Single-Cell Analyses of Human Eosinophils at High Resolution to Understand Compartmentalization and Vesicular Trafficking of Interferon-Gamma.
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-?) 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-? in human eosinophils stimulated or not with tumor necrosis factor alpha (TNF-?) 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-? 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-?-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-? on secretory granules in both unstimulated and stimulated cells. Moreover, EoSVs carrying IFN-? 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-?-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-? mobilization for release. IFN-? extracellular labeling was found at the cell surface, including on extracellular vesicles. Our results provide direct evidence that human eosinophils compartmentalize IFN-? within secretory granules and identify, for the first time, a vesicular trafficking of IFN-? associated with large transport carriers. This is important to understand how IFN-? is trafficked and secreted during inflammatory responses.
Project description:SNARE members mediate membrane fusion during intracellular trafficking underlying innate and adaptive immune responses by different cells. However, little is known about the expression and function of these proteins in human eosinophils, cells involved in allergic, inflammatory and immunoregulatory responses. Here, we investigate the expression and distribution of the Qa-SNARE syntaxin17 (STX17) within human eosinophils isolated from the peripheral blood.Flow cytometry and a pre-embedding immunonanogold electron microscopy (EM) technique that combines optimal epitope preservation and secondary Fab-fragments of antibodies linked to 1.4 nm gold particles for optimal access to microdomains, were used to investigate STX17.STX17 was detected within unstimulated eosinophils. Immunogold EM revealed STX17 on secretory granules and on granule-derived vesiculotubular transport carriers (Eosinophil Sombrero Vesicles-EoSVs). Quantitative EM analyses showed that 77.7% of the granules were positive for STX17 with a mean±SEM of 3.9±0.2 gold particles/granule. Labeling was present on both granule outer membranes and matrices while EoSVs showed clear membrane-associated labeling. STX17 was also present in secretory granules in eosinophils stimulated with the cytokine tumor necrosis factor alpha (TNF-?) or the CC-chemokine ligand 11 CCL11 (eotaxin-1), stimuli that induce eosinophil degranulation. The number of secretory granules labeled for STX17 was significantly higher in CCL11 compared with the unstimulated group. The level of cell labeling did not change when unstimulated cells were compared with TNF-?-stimulated eosinophils.The present study clearly shows by immunanonogold EM that STX17 is localized in eosinophil secretory granules and transport vesicles and might be involved in the transport of granule-derived cargos.
Project description:Eosinophil activation leads to secretion of presynthesized, granule-stored mediators that determine the course of allergic, inflammatory, and immunoregulatory responses. CD63, a member of the transmembrane-4 glycoprotein superfamily (tetraspanins) and present on the limiting membranes of eosinophil-specific (secretory) granules, is considered a potential surface marker for eosinophil degranulation. However, the intracellular secretory trafficking of CD63 in eosinophils and other leukocytes is not understood. Here, we provide a comprehensive investigation of CD63 trafficking at high resolution within human eosinophils stimulated with inflammatory stimuli, CCL11 and tumor necrosis factor α, which induce distinctly differing secretory processes in eosinophils: piecemeal degranulation and compound exocytosis, respectively. By using different transmission electron microscopy approaches, including an immunonanogold technique, for enhanced detection of CD63 at subcellular compartments, we identified a major intracellular pool of CD63 that is directly linked to eosinophil degranulation events. Transmission electron microscopy quantitative analyses demonstrated that, in response to stimulation, CD63 is concentrated within granules undergoing secretion by piecemeal degranulation or compound exocytosis and that CD63 tracks with the movements of vesicles and granules in the cytoplasm. Although CD63 was observed at the cell surface after stimulation, immunonanogold electron microscopy revealed that a strong CD63 pool remains in the cytoplasm. It is remarkable that CCL11 and tumor necrosis factor α triggered increased formation of CD63(+) large vesiculotubular carriers (eosinophil sombrero vesicles), which fused with granules in the process of secretion, likely acting in the intracellular translocation of CD63. Altogether, we identified active, intracellular CD63 trafficking connected to eosinophil granule-derived secretory pathways. This is important for understanding the complex secretory activities of eosinophils underlying immune responses.
