Eosinophils are innate immune leukocytes implicated in the initiation and maintenance of type 2 immune responses, including asthma and allergy. The ability to store and rapidly secrete preformed cytokines distinguishes eosinophils from most lymphocytes, which must synthesize cytokine proteins prior to secretion and may be a factor in the apparent Th2 bias of eosinophils. Multiple studies confirm that human eosinophils from atopic or hypereosinophilic donors can secrete over 30 cytokines with a varying and often opposing immune-polarizing potential. However, it remains unclear whether all of these cytokines are constitutively preformed and available for rapid secretion from eosinophils in the circulation of healthy individuals or are restricted to eosinophils from atopic donors. Likewise, the relative concentrations of cytokines stored within eosinophils have not been studied. Here, we demonstrate that human blood eosinophils are not singularly outfitted with Th2-associated cytokines but rather, constitutively store a cache of cytokines with nominal Th1, Th2, and regulatory capacities, including IL-4, IL-13, IL-6, IL-10, IL-12, IFN-gamma, and TNF-alpha. We demonstrate further rapid and differential release of each cytokine in response to specific stimuli. As agonists, strong Th1 and inflammatory cytokines elicited release of Th2-promoting IL-4 but not Th1-inducing IL-12. Moreover, a large quantity of IFN-gamma was secreted in response to Th1, Th2, and inflammatory stimuli. Delineations of the multifarious nature of preformed eosinophil cytokines and the varied stimulus-dependent profiles of rapid cytokine secretion provide insights into the functions of human eosinophils in mediating inflammation and initiation of specific immunity.

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.