Human Eosinophils Secrete Preformed, Granule‐Stored Interleukin‐4 Through Distinct Vesicular Compartments

Abstract

Secretion of interleukin-4 (IL-4) by leukocytes is important for varied immune responses including allergic inflammation. Within eosinophils, unlike lymphocytes, IL-4 is stored in granules (termed specific granules) and can be rapidly released by brefeldin A (BFA)-inhibitable mechanisms upon stimulation with eotaxin, a chemokine that activates eosinophils. In studying eotaxin-elicited IL-4 secretion, we identified at the ultrastructural level distinct vesicular IL-4 transport mechanisms. Interleukin-4 traffics from granules via two vesicular compartments, large vesiculotubular carriers, which we term eosinophil sombrero vesicles (EoSV), and small classical spherical vesicles. These two vesicles may represent alternative pathways for transport to the plasma membrane. Loci of both secreted IL-4 and IL-4-loaded vesicles were imaged at the plasma membranes by a novel EliCell assay using a fluoronanogold probe. Three dimensional electron tomographic reconstructions revealed EoSVs to be folded, flattened and elongated tubules with substantial membrane surfaces. As documented with quantitative electron microscopy, eotaxin-induced significant formation of EoSVs while BFA pretreatment suppressed eotaxin-elicited EoSVs. Electron tomography showed that both EoSVs and small vesicles interact with and arise from granules in response to stimulation. Thus, this intracellular vesicular system mediates the rapid mobilization and secretion of preformed IL-4 by activated eosinophils. These findings, highlighting the participation of large tubular carriers, provide new insights into vesicular trafficking of cytokines.

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

“…EoSVs, in conjunction with small, classical, round vesicles, represent alternative pathways for transport of granule products to the plasma membrane for extracellular release [44,45]. Both vesicular compartments are immunolabeled positively for typical granule products [43,44].…”

“…It is interesting that cytoplasmic vesiculotubular structures, recently referred to as EoSVs ( Fig. 2A) [44], are sufficiently unique in eosinophils that their presence in the cytoplasm of granulocytes, devoid of specific granules, is useful for lineage assignment of granule-poor, activated cells [50].…”

“…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.…”

“…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.…”

Leukocyte lipid bodies: inflammation-related organelles are rapidly detected by wet scanning electron microscopy

“…Eosinophils activated by this agonist or other physiologic stimuli in vitro, like those recruited during inflammatory processes in vivo, exhibit various morphologic changes associated with activation (27), including the induction of new lipid body formation (7).…”