Galectin-10, the protein that forms Charcot-Leyden crystals, is not stored in granules but resides in the peripheral cytoplasm of human eosinophils

Abstract

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).

Adult‐onset eosinophilic airway diseases

“…In response to type 2 cytokines, terminally differentiated eosinophils are mobilized from a niche in the bone marrow into the blood circulation. In a normal homeostatic condition, the half-life of blood eosinophils is approximately 25 hours, and the majority of senescent (or apoptotic) eosinophils are cleared in the liver and spleen, where they are taken up by macrophages of the reticuloendothelial system. 9 Apoptotic eosinophil clearance by phagocytotic cells (efferocytosis) mediates the resolution of inflammation and is shown to be dependent on the receptor tyrosine kinase Mer.…”

“…20 EETosis is also associated with the crystallization of galectin-10 to form Charcot-Leyden crystals (CLCs), a classical hallmark of eosinophilic inflammation. [23][24][25] CLCs are recognized by the NLRP3 inflammasome, which may sustain inflammation that follows eosinophilic inflammatory processes. 26 Persson et al advocated a novel concept of "CLC crystallopathy" by showing that antibodies directed against key epitopes of the CLC crystallization interface dissolved CLCs and reversed crystal-driven inflammation, IgE synthesis and bronchial hyperreactivity in a humanized mouse model of asthma.…”

“…Interestingly, sputum autoantibodies against eosinophil peroxidase, identified often in severe asthmatics, can directly induce EETosis in vitro 20 . EETosis is also associated with the crystallization of galectin‐10 to form Charcot‐Leyden crystals (CLCs), a classical hallmark of eosinophilic inflammation 23‐25 . CLCs are recognized by the NLRP3 inflammasome, which may sustain inflammation that follows eosinophilic inflammatory processes 26 .…”

Kinetic studies of galectin-10 release from eosinophils exposed to proliferating T cells

“…At approximately the same time‐point, eosinophils with dissolved cytoskeleton and an even cytoplasmic distribution of galectin‐10 were also seen, which we believe to be an early stage of eosinophil cytolysis. A recent study demonstrated that galectin‐10 is stored in the cytoplasm itself and not in the cytoplasmic granules of eosinophils [28], which might explain our observation that the nuclear lobes of eosinophils appear to push accumulated cap‐like formations of galectin‐10 through the cytoplasm prior to the extracellular release of EETs. The timing of events, the fact that the EETs evoked by proliferating T cells were enriched for nuclear DNA and that eosinophils had started to lyse indicate that what we observed was the phenomenon of EETosis, a particular form of cell death by which leukocytes go into cytolysis while simultaneously releasing extracellular DNA traps [24].…”

The Charcot–Leyden crystal protein revisited—A lysopalmitoylphospholipase and more

“…It is now clearer that CLC‐P is not granule associated but is a major, highly abundant protein within the cytoplasm of human eosinophils 49 . By immunofluorescence microscopy and at higher resolution by immunoelectron microscopy, we have localized CLC‐P richly just under the plasma membrane (concentrated in a narrow [∼250 nm wide], circumferential cytoplasmic band) and within the plasma membrane 49 . What may be the function of this protein with this dominant subplasmalemmal location?…”