Extracellular Sombrero Vesicles are Hallmarks of Eosinophilic Cytolytic Degranulation in Tissue Sites of Human Diseases.

Eosinophil sombrero vesicles (EoSVs) are large tubular carriers resident in the cytoplasm of human eosinophils, identifiable by transmission electron microscopy (TEM), and important for immune mediator transport. Increased EoSV formation occurs in activated eosinophils in vitro and in vivo. In tissue sites of eosinophilic cytolytic inflammation, extracellular EoSVs are noted, but their frequency and significance in eosinophil-associated diseases (EADs) remain unclear. Here, we performed comprehensive quantitative TEM analyses and electron tomography to investigate the numbers, density, integrity, and three-dimensional (3D) structure of EoSVs in different biopsy tissues from five prototypic EADs (eosinophilic chronic rhinosinusitis/nasal sinuses, ulcerative colitis/intestines, hypereosinophilic syndrome/skin, dermatitis/skin, and schistosomiasis/rectum). The morphology of extracellular EoSVs was also compared with that of cytoplasmic EoSVs, isolated by subcellular fractionation from peripheral blood eosinophils. We demonstrated that: i) eosinophil cytolysis, releasing intact EoSVs and membrane-bound granules, is a consistent event in all EADs; ii) EoSVs persist intact even after complete disintegration of all cell organelles, except granules (late cytolysis); iii) the EoSV population, composed of elongated, curved, and typical sombreros, and the EoSV 3D architecture, diameter, and density remain unchanged in the extracellular matrix; iv) free EoSVs closely associate with extracellular granules; and v) free EoSVs also associate with externalized chromatin during eosinophil ETosis. Remarkably, EoSVs appeared on the surface of other cells like plasma cells. Thus, eosinophil cytolysis/ETosis can secrete intact EoSVs, alongside granules, in inflamed tissues of EADs, potentially serving as propagators of eosinophil immune responses post-cell death.