Interleukin-3, but not granulocyte–macrophage colony-stimulating factor and interleukin-5, inhibits apoptosis of human basophils through phosphatidylinositol 3-kinase: requirement of NF-κB-dependent and -independent pathways

Authors


R. Robert Schellenberg, MD, University of British Columbia, iCAPTURE Center, McDonald Research Laboratory, St. Paul's Hospital, 1081 Burrard Street, Vancouver, BC, Canada V6Z 1Y6. E-mail: Bschellenberg@mrl.ubc.ca

Summary

Basophils are key effector cells of allergic reactions. Although proinflammatory cytokines, such as interleukin (IL)-3, granulocyte–macrophage colony-stimulating factor (GM–CSF) and IL-5, inhibit eosinophil apoptosis in vitro, little is known about basophil apoptosis, and the signalling mechanisms required for basophil survival remain undefined. To address this issue, we used a novel negative-selection system to isolate human basophils to a purity of > 95%, and evaluated apoptosis by morphology using light and transmission electron microscopy, and by annexin-V binding and propidium iodide incorporation using flow cytometry. In this study, we demonstrated that the spontaneous rate of apoptotic basophils was higher than that of eosinophils as, at 24 hr, 57·6 ± 4·7% of basophils underwent apoptosis compared with 39·5 ± 3·8% of eosinophils. In addition, basophil cell death was significantly inhibited when cultured with IL-3 for 48 hr (84·6 ± 4·9% vehicle-treated cells versus 40·9 ± 3·9% IL-3-treated cells). IL-3 also up-regulated basophil CD69 surface expression. The effects of IL-3 on apoptosis and CD69 surface expression of human basophils were completely blocked by LY294002 (LY), a potent inhibitor of phosphatidylinositol 3-kinase (PI3-K), but only partially inhibited by lactacystin, a proteasome inhibitor that prevents degradation of IκB and NF-κB translocation. These observations reveal the novel finding that IL-3 prevents basophil apoptosis through the activation of PI3-K, which is only partially NF-κB dependent. As basophils are active participants in allergic reactions and IL-3 is one of the abundant proinflammatory cytokines in secretions from allergic tissue, we suggest that IL-3-mediated inhibition of basophil apoptosis may exacerbate the inflammation associated with allergic disorders.

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