Biology

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 f...

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 prot...

Eosinophils are tissue-dwelling leukocytes, present in the thymus, and gastrointestinal and genitourinary tracts of healthy individuals at baseline, and recruited, often in large numbers, to allergic inflammatory foci and sites of active tissue repair. The biological significance of eosinophils is vast and varied. In health, eosinophils support uterine and mammary gland development, and maintain bone marrow plasma cells and adipose tissue alternatively activated macrophages, while in response to tissue insult eosinophils function as inflammatory effector cells, and, in the wake of an inflammatory response, promote tissue regeneration, and wound healing. One common mechanism driving many of the diverse eosinophil functions is the regulated and differential secretion of a vast array of eosinophil-derived cytokines. Eosinophils are distinguished from most other leukocytes in that many, if not all, of the over three dozen eosinophil-derived cytokines are pre-synthesized and stored withi...

Leukocyte lipid bodies are dynamic, functionally active organelles with central roles in inflammation. Here, we report that leukocyte lipid bodies are facilely detected by a versatile, potent technique, termed wet scanning electron microscopy (SEM), which combines the rapid preparation of light microscopy with the resolution of SEM. Using as leukocyte models resting and agonist-stimulated human eosinophils, cells that generate prominent numbers of lipid bodies in inflammatory conditions, we demonstrated that lipid bodies can be rapidly imaged as bright, highly contrasted structures under wet SEM and scored by computerized image processing. Critical advantages of this approach are that it permits cell observation in a fully hydrated system and facilitates lipid preservation. These attributes are especially important because lipid bodies are degraded during routine dehydration processes. Moreover, this technology is advantageous over lipophilic fluorescent probes because it allows sus...

Secretion is a fundamental cell process underlying different physiological and pathological events. In cells from the human immune system such as eosinophils, secretion of mediators generally occurs by means of piecemeal degranulation, an unconventional secretory pathway characterized by vesicular transport of small packets of materials from the cytoplasmic secretory granules to the cell surface. During piecemeal degranulation in eosinophils, a distinct transport vesicle system, which includes large, pleiomorphic vesiculo-tubular carriers is mobilized and enables regulated release of granule-stored proteins such as cytokines and major basic protein. Piecemeal degranulation underlies distinct functions of eosinophils as effector and immunoregulatory cells. This review focuses on the structural and functional advances that have been made over the last years concerning the intracellular trafficking and secretion of eosinophil proteins by piecemeal degranulation during inflammatory resp...