Project description:A key function of human eosinophils is to secrete cytokines, chemokines and cationic proteins, trafficking, and releasing these mediators for roles in inflammation and other immune responses. Eosinophil activation leads to secretion of pre-synthesized granule-stored mediators through different mechanisms, but the ability of eosinophils to secrete extracellular vesicles (EVs), very small vesicles with preserved membrane topology, is still poorly understood. In the present work, we sought to identify and characterize EVs released from human eosinophils during different conditions: after a culturing period or after isolation and stimulation with inflammatory stimuli, which are known to induce eosinophil activation and secretion: CCL11 (eotaxin-1) and tumor necrosis factor alpha (TNF-?). EV production was investigated by nanoscale flow cytometry, conventional transmission electron microscopy (TEM) and pre-embedding immunonanogold EM. The tetraspanins CD63 and CD9 were used as EV biomarkers for both flow cytometry and ultrastructural immunolabeling. Nanoscale flow cytometry showed that human eosinophils produce EVs in culture and that a population of EVs expressed detectable CD9, while CD63 was not consistently detected. When eosinophils were stimulated immediately after isolation and analyzed by TEM, EVs were clearly identified as microvesicles (MVs) outwardly budding off the plasma membrane. Both CCL11 and TNF-? induced significant increases of MVs compared to unstimulated cells. TNF-? induced amplified release of MVs more than CCL11. Eosinophil MV diameters varied from 20 to 1000 nm. Immunonanogold EM revealed clear immunolabeling for CD63 and CD9 on eosinophil MVs, although not all MVs were labeled. Altogether, we identified, for the first time, that human eosinophils secrete MVs and that this production increases in response to inflammatory stimuli. This is important to understand the complex secretory activities of eosinophils underlying immune responses. The contribution of the eosinophil-derived MVs to the regulation of immune responses awaits further investigation.
Project description:Although leukocytes of the innate immune system, including eosinophils, contain within their granules preformed stores of cytokines available for selective and rapid release, little is known about the mechanisms governing the mobilization and secretion of these cytokines. Here we show that a cytokine receptor, the IL-4 receptor alpha chain, mediates eotaxin-stimulated mobilization of preformed IL-4 from eosinophil granules into secretory vesicles. Eosinophils contain substantial intracellular quantities of several granule- and vesicle-associated cytokine receptors, including IL-4, IL-6, and IL-13 receptors as well as CCR3. Both IL-4 and IL-4 receptor alpha chain colocalized in eosinophil granules; and after eotaxin-stimulation, IL-4 receptor alpha chain, bearing bound IL-4, was mobilized into secretory vesicles. These findings indicate that intracellular cytokine receptors within secretory vesicles transport their cognate cytokines requisite for the secretion of cytokines preformed in innate immune leukocytes.
Project description:Rapid secretion of eosinophil-associated RNases (EARs), such as the human eosinophilic cationic protein (ECP), from intracellular granules is central to the role of eosinophils in allergic diseases and host immunity. Our knowledge regarding allergic inflammation has advanced based on mouse experimental models. However, unlike human eosinophils, capacities of mouse eosinophils to secrete granule proteins have been controversial. To study mechanisms of mouse eosinophil secretion and EAR release, we combined an RNase assay of mouse EARs with ultrastructural studies. In vitro, mouse eosinophils stimulated with the chemokine eotaxin-1 (CCL11) secreted enzymatically active EARs (EC(50) 5 nM) by piecemeal degranulation. In vivo, in a mouse model of allergic airway inflammation, increased airway eosinophil infiltration (24-fold) correlated with secretion of active RNases (3-fold). Moreover, we found that eosinophilic inflammation in mice can involve eosinophil cytolysis and release of cell-free granules. Cell-free mouse eosinophil granules expressed functional CCR3 receptors and secreted their granule proteins, including EAR and eosinophil peroxidase in response to CCL11. Collectively, these data demonstrate chemokine-dependent secretion of EARs from both intact mouse eosinophils and their cell-free granules, findings pertinent to understanding the pathogenesis of eosinophil-associated diseases, in which EARs are key factors.
Project description:BACKGROUND:Eosinophil-associated RNases (EARs) are stored preformed in eosinophil cytoplasmic secretory granules and have a key role in eosinophil effector functions in host defence and inflammatory disorders. However, the secretion mechanisms of EARs are poorly understood. OBJECTIVE:Our study aimed to understand the involvement of cytoskeleton machinery in EAR secretion. METHODS:Fresh human and mouse eosinophils were stimulated with CCL11, and the secretion of enzymatically active EARs was detected using an RNase activity assay. The involvement of cytoskeletal elements or microtubules was probed using specific inhibitors. RESULTS:We found that dynamic polymerization of microtubules and cytoskeletal elements, such as Rho and Rac, is required for chemokine-mediated EAR secretion from human and mouse eosinophils. However, inhibition of ROCK (Rho-associated protein kinase) increased EAR secretion in human and mouse eosinophils even in the absence of chemokine stimulation, suggesting ROCK negatively regulates EAR secretion. CONCLUSIONS:Collectively, these data suggest a cytoskeleton-dependent mechanism of EAR secretion from eosinophils, findings that are pertinent to host defence, allergy and other eosinophil-associated diseases.
