CD63 is tightly associated with intracellular, secretory events chaperoning piecemeal degranulation and compound exocytosis in human eosinophils

Abstract

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.

Vesicular trafficking of immune mediators in human eosinophils revealed by immunoelectron microscopy

“…Stimulation with inflammatory stimuli such as CCL11 [5] and tumor necrosis factor alpha (TNF-α) [14] induces increased formation of EoSVs. Moreover, eosinophils naturally activated as observed in hypereosinophilic disorders also exhibit amplified numbers of EoSVs [7].…”

“…Ultrastructural studies showed that the increased genesis of EoSVs within these cells is associated with secretion. In eosinophils stimulated with inflammatory stimuli, not only did the total number of EoSVs increase but also did the number of EoSVs in contact with secretory granules undergoing release of their contents [5, 14]. Ultrastructural images obtained by conventional EM applied to human eosinophils have also indicated that EoSV were projecting from granule boundaries.…”

“…Interestingly, a recent work from our group indicated that EoSVs are able to translocate products not only from but also to granules in response to cell activation, that is, EoSVs have a remarkable ability to interact with granules acting as transient pools of granule-derived mediators [14]. Thus, these transport carriers seem to have a much more complex role within eosinophils, participating actively in the release of granule products as well as in other granule-associated events.…”

“…A recent work using immunonanogold EM and EM quantitative analyses has identified an intracellular trafficking of CD63 associated with secretory granules within activated human eosinophils [14]. In response to eosinophil stimulation with inflammatory mediators (CCL11 and TNF-α), pools of CD63 traffic in the cytoplasm and accumulate within granules undergoing secretion likely acting as a “facilitator” molecule for the release of granulestored products [14].…”

“…The use of an improved approach for ultrastructural detection of proteins in leukocytes (pre-embedding immunonanogold EM) [13] led to the first ultrastructural identification of a vesicle-based transport of interleukin-4 (IL-4) [5] and major basic protein (MBP-1) [7] from eosinophil secretory granules. More recently, this technique located SNARES at secretory granules and vesicles [11] and identified an active intracellular CD63 trafficking connected to eosinophil granule-derived secretory pathways [14]. Here, we review the use of TEM to visualize and understand intracellular trafficking and secretion of immune mediators in eosinophil leukocytes during immune responses.…”