ associated with large transport carriers. This is important to understand how IFN-γ is trafficked and secreted during inflammatory responses.
Highlights
Eosinophils are terminally differentiated cells of the innate immune system with a broad distribution in tissues and varied functions related to both immune homeostasis and immunity [reviewed in Ref. [1,2,3]]
Quantitative transmission electron microscopy (TEM) revealed that both CCL11 and TNF-αactivated eosinophils significantly amplified the numbers of cytoplasmic eosinophil sombrero vesicles (EoSVs) compared to the unstimulated group (Figure 2B), confirming that this vesicular system is actively formed in response to cell activation with inflammatory mediators [12, 16]
Precise immunolocalization of cytokines in cells from the immune system, such as eosinophils, is of critical importance to understand the capabilities of these cells during immune responses
Summary
Eosinophils are terminally differentiated cells of the innate immune system with a broad distribution in tissues and varied functions related to both immune homeostasis and immunity [reviewed in Ref. [1,2,3]]. Eosinophils are sources of cytokines that are mostly stored as preformed pools within secretory (specific) granules, a robust population of large and morphologically distinctive granules, existent in the eosinophil cytoplasm [reviewed in Ref. By mobilizing intracellular stores of preformed cytokines, human eosinophils have the capability of immediate release of these immune mediators in response to cell activation without the necessity for de novo synthesis [reviewed in Ref. The capacity and significance of these innate immune granulocytes to secrete specific cytokines have been recognized for mediating diverse immune-related responses. We showed that high levels of this Th1-associated cytokine are constitutively expressed in human circulating eosinophils and that IFN-γ signals, detected after subcellular fractionation, colocalize in granule-enriched fractions as well as in lighter cytoplasmic fractions [9]. The intracellular compartments involved in the transport and release of this cytokine remain to be established
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