Probing Lymphocyte Responses to Modulation of F0F1-ATPase by BZ-423.

Abstract

Bz-423 is an immunomodulatory 1,4-benzodiazepine that binds to the oligomycin sensitivity conferring protein (OSCP) of the mitochondrial F0F1-ATPase and modulates its activity. Inhibition of the F0F1-ATPase by Bz-423 causes mitochondrial membrane hyperpolarization and superoxide production by the mitochondrial electron transport chain (ETC). Bz-423 ameliorates diseases in mouse models of lupus, arthritis and psoriasis. Unlike conventional immunosuppressants, Bz-423 does not interfere with normal immune function. Concomitant with its specific therapeutic effects, Bz-423 selectively induces apoptosis in pathogenic lymphocytes. As pathogenic lymphocytes are abnormally activated by chronic receptor stimulation, it was hypothesized that activation sensitizes cells to Bz-423. The overall goal of experiments reported in this dissertation was to identify factors that underlie the selectivity of Bz-423 on pathogenic lymphocytes observed in vivo. Towards this goal, anti-IgM and Bz-423 co-treatment in Ramos B cells was established as an in vitro model, and the apoptotic signaling pathways were explored. These experiments found that superoxide and calcium are critical second messengers that increase levels of BH3-only proteins Bim, Bmf, tBid, which in turns activates Bax and Bak. Activated Bax and Bak induce the release of mitochondrial pro-apoptotic factors and subsequent cell death. Bz-423 also activates NFAT. Mechanistic studies using Ramos B cells demonstrate superoxide induced by Bz-423 triggers the opening of CRAC channel, which leads to calcium influx, calcineurin activation, and NFAT activation. NFAT activation leads to transcription of NFAT-dependent genes including IRF4, which may play an important role in receptor editing. Bz-423 also renders cells resistant to subsequent activation, downregulates surface IgM expression, and activates NFAT but not NFB or AP-1. As these changes induced by Bz-423 are characteristics of anergic B cells, Bz-423 may also promote anergy. Anergy, apoptosis, and receptor editing are three main mechanisms to maintain peripheral tolerance and to repress autoimmunity. These effects may underlie efficacy and specificity of Bz-423 in vivo.