Roles of Bcl-2 and caspases in anti-CD30 monoclonal antibody-induced primary human eosinophil apoptosis

Abstract

Background: In vitro studies have suggested that activation of cell surface CD30 by immobilized anti-CD30 monoclonal antibodies (mAb) induces strong apoptosis in human eosinophils. This anti-CD30 mAb-induced eosinophil apoptosis was inhibited by the addition of inhibitors of p38, ERK1/2 mitogen-activated protein kinases (MAPKs), and phosphatidylinositol 3-kinase (PI3K). However, there is little data investigating the role of Bcl-2 and caspases in eosinophil apoptosis induced by anti-CD30 mAb.Objectives: We sought to determine whether anti-CD30 mAb induces human eosinophil apoptosis via Bcl-2 and caspases pathways.Methods: Peripheral blood was drawn from 19 healthy volunteers. Purified human eosinophils were suspended in RPMI 1640 media supplemented with 10% FBS. The CD30 expression on eosinophils was measured at various time points. Eosinophils were then cultured in plates precoated with anti-CD30 mAb (clone Ber-H8), isotype control immunoglobulin G1, interleukin (IL)-5, or dexamethasone. Western blot analysis was performed to determine the expression of Bcl-2, procaspase-8, -9, and -3, and caspase-8, -9, and -3 after cross-linking of CD30. Human eosinophils were also cultured in plates precoated with anti-CD30 mAb (clone Ber-H8) in the presence or absence of caspase-9 or -3 inhibitors. Eosinophil apoptosis was assessed using flow cytometry.Results: The addition of anti-CD30 mAb significantly increased eosinophil apoptosis in vitro compared with controls. In western blot analysis, the addition of anti-CD30 mAb significantly decreased the expression of Bcl-2 and procaspase-9 and -3 and increased the expression of caspase-9 and -3. The addition of caspase-9 or -3 inhibitors at reasonable concentrations decreased anti-CD30 mAb-induced human eosinophil apoptosis in vitro. Procaspase-8 or caspases-8 expression was not changed in response to various stimuli.Conclusions:

Anti-CD30 mAb-induced human eosinophil apoptosis is likely to be mediated through Bcl-2 and caspase-9 and -3. Future studies are warranted to delineate downstream signaling molecules in the Bcl-2 family and caspases in human eosinophil apoptosis.