Infiltração eosinofílica em sítios inflamatórios: estudo ultraestrutural de eosinófilos após a morte celular

Abstract

Eosinophils are multifunctional cells from the immune system with activities associated with the secretion of numerous products stored in their cytoplasmic granules. One of the main mechanisms by which the eosinophil releases the contents of its secretory granules is based on a necrotic cell death process, classically named cytolysis. This consists of the plasma membrane rupture with extracellular deposition of intact and functional secretory granules. Increasing evidence has shown that granules are not the only structures derived from eosinophil cytolysis, but specific vesicles, Charcot-Leyden crystals and DNA nets are also released. Nets are released through ETosis, a more recently described cytolytic cell death mechanism where there is deposition of chromatin together with eosinophil secretory granules, in the extracellular matrix (ECM). The cytolysis process is, therefore, very complex and still poorly understood, mainly in vivo. Many questions about the ultrastructure of both the conventional cytolysis process and mainly about ETosis remain open. The present study aimed to investigate, in situ, the ultrastructural characteristics of eosinophils undergoing cytolysis in human eosinophilic inflammatory diseases. For this, we applied transmission electron microscopy (TEM), the only technique that allows distinguishing in high resolution the eosinophil degranulation mechanisms and its subcellular characteristics, to biopsies of patients diagnosed with eosinophilic chronic rhinosinusitis (ECRS, nasal tissue) and ulcerative colitis (intestinal tissue). Our qualitative and quantitative analyzes showed that cytolysis is one of the most frequent secretory mechanisms of eosinophils in the studied diseases and that ETosis represents a significant portion of these eosinophils. More than 40% of cytolitic eosinophils presented ultrastructural characteristics associated with ETosis (nuclear rounding/delobulation, chromatin decondensation and expansion) and release of chromatin extracellular nets. For the first time, we also identified changes in the nuclear envelope (NE), represented by dilation of the perinuclear space and formation of vesicles derived from the NE. Our analyses showed that the process of ETosis leads not only to the deposition of intact granules, but also to the release of typical human eosinophils’ vesiculotubular carriers, the EoSVs (Eosinophil Sombrero Vesicles). Free intact EoSVs were associated with free granules, inflammatory cells and extracellular DNA nets. EoSVs remain intact after the cytolytic death of human eosinophils with potential functional implications in situ. Finally, we identified free granules deposited in the inflamed matrix with membrane protrusions, a typical morphological characteristic of vesicular formation. Based on this ultrastructural evidence, we propose that EoSVs are generated in situ from free secretory granules, a mechanism by which these granules gradually release their contents in the ECM. Together, our results highlight the mechanisms of cytolytic cell death as an important eosinophil secretory process in eosinophilic diseases and identify other pathways potentially participating in the propagation of the eosinophil response after cell death, with emphasis on the vesicular system derived from eosinophil secretory granules.