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

Abstract

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.

Fig. 1.

Preformed cytokines are stored within intracellular granules. Eosinophil subcellular fractions, prepared and evaluated per Materials and Methods, were analyzed by multiplex analysis for cytokine content. Fractions containing granules and small, less-dense vesicles and cytosol are indicated. Data are from eosinophils from one donor and are representative of studies of eosinophils from three donors.

Fig. 2.

Dose-dependent release of cytokines from eosinophils in response to cytokine stimuli. Eosinophils (3×10⁶/mL) were incubated for 30 min at 37°C with increasing doses of indicated cytokine stimuli. Following stimulation, cell-free supernatants were analyzed by multiplex analysis for the presence of IL-4 (A), IL-13 (B), IL-6 (C), IFN-γ (D), IL-12(p70) (E), IL-10 (F), and TNF-α (G). Data are presented as means (±sd) of duplicate or triplicate wells of one experiment representative of at least three independent experiments. *, P ≤ 0.05, versus nonstimulated controls.

Fig. 3.

Pretreatment with IL-10 does not affect eosinophil cytokine secretion. Eosinophils (3×10⁶/mL) were preincubated for 20 min at 37°C ± 100 ng/mL rIL-10 (P) prior to addition of 100 ng/mL rTNF-α or rIFN-γ for a subsequent 20 min stimulation (S). Cell-free supernatants were analyzed as above. Data are the means (±sd) of duplicate wells from one experiment, representative of three independent experiments.

Fig. 4.

Simultaneous stimulation with multiple cytokines elicits additive effects on eosinophil cytokine secretion. Eosinophils (3×10⁶ per mL) were stimulated with 100 ng/mL IFN-γ, IL-12, and TNF-α, alone or in various combinations, for 30 min at 37°C. Cell-free supernatants were analyzed as above for the presence of IL-4 (A), IL-6 (B), IL-10 (C), IL-13 (D), TNF-α (E), IL-12 (F), and IFN-γ (G). Means (±sd) of stimulation with individual cytokines are stacked and plotted beside results from the appropriate dual stimulation.