Cysteinyl leukotrienes acting via granule membrane-expressed receptors elicit secretion from within cell-free human eosinophil granules

Background—Cysteinyl leukotrienes are recognized to act via receptors (CysLTRs) expressed on cell surface plasma membranes. Agents that block CysLT1R, are therapeutics for allergic disorders. Eosinophils contain multiple preformed proteins stored within their intracellular granules. Cell-free eosinophil granules are present extracellularly as intact membrane-bound organelles in sites associated with eosinophil infiltration, including asthma, rhinitis and urticaria, but have unknown functional capabilities. Objective—We evaluated the expression of CysLTRs on eosinophil granule membranes and their functional roles in eliciting protein secretion from within eosinophil granules. Methods—We studied secretory responses of human eosinophil granules isolated by subcellular fractionation. Granules were stimulated with cysteinyl leukotrienes and eosinophil cationic protein and cytokines were measured in the supernatants. Receptor expression on granule membranes and eosinophils was evaluated by flow cytometry and western blot. Results—We report that receptors for cysteinyl leukotrienes, CysLT1R, CysLT2R and the purinergic P2Y12 receptor (P2Y12R), are expressed on eosinophil granule membranes. Leukotriene (LT) C4 and extracellularly generated LTD4 and LTE4 stimulated isolated eosinophil granules to secrete eosinophil cationic protein. MRS 2395, a P2Y12R antagonist, inhibited cysteinyl leukotrienes-induced eosinophil cationic protein release. Montelukast, likely not solely as an inhibitor of CysLT1R, inhibited eosinophil cationic protein release elicited by LTC4 and LTD4 as well as by LTE4. Conclusion—These studies identify previously unrecognized sites of localization, the membranes of intracellular eosinophil granule organelles, and function for cysteinyl leukotriene-responsive receptors that mediate cysteinyl leukotriene-stimulated secretion from within eosinophil granules, including those present extracellularly

Human eosinophils constitutively express multiple Th1, Th2, and immunoregulatory cytokines that are secreted rapidly and differentially

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-γ, and TNF-α. 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-γ 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

Mature human eosinophils express functional Notch ligands mediating eosinophil autocrine regulation

Eosinophil chemotaxis and survival within tissues are key components in the development of tissue eosinophilia and subsequent effector responses. In this study, we demonstrate a novel mechanism of eosinophil autoregulation affecting migration and survival mediated through Notch signaling. We show for the first time that human blood eosinophils express Notch receptors and Notch ligands, expressions of which are influenced by the presence of eosinophil-activating granulocyte-macrophage colony-stimulating factor (GM-CSF). Evidence of Notch receptor activation and subsequent transcription of the Notch-responsive gene HES1 were observed in GM-CSF–stimulated eosinophils, confirming functionality of eosinophil-expressed Notch-signaling components. Moreover, by inhibiting Notch signaling with γ-secretase inhibitors or Notch receptor–specific neutralizing antibodies, we demonstrate that autocrine Notch signaling enhances stimulus-mediated actin rearrangement and eosinophil chemokinesis, and impairs eosinophil viability. Taken together, these data suggest autocrine Notch signaling, enhanced in response to tissue- or inflammatory-derived signals, influences eosinophil activity and longevity, which may ultimately contribute to the development of tissue eosinophilia and exacerbation or remediation of eosinophil effector functions