Intragranular Vesiculotubular Compartments are Involved in Piecemeal Degranulation by Activated Human Eosinophils

Abstract

Eosinophils, leukocytes involved in allergic, inflammatory and immunoregulatory responses, have a distinct capacity to rapidly secrete preformed granule-stored proteins through piecemeal degranulation (PMD), a secretion process based on vesicular transport of proteins from within granules for extracellular release. Eosinophil-specific granules contain cytokines and cationic proteins, such as major basic protein (MBP). We evaluated structural mechanisms responsible for mobilizing proteins from within eosinophil granules. Human eosinophils stimulated for 30-60 min with eotaxin, regulated on activation, normal, T-cell expressed and secreted (RANTES) or platelet activating factor exhibited ultrastructural features of PMD (e.g. losses of granule contents) and extensive vesiculotubular networks within emptying granules. Brefeldin A inhibited granule emptying and collapsed intragranular vesiculotubular networks. By immunonanogold ultrastructural labelings, CD63, a tetraspanin membrane protein, was localized within granules and on vesicles outside of granules, and mobilization of MBP into vesicles within and extending from granules was demonstrated. Electron tomography with three dimension reconstructions revealed granule internal membranes to constitute an elaborate tubular network able to sequester and relocate granule products upon stimulation. We provide new insights into PMD and identify eosinophil specific granules as organelles whose internal tubulovesicular networks are important for the capacity of eosinophils to secrete, by vesicular transport, their content of preformed and granule-stored cytokines and cationic proteins.

SIRPα/CD172a Regulates Eosinophil Homeostasis

“…Consistent with the hypothesis that the cytoplasmic domain of SIRPa/ CD172a plays an inhibitory role in degranulation, the SIRPa Cyto 2/2 mice showed a reduced number of eosinophils in the small intestinal lamina propria and an increased surface expression of the degranulation marker CD63 (Fig. 4), which is normally present in the membrane of intracellular crystalloid granules and appears on the cell surface during the piecemeal degranulation of human eosinophils (17,44). In addition, the SIRPa Cyto 2/2 mice showed an increased frequency of extracellular MBP + CD63 + granules and MBP + CD63 + amorphous materials in the lamina propria, increased collagen deposition in the submucosa, and smooth muscle hypertrophy in the jejunum, which are characteristic features of the increased tissue remodeling induced by eosinophil degranulation (45).…”

Mechanisms of eosinophil secretion: large vesiculotubular carriers mediate transport and release of granule-derived cytokines and other proteins

“…Recent data have brought conclusive evidence for the participation of morphologically distinct, large, membrane-bound, tubular compartments in the eosinophil secretory route [43][44][45]. Although these vesiculotubular structures have long been recognized in the cytoplasm of eosinophils [48][49][50] (Fig.…”

“…Both vesicular compartments are immunolabeled positively for typical granule products [43,44]. EPO-loaded vesicles and tubules were detected initially within eosinophils, which developed from human-cord blood mononuclear cell cultures supplemented with IL-5 [51].…”

“…How these transport vesicles are formed and mediate the rapid and specific secretion of eosinophil proteins are questions, which have been addressed recently. In addition to small, classical, round vesicles, it has now become apparent that the eosinophil secretory pathway is mediated by morphologically distinct, large pleiomorphic vesiculotubular carriers, termed eosinophil sombrero vesicles (EoSVs), which are responsible for moving proteins between granules and the plasma membrane [43][44][45]. During eosinophil secretion, EoSVs are actively formed from granules and direct differential release of proteins.…”

Reply to Eosinophil cytolysis and release of cell-free granules

“…A second type of degranulation, piecemeal degranulation, is a process that was first defined by the electron microscopic observations of Dvorak and colleagues 3 , who noted that, similarly to what had been observed for basophils, eosinophil granules do not empty as completely or as explosively as would be anticipated if exocytosis were taking place 4 . As discussed in detail in the original Review 1 , recent studies by Spencer, Melo, Weller and colleagues 5,6 have identified tubulovesicular subcellular structures and have elucidated receptorchaperone systems that are the among the important features underlying piecemeal degranulation.…”