Project description:Eosinophil secretory (specific) granules have a unique morphology and are both a morphologic hallmark of eosinophils and fundamental to eosinophil-mediated responses. Eosinophil mediators with multiple functional activities are presynthesized and stored within these granules, poised for very rapid, stimulus-induced secretion. The structural organization and changes of eosinophil specific granules are revealing in demonstrating the complex and diverse secretory activities of this cell. Here, we review our current knowledge on the architecture, composition, and function of eosinophil specific granules as highly elaborated organelles able to produce vesiculotubular carriers and to interplay with the intracellular vesicular trafficking. We reconsider prior identifications of eosinophil cytoplasmic granules, including "primary," "secondary," "microgranules," and "small granules"; and consonant with advances, we provide a contemporary recognition that human eosinophils contain a single population of specific granules and their developmental precursors and derived secretory vesicles.
Project description:A predominant protein of human eosinophils is galectin-10 (Gal-10), also known as Charcot-Leyden crystal protein (CLC-P) because of its remarkable ability to form Charcot-Leyden crystals (CLCs), which are frequently found in tissues from patients with eosinophilic disorders. CLC-P/Gal-10 is highly expressed in human eosinophils and considered a biomarker of eosinophil involvement in inflammation. However, the intracellular sites where large pools of CLC-P/Gal-10 constitutively reside are still unclear, and whether this protein is derived or not from eosinophil granules remains to be established. Here, we applied pre-embedding immunonanogold transmission electron microscopy combined with strategies for optimal antigen and cell preservation and quantitative imaging analysis to investigate, for the first time, the intracellular localization of CLC-P/Gal-10 at high resolution in resting and activated human eosinophils. We demonstrated that CLC-P/Gal-10 is mostly stored in the peripheral cytoplasm of human eosinophils, being accumulated within an area of ∼250 nm wide underneath the plasma membrane and not within specific (secretory) granules, a pattern also observed by immunofluorescence. High-resolution analysis of single cells revealed that CLC-P/Gal-10 interacts with the plasma membrane with immunoreactive microdomains of high CLC-P/Gal-10 density being found in ∼60% of the membrane area. Eosinophil stimulation with CCL11 or TNF-α, which are known inducers of eosinophil secretion, did not change the peripheral localization of CLC-P/Gal-10 as observed by both immunofluorescence and immuno-EM (electron microscopy). Thus, in contrast to other preformed eosinophil proteins, CLC-P/Gal-10 neither is stored within secretory granules nor exported through classical degranulation mechanisms (piecemeal degranulation and compound exocytosis).
Project description:Intracellular granules in several types of leukocytes contain preformed proteins whose secretions contribute to immune and inflammatory functions of leukocytes, including eosinophils, cells notably associated with asthma, allergic inflammation, and helminthic infections. Cytokines and chemokines typically elicit extracellular secretion of granule proteins by engaging receptors expressed externally on the plasma membranes of cells, including eosinophils. Eosinophil granules, in addition to being intracellular organelles, are found as intact membrane-bound structures extracellularly in tissue sites of eosinophil-associated diseases. Neither the secretory capacities of cell-free eosinophil granules nor the presence of functional cytokine and chemokine receptors on membranes of leukocyte granules have been recognized. Here, we show that granules of human eosinophils express membrane receptors for a cytokine, IFN-gamma, and G protein-coupled membrane receptors for a chemokine, eotaxin, and that these receptors function by activating signal-transducing pathways within granules to elicit secretion from within granules. Capacities of intracellular granule organelles to function autonomously outside of eosinophils as independent, ligand-responsive, secretion-competent structures constitute a novel postcytolytic mechanism for regulated secretion of eosinophil granule proteins that may contribute to eosinophil-mediated inflammation and immunomodulation.
Project description:Eosinophils and secretory leukocyte protease inhibitor (SLPI) are both associated with Th2 immune responses and allergic diseases, but whether the fact that they are both implicated in these conditions is pathophysiologically related remains unknown. Here we demonstrate that human eosinophils derived from normal individuals are one of the major sources of SLPI among circulating leukocytes. SLPI was found to be stored in the crystalline core of eosinophil granules, and its dislocation/rearrangement in the crystalline core likely resulted in changes in immunostaining for SLPI in these cells. High levels of SLPI were also detected in blood eosinophils from patients with allergy-associated diseases marked by eosinophilia. These include individuals with eosinophilic granulomatosis with polyangiitis (EGPA) and atopic dermatitis (AD), who were also found to have elevated SLPI levels in their plasma. In addition to the circulating eosinophils, diseased skin of AD patients also contained SLPI-positive eosinophils. Exogenous, recombinant SLPI increased numbers of migratory eosinophils and supported their chemotactic response to CCL11, one of the key chemokines that regulate eosinophil migratory cues. Together, these findings suggest a role for SLPI in controlling Th2 pathophysiologic processes <i>via</i> its impact on and/or from eosinophils.